2014 Book InflammationAndCancer PDF
2014 Book InflammationAndCancer PDF
2014 Book InflammationAndCancer PDF
Bharat B. Aggarwal
Bokyung Sung
Subash Chandra Gupta Editors
Inflammation
and Cancer
Advances in Experimental Medicine and Biology
Volume 816
Editorial Board
Irun R. Cohen, The Weizmann Institute of Science, Rehovot, Israel
Abel Lajtha, N.S. Kline Institute for Psychiatric Research, Orangeburg, NY, USA
John D. Lambris, University of Pennsylvania, Philadelphia, PA, USA
Rodolfo Paoletti, University of Milan, Milan, Italy
13
Editors
Bharat B. Aggarwal
Bokyung Sung
Subash Chandra Gupta
Department of Experimental Therapeutics
The University of Texas M.D. Anderson
Cancer Center
Houston, TX
USA
It was Aulus Cornelius Celsus, a physician in first-century Rome, who first defined
inflammation as calor (heat), dolor (pain), rubor (redness), and tumor (swell-
ing). However, it was Rudolf Virchow who in the mid-1800s linked inflammation
with atherosclerosis, rheumatoid arthritis, multiple sclerosis, asthma, Alzheimer’s
disease, cancer, and other chronic diseases. The suffix “-itis” was introduced to indi-
cate inflammation in words such as bronchitis (inflammation of the bronchus) and
colitis (inflammation of the colon). Extensive research has revealed that inflam-
mation precedes most cancers; for example, cancers of the liver, lung, colon,
cervix, pancreas, stomach, and prostate are preceded by hepatitis, bronchitis,
colitis, cervicitis, pancreatitis, gastritis, and prostatitis, respectively.
Within the past three decades, researchers have determined the molecular basis
of most kinds of inflammation. Furthermore, various cell-signaling pathways
that lead to inflammation have also been relatively well defined, leading to the
development of various therapeutics that can modulate these pathways and thus
alter the course of disease.
The current monograph deals with the role of inflammation in cancer, and some
of the leaders in the field have contributed to this volume. We would like to thank
these experts for their contributions and the publisher for giving us the opportunity
to edit this volume.
Bharat B. Aggarwal
Bokyung Sung
Subash Chandra Gupta
v
Contents
vii
viii Contents
Kazım Şenol, Murat Bulut Özkan, Selahattin Vural and Mesut Tez
10 The Role of Inflammation in Gastric Cancer. . . . . . . . . . . . . . . . . . . . 235
Editors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473
Contributors
ix
x Contributors
James Finke Cleveland Clinic Lerner College of Medicine, Case Western Reserve
University, Cleveland, OH, USA
Tamer M. Fouad Department of Breast Medical Oncology, Morgan Welch
Inflammatory Breast Cancer Research Program and Clinic, The University of
Texas MD Anderson Cancer Center, Houston, TX, USA; Department of Medical
Oncology, The National Cancer Institute, Cairo University, Cairo, Egypt
Ciara Freeman Department of Haematology, Barts and the Royal London NHS
Trust, London, UK
Helmut Friess Department of Surgery, Klinikum rechts der Isar, Technische
Universität München, Munich, Germany
Georgios Gakis Department of Urology, University Hospital Tübingen,
Eberhard-Karls University, Tübingen, Germany
Khushboo Gandhi Maru Lab, Advanced Centre for Treatment, Research and
Education in Cancer (ACTREC), Tata Memorial Centre (TMC), Kharghar, Navi
Mumbai, India
Francis J Giles Northwestern Medicine Developmental Therapeutics Institute,
Robert H. Lurie Comprehensive Cancer Center of Northwestern University,
Chicago, USA
Annunziata Gloghini Department of Diagnostic Pathology and Laboratory
Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
Mónica Gomes Instituto Português de Oncologia do Porto Francisco Gentil, EPE,
Grupo de Oncologia Molecular—CI, Porto, Portugal; ICBAS, Abel Salazar I nstitute
for the Biomedical Sciences, University of Porto, Porto, Portugal; LPCC Research
Department-Portuguese League Against Cancer (NRNorte), Porto, Portugal
Simone Hausmann Department of Surgery, Klinikum rechts der Isar, Technische
Universität München, Munich, Germany
Heidi A. Hempel Department of Pathology, The Johns Hopkins University School
of Medicine, Baltimore, MD, USA
Naveena B. Janakiram Center for Cancer Prevention and Drug Development,
Department of Medicine, Hematology Oncology Section, PCS Cancer Center,
University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
Kenneth M. Johnson Department of Pharmacology and Toxicology, The
University of Texas Medical Branch (UTMB), Galveston, TX, USA
Takahiro Kogawa Department of Breast Medical Oncology, Morgan Welch
Inflammatory Breast Cancer Research Program and Clinic, The University of Texas
MD Anderson Cancer Center, Houston, TX, USA
Bo Kong Department of Surgery, Klinikum rechts der Isar, Technische Universität
München, Munich, Germany
Contributors xi
Abstract Lung cancer remains a serious public health problem and is the first
cause of cancer death worldwide, and the overall 5-year survival rate for all stages
is 14–17 % for Non-small-cell lung cancer and 6 % for small-cell lung cancer.
Clinical and epidemiologic studies have suggested a strong association among
chronic infection, inflammation, and cancer. Immune system plays a critical role
in maintaining tissue homeostasis, cell turnover, tissue remodeling, and preventing
In 2008, about 12.7 million cancer cases and 7.6 million cancer deaths are
estimated to have occurred in this year in worldwide, with 56 % of cases and
64 % of the deaths in the economically developing world (Jemal et al. 2011).
Lung cancer was found to be the most commonly diagnosed cancer as well as
the primary cause of cancer-related mortality for males worldwide and the sec-
ond leading cause of cancer-related deaths for women (Jemal et al. 2011; Siegel
et al. 2012). For the year 2012, it is estimated that lung cancer will account for
26 % of all female cancer deaths and 29 % of all male cancer deaths (Siegel et al.
2012). Breast cancer in females and lung cancer in males are the most frequently
diagnosed cancers and the leading cause of cancer death for each sex in both eco-
nomically developed and developing countries, except lung cancer is preceded by
prostate cancer as the most frequent cancer among males in economically devel-
oped countries (Jemal et al. 2011).
Lung cancer was the most commonly diagnosed cancer as well as the leading
cause of cancer death in males in 2008, globally. Among females, it was the fourth
most commonly diagnosed cancer and the second leading cause of cancer death
(Jemal et al. 2011; Ferlay et al. 2010). Lung cancer accounts for 13 % (1.6 mil-
lion) of the total cases and 18 % (1.4 million) of the deaths in 2008 (Ferlay et al.
2010; Jemal et al. 2011).
The observed variations in lung cancer rates and trends across countries or
between males and females within each country largely reflect differences in the
stage and degree of the tobacco epidemic (Jemal et al. 2011).
Lung cancer can be divided into two major groups: small-cell lung cancer
(SCLC) and non-small-cell cancer (NSCLC) (Hoffman et al. 2000; Molina et al.
2008), NSCLC accounts for approximately 85 % of all cases of lung cancer
1 The Role of Inflammation in Lung Cancer 3
(Molina et al. 2008; Araujo et al. 2007). These lung cancer cells can again be
categorized based on their histological characteristics as squamous cell carci-
noma, large cell carcinoma, and adenocarcinoma (Tang et al. 2013). NSCLC
spreads slower than SCLC, so many patients who are diagnosed at an earlier stage
are potentially curable, though NSCLC may often relapse at other metastatic site.
Furthermore, NSCLC is generally less responsive to chemotherapy than SCLC,
so that even with surgical resection at early diagnosis, approximately 50 % of
NSCLC patients face recurring cancers (Tang et al. 2013).The 1-year survival rate
for lung cancer was 43 % in 2003–2006. However, despite extensive preclinical
and clinical research, the overall 5-year survival rate for all stages is still as low as
14–17 % for NSCLC (Araujo et al. 2007; Peebles et al. 2007) and even lower in
SCLC (6 %) (Wu et al. 2012).
In recent years, knowledge concerning the molecular mechanisms underly-
ing cellular transformation and development of cancer has been greatly expanded
(Araujo et al. 2007). Alteration of the major cell signaling and regulatory path-
ways either by overexpression or gene sequence variation is a frequent event in
lung cancer. These changes include alterations in receptor tyrosine kinases (TKs),
such as epidermal growth factor receptor (EGFR), and alterations in angiogenesis
pathways, apoptosis, proteasome regulation, and cell cycle control, among others
(Molina et al. 2008).
The EGFR is a tyrosine kinase that contributes to the regulation of cellular
homeostasis. It is a 170-KDa membrane protein that stimulates downstream cell
proliferation, survival, and tumorigenesis (Wheeler et al. 2010; Cohen 1965).
EGFR has been implicated in the growth of several human epithelial malignancies,
including lung cancer. It is overexpressed in several cancers, including approxi-
mately 40–80 % of NSCLC, which made EGFR a popular target for new drug
treatment exploration (Tang et al. 2013).
The ALK tyrosine kinase receptor has gained much attention recently as a
newly emerging relevant biomarker and therapeutic target in NSCLC (Wu et al.
2012). The activation of ALK is primarily through the formation of fusion genes.
EML4-ALK translocation is the most common ALK gene rearrangement. This
rearrangement in NSCLC patients is mainly found in younger non-smoking
patients with adenocarcinoma (Wu et al. 2012; Kwak et al. 2010). EML4-ALK
rearrangements are mutually exclusive with EGFR or KRAS mutations (Wu et al.
2012; Li et al. 2013). It has been reported that approximately 2–11 % of tumors
carrying positive EML4-ALK (Li et al. 2013).
KRAS mutations are a negative predictor of response to EGFR TKs, mainly
accounting for primary resistence (Linardou et al. 2008; de Mello et al. 2011).
Most KRAS mutations in lung adenocarcinoma are associated with smoking.
KRAS positive mutations are limited to NSCLC and are mutually exclusive to
mutations in EGFR and ALK (Linardou et al. 2008; Wu et al. 2012).
Lung cancer is a very aggressive cancer and its treatment still remains a chal-
lenge for health professionals. Conventional treatments are based on surgery, radi-
ation therapy, and chemotherapy. The selection of therapeutic regimen is based on
the cancer type (small-cell or non-small-cell), stage of disease, patient’s functional
4 M. Gomes et al.
ability, and genetic characterization (Wu et al. 2012; Tang et al. 2013; Hoffman
et al. 2000).
The majority of stage I through stage IIIA lung cancer patients generally choose
surgery as their primary option. Another popular option is preoperative chemo-
therapy, which has been shown to improve survival rate in patients with NSCLC.
Patients who require complete resection and no preoperative chemotherapy usually
invest in adjuvant chemotherapy. For patients with unresectable NSCLC, RT and
chemotherapy are excellent options for treatment (Tang et al. 2013). Further, cer-
tain agents have been combined with the chemotherapy to enhance its effects. The
anti-vascular endothelial growth factor agent, bevacizumab, for example, when
combined with chemotherapy, has resulted in increased survival rate when com-
pared to chemotherapy treatment alone (Tang et al. 2013).
For first-line chemotherapy, a platinum-based two-drug combination is sug-
gested for patients (Azzoli et al. 2009; Molina et al. 2008). Studies show that the
cisplatin, when used in combination chemotherapy, is associated with improved
response rates, no change in survival rate, and increased toxicity when compared
with the carboplatin (Tang et al. 2013). Also, another drug bevacizumab has dem-
onstrated great potential when used in combination with carboplatin or paclitaxel
in NSCLC patients (Tang et al. 2013; Molina et al. 2008).
The second-line chemotherapy treatment options, after primary treatment fails
to yield effective results, do differ from the first-line drugs. Approximately 30 %
of NSCLC patients who undergo first-line cancer treatment are candidates for
second- or third-line therapeutics. The first agent that was approved for second-
line therapeutics was docetaxel (Fossella et al. 2000). Other drugs that were also
soon approved include pemetrexed, erlotinib, and gefitinib (Tang et al. 2013).
Undergoing research is currently evaluating other possible strategies for second-
line therapeutics.
cancerous tissue often arose at sites of chronic inflammation and that inflammation
cells were present in the resect tumors (Bremnes et al. 2011; Mantovani et al. 2008;
Balkwill and Mantovani 2001). In contrast, Burnet proposed, in 1970, the concept
of immunological surveillance: the immune system spontaneously identifies and
eliminates cancer cells, thus protecting against tumor development (Bremnes et al.
2011; Van Ginderachter et al. 2006) (Fig. 1.1).
During the last decades, and according to Virchow hypothesis, epidemiological
studies have shown that individuals prone to chronic inflammatory diseases have
an increased risk of cancer development and that the underlying infections and
inflammatory responses have been linked to 15–20 % of all cancer deaths world-
wide (Bremnes et al. 2011).
The ultimate recognition of inflammation as a major player in cancer develop-
ment was reinforced with the 2011 update article on cancer hallmarks by Hanahan
and Weinberg, where it was classified as an enabling characteristic of tumors
(Mantovani et al. 2008; Hanahan and Weinberg 2011).
These evidence gathered over the last years showed that inflammation con-
tributes to the appearance of multiple cancer hallmark capabilities by supplying
important molecules to the tumor microenvironment. Those molecules include
growth factors that sustain the proliferative signaling, survival factors that limit
apoptosis, pro-angiogenic factors, extracellular matrix-modifying enzymes that
favor angiogenesis, invasion, and metastasis (Ben-Baruch 2006). Furthermore,
inflammation manifestations are observed at the earliest stages of tumor progres-
sion and are capable of nurturing insipient neoplasias into developed cancers.
In addition, inflammatory cells can release a number of chemicals, such as ROS,
6 M. Gomes et al.
that are actively mutagenic and promote malignancy even further (Hanahan and
Weinberg 2011).
Immune system plays critical roles in maintaining tissue homeostasis, cell
turnover, tissue remodeling, and preventing infection and cell transformation. It
is composed of two distinct compartments mediating innate and adaptive immune
response. Each compartment has, through a diversity of cells and soluble mediators,
advanced communication networks, which enable rapid and effective responses to
tissue injury (Bremnes et al. 2011).
Although it is clear that inflammation itself is not the cause of the onset of can-
cer cell proliferation, a sustained atmosphere rich in inflammatory cells, growth
factors, and promoters activated stromal DNA damage may enhance and/or pro-
mote risk for the emergence of malignancies. This tumor microenvironment is
composed not only by resident tissue cells such as fibroblasts and endothelial cells
but also by infiltrating host leukocytes (Ben-Baruch 2006). Tumor cells produce
several cytokines and chemokines that attract leukocytes. Several inflammatory
cytokines have been implicated to mediate different steps in the pathway leading
to carcinogenesis. Increased serum levels of pro-inflammatory interleukins IL-1β,
IL-6, IL-8, IL-12, and IL-18 have been observed in different types of cancer,
including lung cancer (Tsai et al. 1999; Srivani and Nagarajan 2003; Michalaki et
al. 2004; Ye et al. 2007; Azevedo et al. 2011).
On the other hand, the pleiotropic anti-inflammatory interleukins, IL-4 and
IL-10, stimulate the growth of many tumors, such as ovarian, prostate, and lung
although they have an inhibitory effect on growth or invasion of other types of
cancer (Toi et al. 1992; Takeshi et al. 2005; Lan et al. 2006; Gomes et al. 2012)
(Fig. 1.2).
The inflammatory component in the development of the neoplasm includes a
diverse leukocyte population, which stand macrophages (abundant in many types
of tumors), lymphocytes, natural killer (NK) cells, neutrophils, and dendritic cells.
These components are considered inflammatory tumor key factors in pro-
moting tumor progression due to its ability to release a variety of cytokines,
chemokines, cytotoxic mediators such as reactive oxygen species, metallopro-
teinases (MMPs) and agents perforator membrane, and soluble mediators of cell
death, such as TNF-α (Tumor Necrosis Factor-α), interleukins (IL), and interfer-
ons (IFNs) (Coussens and Werb 2002).
Many of the mediators released during chronic inflammation promote unregu-
lated cell proliferation and invasion, induce angiogenesis, and increase mutagen-
esis. Due to these characteristics, the transformation and initiation of a malignant
phenotype may occur and tumor progression may be promoted. In addition, many
of the factors released by inflammatory cells may lead directly or indirectly to a
marked suppression of the immune response, which otherwise could have an
important role in tumor eradication (Ben-Baruch 2006).
The lung cancer tumor microenvironment is composed of extracellular matrix,
tumor cells, fibroblasts, inflammatory cells, vascular and lymphatic endothelial
cells, growth factors, cytokine, chemokines, hormones, proteases, among oth-
ers. Fibroblasts exist normally in the connective tissue and produce extracellular
1 The Role of Inflammation in Lung Cancer 7
Fig. 1.2 The tumor microenvironment associated with inflammation and its consequence in can-
cer processes (adapted from Serefoglou et al. 2008)
matrix and collagen. They are essential in wound repairing but when exposed to
cigarette smoke, they secrete pro-inflammatory mediators such as prostaglandin-E2
(PGE2), IL-8, IL-6, and MCP-1, leading to a prolonged inflammatory response
(Martey et al. 2004). Macrophages are recruited by granulocyte colony-stimulating
factor (G-CSF), GM-CSF, macrophage-stimulating protein (MSP), vascular
endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and
macrophage migration inhibitory factor (MIF).
It is known that macrophages can be activated in response to microbiological
agents and in particular cytokines interferon-γ (IFN-γ) (classical macrophage acti-
vation). However, it was recently discovered that anti-inflammatory molecules,
such as glucocorticoid hormones and cytokines IL-4, IL-13, and IL-10, induce a
different program activation of macrophages (alternative macrophage activation)
(Sica et al. 2006; Mantovani et al. 2002, 2004). Tumor-associated macrophages
(TAM) that interact with tumor cells to produce cytokines and growth factors
that influence tumor development have two different phenotypes: M1 and M2
(O’Callaghan et al. 2010). The M1 macrophages have been associated with better
prognosis in NSCLC, are efficient immune cells and are associated with an anti-
tumor behavior (Bremnes et al. 2011). Nonetheless, the most prevalent phenotype
is the M2, which promotes tumor growth, angiogenesis, invasion, and metastasis.
8 M. Gomes et al.
There are some events that are required to drive from initiated cells to malignant
tumors (Hanahan and Weinberg 2011). The infiltration of immune cells to tumors
may repress tumor growth (Dunn et al. 2002). However, the increasing concern
hypothesis is that inflammatory cells act as tumor promoters in inflammation-
associated cancers (Smyth et al. 2004). Accumulated mutations in epithelial cells
lead to dysregulation of their growth and migration. These dysregulated may also
produce cytokines and chemokines to attract immune cells to facilitate cancer
development (Lin and Pollard 2004; Coelho et al. 2006).
Several studies on tumor–host interaction have highlighted the importance of
inflammatory response in the early steps of carcinogenesis as well as in estab-
lished progressive tumors and are beginning now to identify the contribution of
polarized inflammatory responses in cancer progression (Sica et al. 2006; Wang
and Joyce 2010).
TNF is a transforming agent for carcinogen-treated fibroblast. Two weeks of
exposure to the cytokine in vitro is sufficient to render cells capable of tumor
formation in nude mice (Komori et al. 1993). The molecular basis may involve
induction of reactive oxygen. Reactive oxygen in the form of NO is often gener-
ated by inflammatory cytokine induction of NO synthase. NO is an important reg-
ulatory molecule in both inflammation and cancer development (Lu et al. 2006).
NO can directly oxidize DNA, resulting in mutagenic changes, and may damage
some DNA repair proteins (Jaiswal et al. 2000). In study of Yan and co-workers,
they revealed that TNF-α is potent mutagen that causes DNA damage through
the induction of ROS (Yan et al. 2006; Aggarwal et al. 2006). This study brings
10 M. Gomes et al.
up two new concepts, a mechanism through which a cytokine can induce genetic
instability and the involvement of the TNF-α-mediated DNA damage pathway in
inflammation-associated carcinogenesis (Yan et al. 2006). DNA damage can be
induced by conventional mutagens, such as radiation and chemicals, or endoge-
nous from errors in DNA replication or ROS produced cell metabolism. Yan and
co-workers found that endogenous cytokine TNF-α is potent mutagen by virtue of
its ability to induce ROS (Yan et al. 2006). Therefore, TNF-α drives tumor devel-
opment by promoting the accumulation of mutations and survival of precancerous
or transformed cells (Yan et al. 2006).
A large number of studies suggest that TNF and chemokines are candidate linking
molecules between inflammation and cancer (Lu et al. 2006). The TNF, which is
produced mainly by activated macrophages but also by tumor cells, binds to mem-
brane-bound homotrimeric receptors (Lu et al. 2006).
It is well established the critical role of TNF-α in chronic inflammatory dis-
eases, and its tumor-promoting effects have been demonstrated (Lin and Karin
2007). The production of TNF-α by tumor cells or inflammatory cells in the tumor
microenvironment can promote tumor cell survival through the induction of genes
encoding NF-кB-dependent anti-apoptotic molecules (Lin and Karin 2007; Luo
et al. 2004). Furthermore, TNF-α promotes cell survival and thereby reduces
asbestos-induced cytotoxicity, increasing the pool of asbestos-damaged mesothe-
lial cells that are susceptible to malignant transformation (Yang et al. 2006; Lin
and Karin 2007). Other actions of TNF-α that might enhance tumor progression,
as opposed to tumor initiation, include promotion of angiogenesis and metasta-
sis, as well as impairment of immune surveillance by strongly suppressing many
T cell responses and the cytotoxic activity of activated macrophages (Elgert et al.
1998; Lin and Karin 2007).
Transforming growth factor-β (TGFβ), an immunosuppressive cytokine (Flavell
et al. 2010), with a pleiotropic role in tumor biology, is a cytokine frequently over-
expressed in many cancers, including NSCLC (Bruno et al. 2013; Teixeira et al.
2011; Siegel and Massague 2003). TGFβ belong to widely expressed family of
cytokines with pleiotropic effects on a variety of cellular functions such as cell
growth, proliferation, differentiation, and apoptosis (Luo et al. 2010).
TGFβ also has a role in the tumor microenvironment immune cell polariza-
tion, including macrophages, neutrophils, and NK cells associated with tumor
immune evasion (Flavell et al. 2010; Siegel and Massague 2003). High expres-
sion of TGFβ is characteristic of NSCLC and predictive of poor survival
(Teixeira et al. 2011).
Interleukin-10 (IL-10) is a multifunctional cytokine with both immunosuppres-
sive and anti-angiogenic functions and consequently has both tumor-promoting
1 The Role of Inflammation in Lung Cancer 11
and tumor-inhibiting properties (Shih et al. 2005). Raised levels of serum and
peri-tumoral IL-10 production have been reported in many malignant (Dummer
et al. 1995), including lung cancer (Shih et al. 2005), which have been inter-
preted in support of a role for IL-10 in tumor escape from the immune response.
Furthermore, increased production of immunosuppressive IL-10 by NSCLC
and increased serum concentrations of IL-10 in NSCLC patients have both been
shown recently to correlate with reduced survival (Shih et al. 2005). Serum levels
of IL-10 were found to be elevated in NSCLC patients when compared to healthy
controls; moreover, IL-10 serum levels were demonstrated to be higher in patients
with metastatic disease as opposed to the values recorded in patients with undis-
seminated cancer (De Vita et al. 2000). IL-10 promotes tumor malignancy by
promoting T cell apoptosis and tumor cell survival (Wang et al. 2012). In lung
carcinomas, IL-10 production can inhibit tumor cell susceptibility to cytotoxic
T-lymphocyte-mediated killing (Asselin-Paturel et al. 2001). IL-10 transgenic
cytotoxic mice injected with Lewis lung carcinoma cells developed larger tumors
than control mice, suggesting that the production of IL-10 prevents the develop-
ment of an effective immune response against the tumor cells (Montuenga and
Pio 2007).
have not been fully understood (Chen et al. 2011). There is considerable evidence
that NF-кB is constitutively activated in a variety of solid tumors, including pros-
tate, breast, cervical, pancreatic, and lung cancer (Chen et al. 2011; Karin 2008).
Although lung tumors are histologically heterogenic, tumor samples obtained
from lung cancer patients showed high levels of NF-кB activation in both SCLC
and NSCLC and are significantly associated with disease advancement in TNM
stages and poor prognosis in lung cancer patients (Chen et al. 2011). Inhibiting
NF-кB with different approaches such as siRNA, IKK inhibitors, and IkappaB
super suppressor inhibited lung cancer cell’s survival and proliferation (Chen
et al. 2011; Karin 2008).
12 M. Gomes et al.
The tumor vasculature is derived from sprouting of local blood vessels (angiogen-
esis) and circulating vasculogenic progenitor cells derived from the bone marrow
(vasculogenesis). The new vessels are often irregular and leaky due to lack of the
pericyte cover, with the result that tumor cells can penetrate them more easily. As
compared to blood capillaries, lymphatic endothelial cells have even less devel-
oped junctions with frequently large interendothelial gaps and impaired basement
membranes (Kessenbrock et al. 2010). The invasive margin is a critical area for
stimulation of angiogenesis and lymphangionesis in tumors, which contributes to
tumor invasion and metastasis (Padera et al. 2002).
The major MMPs involved in tumor angiogenesis are MMP-2, MMP-9, and
MMP-14, and to a lesser extent MMP-1 and MMP-7 (Kessenbrock et al. 2010;
Rao et al. 2005).
MMPs are a family of proteolytic enzymes that are capable of degrading vari-
ous components of the extracellular matrix (Liu et al. 2012). They are involved
in all stages of cancer progression, not only in the process of tumor invasion and
metastasis (Hu et al. 2005) but also in as proliferation, adhesion, migration, dif-
ferentiation, angiogenesis, senescence, autophagy, apoptosis, and evasion of the
immune system (Gonzalez-Arriaga et al. 2012; Deryugina and Quigley 2006).
The expression of these MMPs by tumor cells may help to increase the invasive
potential of tumor cells by allowing the remodeling of the extracellular matrix
1 The Role of Inflammation in Lung Cancer 13
Fig. 1.3 Overview of
the role of inflammatory
molecules in development of
lung cancer
(Hu et al. 2005; Gomes et al. 2011). The proteolytic activity is also required for
a cancer cell to invade a nearby blood vessel (intravasation) and then extravasate
at a distant location and invade the distant tissue in order to seed a new metastatic
site (Roy et al. 2009). Increased expression of MMP-2 and MMP-9 was shown to
correlate with an invasive phenotype of cancer cells (Vihinen and Kahari 2002).
Several recent reports confirmed lung neoplastic cells produce both and their
inhibitors (Brown et al. 1993).
MMPs have also been implicated in the epithelial to mesenchymal transition
(EMT), a hallmark of cancer progression to metastasis (Thiery 2002; Roy et al.
2009; Rao et al. 2005). During EMT, tumor cells acquire migratory characteris-
tics and more readily invade into surrounding tissues and metastasize to secondary
sites (Roy et al. 2009; Rao et al. 2005).
Several studies have reported that plasma and/or serum levels of MMP-9 and
TIMP-1 are elevated in patient with stage III or IV lung cancer, when compared
with those in patients with nonmalignant lung diseases (Jumper et al. 2004; Koc
et al. 2006). Retrospective studies of NSCLC tissue found that MMP-7 expression
was higher in squamous cell carcinomas than in adenocarcinomas and correlated
with significantly lower overall in patients (Liu et al. 2007). In the normal lung,
MMP-9 is not produced by resident cells, but under various forms of stimulation,
bronchial epithelial cells, alveolar type II cells, fibroblasts, smooth muscle cells,
and endothelial cells produce MMP-9 (Atkinson and Senior 2003). Leukocytes in
the lung can also be a source of MMP-9. Macrophages, eosinophils, mast cells,
lymphocytes, NK cells, and dendritic cells all can produce MMP-9 (Atkinson and
Senior 2003). Lung cancer cells, both primary and metastatic, can express MMP-9
constitutively, which may correlate with metastatic potential (Atkinson and Senior
2003; Zucker et al. 1992; Baruch et al. 2001) (Fig. 1.3).
14 M. Gomes et al.
In study of Zeni and co-workers, they show that expression of IL-10 is increased in
TAMs of patients with stage II, III, and IV NSCLC compared with those with stage
I NSCLC. In adition, IL-10 positive TAM percentage was higher in patients with
lymph node metastases than in those without lymph node metastases. Moreover,
higher IL-10 expression by TAMs was associated with shorter overall survival
(Zeni et al. 2007). This study was the first time they showed, in NSCLC, TAMs
express IL-10 and that its expression correlates with both disease progression and
prognosis (Zeni et al. 2007). By the other hand, the study of Hatanaka and co-work-
ers shows that NSCLC patients with high IL-10-expressing tumors showed poorer
prognosis than those without IL-10 expression (Hatanaka et al. 2000).
Another important molecule is RANTES (Regulated on Activation, Normal
T cell Expressed and Secreted); also, CCL5 is a known chemotactic cytokine that
is produced by many cell types, including T-lymphocytes, monocytes, platelets,
eosinophils, epithelial cells, dendritic cells, and mast cells (Umekawa et al. 2013).
RANTES has been used as a prognostic indicator in both breast and cervical can-
cers, and high levels of RANTES in these malignancies correlate with poor out-
come (Borczuk et al. 2008; Niwa et al. 2001).
Umekawa and co-workers showed that, in NSCLC patients, high level of
plasma RANTES at diagnosis was associated with the severity of general fatigue.
Low level of plasma RANTES at diagnosis was significantly associated with long-
term survival. Thus, patients with high systemic inflammation, as represented
by RANTES, may experience severe general fatigue and shorter survival time
(Umekawa et al. 2013). In another study, Moran and co-workers found a correla-
tion between increased RANTES expression and tumor lymphocytic response in
lung cancer patients (Moran et al. 2002).
De Vita, in 1998, has evaluated serum levels of IL-6 in a group of advanced
NSCLC patients and found that patients who respond to cisplatin-based chemo-
therapy have lower serum IL-6 levels when compared with unresponsive patients.
Their data suggest that NSCLC patients with high levels of IL-6 have a worse clin-
ical outcome and may manifest resistance to cisplatin chemotherapy (De Vita et al.
1998). However, in study of Chang and co-workers, they failed to demonstrate that
exogenous or endogenous IL-6 could influence cisplatin or etoposide sensitivity of
the tested NSCLC cells at cellular level (Chang et al. 2005).
cancer mortality (Zhan et al. 2013; Vaish and Sanyal 2011; Setia and Sanyal
2012). Both clinical and experimental studies support the anti-neoplastic effects
of NSAIDs mediated by regulation of COX-2 levels and induction of apoptosis
(Haynes et al. 2003). A daily intake of NSAIDs for 1 or 2 years is reported to
reduce 60–68 % of relative risk of lung cancer (Harris et al. 2007). The best
known target of NSAIDs, including aspirin, is the enzyme COX-2, a key enzyme
involved in the production of prostaglandins and other eicosanoids from arachi-
donic acid (Menter et al. 2010; Pereira et al. 2010). Due primarily to the action
of COXs on the free arachidonic acid (AA) liberated from membrane phospho-
lipids, overproduction of PGE2 which is predominantly generated by upregula-
tion of COX-2 is associated with a variety of carcinogenic mechanisms (Mao
et al. 2005, 2011).
The association between COX-2 overexpression and survival in lung cancer
patients has been studied for over a decade (Dalwadi et al. 2005). COX-2 expres-
sion has also been shown to be a poor prognostic indicator in non-small-cell lung
cancer (Khuri et al. 2001; Li et al. 2011). Inhibition of COX-2 and thus of PGE2
synthesis suppresses lung tumorigenesis in animal models (Mao et al. 2011).
According to these evidences, COX-2 is one of the targets under investigation
for lung cancer therapy and chemoprevention (Dubinett et al. 2003; Lee et al.
2007). Some reports indicate that the regular use of aspirin and other is associated
with reduced risks of developing lung cancer in animal models and in smokers
(Smith et al. 2006; Brody and Spira 2006; Peebles et al. 2007). Later epidemio-
logic studies have confirmed the chemopreventive effect of NSAIDs in colorectal
cancer (Gupta and Dubois 2001). More recently, it has become clear that effects
of aspirin may not be restricted to gastrointestinal tract cancers, but may also be
relevant in the prevention of breast cancer and lung cancer (Ballaz and Mulshine
2003). In animal model of lung cancer, anti-inflammatory treatment resulted in a
significance (34–52 %) reduction of tumor multiplicity (i.e., in number of tumors
per animal), although treatment with anti-inflammatory drugs did not completely
inhibit tumor growth (Rioux and Castonguay 1998; Duperron and Castonguay
1997; Ballaz and Mulshine 2003).
Strong lines of evidence suggest that the chemopreventive properties of chronic
NSAID administration are based on their COX-inhibitory activity. Overexpression
of COX-2 is associated with poorer prognosis in some cancers, including NSCLC
(Brabender et al. 2002; Ballaz and Mulshine 2003).
However, increasing evidences showing that NF-кB plays a critical role in lung
cancer development suggest NF-кB as a target for lung cancer chemoprevention
(Chen et al. 2011). Interestingly, some agents that have lung cancer preventive
potential, including NSAIDs and dietary compounds, possess inhibitory activity
on NF-кB (Cuzick et al. 2009). Oral administration of pomegranate fruit extract,
which inhibits NF-кB, significantly reduced multiplicity of lung tumor induced
by benzo(a)pyrene and N-nitroso-tris-chloroethylurea (Khan et al. 2007a, b).
Chemoprevention involves prolonged use of preventive agents. The long-time use
of the NF-кB inhibitors or anti-inflammatory drugs is likely to result in un-tolera-
ble side effects (Karin 2006). Thus, dedicated single NF-кB inhibitors are unlikely
to be used as chemoprevention agents (Cuzick et al. 2009). It has been proposed
16 M. Gomes et al.
Inflammation can affect all hallmarks of tumor development and prognosis as well
as the response to therapy. During the inflammation progress, various types of leu-
kocytes, lymphocytes, and other inflammatory cells are activated and attracted to
the inflamed site by a signaling network involving a great number of growth fac-
tors, cytokines, and chemokines (Lu et al. 2006).
In the NSCLC microenvironment, there is a complex interaction between
immune cells and tumor cells as well as other stromal cell types and tissue compo-
nents. The distribution of these cells and the expression of different inflammatory
molecules throughout the tumor microenvironment are, to various extents, related
to tumor progression and survival.
We believe that further studies are needed and further research in order to find
new predictive and prognosis biomarkers in NSCLC. Also needed are new meas-
ures to reduce the risk of cancer. Thereby, prevention is a much better and more
economical way to fight cancer than treating an already advanced and often incur-
able disease.
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Chapter 2
The Role of Inflammation in Colon Cancer
Abstract Colorectal cancer (CRC) is the one of the leading causes of cancer-related
deaths in the world. CRC is responsible for more than 600,000 deaths annually and
incidence rates are increasing in most of the developing countries. Epidemiological
and laboratory investigations suggest that environmental factors such as western
style dietary habits, tobacco-smoking, and lack of physical activities are considered
as risks for CRC. Molecular pathobiology of CRC implicates pro-inflammatory
conditions to promote the tumor malignant progression, invasion, and metasta-
sis. It is well known that patients with inflammatory bowel disease are at higher
risk of CRC. Many evidences exist reiterating the link between Inflammation
and CRC. Inflammation involves interaction between various immune cells,
inflammatory cells, chemokines, cytokines, and pro-inflammatory mediators, such
as cyclooxygenase (COX) and lipoxygenase (LOX) pathways, which may lead to
signaling towards, tumor cell proliferation, growth, and invasion. Thus, this review
will focus on mechanisms by which pro-inflammatory mediators and reactive oxy-
gen/nitrogen species play a role in promoting CRC. Based on these mechanisms,
various preventive strategies, involving anti-inflammatory agents, such as COX
inhibitors, COX-LOX inhibitors, iNOS inhibitors, natural supplements/agents, and
synthetic agents, that blocks the inflammatory pathways and suppress CRC are dis-
cussed in this review.
N. B. Janakiram (*) · C. V. Rao (*)
Center for Cancer Prevention and Drug Development, Department of Medicine, Hematology
Oncology Section, PCS Cancer Center, University of Oklahoma Health Sciences Center,
Oklahoma City, OK 73104, USA
e-mail: [email protected]
C. V. Rao
e-mail: [email protected]
Abbreviations
IL-1β Interleukin-1β
AA Arachidonic acid
NO-NSAID NO-releasing NSAID
IL-4 Interleukin 4
COXibs COX-2-specific inhibitors
FAP Familial adenomatous polyposis
L-NAME L-nitro arginine methyl ester
Se-PBIT Selenium [S,S’-1,4-phenylenebis(1,2-ethanediyl) bis-isothiourea]
GI Gastrointestinal
MIP Macrophage inflammatory protein
MCP Monocytes chemo attractant protein
ABC ATP-binding cassette
DMH Dimethyl hydrazine
MDFs Mucin depleted foci
DSS Dextran sulfate sodium
EPA-FFA Eicosapentaenoic acid-free fatty acid
ONA Oleanolic acid
OT 18α-olean-12-ene-3β-23,28-triol
18R-RvE1 5,12,18R-trihydroxy-EPA
LXA4 lipoxins A4
ABC ATP-binding cassette
Inflammatory bowel disease (IBD) may lead to colitis-associated cancer (CAC), which
is a usually difficult-to-treat cancer having high mortality (Feagins et al. 2009). It is
reported that more than 20 % of IBD patients develop cancer and 50 % of these will
28 N. B. Janakiram and C. V. Rao
die of colon cancer (Lakatos and Lakotos 2008). These patients are reported to have
increased inflammatory infiltration and increased expression of inflammatory genes
(Atreya and Neurath 2008; Atreya et al. 2008; Waldner and Neurath 2008; Clevers
2004). A higher risk for colon cancer is observed in IBD patients who have a fam-
ily history of CRC (Askling et al. 2001). This increased risk suggests an overlap of
signaling pathways and mechanisms that drive cancer development in CAC and CRC.
Anti-inflammatory therapy has been reported to reduce the risk or prevent CRC and
colitis-related CRC in several observational studies. Non-steroidal anti-inflammatory
drugs (NSAIDs) have been reported to reduce colorectal neoplasia by 40–50 % (Thun
et al. 1991; Smalley and Dubois 1997) and also have been reported to reduce CRC
mortality odds by 49 % in a population of US military veterans with IBD (Bansal and
Sonnenberg 1996). A recent meta-analysis of 9 observational studies reported that use
of 5-aminosalicylic acid (5-ASA), mesalamine reduced the odds of colitis-related CRC
by 49 % (Velayos et al. 2006). It is noteworthy that anti-inflammatory drugs such as
5-aminosalicylate-based compounds have remained in the mainstream for the treat-
ment of IBD patients for >50 years. The findings in the human studies confirm obser-
vations in animal models, which show that NSAIDs reduce the occurrence of intestinal
neoplasia. More than 90 % of 110 preclinical animal studies examining the effects of
NSAIDs on tumorigenesis reported an anti-neoplastic effect (Hawk and Levin 2005).
The large volume of compelling data on the use of anti-inflammatory agents/NSAIDs
to reduce the risk of CRC suggests a potential role of inflammation in CRC.
2.3 Inflammation in CRC
Pathological analysis of CRC shows infiltration with various types of cells that func-
tion in innate immunity, such as neutrophils, mast cells, natural killer (NK) cells,
dendritic cells (DC), and tumor-associated macrophages (Atreya and Neurath 2008).
2 The Role of Inflammation in Colon Cancer 29
COX-2 is the inducible COX gene that mediates prostaglandin synthesis and pro-
inflammatory functions. The expression of COX-2 is elevated in 50 % of ade-
nomas and in 85 % of adenocarcinomas. In human intestinal tumors, COX-2 is
expressed in epithelial and stromal cells; it usually is induced by interleukin 1β
(IL-1β) and TNF-α. Over-expression of COX-2 increased azoxymethane (AOM)-
induced tumor formation (Al-Salihi et al. 2009); and COX-2 deficiency signifi-
cantly diminished tumorigenesis in mouse models of colon cancer (Chulada et al.
2000a, b; Oshima et al. 1996a, b), confirming a role for COX-2 in tumorigenesis.
The pro-inflammatory and pro-tumorigenic effects of COX-2 are mediated by its
major end product, PGE2, which stimulates tumor cell proliferation/growth, angi-
ogenesis, and survival and inhibits apoptosis in CRC (Wang and Dubois 2006;
Castellone et al. 2006). PGE2 activates a number of oncogenic signaling pathways,
including β-catenin/transcription factors (TCF), Ras, and the phosphatidylino-
sitol 3-kinase (PI3K) pathways. The generation of microsomal prostaglandin E
synthase (mPGES-1)-deficient mice has revealed a dominant role of this enzyme
in PGE2 generation relevant to promotion of inflammation (Trebino et al. 2003).
The mPGES-1-derived-PGE2 exhibits similar inflammatory responses during
tumor growth. mPGES-1 deficiency was linked to reduced vascular endothelial
growth factor (VEGF). Together, these findings show that deletion or inhibition of
mPGES-1 markedly reduces inflammatory responses in mouse models and eventu-
ally may lead to inhibition of tumor cell proliferation.
PGD2, another important metabolite of COX-2, appears to be a negative regu-
lator of tumorigenesis; it has been demonstrated to possess anti-tumor proper-
ties (Murata et al. 2008). It is produced locally by inflammatory cells at sites of
inflammation; and its receptor (DP1) also is expressed highly in tumor endothelial
cells. The DP1 receptor is expressed on DCs that play a key role in initiating an
adaptive immune response to foreign antigens (Hammad et al. 2003). These stud-
ies suggest that different COX-2-derived prostaglandins have opposing effects on
2 The Role of Inflammation in Colon Cancer 31
inflammation and tumor cell proliferation and that selective modulation of these
prostaglandins may prevent tumor growth in CRC.
Among the LOX pathways, 5-LOX and 12-LOX pathways are closely related to
inflammation and carcinogenesis; however, metabolites from another LOX path-
way, 15-LOX are linked positively and shown to inhibit inflammation and carcino-
genesis. A number of reports suggest the involvement of 5-LOX in early stages of
CRC (Qiao et al. 1995; Bortuzzo et al. 1996; Avis et al. 2001; Ding et al. 2003;
Tong et al. 2005). Hong et al. (1999) reported high expression of 5-LOX and
5-LOX-activating protein in cancer cell lines. High expression of 5-LOX and its
receptors was observed in CRC patients showing poor prognosis (Ohd et al. 2003).
Accumulation of 5-HETE and LT upon activation of 5-LOX resulted in cancer cell
proliferation (Ding et al. 1999). COX and LOX pathways are both linked in such
a way that disturbance in one pathway may lead to over-expression of the other
pathway; thus, balanced inhibition of these two pathways is favorable for inhibit-
ing CRC (Byrum et al. 1997; Griffiths et al. 1997; Goulet et al. 1994). Many stud-
ies have suggested that removal of 5-LOX and 5-LOX-activating protein (FLAP)
results in increased expression of COX metabolites (Byrum et al. 1997; Goulet et
al. 1994). These studies provide evidence for an important role of 5-LOX in CRC
and suggest the potential for chemoprevention and treatment for CRC. Thus, tar-
geting both COX-2 and 5-LOX pathways together and increasing production of
LX and RVs is a better approach for prevention/treatment of CRC.
intermediates that can induce DNA damage and mutation in adjacent epithelial cells.
These changes can stimulate ROS production within the epithelial cells may cause
epigenetic silencing of tumor suppressor genes (Meira et al. 2008; Westbrook et al.
2009). The discovery of NO as a product of immune system cells has implicated this
chemical in the mechanism of carcinogenesis (Tamir and Tannenbaus 1996). Produced
NO can interact with O2.− resulting in the propagation of the highly reactive species
peroxynitrite (Oshima and Bartsch 1994). Peroxynitrite, which is formed from the
reaction between O2.− and NO, reacts with all classes of biomolecules and thereby is
thought to be involved in many pathologic phenomena (Bartosz 1996). NO and perox-
ynitrite concentrations were reported to be increased in cancerous samples (Haklar et
al. 2001). NO is produced by three isoforms of nitric oxide synthase (NOS) [neuronal
nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), and induc-
ible NOS (iNOS)]. nNOS and eNOS are constitutive NOS isoforms, whereas iNOS is
induced upon exposure to inflammatory stimulation. iNOS is expressed in many cells:
extravascular resident leukocytes (macrophages), intravascular and/or infiltrating leu-
kocytes (neutrophils and monocytes), endothelium, and parenchymal cells, including
intestinal epithelium. Its production is stimulated by lipopolysaccharide (LPS), TNF-α,
or interleukin-1β (IL-1β). The role of NO in colon cancer is controversial. Increased
production of NO for a limited time is considered to produce positive results in inhib-
iting CRC, whereas chronic and continuous production of NO produced by iNOS is
implicated in neoplastic transformation, a very crucial step during carcinogenesis.
Studies with iNOS knockout mice suggested a positive role for iNOS in inducing pol-
yps in adenomatous polyposis coli (APC) min/+ mice (Hofseth et al. 2003; Crowell
et al. 2003; Ahn and Ohshima 2001; Nam et al. 2004). High expression of iNOS in
CRC xenografts suggested an inhibitory role for iNOS in tumor growth (Xu et al.
2002). In various preclinical studies in animal models, we noted that iNOS inhibitors
show promise for inhibiting CRC (Rao et al. 1999, 2002). In summary, both increased
expression and decreased expression of NO is observed to have beneficial effects on
CRC. Carefully designed, detailed studies to understand the role of NO during inflam-
mation are necessary in order to understand how to modulate its effects in CRC.
Interactions between NO and COX-2 are well documented, and combinations of iNOS
inhibitors and COX-2 inhibitors have provided inhibition of invasive adenocarcinomas
in animal models of colon cancer (Janakiram and Rao 2012).
in causing CRC, thus natural constituents in these and other foods may contribute to
reduce cancer risk or prevention. Here, we discuss various synthetic and natural bio-
active compounds that can activate or deactivate signaling cascades implicated during
tumor development and that may exhibit chemopreventive properties.
Preclinical and clinical evidences suggest the presence of high levels of prostaglan-
dins, such as PGE2 as mentioned earlier, which affect tumor cell proliferation by
suppressing immune responses (Marnett 1992; Smith 1992). Hence, it is reasonable
to use NSAIDs that can suppress the synthesis of these prostaglandins by inhibiting
COX enzymes may in turn suppress tumor development and growth in colon.
Epidemiological studies, intervention trials, and animal studies have provided
compelling data for inhibition of colorectal carcinogenesis by aspirin and other
NSAIDs (Giovannuci 1999; Brown and Dubois 2005). The first epidemiological
report suggested use of aspirin to decrease risk for CRC (Kune et al. 1988). Most of
the subsequent case–control studies and prospective studies supported these results
(Thun et al. 1991; Freedman et al. 1998; La Vecchia et al. 1977; Muscat et al. 1994;
Peleg et al. 1994; Suh et al. 1993; Giovannucci et al. 1994; Schreinemachers and
Everson 1994; Chan et al. 2005). The relative risks were very consistent in reducing
the risk to 50 % in aspirin users compared with non-aspirin users. Studies on pre-
cursors of CRC such as adenomatous polyps have shown similarly decreased risks
(Suh et al. 1993; Greenberg et al. 1993; Logan et al. 1993; Martinez et al. 1995).
Whereas, the risk reduction of CRC is linked to the dose intake and also the dura-
tion of aspirin use which is explored in a subset of studies. Across-study compari-
sons indicate a dose—response relationship between aspirin and CRC or other cancer
types (Harris et al. 2005). A greatest risk reduction was seen among women who
took more than two aspirin tablets daily reported by the Nurses’ Health Study sup-
port a strong dose—response relationship with colon cancer (Chan et al. 2005). Ten
years of consistent aspirin use seems to be having reduced risks of CRC which is evi-
denced and seems consistent (Chan et al. 2005). Further, the role of NSAIDs/aspirin
use is substantially strengthened in secondary prevention for reduction of metachro-
nous lesions among patients with primary colorectal adenomas or CRC by two ran-
domized controlled trials. In this trial, aspirin had a modest effect on patients with
previous adenomas in reducing the risk of developing new adenomas or cancer that
differed by dose. In this study, a lower dose (81 mg/day) showed better response of
19 % reduced risk of adenomas than a higher dose (325 mg/day) (Sandler et al. 2003;
Baron et al. 2003). Aspirin use as a chemopreventive agent is strongly supported
by these trails against colorectal carcinogenesis among individuals with a known
increased risk as a result of previous disease.
We and others have previously shown that several COX inhibitors, such as indo-
methacin, piroxicam, aspirin, ibuprofen, and sulindac suppress colon carcinogenesis
in AOM-induced F344 rats (Reddy et al. 1993, 1987; Metzger et al. 1984; Narisawa
34 N. B. Janakiram and C. V. Rao
Fig. 2.1 The figure depicts the various pathways observed which initiates inflammation and
tumor cell proliferation. Arachidonic acid metabolism leads to formation of both pro-inflam-
matory and anti-inflammatory metabolites. 5-LOX pathway leads to formation of leukotrienes
which are known for their pro-inflammatory and pro-tumorigenic role. Triterpenoids are reported
to show inhibitory effects on formation of leukotrienes. COX-2 in the presence of aspirin will
lead to formation of epilipoxins (epiLXA4), which are anti-inflammatory and anti-tumorigenic in
functions. Naturally 5-LOX pathway leads to formation of lipoxins, which show similar func-
tions as that of epiLXA4. COX-1 and COX-2 pathway leads to the formation of eicosanoids,
PGI2, PGF2, TXA2, PGD2, and PGE2. PGE2 has been found to play a vital role during inflamma-
tion, development of Tregs, formation of tolerogenic DCs, tumor cell proliferation, and growth.
NSAIDs and natural agent like curcumin are reported to have inhibitory effects on the formation
of eicosanoids. LPS, IL-1β, and TNF-α are known to be involved in formation of nitric oxide
(NO), and IL-6 is a known inflammatory cytokine involved during tumorigenesis. NO formed can
initiate the inflammation and tumor formation by itself or by interacting COX-2 pathway. iNOS
inhibitors and triterpenoids are shown to inhibit NO formation, and resveratrol and diosgenin
inhibit pro-inflammatory cytokines. The free radical formation by macrophages which can cause
DNA damage and eventually tumor cell proliferation by down-regulating p21, p53, and BAX are
observed. Curcumin and resveratrol are reported to restore p21, p53, and p53 and inhibit tumor
cell proliferation
et al. 1993; Pollard and Luckert 1984; Moorghen et al. 1988). Indomethacin was
reported to inhibit tumor growth in chemically induced large-bowel tumors in rats
(Kudo et al. 1980). Similar results were observed in other preclinical studies (Pollard
and Luckert 1980, 1981; Narisawa et al. 1981). Due to GI toxicities of indometha-
cin, we developed and tested a potentially less toxic derivative, NO-indomethecin,
in AOM-induced carcinoma models. Nitric oxide-releasing non-steroidal anti-
inflammatory drugs (NO-NSAID) are promising chemoprevention agents; unlike
conventional NSAIDs, they seem to be free of appreciable adverse effects, while
they retain the beneficial activities of their parent compounds. NO-indomethecin sig-
nificantly suppressed AOM-induced tumor multiplicity and incidence in F344 rats
(Rao et al. 2006). Its activity is related to suppression of COX, iNOS, and β-catenin
levels (Fig. 2.1).
2 The Role of Inflammation in Colon Cancer 35
Rigau et al. (1991) have demonstrated that colon mucosal samples from
patients on long-term sulindac therapy have a reduced PG-biosynthetic capac-
ity. In a randomized, placebo-controlled, double-blind crossover study in patients
with familial adenomatous polyposis (FAP), administration of sulindac at a dose
of 300 mg/day for 6–12 months caused disappearance of all polyps (Laybayle
et al. 1991). Most of the clinical trials with FAP patients on long-term treatment
with sulindac reported a reduction in the number and size of adenomas (Belliveau
and Graham 1984; Waddel et al. 1989; Laybayle et al. 1991; Spagnesi et al. 1994;
Winde et al. 1993; Giardello et al. 1993). Dietary administration of sulindac inhib-
ited dimethyl hydrazine (DMH)-induced colon tumor incidence and multiplicity
in mice (Moorghen et al. 1998; Skinner et al. 1991). In these experiments, sulin-
dac was administered along with DMH throughout the study; however, administra-
tion of sulindac to mice seventeen weeks after DMH administration showed no
reduction in colon tumor growth or development. Oral administration of sulindac
(10 mg/kg) twice daily inhibited DMH-induced primary colon tumor development
and growth in rats. Ahnen et al. (1994) showed that dietary administration of sulin-
dac and its metabolite sulindac sulfone significantly inhibited AOM-induced colon
carcinogenesis in F344 rats. We found that sulindac was effective at both initia-
tion and postinitiation stages of colon tumor formation in F344 rats (Rao et al.
1995). This study suggested that its inhibitory function may be due to its modula-
tory effects on AA metabolism. In another study by Suh et al. (2011), the NSAIDs
sulindac and naproxen, individually and in combination with atorvastatin, caused
significant reduction in AOM-induced colon tumors in F344 rats. The NSAID-fed
animals showed reduction in inflammatory markers iNOS and COX-2 as well as in
phospho-p65 and in the pro-inflammatory cytokines TNF-α, IL-1β, and interleukin
4 (IL-4). Hence, use of NSAIDs in combination with statins was suggested for
retaining efficacy with less/no GI toxicity.
The concern over gastric toxicity associated with aspirin use led to efforts to
develop COX-2-specific inhibitors (COXibs such as rofecoxib, celecoxib) (Gupta
and Dubois 2001). Available literature provides strong evidence for a role of
COX-2 in inflammation and carcinogenesis. Several studies using COX-2 knock-
out or disrupted genes in mouse models of FAP or in chemically induced colon
carcinogenesis in rats indicated that COX-2 selective inhibitors, such as rofecoxib
and celecoxib, inhibit formation of intestinal adenomas (Dannenberg et al. 2005;
Rao and Reddy 2004; Oshima et al. 1996a, b, 2001; Chulada et al. 2000a, b;
Jacoby et al. 2000; Boolbol et al. 1996; Mahmoud et al. 1998; Kawamori et al.
1998; Reddy and Rao 2002). In a clinical trial, FAP patients treated twice daily
with 400 or 200 mg celecoxib had 31 and 12 % reduction, respectively, in polyp
number (Arber et al. 2006). In this clinical trial, celecoxib, at a dose of 400 mg
daily, reduced advanced adenoma formation in the colon by almost 50 % com-
pared with the placebo through a 3-year treatment period (Arbor et al. 2006).
Although introduction of COXibs was successful in reducing GI toxicity, these
drugs were associated with cardiovascular toxicity due to high selectivity toward
COX-2 (Smith et al. 2000; Silverstein et al. 2000; Laine et al. 2003; Bresalier
et al. 2005; Nussmeier et al. 2005; Solomon et al. 2005). An initial study indicated
36 N. B. Janakiram and C. V. Rao
High NOS activity and high levels of NO are observed in AOM-induced colonic
tumors in rats (Rao et al. 1999, 2002), in Crohn’s disease (Rachmilewitz et al. 1995)
2 The Role of Inflammation in Colon Cancer 37
Ziegler et al. (2004) reported null results with resveratrol in APCMin/+ mice.
Even though this study used high doses of resveratrol (4, 20, and 90 mg/kg body
weight) in pellet form, no difference was observed in incidence of intestinal
tumors. Another Apc min mouse study reported a 27 % decrease in adenoma for-
mation by 60 mg/kg of resveratrol administered in the diet (Sale et al. 2005). This
reduction was attributed to decreases (of 58 and 62 % compared with intestinal
mucosa from mice on control diet) in PGE2, which is involved in the maintenance
of the malignant phenotype (Sale et al. 2005). Evidence from all of these stud-
ies suggests that resveratrol has potential prevention and therapeutic properties and
needs further evaluation for its dosage and clinical efficacy in CRC.
Diosgenin, a natural steroidal saponin found predominantly in fenugreek and
wild yams, has diverse biological properties (Raju and Mehta 2009). The com-
mercial synthesis of steroid products, such as cortisone, pregnenolone, and pro-
gesterone, involves use of diosgenin as a precursor (Raju and Mehta 2009). It
is considered safe since it is neither synthesized nor metabolically converted
into steroid by-products in the mammalian body. In preliminary studies with
human subjects, diosgenin has been found to be effective against hyperglycemia
(McAnuff et al. 2005), hypercholesterolemia (Juarez-Oropeza et al. 1987; Son
et al. 2007), and hypertriacylglycerolemia (Kwon et al. 2003). Significant anti-
inflammatory functions have been demonstrated in relevant animal models. It is
used in rats to heal the GI toxicity generated by indomethacin treatment. Its anti-
inflammatory role has been explored further by Yamada et al. (1997). Preclinical
animal studies with AOM-induced ACFs in F344 rats suggested that diosgenin
possesses chemopreventive efficacy in CRC. Administration of 0.1 or 0.05 %
diosgenin in the diet during initiation, postinitiation, or promotion stages of colon
carcinogenesis dose-dependently decreased ACF formation (Raju et al. 2004).
Another study investigated the preventive effects of diosgenin (20, 100, or 500 mg/
kg) on AOM/dextran sodium sulfate (DSS)-induced CRC in mice. Diosgenin at
very low doses significantly inhibited (53, 46, and 40 %, respectively) colonic
mucosal ulcers and dysplastic crypts induced by AOM/DSS treatment and also
reduced expression of inflammatory cytokine genes, including IL-1β, IL-6,
IL-12b, and TNF-α, which are significantly elevated in the colonic mucosa of mice
treated with AOM/DSS (Fig. 2.1). These studies suggest that diosgenin is a potent
bioactive molecule possessing both anti-inflammatory and anti-tumorigenic prop-
erties that make it ideal for further investigation of its molecular and anti-neoplas-
tic functions in human clinical trials.
Triterpenoids are isolated from various medicinal plants and have been stud-
ied for their anti-inflammatory properties. Mostly these compounds are non-toxic
and have made their way into cosmetics and health products (Liu 1995). Recently,
interest in understanding and elucidating the biological roles of triterpenoids for
their hepatoprotective, analgesic, anti-tumor, anti-inflammatory, and immunomod-
ulatory effects is increasing (Mahato and Sen 1997; Liu 1995). These agents are
broken down in the gut to release triterpene metabolites, which are integrated
into the intestinal cell membranes, absorbed, and lead to modulation of signal-
ing pathways. These molecules inhibit expression of inflammatory genes such as
2 The Role of Inflammation in Colon Cancer 41
COX-2, iNOS and various inflammatory cytokines that are known enhancers of
carcinogenesis (Janakiram et al. 2008; Rao et al. 2002, Raju et al. 2004) (Fig. 2.1).
Recently, triterpene analogs that are more potent than the original parent mol-
ecules have been synthesized. Kawamori et al. (1995) found that oleanolic acid
(ONA), a crude plant extract of triterpenoid at a dose of 200 ppm, was effective in
reducing ACF in the intestine of F344 rats. We reported the anti-neoplastic prop-
erties of ONA and the analog 18alpha-olean-12-ene-3 beta-23,28-triol (OT) in
AOM-induced ACFs in F344 rats (Janakiram et al. 2008). These triterpenoids sig-
nificantly suppressed carcinogen-induced colonic preneoplastic lesions at dietary
doses of 750 and 1,500 ppm of ONA, and 250 and 500 ppm of OT and without
any toxicity. ONA inhibited 52 % of total AOM-induced ACFs and ~66 % of ACF
with four or more crypts. OT inhibited up to 48 % of total AOM-induced ACF
formation and 60 % of ACF with four or more crypts at very low doses compared
with those of ONA. These studies support chemopreventive effects of triterpe-
noids in CRC and suggest that an in-depth evaluation of these agents in clinical
trials should be carried out to assess pharmacokinetics, bioavailability, and anti-
neoplastic functions.
Epidemiological, experimental, and clinical studies provide evidence for anti-
CRC activity of omega (ω)-3 PUFAs. Evidence from animals and humans sug-
gest that ω-3 PUFAs may play an inhibitory role during different stages of CRC,
from primary CRC prevention to “tertiary” prevention after treatment of CRC and
advanced metastatic disease. Out of 8 reported clinical studies of ω-3 PUFAs sup-
plementation, 6 reported protective effects. In patients with a previous history of
sporadic colorectal adenomas, oral supplementation with ω-3 PUFA has resulted
in a 13–70 % reduction in intestinal epithelial cell proliferation as compared to
placebo groups (Cockbain et al. 2012). A phase III randomized, double-blind, pla-
cebo-controlled trial investigated treatment with eicosapentaenoic acid-free fatty
acid (EPA-FFA) in 58 patients with FAP who had previously undergone colec-
tomy and ileorectal anastomosis and showed a 22.4 % reduction in polyp number
compared with placebo (West et al. 2010). Colon cancer xenograft studies showed
consistent protective effects (40–60 % reduction in xenograft size) in mice sup-
plemented with ω-3 PUFAs as compared to untreated mice (Boudreau et al. 2001;
Kato et al. 2002; Calviello et al. 2004). Similar beneficial results were reported
from studies with CRC cell allograft tumors (Mund et al. 2007; Cannizzo and
Broitman 1989; Togni et al. 2003; Pizato et al. 2005). In spite of these encouraging
data, no published studies yet have investigated the anti-neoplastic effect of u-3
PUFAs in patients with primary or metastatic CRC.
Fish and fish oil are rich sources of the ω-3 PUFAs EPA and DHA. The metab-
olites derived from these PUFAs result in formation of 3-series prostaglandins,
which are anti-inflammatory rather than pro-inflammatory and also may possess
anti-tumor properties. A report of a switch from 2 series PGE2 to 3 series PGE3
was demonstrated in colonic mucosa of rats treated with fish oil (Vanamala et al.
2008). The recently discovered anti-inflammatory lipid mediators RVs and LX
derived from EPA and DHA are gaining importance for their anti-neoplastic func-
tions. RVs derived from EPA are called as “E” series. Protectins are also generated
42 N. B. Janakiram and C. V. Rao
from precursors of omega 3-PUFAs. RVs or protectins from DHA, named “D”
series, possess anti-inflammatory and immunomodulatory properties. The concen-
tration required for these lipid mediators to exhibit any biological activity is in the
nanomolar or picomolar range. Acetylation of aspirin by COX-2 in the presence
of EPA results in formation of 5,12,18R-trihydroxy-EPA (18R-RvE1) (Janakiram
et al. 2011) (Fig. 2.1). Ingestion of aspirin and EPA generated 18R-RvE1 that was
detectable in plasma of healthy volunteers (Oh et al. 2011). The anti-inflammatory
role of RvE1 is well documented in a mouse model of DSS-induced colitis; it acts
through inhibition of phosphorylation of NF-κB (Ishida et al. 2010). Another study
reported a protective role of RvE1 in mouse colitis through induction of intestinal
alkaline phosphatase (Campbell et al. 2010). EPA and DHA exhibited protective
effects against colitis in a rat model by restoring the number of mature, mucin-
filled goblet cells (Arita et al. 2005). Two other studies also reported protective
effects of RvE1 against colitis induced by DSS and 2, 4, 6-trinitrobenzene sulfonic
acid (Nieto et al. 2002; Ishida et al. 2009).
Lipoxin A4 (LXA4) was shown to inhibit neutrophil chemotaxis, adherence,
transmigration, and activation during resolution of inflammation and suppression
of IL-8 production by epithelia and leukocytes and to cause clearing of neutro-
phils by up-regulation of monocyte ingestion (Serhan 1997, 2002; Canny et al.
2002). Decreased LXA4 expression was shown in a DSS-induced colitis model
(Gewirtz et al. 2002). Protective effects of LXA4 analogs were observed in DSS
and other chemically induced colitis animal models (Gewirtz et al. 2002; Fiorucci
et al. 2001). The protective effects of these analogs are attributed for their LXA4
receptor-mediated inhibitory effects on pro-inflammatory signaling pathways.
15-epi-LXA4 is formed in the presence of aspirin; and some of the preventive
or therapeutic effects of aspirin-like NSAIDs may be through these 15-epi-LX
(Claria and Serhan 1995) (Fig. 2.1). The anti-inflammatory functions of these lipid
mediators suggest a potential chemopreventive therapeutic strategy for inflamma-
tion-related diseases like CRC.
2.8 Conclusions
and LOX inhibition, including natural agents like curcumin or synthetic agents
like licofelone, to achieve safer toxicity profiles while retaining significant inhibi-
tion of CRC. Additional natural bioactive anti-inflammatory compounds are being
identified to provide beneficial effects against colitis-induced inflammation and
CRC. Many of these agents are well tolerated and may provide safe alternatives
to existing, more toxic compounds. Increased consumption of EPA- and DHA-rich
foods may reduce inflammation and its related CRC conditions. And other novel
lipid mediators, such as LX, RVs and their analogs, need to be evaluated in CRC
models for their effects on colon mucosal immunity against development of CRC.
Acknowledgements We want to acknowledge the Grant support NCI-R01-94962; NCI-
CN-53300 for the work quoted in this chapter and Dr. Julie Sando for her scientific and language
editing of this chapter.
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Abstract Inflammatory breast cancer (IBC) is the most aggressive form of breast
cancer. Despite extensive study, whether inflammation contributes to the tumori-
genicity or aggressiveness of IBC remains largely unknown. In this chapter, we
will review the potential role played by inflammation in IBC based on the results
of in vitro, in vivo, and patient studies. Current evidence suggests that several
major inflammatory signaling pathways are constitutively active in IBC and breast
cancer. Among them, the NF-κB, COX-2, and JAK/STAT signaling systems seem
to play a major role in the tumorigenesis of IBC. Inflammatory molecules such
as interleukin-6, tumor necrosis factor alpha (TNF-α), and gamma interferon have
been shown to contribute to malignant transformation in preclinical studies of
IBC, while transforming growth factor-β, interleukins 8 and 1β, as well as TNF-α
appear to play a role in proliferation, survival, epithelial–mesenchymal transition,
invasion, and metastasis. In this chapter, we also describe work thus far involving
inhibitors of inflammation in the development of prevention and treatment strate-
gies for IBC.
3.1 Introduction
Breast cancer is the second most common cancer, following skin can-
cer, among women in America. The American Cancer Society estimates
that there will be 232,670 new cases of breast cancer among women in the
United States in 2014 (ACS 2014). It is also the second leading cause of
death from cancer in women, approximately with 40,000 predicted deaths for
2014. Inflammatory breast cancer (IBC) is the most aggressive form of breast
cancer. Although it accounts for an estimated incidence rate of up to 5 %
of breast cancers (Anderson et al. 2005; Hance et al. 2005; Jaiyesimi et al.
1992), IBC is responsible for a disproportionate 8–10 % of all breast cancer-
related deaths (Hance et al. 2005).
The word “inflammatory” was first applied to the IBC subtype of breast
cancer by Haagensen (1971). His description was based on certain presenting
features that are unique to this subgroup of patients. IBC presents with rapid
onset of breast erythema occupying at least one-third of the breast, accompa-
nied by breast edema leading to the characteristic peau d’orange appearance
of the skin. Other features include breast enlargement, pain, and tenderness.
Approximately 50 % of patients do not present with a palpable mass or radio-
graphic evidence of cancer (Ueno et al. 1997; Yang et al. 2008). Almost all
IBC patients present with lymph node metastasis at the time of diagnosis, and
approximately 30 % present with distant metastasis (Jaiyesimi et al. 1992; Li
et al. 2011).
For diagnosing IBC, consensus guidelines recommend at a minimum a core
biopsy to enable detection of invasive carcinoma and to allow marker study
(hormone receptors and HER2). A skin punch biopsy to confirm the presence of
dermal lymphatic invasion, one of the hallmarks of IBC, is also strongly recom-
mended in suspected cases (Dawood et al. 2011).
Treatment of IBC, as for other types of breast cancer, involves a multidisci-
plinary approach that includes surgery, radiation therapy, and medical oncol-
ogy. Patients are stratified according to extent of disease and the molecular
subtype. This approach has been associated with a significant reduction in can-
cer-related mortality (Ueno et al. 1997; Kesson et al. 2012). Currently, the most
active anti-cancer agents include anthracycline and taxanes, in addition to anti-
HER2 therapy and endocrine therapy. However, compared with other types of
breast cancer, treating IBC has proved to be more challenging mainly because
of its rapidly aggressive nature combined with the lack of effective targeting
therapy.
Despite extensive study, whether inflammation contributes to the tumorigenic-
ity or aggressiveness of IBC remains unknown. In this chapter, we will review the
potential role played by inflammation in IBC based on the results of in vitro, in
vivo, and patient studies. We will also describe work thus far involving inhibitors
of inflammation in the prevention and treatment of IBC.
3 The Role of Inflammation in Inflammatory Breast Cancer 55
Several studies have suggested complex cross talk between NF-κB and estrogen
receptor (ER) in IBC as well as in breast cancer in general. In a separate study,
Van Laere et al. reported that NF-κB activation in IBC tumors was associated with
ER downregulation, which was linked to both EGFR and/or HER2 overexpression
and MAPK hyperactivation (Laere et al. 2007). Additionally, ER seems to be capa-
ble of inhibiting both the constitutive and the inducible activation of NF-κB in a
dose-dependent manner (Biswas et al. 2004). On the other hand, studies also seem
to suggest that in ER-positive patients, cross talk between ER and NF-κB occurs
that may either be transrepressive or positive (Kalaitzidis and Gilmore 2005).
Theoretically, this could explain how in luminal B subtypes and some ER-positive
IBC patients, resistance to hormonal therapy and poorer outcome could result
from positive cross talk between NF-κB and ER leading to enhanced ER-mediated
expression of genes involved in cell proliferation, survival, and resistance.
COX family The cyclooxygenase (COX) family of enzymes consists of two
members, COX-1 and COX-2. Both enzymes catalyze the conversion of arachi-
donic acid to prostanoids and are also responsible for the generation of eicosanoid
products, which are important mediators of pain and inflammation. Tissue upreg-
ulation of COX-2 can be triggered by several stimuli, including growth factors
and oncogenes (Williams et al. 1999). Aberrant activation of COX/prostaglandin
signaling is common in many cancers, especially in colon cancer, where COX-2
is overexpressed in 85 % of tumors (Brown and DuBois 2005). This has also been
the case with breast cancer; enzyme levels have been found to be increased in
40 % of breast tumor tissues examined (Yoshimura et al. 2003).
Several studies have documented the role that the PTGS2 gene, which encodes
COX-2, plays in cell proliferation, invasion, angiogenesis, and metastasis (Wang and
Dubois 2004; Menter et al. 2010). Overexpression of COX-2 in breast cancer cor-
relates with a more aggressive breast cancer profile that is characterized by higher
proliferation rates, larger tumors, higher pathologic grade, hormone receptor nega-
tivity, and HER2 overexpression (Ristimaki et al. 2002; Subbaramaiah et al. 2002).
Compared with non-IBC tumors, the PTGS2 gene is differentially overexpressed in
IBC, and it is identified as a key component in the molecular signature for IBC (Laere
et al. 2005, 2007). Moreover, prostaglandin E2 (PGE2), which is a product of COX-2
enzymatic activity, is known to be upregulated in primary IBC tumors and metastatic
lesions (Robertson et al. 2008). These findings emphasize the role of the COX-2 path-
way in IBC and its potential use as a target for disease prevention and treatment.
JAK/STAT signaling The JAK/STAT signaling system is the main pathway for
a variety of cytokines, including interferon and interleukins (e.g. IL-6), as well as
growth factors or other chemical messengers. Depending on both the context and
the integrity of the pathway, JAK/STAT signaling can stimulate proliferation and
cell migration versus differentiation and apoptosis (Rawlings et al. 2004).
STAT3 is known to be constitutively activated in >50 % of breast cancers and
tumor-derived cell lines (Garcia et al. 1997; Diaz et al. 2006). Using small inter-
fering RNA (siRNA) to block STAT3 in both cell culture and xenograft models
of breast cancer, investigators were able to show increased apoptosis through the
Fas-mediated intrinsic apoptotic pathway, as well as reduced expression of the
transmembrane molecule B-cell lymphoma-extra large (Bcl-xL), which promotes
3 The Role of Inflammation in Inflammatory Breast Cancer 57
Accumulating DNA mutations play a causal role in the process of malignant trans-
formation. Oncogenic insults may result in activation of oncogenes, loss of tumor
suppressor genes, or the constitutive activation of membrane receptors or lead to the
alteration of critical cellular processes such as the cell cycle or apoptosis (Hanahan
and Weinberg 2000). In this setting, inflammation may contribute to carcinogenesis
through the activation of the DNA damage response system in response to major
oncogenic insults (Martin et al. 2011; Hartman et al. 2011). Moreover, viral infec-
tions, such as human papillomavirus (HPV), whose genome has been detected in
breast cancer tissue, may also cause DNA damage, resulting in activation of the
DNA damage response pathway, and stimulate the formation of a pro-inflammatory
tumor microenvironment (Moody and Laimins 2009; Kan et al. 2005).
Interleukin-6 (IL-6), which is overexpressed in the SUM149 preclinical model
of IBC (Golen et al. 2000), plays a potent role in malignant transformation. IL-6
was able to convert a non-transformed mammary epithelial cell line (MCF-10A) to
the transformed state in 24–36 h (Iliopoulos et al. 2009, 2010).
Additionally, HER2 overexpression, which is known to occur in up to 60 % of
IBC tumors and 25 % of non-IBC tumors, is associated with poor outcome (Guerin
et al. 1989; Kallioniemi et al. 1991). HER2 amplification was associated with
marked increase in IL-6 in breast cancer cells and induced STAT3 activation, sug-
gesting a HER2-IL-6-STAT3 signaling pathway could play a critical role in tumori-
genesis (Hartman et al. 2011).
58 T. M. Fouad et al.
On the other hand, cancer stem cells (CSCs), also known as tumor-initiating
cells, are highly tumorigenic cells and are enriched in IBC tumors as well as in
preclinical models of IBC (Laere et al. 2010; Charafe-Jauffret et al. 2010; Xiao
et al. 2008). IL-6 acts as a growth factor for CSCs and is sufficient to convert non-
stem cancer cells to CSCs (Iliopoulos et al. 2011). IL-6 gene expression was found
to promote self-renewal, as well as invasive potential, in both normal and MCF-
7-derived spheroids (Sansone et al. 2007). IL-6 is also at the center of epigenetic
regulation of stem cells (D’Anello et al. 2010; Hodge et al. 2005). IL-6 thus plays
a critical role in mediating the epigenetic switch that involves NF-κB and STAT3
and links inflammation to cell transformation (Iliopoulos et al. 2009, 2010).
Additional inflammatory signaling involved in the regulation of CSCs includes
the tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) path-
ways, both of which are upregulated in breast CSCs (Murohashi et al. 2010). Both
cytokines are also able to activate the NF-κB pathway (Cheshire and Baldwin
1997; Hayden and Ghosh 2008; Matsumoto et al. 2005). Treatment with the
chemokine IL-8 resulted in increased mammosphere formation, whereas IL-8
receptor/CXCR1 blockade depleted the breast CSC population both in vitro and
in xenografts (Murohashi et al. 2010; Charafe-Jauffret et al. 2009; Ginestier et al.
2010). Expression of CCL5/RANTES was also found to be upregulated in breast
CSC populations (Murohashi et al. 2010).
One of the hallmarks of cancer cells is their capacity to acquire resistance to apop-
totic signals (Hanahan and Weinberg 2000). Transforming growth factor (TGF)-β is
a pro-apoptotic cytokine that normally induces cell cycle arrest in the early phases of
tumorigenesis. The mechanisms by which cancer cells escape the inhibitory effects of
TGF-β are not fully understood but may include inactivating mutations or homozy-
gous deletions(Kaklamani et al. 2003; Pasche et al. 2004; Dunning et al. 2003) or
upregulation in oncogenic expression (Zhang et al. 2003). Once the pro-apoptotic
functions of TGF-β are subverted, its tumorigenic potential becomes unhindered, thus
stimulating growth, invasion, and angiogenesis (Biswas et al. 2007; Lei et al. 2002).
Likewise, although TNF-α promotes apoptosis in MCF-7 cells (Simstein et al.
2003), a process similar to TGF-β subverts the pro-apoptotic effect of TNF-α
(Rivas et al. 2008). HER2 amplification, which is present in up to 60 % of IBC
patients, was shown to confer resistance to TNF-α-induced apoptosis in breast
cancer cell lines mainly through an Akt/NF-κB anti-apoptotic cascade (Zhou et al.
2000). Likewise, increased expression of claudin-1 was able to reverse TNF-α-
induced apoptosis in the MCF-7 breast cancer cell line (Liu et al. 2012). These
studies suggest that there are multiple pathways by which breast cancer cells can
overcome TNF-α-induced apoptosis, thus promoting cancer cell survival and
unleashing the tumorigenic potential of TNF-α.
3 The Role of Inflammation in Inflammatory Breast Cancer 59
One of the challenges facing IBC research is the development of preclinical mod-
els that accurately recapitulate the aggressiveness of the disease. Currently, there
is a need for better immunocompetent mouse models of IBC that allow assess-
ment of the molecular and inflammatory mechanisms underlying the disease and
the development of effective therapeutic targets.
In a recent study that looked at the role of NF-κB signaling in conferring
self-renewal to breast cancer cells, three types of IBC SUM149 cells were pre-
pared and injected into the mammary fat pads of nude mice. These included cells
expressing IκBα-SR at low or high density or an empty vector (Kendellen et al.
2013). Investigators assessed self-renewal by measuring the ability of cells when
injected at limiting dilutions to establish primary tumors. Cells with deficient NF-
κB signaling produced smaller tumors at a much later onset compared to those
with empty vector, whereas the low density of SUM149 cells expressing IκBα-SR
did not form tumors. The ability to self-renew appears to require both intact
canonical and non-canonical NF-κB pathways (Kendellen et al. 2013). This dem-
onstrates the importance of NF-κB for tumorigenesis in xenograft models.
Cyclooxygenase-2 (COX-2) is over-expressed in mammary tumors derived
from rodent models of breast cancer. Enhanced COX-2 expression was found to be
sufficient to induce mammary gland tumorigenesis in the mouse mammary tumor
virus (MMTV)/COX-2 transgenic mouse strain, thus providing evidence for its in
vivo oncogenicity (Liu et al. 2001). Additionally, mammary gland involution after
weaning was delayed in the transgenic animals compared to controls, suggesting
that apoptosis suppression was also involved (Liu et al. 2001).
62 T. M. Fouad et al.
Studies have also demonstrated that tumor formation in these models can be sup-
pressed either pharmacologically by using anti-inflammatory drugs, including COX
inhibitors, or through genetic ablation (Howe 2007; Howe et al. 2001; Howe et al.
2005). COX inhibitors were also evaluated in HER2/neu transgenic mice, which are
also ER negative. Celecoxib administration was able to significantly delay tumor
formation in the animal model (Howe et al. 2002; Lanza-Jacoby et al. 2003).
To examine the consequences of knocking out COX-2, investigators adopted
an approach used in intestinal cancer models (Oshima et al. 1996), by cross-
ing COX-2 knockout mice with mammary tumor virus/neu deletion mutant
(MMTV/NDL) mice and comparing tumor multiplicity to HER2/neu transgenic
mice that were COX-2 wild type, heterozygous, and null (Howe et al. 2005).
Tumor multiplicity and size were significantly reduced in COX-2 knockout mice
(heterozygous and null) compared to controls (Howe et al. 2005). Additionally, the
authors observed that COX-2 null animals were associated with reduced expres-
sion in several angiogenesis factors, which led to a reduction in mammary blood
vessel formation. Together, these studies suggest that an intact COX-2 pathway is
both necessary and sufficient for the induction of tumorigenesis.
Similarly, the tumorigenicity of TGF-β was assessed by developing a dox-
ycycline-inducible triple transgenic mice model in which doxycycline can be
used to induce TGF-β1 expression in polyomavirus middle T antigen (PyVmT)
transformed mammary tumors (Muraoka-Cook et al. 2004). TGF-β1 stimulation
resulted in rapidly accelerated metastatic progression with >ten-fold increase in
lung metastases in as little as 2 weeks. Antisense-mediated inhibition of TGF-β1
resulted in decreased cell motility, survival, anchorage-independent growth, tumo-
rigenicity, and metastasis. Similarly, Criswell et al. looked at the role of TGF-β
type III receptors in inducing EMT, cancer cell motility, and invasion of metastatic
cancer cells through a similar transgenic model (Criswell et al. 2008).
To address the role of IL-6 in cancer proliferation, investigators looked at
whether expression of IL-6 in MCF-7 cells would alter tumor growth rates in
immunocompromised mice. Xenografts expressing IL-6 underwent rapid engraft-
ment and expansion relative to MCF-7 xenografts that did not express IL-6 (Sasser
et al. 2007). On the other hand, using siRNA to knock down STAT3 expression in
nude mice, investigators were able to suppress breast cancer cell growth compared
with controls. pRNAi-STAT3 also led to downregulation of STAT3 and Bcl-xL, as
well as upregulation of Fas and induction of apoptosis via expression of cleaved
caspase-3 (Kunigal et al. 2009; Matthews et al. 2007).
The rarity of IBC as a disease has not allowed the role of inflammation to be
systematically examined in clinical studies involving IBC patients; however,
numerous clinical reports and observational studies have addressed the role of
inflammation in breast cancer in general.
3 The Role of Inflammation in Inflammatory Breast Cancer 63
(Singh et al. 2010). CTCE-9908 as a single agent inhibited skeletal metastases but
failed to prevent primary tumor growth or pulmonary metastasis.
Owing to their unacceptable cardiotoxicity, the use of selective COX-2 inhibi-
tors has been limited despite initial enthusiasm regarding promising epidemio-
logic results and their anti-cancer activities (Psaty and Furberg 2005; Graham
et al.2005). Interest has instead shifted to searching for alternative COX-2-targeted
agents. One such target is the family of prostanoid receptors, particularly EP4, that
bind with PGE2, which is a product of COX-2 (Jones et al. 2009). EP4 was found
to mediate invasion and metastasis in both inflammatory and non-IBCs, and EP3
suppressed angiogenesis in IBC tumors (Robertson et al. 2008, 2010). None of the
available EP4 antagonists have yet been tested in cancer patients.
Apricoxib is a novel selective COX inhibitor analog that is currently under evalu-
ation in breast cancer in combination with lapatinib and capecitabine in the treatment
of HER2/neu-positive advanced breast cancer (Health NIo 2001). Tranilast is another
compound under investigation and is known as a potent inhibitor of PGE2. It was
shown to suppress tumorigenesis in both xenograft mammary tumors and human triple-
negative breast cancer cells (Chakrabarti et al. 2009; Subramaniam et al. 2010, 2011).
Another agent under evaluation in breast cancer is fish oil. Fish oils con-
tain the omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic
acid (EPA), which ultimately lead to the inhibition of inflammation in the body
(Weaver et al. 2009). This is suspected to occur through inhibition of COX/PGE2
production (Wendel and Heller 2009).
Recently, a new role for statins as a preventive agent against cancer has
emerged. Recent evidence has suggested that in addition to their lipid-lowering
effects, statins exert powerful anti-inflammatory effects by acting on multiple
inflammatory gene pathways (Jain and Ridker 2005). The anti-cancer effects of
statins have also been linked to the mevalonate pathway, which in turn leads to
the inhibition of many downstream growth factors (Nielsen et al. 2012). Large
observational studies have pointed toward the potential role this pathway has
against cancer in general as well as breast cancer specifically (Nielsen et al. 2012;
Ahern et al. 2011). Brewer et al. have also addressed the potential role of statins in
improving the survival of patients with IBC (Brewer et al. 2012).
Curcumin is the principal derivative of turmeric, the popular Indian spice and a
member of the ginger family. Various preclinical studies have looked into its role
in breast cancer as a chemosensitizer and radiosensitizer to agents such as doxo-
rubicin, 5-fluorouracil, and paclitaxel (Goel and Aggarwal 2010). Curcumin is a
known potent inhibitor of NF-κB, STAT3, and COX-2 as well as other growth fac-
tors and anti-apoptotic proteins (Goel and Aggarwal 2010).
Current evidence supports that inflammation plays a central role in the process of
tumor formation in IBC at various levels. Several important inflammatory gene
pathways are differentially upregulated in IBC and contribute to the formation
66 T. M. Fouad et al.
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3 The Role of Inflammation in Inflammatory Breast Cancer 73
Abstract Malignant brain tumors are among the most lethal of human tumors,
with limited treatment options currently available. A complex array of recurrent
genetic and epigenetic changes has been observed in gliomas that collectively
result in derangements of common cell signaling pathways controlling cell sur-
vival, proliferation, and invasion. One important determinant of gene expression
is DNA methylation status, and emerging studies have revealed the importance
of a recently identified demethylation pathway involving 5-hydroxymethylcyto-
sine (5hmC). Diminished levels of the modified base 5hmC is a uniform finding
in glioma cell lines and patient samples, suggesting a common defect in epigenetic
reprogramming. Within the tumor microenvironment, infiltrating immune cells
increase oxidative DNA damage, likely promoting both genetic and epigenetic
changes that occur during glioma evolution. In this environment, glioma cells are
selected that utilize multiple metabolic changes, including changes in the metab-
olism of the amino acids glutamate, tryptophan, and arginine. Whereas altered
metabolism can promote the destruction of normal tissues, glioma cells exploit
these changes to promote tumor cell survival and to suppress adaptive immune
Tumors of the central nervous system (CNS) were first classified by the World
Health Organization (WHO) on the basis of histopathology, clinical, and diag-
nostic criteria in 1979. In 1993, immunohistochemical criteria were added, and in
2000, some genetic profiles, epidemiological data, clinical signs and symptoms,
imaging, and other predictive factors were added to the classification. As of 2007,
additional types had been added, creating an array of more than 130 tumor types
and subtypes (Louis et al. 2007; Huttner 2012). The WHO grading scale is based
upon histology and includes a “malignancy scale” useful in predicting biological
behavior and in selecting treatment strategies. Of the “gliomas,” Grade I tumors
have low proliferative potential and include all pilocytic astrocytomas and gener-
ally can be cured surgically. Grade II tumors demonstrate some infiltration and
are likely to recur following surgery. Grade III tumors demonstrate evidence of
malignancy, and patients with these tumors generally receive radiation and chemo-
therapy. Grade IV tumors are malignant, mitotically active and are likely to show
evidence of necrosis and vascular proliferation. Grade IV tumors progress rap-
idly, usually with fatal outcome. CNS tumors can be classified broadly as glio-
mas, including astrocytomas, oligodendrogliomas, and ependymomas, or non-glial
tumors, including meningiomas, pituitary tumors, and medulloblastoma.
Glioblastoma multiforme, a Grade IV tumor, is the most common primary
malignant brain tumor diagnosed in the USA and is recognized for its aggressive
growth, recurrence, resistance to therapy, and short median survival (Fig. 4.1).
As glioblastomas account for approximately 80 % of malignant brain tumors
(CBTRUS 2011), they will be the focus of this review on inflammation and brain
cancer. Glioblastomas primarily affect adults with a peak incidence between 40
and 70 years. Most glioblastomas arise in older individuals as primary tumors (pri-
mary glioblastoma) in the cerebral hemispheres and demonstrate microvascular
proliferation and necrosis. A smaller number (<10 %) likely arose from tumors
of a lower grade in younger patients that progressed (secondary glioblastoma)
(Huttner 2012).
Current treatment strategies include surgical resection, radiation therapy, and
chemotherapy, primarily with the alkylating agent temozolomide.
4 The Role of Inflammation in Brain Cancer 77
ligand
RTKs:
EGFR
ligand M p14ARF
ERBB2
PDGFRA
MET
ROS
M NF1 RAS
cross talk
RAS DNA DNA damage M TP53
repair
ERK mTOR
Proliferation Rb pathway
mutation amplification
Survival
CDKN2A CDK6 mutation
Invasion
CDKN2B CCND2 RB1 G1 S phase
M progression
CKKN2C CDK4
M
(Rajasekhar et al. 2003; Aksamitiene et al. 2011; Chen et al. 2012). Members of the
RTK family significant in glioblastoma include amplification of EGFR, EGFRvIII
ERBB2, PDGFRA, and MET. The binding of ligands to these membrane recep-
tors stimulates the intrinsic tyrosine kinase activity of the RTK, leading to receptor
autophosphorylation and activation of Ras by GTP binding. The RAS signaling kinase
cascade can then proceed down the RAS/PI3K/PTEN/AKT/mTOR or the RAS/RAF/
MEK/ERK pathways. Ultimately, either a series of transcription factors are activated
that promote transcription of a subset of genes, or existing mRNAs are differentially
recruited into polysomes. Although these pathways are often presented as being
discrete, substantial cross talk occurs, creating a complicated signaling network.
Under normal conditions, these signaling pathways allow a cell to communicate
with its environment and to respond appropriately. Changes in protein levels or muta-
tions in key genes can result in the disruption of normal signaling. In glioblastoma,
signaling through these pathways is often perturbed by amplification or mutation
of the RTK member, EGFR. Mutations or amplification in other RTKs are less fre-
quently observed. The neurofibromatosis 1 (NF1) and PTEN (phosphatase and tensin
homolog deleted on chromosome 10) gene products act as regulators of each arm of
this pathway, and both are known to be mutated or deleted in a substantial number of
glioblastomas. Loss or alteration of these regulators results in partial or complete loss
of regulation of the signaling pathway. Mutations in RAS, PI3K, or other members
of the network could result in constitutive activation of the signaling cascade.
4 The Role of Inflammation in Brain Cancer 79
The TP53 gene product is an important tumor suppressor that reduces the number
of mutant cells in a population by directing cells with substantial DNA damage
toward senescence or apoptosis through transcription-dependent or -independent
mechanisms. The TP53 gene is frequently mutated in human tumors and is the
most frequently mutated gene in glioblastoma. Protein levels of p53 are regu-
lated by the mouse double minute 2 homolog (MDM2) and double minute 4 pro-
tein (MDM4) ubiquitin ligases, which mark p53 for proteosomal degradation, as
well as inhibit p53-mediated transcriptional activation. The amplification of either
of these ubiquitin ligases can diminish p53 levels and thus abrogate p53 function.
Another member of the p53 network is p14Arf transcribed from an alternate read-
ing frame of the CDNK2A locus that inhibits MDM2; therefore, deletion or muta-
tion of p14Arf would result in increased MDM2 levels and increased p53 degradation
(Riemenschneider et al. 1999; Zheng et al. 2008; Stegh and DePinho 2011).
4.1.2.3 The RB Pathway
Wild-type adenoviruses similarly exploit the critical role of pRB upon infection
of human cells by expressing the virally encoded E1A protein. This protein binds to
pRB, releasing E2F and allowing progression of quiescent cells into the cell cycle,
therefore promoting viral replication. An oncolytic adenovirus has been engineered
in which a 24 base pair deletion has been inserted into the E1A region (Δ24). This
virus will replicate only in RB-deficient cells, resulting in cell lysis (Fueyo et al.
2000). Adenovirus normally binds to the coxsackie and adenovirus receptor (CAR)
present on the surface of some human cells. However, a mutation can be created that
prevents binding to this receptor. In its place, an RGD-4C peptide coding sequence
has been inserted, effectively retargeting the adenovirus to cells that express inte-
grins αvβ3,5, which are frequently upregulated in a number of high-grade tumors
including glioblastoma (Piao et al. 2009). Thus, the combined Δ24-RGD oncolytic
adenovirus will selectively destroy glioblastoma tumor cells with defects in the RB
pathway and increased expression in integrins. Clinical trials are currently underway
with this viral vector approach.
O
CH2OH
HN
O N
APOBEC TDG
deamination glycosylase
? cleavage
O N
irreproducible (Ooi and Bestor 2008). Two significant findings reported in 2009
redirected studies on DNA demethylation to an oxidation product of 5mC, 5hmC. In
one study, 5hmC was identified as a “sixth base” in the DNA of mammalian Purkinji
neurons (Kriaucionis and Heintz 2009). Simultaneously, it was reported that mam-
malian 10–11 translocation (TET) enzymes requiring α-ketoglutarate, Fe(II), and
oxygen could convert 5mC to 5hmC (Tahiliani et al. 2009). These reports have since
led to a flood of reports on potential epigenetic reprogramming pathways in mam-
malian DNA with demonstrated defects in human cancer, including glioblastoma.
The pyrimidine methyl groups of both thymine and 5mC in DNA can be chem-
ically oxidized by endogenous reactive oxygen species (ROS) with formation of
5-hydroxymethyluracil (5hmU) and 5hmC (Mellac et al. 1993; Privat and Sowers
1996; Tardy-Planechaud et al. 1997; Burdzy et al. 2002), respectively. The oxida-
tion of 5mC to 5hmC is known to interfere with the binding of proteins contain-
ing methyl-binding domains, including MeCP2 (Valinluck et al. 2004), and also
with methyl-directed methylation of hemimethylated DNA by methyltransferases,
including mammalian DNMT1 (Valinluck and Sowers 2007). This confirms earlier
suggestions that 5hmC might be an intermediate in a DNA demethylation path-
way (Rusmintratip and Sowers 2000). Potential mechanisms are currently being
explored for downstream events in the processing of 5hmC, including enzymatic
deamination to 5hmU, followed by removal by the thymine-DNA glycosylase
(TDG) (Guo et al. 2011; Cortellino et al. 2011), and further enzymatic oxida-
tion by TET family members to 5-formylcytosine (5foC) and 5-carboxylcytosine
(5caC), also removed by TDG (He et al. 2011).
Recent studies have measured 5hmC levels in both normal brain tissues and in
tumors by both liquid chromatography mass spectrometry (LC-MS) and locali-
zation by immunohistochemistry (IHC). In the mouse tissue, 5mC is found in all
tissues, with the highest levels in neurons within the CNS (Munzel et al. 2010;
Globisch et al. 2010; Munzel et al. 2011). In human adult and embryonic tissues,
the highest levels of 5hmC are found in terminally differentiated cells. Less dif-
ferentiated cells and stem cell compartments had lower 5hmC levels (Haffner et al.
2011). In the normal adult brain, 5hmC is abundant in cells of the cortex and white
matter; however, reduced levels are found in gliomas, with lower levels in tumors
of higher grade (Kraus et al. 2012; Orr et al. 2012). In adult GBM, low 5hmC lev-
els are related to reduced survival (Orr et al. 2012), suggesting that the formation
and metabolism of 5hmC represent critical events in the development of GBM and
might reveal future treatment strategies. It is as yet unknown if 5hmC functions
primarily as an intermediate in a 5mC demethylation pathway or if 5hmC might
have an independent function in modulating specific DNA–protein interactions
with currently unidentified proteins (Yildirim et al. 2011).
It is as yet unknown what metabolic events result in the loss of 5hmC, though
several mechanisms are under investigation (Fig. 4.3). A convergence between
5hmC formation and glioblastoma somatic mutation has been shown for the isoci-
trate dehydrogenase gene (IDH1/2). IDH mutations (Sahm et al. 2011; Xu et al.
2011; Jin et al. 2011; Liu et al. 2012) are frequently found in secondary glioblasto-
mas, although rarely in primary tumors. The IDH gene product converts isocitrate
4 The Role of Inflammation in Brain Cancer 83
IFN-γ macrophages
quinolinate
glutamate annexin-1
ROS mutation
mutation hypermethylation growth invasion
Cell of Origin Tumor Tumor Tumor Glioma
kynurenine quinolinate
IL-8 kynurenine
HOCl
arginase 1 arginine
(Rossi et al. 1987; Fossati et al. 1999; Murat et al. 2009; Charles and Holland
2010; Yang et al. 2011; Sen 2011; Huysentruyt et al. 2011; Curran and Bertics
2012; Charles et al. 2012). Interactions in the tumor microenvironment are pre-
sented diagrammatically in Fig. 4.4.
Among the many genes often mutated in glioblastoma, the p53 gene is the most
frequently mutated and has been studied the most extensively. The most prevalent
mutations in p53 of brain tumors are single-base changes, with ~85 % simple sub-
stitutions (Greenblatt et al. 1994). Among these, transition mutations (GC to AT or
AT to GC) comprise ~65 % of the substitutions. A significant number (~30 %) of
the GC to AT transitions are within a CpG dinucleotide, and CpG dinucleotides in
the p53 gene are generally methylated (Tornaletti and Pfeifer 1995). Therefore, a
86 J. L. Sowers et al.
significant number of p53 mutations arise from the hydrolytic deamination of 5mC
to thymine, a base change that is difficult to repair. The spectrum of single-base
mutations found in the p53 gene outside of CpG dinucleotides in brain tumors is
consistent with cycles of endogenous damage and repair cycles.
The T-G mispair formed by hydrolytic deamination of 5-methylcytosine can
be repaired by four human glycosylases: UNG2, SMUG1, MBD4, and TDG
(Vasovcak et al. 2012). Failure to repair the T-G mispair would result in the
observed GC to AT transition mutations frequently observed at p53 CpG sites
in glioblastoma. The TDG glycosylase might be of particular importance as it is
involved in epigenetic reprogramming/demethylation as previously discussed
(Fig. 4.3), and TDG expression is apparently regulated by p53 (da Costa et al.
2012) and is essential for maintaining epigenetic stability (Cortázar et al. 2011).
The TDG glycosylase represents an intersection between p53 mutation and epige-
netic reprogramming in glioblastoma.
tumor tissue and in plasma are significantly higher in glioblastoma patients versus
control (Atukeren et al. 2010). Elevated MPO levels have been measured inside
tumors and in peritumoral cerebrum using a gadolinium-based MRI method in
rodent gliomas (Kleijn et al. 2011). Although MPO is likely derived from infiltrat-
ing neutrophils, it has been reported that astrocytes can aberrantly express MPO in
a mouse model of Alzheimer’s disease (Maki et al. 2009). Further studies examin-
ing halogenated DNA bases in human glioblastoma are warranted.
Emerging evidence indicates that astrocytes and neutrophils interact with one
another in a normal process of neuroinflammatory homeostasis. Following injury
or infection in the brain, neutrophils and other peripheral immune cells infiltrate
the brain parenchyma. Through interacting with normal astrocytes in the brain,
infiltrating immune cells can destroy infectious agents, eliminate necrotic tissues,
and stimulate tissue repair. Recent studies in mice have revealed some interact-
ing pathways (Xie et al. 2010). Neutrophils undergo spontaneous apoptosis to
limit inflammatory damage mediated by ROS, including HOCl generated by
MPO. Direct contact between neutrophils and astrocytes may prolong neutrophil
survival and reduce necrosis, which results in the dumping of matrix metallopro-
teinases (MMPs) and MPO into the tissues. The antimicrobial activity of neutro-
phils requires an NADPH-dependent oxidative burst generating H2O2, followed
by MPO-mediated conversion to HOCl. Cell–cell contact between astrocytes and
neutrophils can decrease both the production of ROS in neutrophils and the release
of MPO by neutrophils (Xie et al. 2010).
While neutrophils may interact effectively with normal brain cells in destroying
invading pathogens or promoting tissue turnover and repair, HOCl generated by
neutrophils is indiscriminate in its chemical reactivity and can damage the DNA of
normal cells, including astrocytes, promoting both genetic and epigenetic changes
as described previously. MPO-positive neutrophils are frequently identified within
human glioma tissues; though their role in tumor initiation and progression has
not yet been established. The presence of neutrophils increases with tumor grade,
as approximately 85 % of Grade IV gliomas samples show significant infiltration
88 J. L. Sowers et al.
4.3.2.2 Annexin1 → Neutrophils
Eosinophils stimulated with GM-CSF release TGF-α, a ligand that can promote gli-
oma proliferation via the EGF receptor (Curran and Bertics 2012; Curran et al. 2011).
Tryptophan is an essential amino acid that serves as a building block for protein
synthesis but also functions as a precursor for other biochemical mediators including
serotonin. Tryptophan also undergoes metabolism through the kynurenine pathway
(Fig. 4.5), ultimately generating quinolinic acid needed for the synthesis of nicoti-
namide adenine dinucleotide (NAD+). Several of the metabolites formed along this
pathway, including kynurenine, kynurenic acid, and quinolinic acid, have activities
that can result in neuroprotection or pathophysiology. Several reviews have been
published on the role of the kynurenine pathway in the brain (Guillemin et al. 2001;
Schwarcz and Pellicciari 2012; Guillemin et al. 2007; Schwarcz et al. 2012; Adams
et al. 2012; Vécsei et al. 2013).
The kynurenine pathway is triggered by inflammatory cytokines found in the
glioma tumor microenvironment including IFN-α, IFN-γ, TNF-α, TGF-β, IL-4,
and IL-23 (Mándi and Vécsei 2012). These cytokines induce the first enzyme of
the kynurenine pathway, indoleamine 2,3-dioxygenase (IDO), which converts
tryptophan to kynurenine. However, induction of the enzymes along the kynurenine
pathway can be both cytokine and cell-type specific. In human mesenchymal stem cells,
both IFN-β and IFN-γ upregulate expression of mRNAs for all of the enzymes along
90 J. L. Sowers et al.
CO2H H
tryptophan
NH2 hydroxylase HO NH2
N N
tryptophan serotonin
H H
IFN-γ indoleamine
dioxygenase
induced
CO2H
kynurenine OH
NH2 aminotransferase
O
NH2 kynurenine N CO2H
kynurenic
neuroprotective
acid
kynurenine
hydroxylase
α-ketoglutarate
CO2H dependent
NH2
O
NH2 3-hydroxykynurenine
OH
kynureninase
the pathway, and IFN-γ upregulates all pathway enzymes substantially in macrophages.
In contrast, in mouse neural stem cells, IFN-g upregulates the initiating enzyme, IDO,
kynurenine aminotransferase that converts kynurenine to kynurenic acid, and quinoli-
nate phosphoribosyltransferase (QPRT) that converts quinolinic acid to nicotinic acid
ribonucleotide, a necessary substrate for (NAD+) synthesis (Croitoru-Lamoury et al.
2011). Astrocytes lack kynurenine hydroxylase, such that activation of the kynurenine
pathway in these cells generates kynurenine and kynurenic acid, but not quinolinic acid
(Guillemin et al. 2001).
The upregulation of some, but not all of the kynurenine pathway enzymes in neurons
suggests that some of the initial metabolites including kynurenine and kynurenic acid
could be beneficial to neurons, but that quinolinic acid is not. However, quinolinic
acid generated by other cells might be utilized as a substrate for replenishing (NAD+)
in neurons. Therefore, examining the kynurenine pathway within the context of the
inflammatory tumor microenvironment might provide important clues not revealed by
examining the same pathway in a single cell type in isolation.
Although glutamate is excitotoxic to neurons, glioma cells not only have a much
higher threshold for glutamate damage, but glutamate binding to glioma-specific
receptors can also promote glioma cell proliferation. Multiple groups have recently
reported that glioma cells and glioblastoma-derived brain tumor initiating cells
(BTICs) have Ca2+ permeable AMPA receptors. AMPA receptors are tetrameric and
are composed of GluR1 through GluR4 subunits, and GluR2-lacking receptors are
Ca2+ permeable. AMPA receptors on BTICs are comprised of GluR1 and GluR4
subunits (Oh et al. 2012), whereas AMPA receptors on the glioblastoma cell line
U87MG contain the GluR2 subunit and are Ca2+ impermeable (Ekici et al. 2012).
Human surgical glioma tissues contain Ca2+ permeable AMPA receptors, and gluta-
mate activation promotes cell growth and mobility (Lyons et al. 2007) by activation
of the Akt pathway (Ishiuchi et al. 2007; Schunemann et al. 2010).
4 The Role of Inflammation in Brain Cancer 95
At the time of presentation, high-grade gliomas are aggressive tumors that display
aberrant vascularization with infiltrating immune cells creating a heterogeneous
tumor microenvironment. Multiple recurrent genetic and epigenetic abnormalities are
observed in glioma cells that converge on several key signaling pathways involved
in tumor cell survival, proliferation, and invasion. Despite the significant increases
in understanding of the molecular changes found in glioma cells, current therapy
options remain focused on surgical resection, radiation therapy, and chemotherapy
with the alkylating agent temozolomide, and survival is still measured in months.
Very little is currently known about agents or events that might drive the genetic
and epigenetic changes leading to the evolution of high-grade glioma, and it is
unknown if the multitude of observed changes must occur in a particular sequence.
It is likely that initial metabolic changes within tumor initiating cells increase intra-
cellular oxidative stress, leading to increased oxidative DNA damage. Increased
DNA damage, coupled with diminished DNA repair could then drive subsequent
mutations. Within the inflammatory tumor microenvironment, reactive oxygen,
nitrogen and halogen species contributed by activated macrophages, neutrophils,
and eosinophils likely contribute to further mutations and epigenetic changes as
glioma cells progress.
96 J. L. Sowers et al.
The genetic and epigenetic changes in glioma cells account for the survival, pro-
liferation, and invasion of tumors. Yet, these changes alter cellular metabolism in
ways that could be exploited for the future development of targeted chemotherapy
focused against metabolism. Altered metabolism within the tumor cells is also cou-
pled to changes within the tumor environment that influence the impact of the gli-
oma cells on normal neurons and immune cells. Although a multitude of genetic and
epigenetic changes can be found in glioma cells, a uniform finding in glial tumors is
the absence of the modified DNA base, 5hmC. This recently identified modified base
is believed to be an intermediate in an enzymatic demethylation pathway required
for epigenetic reprogramming. The uniform loss of 5hmC in glioma cells lines and
human tissues suggests a common defect central to gliomagenesis.
Emerging studies indicate that metabolic activity of three amino acids, glutamate,
tryptophan, and arginine, and these metabolic alterations have profound effects on
glioma progression. Whereas normal glial cells sequester glutamate, maintaining
low extracellular levels, glioma cells export glutamate that is excitotoxic to neurons.
Subsequent neuronal death results in further increases in extracellular glutamate,
promoting a cascade of neuron loss and tissue destruction. In contrast with normal
neurons, glutamate drives glioma progression by binding to glutamate AMPA recep-
tors, activating cell signaling pathways that drive proliferation.
Tissue destruction results in increases in inflammatory mediators including
IFN-γ. Inflammatory cytokines upregulate enzymes of tryptophan metabolism
within the kynurenine pathway. In most cell types, including glioma cells, IFN-γ
upregulates the first enzyme of this pathway, IDO, which converts tryptophan to
kynurenine. Kynurenine binds to the AhR on T cells diminishing antitumor T-cell
responses and promoting the formation of Tregs. Activation of the entire kynure-
nine pathway in macrophages results in the conversion of tryptophan to quino-
linic acid. Although quinolinic acid is toxic to human neurons, and is commonly
used in experimental models of excitotoxic neurodegeneration, quinolinic acid
promotes glioma cell survival and proliferation. Increased oxidative stress within
glioma cells results in increased DNA damage which in turn results in upregula-
tion of PARP-1 and consumption of NAD+. Diminished NAD+ levels can lead to
energy failure and cellular death; however, quinolinic acid generated by other cells
can serve as a precursor for NAD+ synthesis in glioma cells.
Chemoattractants generated within the tumor microenvironment are able to facil-
itate the invasion of neutrophils. Reactive molecules from activated neutrophils can
lead to further tissue destruction and glioma mutagenesis. Neutrophil degranulation
also dumps arginase 1, which converts arginine to ornithine. Diminished arginine
concentrations reduce macrophage-generated NO and profoundly suppress T-cell
immune responses. Through both tryptophan and arginine metabolic pathways,
inflammation within the tumor microenvironment promotes immunosuppression
and promotes tumor evolution.
Substantial further studies are required to understand the complex interactions
between the multiple cell types associated with gliomas, as well as how genetic
and epigenetic changes within glioma cells are both likely induced by the inflam-
matory environment and exploited by glioma cells to promote tumor cell survival
4 The Role of Inflammation in Brain Cancer 97
with collateral damage to normal tissues. Studying tumor cells in isolation may
provide targets for metabolic intervention and future chemotherapy development.
However, a more complete understanding of the complex interactions between the
tumor cells and surrounding normal tissues could lead to strategies to redirect the
host response against these rapidly growing and lethal human tumors.
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4 The Role of Inflammation in Brain Cancer 103
M. Bonomi (*)
Head and Neck Oncology Program, Wake Forest School of Medicine, Winston-Salem,
North Carolina, USA
e-mail: [email protected]
A. Patsias · M. Posner · A. Sikora
Head and Neck Oncology Program, Mount Sinai School of Medicine, New York, USA
M. Posner
e-mail: [email protected]
homeostasis and repair may provide a logical framework for understanding the
connection between the inflammatory response and cancer. The cells and molecules
outlined here represent potential targets for the treatment of head and neck cancer.
5.1 Introduction
Head and neck squamous cell carcinoma (HNSCC) originates in the mucosa of
5 major anatomic subsites: the oral cavity, oropharynx, larynx, hypopharynx, and
nasopharynx. It is the sixth most common cancer worldwide, with approximately
650,000 new cases reported annually. Aggravating factors are tobacco smoking,
alcohol consumption, betel chewing, and human papilloma virus (HPV) infection
(Curado and Hashibe 2009). In the United States, there were 49,000 new cases in
2010, with 11,000 deaths (Jemal et al. 2010). Despite the overall decreased inci-
dence of HNSCC in the United States over the past 3 decades, researchers have
observed a significant increase in the incidence of squamous cell malignancies of
the base of tongue, and tonsil, particularly in young-to-middle-age patients likely
due to rising incidence of HPV-associated HNSCC (Shiboski et al. 2005).
Despite treatment advances in multimodality therapy with surgery, radiother-
apy, and chemotherapy, 5-year survival is still poor for patients with locoregionally
advanced disease (Forastiere et al. 2003; Posner et al. 2007; Vermorken et al. 2007).
The genetic alteration of cells in wide preneoplastic fields (field cancerization)
results in locoregional recurrence and second primary cancer. Half of all individu-
als still die from their disease. The characterization of the mechanisms involved
in the metastasis formation and the identification of markers allowing identifying
patients with biologically aggressive tumors is of great interest for the effective
management of HNSCC patients.
Cigarette smoke (CS) causes considerable morbidity and mortality by inducing
cancer, chronic lung and vascular diseases, and oral disease. Despite the well-rec-
ognized risks associated with smoking, the habit remains unacceptably prevalent.
Several toxins present in CS have immune modulatory effects. CS also contains
trace amounts of microbial cell components, including bacterial lipopolysaccha-
ride. These and other CS constituents induce chronic inflammation at mucosal
surfaces and modify host responses to exogenous antigens. Mucosal damage from
chronic tobacco and alcohol exposure has been well characterized, both in terms
of its clinicopathologic course and the underlying molecular derangements respon-
sible for tumor development. Premalignant lesions, including leukoplakia and
erythroplakia, progress to invasive carcinomas along a well-described pathologic
sequence (Perez-Ordonez et al. 2006).
Molecular events that undergird this process include increasing cytogenic abnor-
malities, inactivation of tumor suppressor genes, and changes in intracellular signaling
pathways that induce cellular immortalization. The effects of CS on immunity are far-
reaching and complex; both pro-inflammatory and suppressive effects may be induced.
The net effect of CS on immunity depends on many variables, including the dose and
5 The Role of Inflammation in Head and Neck Cancer 109
type of tobacco, the route, and chronicity of exposure, and the presence of other factors
at the time of immune cell stimulation, such as Toll receptor ligands or other inflam-
matory mediators. CS impairs innate defenses against pathogens, modulates antigen
presentation, and promotes autoimmunity. CS also impairs immunity in the oral cavity
and promotes gingival and periodontal disease and oral cancer. The recognition of spe-
cific mechanisms by which CS affects host immunity is an important step toward elu-
cidating mechanisms of tobacco-induced disease and may identify novel therapeutic
approaches for the management of smoking-related diseases (Lee et al. 2012).
Human papilloma virus-related oropharyngeal carcinoma (HPVOPC) clinically
behaves differently than tobacco- and alcohol-induced HNSCC. Inflammation
and immunosuppression are likely to also play a critical role in HPVOPC. These
patients tend to present at a younger age, and without a history of excessive
tobacco or alcohol use. Overall, HPVOPC patients also have better outcomes, with
tumors more responsive to both surgical and non-surgical therapies and a lower
risk of dying from disease. The relationship of HPVOP to inflammation remains
largely unexplored (Chung and Gillison 2009; Ang et al. 2010).
HPV inactivates the same pathways via direct viral effects. The HPV is a
circular, double-stranded DNA virus that encompasses many different subtypes,
with HPV-16 and HPV-18 being the most common oncogenic variants in HPVOPC.
Using in situ hybridization, HPV-16 DNA has been found in up to 72 % of oro-
pharyngeal cancer specimens where this association remains the highest (D’Souza
et al. 2007). On a molecular level, HPV gains access to the intracellular compart-
ment of mucosal squamous cells and integrates into host DNA. The integrated virus
subsequently expresses oncoproteins E6 and E7, which act synergistically to target
the tumor suppressor genes p53 and pRb for ubiquitin-mediated intracellular degra-
dation, resulting in genomic instability and oncogenic transformation as the normal
cell-cycle regulatory points are inactivated (Chung and Gillison 2009).
Experimental tumor model studies show that non-steroidal anti-inflammatory
drugs (NSAIDs) impair the growth and development of HNSCC, indicating poten-
tial as a chemopreventive agent. Furthermore, regular use of NSAIDs and aspirin
has been shown to reduce the risk of other cancers.
Biologically, NSAIDs act as non-specific inhibitors for the pro-inflammatory
cyclooxygenase enzymes (COX-1 and COX-2), which are involved in the conver-
sion of arachidonic acid (AA) to prostaglandins (PG). COX-1 is present in most
tissues and is involved in the production of PGs required for many normal physi-
ologic functions, while COX-2 is found only in a limited number of cell types and
is induced by stimulatory factors implicated with inflammation and many cancers.
Overexpression of COX-2 and PGs have been reported in a variety of cancer
sites, including HNSCC, with increased levels reported in both tumor tissue and
adjacent epithelium in HNSCC but not normal epithelium. Studies also suggest a
correlation between COX-2 expression and head and neck tumor size and progno-
sis, with higher expression correlating with poorer outcome (Wilson et al. 2011).
The downstream actions of PGs, such as increased cell proliferation, cell mobility
and invasion, neo-angiogenesis, and the inhibition of apoptosis, are known to play
important roles in cancer development. The mechanism by which NSAIDs inhibit
tumor development is not clearly understood, although it is thought that they may
act through the inhibition of COX-2 and consequently the synthesis of PGs and
their pro-cancerous downstream effects (Wilson et al. 2011).
It has been shown that the EGFR and COX-2 have an important role in the biology
of HNSCC. Overexpression of COX-2 is associated with a poor prognosis in HNSCC,
and COX-2 inhibitors have demonstrated synergy when combined with EGFR inhibi-
tors in preclinical models (Chen et al. 2004; Chung et al. 2011). Inflammatory media-
tors can promote epithelial–mesenchymal transition (EMT), a process by which
epithelial cells lose their cell polarity and cell–cell adhesion, and gain migratory and
invasive properties to become mesenchymal cells. This process is responsible for the
increase resistance to EGFR-TKIs in HNSCC. These studies provide a strong ration-
ale for combining a COX-2 inhibitor with an EGFR TKI (Kao et al. 2011).
Recent advances in the understanding of the oncogenesis of HNSCC have
revealed multiple deregulated signaling pathways. Transforming growth factor-β
(TGF-β) and PTEN/PI3K/Akt/mTOR pathways are among the most frequently
altered signaling routes. Both pathways have central roles in numerous cellular
5 The Role of Inflammation in Head and Neck Cancer 111
During the process of tumor dissemination, tumor cells lose their epithelial
characteristics [inhibition of E-cadherin (Cdh1)] to the profit of mesenchymal
properties (expression of Snail1 and increased migratory abilities), allowing
them to invade blood and lymphatic systems and establish new colonies in dis-
tant organs (Thiery et al. 2009). Other inflammatory mediators in addition to
COX-2 have been shown to modulate EMT. Indeed in HNSCC cell lines, IL-1β
was reported to stimulate Snail1 and inhibit Cdh1 expression (John et al. 2009).
Among inflammatory actors, IL-32 was reported to modulate cytokine expression
and to be up-regulated by TNF-α, IL-1β and IL-6 (Shioya et al. 2007; Kim et al.
2005). IL-8 and GRO1 serve as chemoattractants for neutrophils, monocytes, and
endothelial cells, which are all major constituents of the inflammatory and angio-
genesis response, and their expression promotes aggressive growth and metastasis
(Van Waes 2007). In addition, IL-1 and IL-6 are potent inducers of HGF pro-
duction by stromal cells, such as fibroblasts, further enhancing IL-8 and VEGF
expression (Worden et al. 2005). Several cytokines and growth factors also acti-
vate signal pathways that promote the malignant phenotype. TNF-α, IL-1, HGF,
and their receptors promote activation of the mitogen-activated protein kinase-acti-
vator protein-1 (MAPK-AP-1), nuclear factor-kappa B (NF-κB), and phosphati-
dylinositol-3 kinase (PI3K)/Akt pathways (Van Waes 2007). Epidermal growth
factor (EGF) and IL-6 activate signal transducer and activating transcription fac-
tor-3 (STAT3) in HNSCC cells (Lee et al. 2008).
An increasing number of studies have recently focused on the role of cytokine
networks, including IL-6, in the pathogenesis and progression of oral malignancy.
In particular, clinical studies reported elevation of IL-6 levels in serum and saliva
of patients with oral and other cancers of the head and neck compared with age-
matched control subjects and their significant relation with staging and response
to therapy (Chen et al. 1999; Bigbee et al. 2007). The expression of IL-6 and
IL-8 genes was shown, via large-scale gene expression profiling on laser-cap-
tured microdissected oral cancer and normal oral epithelial cells, to be uniquely
associated with HNSCC (Alevizos et al. 2001). IL-6 seems to contribute to oral
cancer pathogenesis through different mechanisms and biologic processes. An in
vitro study showed that IL-6 can stimulate HNSCC cells to enhanced secretion of
matrix metalloproteinases 1 and 9, which play a major role in infiltrative growth,
metastasis, and neo-angiogenesis (Sundelin et al. 2005). IL-6 may also modulate
a variety of keratinocytes pathways including cell growth, survival, and differen-
tiation. In particular, IL-6 has been shown to stimulate proliferation of cultured
human keratinocytes in psoriatic skin (Nibali et al. 2012). Furthermore, IL-6 can
activate transcription factors such as signal transducer and activator of transcrip-
tion (STAT)-1 and STAT-3, which in turn have been observed in various tumors
(Hirano et al. 2000). A recent study showed that IL-6 can also promote tumorigen-
esis by causing DNA hypomethylation as well as aberrant promoter hypermethyla-
tion changes, which can lead to epigenetic changes in gene expression of HNSCC
cells (Gasche et al. 2011). Furthermore, in vitro studies demonstrated that oral
keratinocytes can produce IL-6 in response to a number of environmental factors
114 M. Bonomi et al.
well known to increase oral cancer risk such as areca nut and tobacco smoking
(Jeng et al. 2003). Indeed, biopsies from individuals with oral submucous fibrosis
showed increased expression of IL-6 in the epithelium and underlying inflamma-
tory infiltrate, as well as in peripheral blood mononuclear cells (Haque et al. 2000).
IL-32 is one of the cytokines with pro-inflammatory activities implicated in
inflammatory disorders, such as rheumatoid arthritis, mycobacterium tuberculo-
sis infections, and inflammatory bowel disease (Shioya et al. 2007; Heinhuis et al.
2011). On a retrospective study of 65 patients with HNSCC, it was shown that
patients with tumors expressing high amounts of IL32 had a worse disease-free
survival and overall survival in comparison with individuals with weak IL32 tumor
expression. In addition, in vitro data linked IL32 expression to metastatic poten-
tial (Guenin et al. 2013). The inverse correlation between IL32 and p53 expres-
sion found in this study was also found in patients with hepatocarcinoma (Kang
et al. 2012). The increased p53 expression induced by IL-32 inhibition could origi-
nate from the loss of Snail1 which would not be able to form a complex with p53
leading to its degradation through a transcription-independent mechanism (Lee
et al. 2009). Alternatively, IL-32 inhibition was reported to decrease NF-κB which
is a well-described p53 inhibitor and an activator of Snail1 expression (Gurova
et al. 2005; Tergaonkar and Perkins 2007; Zhang et al. 2011). Therefore, IL-32
down-regulation might allow p53 re-expression through NF-κB and Snail1 inhibi-
tion (Kim et al. 2011). We can speculate that IL32 plays a pivotal role in tumor
responses to inflammatory mediators and enhances cell invasiveness properties
through a nuclear NF-κB/Snail1 axis in which intermediary actors have to be iden-
tified. This is supported by its nuclear localization found in the more aggressive
tumors (Guenin et al. 2013).
TGF-β belongs to a superfamily of multifunctional cytokines that regulate cell
proliferation, differentiation, migration, adhesion, and apoptosis, thereby influ-
encing important physiologic processes such as embryonic development, immune
function, and carcinogenesis (Derynck and Zhang 2003; Massague 2008). The
three mammalian TGF-β isoforms, TGF-β1, TGF-β2, and TGF-β3, exert their
functions through a cell-surface receptor complex composed of type I (TGFBR1)
and type II (TGFBR2) serine/threonine kinase receptors. Upon ligand binding,
TGFBR2 recruits and phosphorylates TGFBR1, which in turn phosphorylates
Smad2 or Smad3. Phosphorylated Smad2 or Smad3 binds to Smad4, and then,
these complexes translocate from the cytoplasm into the nucleus. This results in
the transcriptional activation of TGF-β-responsive genes that mediate the effects of
TGF-β at the cellular level. In addition to Smad-mediated signaling, receptor acti-
vation also induces other downstream targets, including Ras, RhoA, TAK1 (TGF-
β-activated kinase-1), MEKK1, PI3K, and PP2A, to produce the full spectrum of
TGF-β response (Moustakas and Heldin 2009; Zhang 2009).
The effects of TGF-β signaling on carcinogenesis largely depend on the tissue
of origin and the tumor type. In most types of human cancer, TGF-β has a para-
doxical role in cancer development by way of functioning as a tumor suppressor
during the early stages (Engle et al. 1999) and as a tumor promoter during the
later stages (Piek and Roberts 2001; Tang et al. 2003). Several reports have noted
5 The Role of Inflammation in Head and Neck Cancer 115
that mutations and polymorphisms of TGFBR1 and Smads are associated with
HNSCC, (Chen et al. 2001; Xie et al. 2003; Pasche et al. 2005) suggesting that
TGF-β functions as a potent tumor suppressor. However, it is not clear whether
alterations in TGF-β signaling act alone or in concert with alterations in other
pathways to promote a pro-oncogenic phenotype in advanced late-stage HNSCC.
As noted above, The PI3K/Akt pathway is important for suppressing apopto-
sis, and promoting cell growth and proliferation. In HNSCC, hyper activation of
PI3K can be induced by mutations or by enhanced activity of its upstream activa-
tors, including the activation of Ras oncoproteins or inactivation of phosphatase and
tensin homolog (PTEN) deleted on chromosome 10 (Molinolo et al. 2009). PTEN
is a potent tumor suppressor gene and a negative regulator of the PI3K/Akt path-
way. As PTEN mutations were identified in 0–16 % of HNSCCs, loss of PTEN
expression was observed in 29 % of tongue cancers and loss of heterozygosity of
the PTEN locus was identified in 40 % of HNSCCs (Henderson et al. 1998; Shao
et al. 1998; Lee et al. 2001). Additionally, 47 % of HNSCC cases showed at least
one molecular alteration in the PI3K/Akt pathway, including PI3 KCA and AKT2
amplification, p110α overexpression and PTEN protein down-regulation. This
suggests the critical role of the PTEN/PI3K/Akt signaling pathways in the car-
cinogenesis of HNSCC (Pedrero et al. 2005). It seems that there may be negative
cross talk between the TGF-β tumor suppressor and the PI3K/Akt pathways (Bian
et al. 2012). It was shown that defects in the TGF-β and PI3K/Akt signaling path-
ways are common in human HNSCCs. Activation of the PI3K/Akt pathway due to
PTEN deletion initiates tumor formation by increasing proliferation in the head and
neck epithelia. However, PTEN deletion alone is not sufficient to induce invasive
HNSCC due to the induction of premature senescence by p-Akt in the presence of
the tumor suppressor TGF-β. In combination with the additional loss of TGFBR1,
which blocks tumor inhibition by TGF-β signaling, premalignant cells cannot
undergo cellular senescence and will progress into cancer cells (Bian et al. 2012).
Studies on a 2cKO mouse model showed that TGFBR1 and PTEN work col-
laboratively in suppressing tumor progression. The loss of TGFBR1/PTEN func-
tion is associated with increased cell proliferation, loss of apoptosis, and increase
levels of Cyclin D1 (CCND1) in head and neck cancer (Bian et al. 2012).
The multifunctional cytokine TGF-B has different effects in premalignant and
malignant cells. In epithelial cells, TGF-B has a tumor-suppressor effect via its
autocrine interaction with other signaling pathways. On the other hand, in tumor
cells, TGF-B increases tumor proliferation via its paracrine effects which include
but not limited to inflammation, angiogenesis, and escape form immunosurveil-
lance (De Wever and Mareel 2003).
The interaction between different pathways, transcription factors, and multi-
funcitonal cytokines is far more complex than previously thought. For instance,
in a head and neck mouse model, it was recently shown that the deletion of
TGFBR1/PTEN is associated with the activation of the NF-kB pathway. As a
result of this interaction, several genes that are associated with an inflammatory
state are also over-expressed (i.e., Cxc11, Cxcl5, Ptgs2). This pro-inflamma-
tory state is responsible for the recruitment of myeloid-derived suppressor cells
116 M. Bonomi et al.
(MDSCs), which increases the angiogenesis and immune suppressive state within
the tumor stroma (Bian et al. 2012). The disruption of the TGF-B signaling path-
way can lead to similar findings (Lu et al. 2006; Bierie et al. 2008). These data
support the concept that the tumor stroma has a pivotal role in the development
and progression of head and neck cancer (Bian et al. 2012).
Neuroblast differentiation-associated protein AHNAK, also known as
desmoyokin, is a protein that in humans is encoded by the AHNAK gene. AHNAK
was originally identified in 1989 (in bovine muzzle epidermal cells) and named
desmoyokin due to its localization pattern (that resembled a yoke) in the desmo-
somal plaque. It is a protein of exceptionally large size (700 kDa) that is expressed
in a variety of cell types (Shtivelman et al. 1992). This protein has the ability to
shuttle between various subcellular compartments. For instance, it has been shown
that AHNAK can translocate from the cytoplasm to the plasma membrane of
keratinocytes in a manner dependent on Ca2+ and protein kinase C (Hashimoto
et al. 1995). Furthermore, AHNAK was shown to contain a nuclear export sig-
nal (NES) sequence which allowed it to be excluded from the nuclei of epithelial
cells following cell–cell contact and activation of protein kinase B, respectively
(Sussman et al. 2001). At functional level, AHNAK was shown to be involved in
various cellular processes, including calcium regulation and organization of the
actin cytoskeleton (Haase et al. 1999; Gentil et al. 2001). In tumor cells, AHNAK
was recently found to be essential for pseudopodia formation and tumoral
migration/invasion (Shankar et al. 2010). Other recent studies proposed that the
AHNAK gene might be involved in mutagenic transformation of colon epithelial
cells and thus carcinogenesis (Tanaka et al. 2008). It is well established that solid
tumors display an inflammatory microenvironment characterized by large numbers
of tumor-infiltrating immune cells (Coussens and Werb 2002). Within this micro-
environment, the immune cells of the host are reprogrammed by the tumor cells to
acquire pro-tumoral activities. Although less characterized than tumor-associated
macrophages (TAMs) or tumor-infiltrating lymphocytes (TILs), tumor-infiltrating
neutrophils are emerging as important players in the pathophysiology of cancer.
Within the tumor tissue, neutrophils can modulate several cellular processes which
may ultimately lead to tumor progression. Neutrophils were shown to modulate
angiogenesis in several murine tumor models (Nozawa et al. 2006; Jablonska
et al. 2010; Bekes et al. 2011) and were recently associated with angiogenesis pro-
gression in hepatocellular carcinoma patients (Kuang et al. 2011). Further stud-
ies showed that neutrophils could directly modulate the biology and functions of
tumor cells by promoting their migration, invasion or proliferation (Gregory and
Houghton 2011).
There is an association of high numbers of tumor-infiltrating neutrophils with
advanced disease and poor clinical outcome in patients with different types of
cancer, such as renal cancer, hepatocellular cancer, non-small-cell lung carcinoma
(NSCLC), or melanoma (Dumitru et al. 2012).
In head and neck cancer patients, it was demonstrated that a high neutrophilic
infiltration of the tumor tissue was correlated with high tumor stage and poor sur-
vival (Trellakis et al. 2011). In vitro studies indicated a direct interaction between
5 The Role of Inflammation in Head and Neck Cancer 117
neutrophils and head and neck cancer cells by showing that neutrophils were
primed by the tumor cells to release pro-inflammatory factors, which promoted
tumoral migration in a feedback manner (Dumitru et al. 2011, 2012). Selected solu-
ble inflammatory mediators, such as cytokines, chemokines, and metabolites of the
AA pathway, have been found to change the function and differentiation of immune
cells (Lin and Karin 2007). Among these molecules, macrophage migration inhibi-
tory factor (MIF) is emerging as an important regulator of inflammation in cancer
(Bucala and Donnelly 2007). A number of studies found that high levels of MIF
in the tumor tissues or serum of patients with different types of cancer were asso-
ciated with advanced disease and poor clinical outcome (Grieb et al. 2010). It was
also demonstrated that overexpression of tumoral MIF was associated with poor over-
all survival in patients with orohypopharyngeal cancer (Dumitru et al. 2011). More
importantly, MIF was identified as one of the missing links in the tumor-neutrophil
interaction and showed that head and neck cancer cells released MIF which subse-
quently enhanced the pro-inflammatory functions of neutrophils to promote tumoral
migration (Dumitru et al. 2011). AHNAK overexpression is associated with poor
survival in these patients. Interestingly, in patients with HNSCC, it was found that
high levels of AHNAK together with high MIF expression or high neutrophilic infil-
tration, respectively, were strongly associated with poor survival. Synchronous high
levels of MIF and tumor-infiltrating neutrophils had stronger predictor values over
the individual markers as well. Finally, patients with high levels of all three mark-
ers displayed the shortest survival in the entire patient cohort (Dumitru et al. 2013).
These findings suggest that AHNAK might cooperate with MIF and/or neutrophils to
enhance progression of HNSCC. There is data regarding direct interactions between
HNSCC-derived MIF and neutrophils both in vitro and in vivo. It was shown that
HNSCC-derived MIF enhanced neutrophil chemotaxis in vitro and that tumoral MIF
levels correlated with the neutrophilic infiltration in tissues from orohypopharyngeal
carcinoma patients (Dumitru et al. 2011). Since MIF is a known ligand for CXCR2,
one of the major chemokine receptors on neutrophils, (Bernhagen et al. 2007) MIF-
mediated recruitment might be a critical mechanism for infiltration of HNC tissues
by neutrophils. It was further demonstrated that HNC-derived MIF stimulated neutro-
phils to release large amounts of pro-inflammatory factors, among which CCL4 and
MMP9 (Dumitru et al. 2011). Neutrophils enhance the motility, migration, and inva-
sion of tumor cells via—not fully identified—soluble factors and molecular mecha-
nisms (Dumitru et al. 2012). Interestingly, AHNAK was recently linked to regulation
of tumoral migration/invasion. It seems that AHNAK is essential for rearrangement of
the actin cytoskeleton and pseudopodia formation (Shankar et al. 2010).
HPV-HNSCC differs from tobacco-related head and neck cancers in several
ways. The patients tend to be younger in age, lack a significant tobacco and/or
alcohol history, and have improved clinical outcomes. The virus-related tumors
arise from the deep crypts within the lymphoid tissue of the tonsil and base of
tongue and the majority can be distinguished from tobacco-related HNSCC
by the characteristic infiltration of lymphocytes in the stroma and tumor nests.
Nevertheless, despite this profound inflammatory response, HPV-HNSCCs are
able to evade immune surveillance, persist, and grow (Gillison et al. 2008).
118 M. Bonomi et al.
Various mechanisms have been proposed for the resistance of human solid
tumors to immune recognition and obliteration, including the recruitment of regula-
tory T cells, MDSCs, and local secretion of inhibitory cytokines. Recent evidence
suggests that tumors develop physiologic mechanisms of tissue protection from
inflammatory destruction via up-regulation of immune inhibitory ligands. Antigen-
induced activation and proliferation of T cells are regulated by the temporal expres-
sion of both co-stimulatory and co-inhibitory receptors and their cognate ligands
(Topalian et al. 2012).
In the context of cancer, in which immune responses are directed against
antigens specifically or selectively expressed by tumor cells, these immune
checkpoints can represent major obstacles to the generation and maintenance of
clinically meaningful anti-tumor immunity. CTLA-4 and programmed cell death-1
(PD-1) are two such checkpoint receptors being actively targeted in the clinic
(Lyford-Pike et al. 2013).
It has been shown that in HPV-HNSCCs that are highly infiltrated with lym-
phocytes, PD-L1 expression on both tumor cells and CD68(+) TAMs is geograph-
ically localized to sites of lymphocyte fronts, whereas the majority of CD8þ TILs
express high levels of PD-1, the inhibitory PD-L1 receptor. Significant levels of
mRNA for IFN-γ, a major cytokine inducer of PD-L1 expression, were found in
HPV(+) PD-L1(+) tumors. These findings support the role of the PD-1: PD-L1
interaction in creating an “immune-privileged” site for initial viral infection and
subsequent adaptive immune resistance once tumors are established and suggest a
rationale for therapeutic blockade of this pathway (Lyford-Pike et al. 2013).
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Chapter 6
The Role of Inflammation in Pancreatic
Cancer
esophagus and esophageal cancer, hepatitis and liver cancer. The fibroinflam-
matory stroma of chronic pancreatitis resembles that of pancreatic cancer, and
patients with familial chronic pancreatitis have a 26-fold increased risk of devel-
oping pancreatic cancer compared with the normal population—probably owing
to chronic inflammation (Lowenfels et al. 1993). In this chapter, key pathways
involved in this multifaceted interaction between inflammatory cells and pancre-
atic cancer are summarized.
The poor prognosis of pancreatic cancer patients is mostly due to late diagnosis and
the absence of effective therapy options. Considering that three quarters of patients
are not candidates for surgery at the time of first diagnosis, the most commonly
employed therapy consists of radio- and chemotherapy. In the last decade, gemcit-
abine-based chemotherapy became the reference treatment since studies compar-
ing gemcitabine with 5-fluorouracil showed similar survival advantages but better
quality of life with the former (Neoptolemos et al. 2010). Different combinations of
gemcitabine with other cytotoxic drugs could not improve the survival of patients
significantly compared to gemcitabine treatment alone. Even the highly toxic regi-
men Folfirinox, consisting of irinotecan, oxaliplatin, 5-fluorouracil and folinic acid,
resulted in a median survival of less than one year in patients with advanced pan-
creatic cancer (Conroy et al. 2011). Many targeted agents, including angiogenesis
inhibitors, which have shown success in the preclinical setting, failed to prolong
survival of pancreatic cancer patients in the clinical setting (Erkan et al. 2012). The
6 The Role of Inflammation in Pancreatic Cancer 131
only FDA approved targeted agent Erlotinib (a tyrosine kinase inhibitor that acts
on human epidermal growth factor receptor type 1 [HER1/EGFR]) is no exception.
This agent only increases median survival from 5.91 months (gemcitabine and pla-
cebo) to 6.24 months (gemcitabine and erlotinib) in patients with advanced pancre-
atic cancer (Moore et al. 2007).
Recently, the abundant fibrotic stroma of pancreatic cancer is shown to form
a mechanical barrier for the effective delivery of chemotherapeutic agents. This
observation has led to the emergence of anti-fibrotic therapies which appear to be
effective in the preclinical setting. For example, the inhibition of the hedgehog
signaling pathway in a genetically engineered mouse model of pancreatic cancer
showed a better penetrance of the tumor with gemcitabine and a longer survival of
the mice (Olive et al. 2009). However, the first phase II trial (IPI-926-03) has to be
stopped after interim analysis due to increased mortality in the therapy arm (Erkan
2013a). These results hint that the problem in pancreatic cancer is multifaceted,
and a successful therapy should aim at correcting several defects at genetic, epige-
netic, and microenvironmental levels.
After the separation from the inhibitory protein IκB, the subunits can translocate
to the nucleus, bind to κB-sequences within promoter regions and regulate the tran-
scription of different genes involved in survival, inflammation as well as its own
inhibitor IκB-α (Pahl 1999; Hayden and Ghosh 2011).
In many studies, it could be shown that the NFκB pathway is activated
in early stages of pancreatitis and enhances the pro-inflammatory response
through the activation of anti-apoptotic and inflammatory genes (Gukovsky
et al. 1998; Steinle et al. 1999; Karin 1998). In a recent paper published by
Huang and colleagues, it was demonstrated that the level of NFκB correlates
with the severity of acute pancreatitis. Furthermore, the group displayed that
long periods of activated NFκB in pancreatic acinar cells lead to a chronic pan-
creatitis characterized by severe pancreatic damage, immune cell infiltration,
and fibrosis (Huang et al. 2013). Another group showed that the deletion of
the IκB kinase IKK2 in all pancreatic epithelial cells prevented the formation
of PanIN lesions in PdxCre/+; LSL-KrasG12D/+ mice (Maniati et al. 2011) thus
indicating that the NFκB pathway plays an important role in the carcinogenesis
of pancreatic cancer.
that inhibition of IL-6 or STAT3 can reduce PanIN progression and diminishes the
development of PDAC. These results from in vitro and in vivo studies emphasize
the importance of the IL-6–STAT3 axis in the initiation as well as progression of
pancreatic cancer.
Toll-like receptors (TLRs) belong to the pattern recognition receptors and are
mainly expressed on innate immune cells as well as on neoplastic tissues (Huang
et al. 2008). Ligands for the Toll-like receptors include conserved patterns of
bacterial and viral origin also referred to as pathogen-associated molecular pat-
terns (PAMPs) as well as damage-associated molecular patterns (DAMPs). A
recent paper published by Ochi et al. (2012) showed that the TLR7 is not only
overexpressed in the epithelial compartment in pancreatic cancer but also in the
tumor stroma in mice and humans. Using a mouse model of pancreatic cancer
(p48Cre/+; KrasG12D/+), the group showed that TLR7 ligation accelerated the
development of pancreatic cancer and inhibition of TLR7 was able to inhibit
pancreatic tumorigenesis. The activation of TLR7 induced STAT3 activation and
interacted with Notch, canonical NFκB, and MAP kinase pathways. Another
group showed that the inflammatory substance lipopolysaccharide (LPS) which
activates TLR4 increased the invasive behavior of pancreatic cancer cell lines
Panc-1 and AsPC-1 through the activation of the NFκB signaling pathway. These
results demonstrate the interplay between TLR4 and NFκB signaling may be
one of the pathways linking inflammation and PDAC progression in vitro (Ikebe
et al. 2009).
In response to acinar damage, the expression of the cytokine tumor necrosis f actor
alpha (TNF-α) is induced. In human pancreatic cancer cell lines, it could be shown
that the treatment of these cell lines with TNF-α was able to induce the expres-
sion of epidermal growth factor receptor (EGFR) and its ligand, transforming
growth factor α (TGF-α) (Schmiegel et al. 1993). In vitro studies by Means et al.
6 The Role of Inflammation in Pancreatic Cancer 135
the further demonstrated the importance of the EGFR signaling pathway in the
transformation of acinar cells toward a malignant phenotype. Treatment of wild-
type acinar cells with TGF-α resulted in transformation of acinar cells into a ductal
phenotype which was accompanied by loss of acinar markers and expression of
ductal markers like cytokeratin 19 (Means et al. 2005).
NFκB exerts its anti-apoptotic effects. Several studies were able to show that
NFκB is also involved in the regulation of cyclin D1 expression (Yamamoto and
Gaynor 2001). In a recent study, it was shown that downregulation of the NFκB
subunit p65 in pancreatic cancer cells leads to a subsequent downregulation of
the pro-apoptotic gene BcL-2 as well as to the cell cycle gene cyclin D1 lead-
ing to growth inhibition of the pancreatic cancer cell line BxPC-3 (Kong et al.
2010). Another study showed that blocking the EGFR pathway in the pancre-
atic cancer cell line MDA Panc-28 resulted in a decreased NFκB binding activ-
ity as well as a reduced expression of the pro-apoptotic gens BcL-xL and Bfl-1
(Sclabas et al. 2003). The pro-inflammatory cytokine IL-6 was also shown to
contribute to survival of pancreatic cancer cells by upregulating BcL-2 and
BcL-xL. This effect could be reverted by the use of an IL-6 antibody (Miyamoto
et al. 2001).
Many inflammatory molecules have been indicated to play a role in invasion, metas-
tasis, and angiogenesis of PDAC. One of these is the pro-inflammatory cytokine
IL-1α which is produced by pancreatic cancer cells. In recent studies, it could be
demonstrated that IL-1α promotes proliferation, adhesion, and migration of the pan-
creatic cancer cell lines BxPC-3, SW1990, and Capan-2 through the upregulation
of the integrin subunits α6 and β1 and the uPAR. The above-mentioned effects are
associated with the activation of RAS and the downstream ERK signaling pathway.
By using inhibitory antibodies against α6, β1, and uPA, the group showed that the
activation of the ERK signaling as well as proliferation, adhesion, and migration of
pancreatic cancer cell lines was prevented (Sawai et al. 2006). In an additional study,
it was elucidated that IL-1α produced by pancreatic cancer cells is able to induce
the expression of hepatocyte growth factor (HGF) by fibroblasts (Xu et al. 2010).
In co-culture experiments with pancreatic cancer cells and fibroblasts, the group
showed not only the IL-1α-dependent expression of HGF by fibroblasts but also
an increased invasive and proliferative behavior of pancreatic cancer cells as well
as of human umbilical vein endothelial cells (HUVECs). This can be explained by
binding of HGF to its receptor c-met/HGF on the surface of pancreatic cancer cells
and thus fostering the observed behavior of pancreatic cancer cells (Xu et al. 2010).
Another study demonstrated that forced expression of IL-1α in the pancreatic can-
cer cell line MiaPaCa-2 activated the NFκB signaling pathway as assessed by an
increase in NFκB downstream targets. As a result of the forced expression of IL-1α
and subsequent NFκB activation, the cells gained an invasive phenotype. However,
when the NFκB pathway was inactivated by the expression of a dominant negative
IκB protein, the metastatic behavior was prevented. The same behavior of the cells
was observed when IL-1α was silenced in the metastatic pancreatic cancer cell line
L3.6pl, indicating that IL-1α-induced NFκB expression is contributing to the meta-
static phenotype of pancreatic cancer cells (Melisi et al. 2009).
In many cancer types, the pro-inflammatory cytokine IL-1β has been indicated
to influence metastasis and tumor growth (Apte et al. 2006). IL-1β together with
IL-1α belongs to the IL-1 family and has been shown to induce the expression of
138 S. Hausmann et al.
Not only in vitro studies were able to show that the inflammatory mediator
STAT3 is linked with pancreatic precursor lesion formation, but also in vivo stud-
ies demonstrated the role of STAT3 in the development of preneoplastic lesions
(Corcoran et al. 2011; Fukuda et al. 2011; Lesina et al. 2011; Li et al. 2011).
Corcoran et al. (2011) showed that STAT3 is necessary both for the development
of precursor lesions [i.e., ADM, pancreatic intraepithelial neoplasia (PanIN)],
and progression to PDAC. Fukuda et al. (2011) confirmed that STAT3, which
is overexpressed in epithelial cells after cerulein-induced inflammation in a
KrasG12D mouse model, helps to initiate tumor development and progression.
Blocking of STAT3 has led to attenuation of precursor lesion formation and pro-
liferation as well as increased apoptosis, proving the contribution of STAT3 to
cancer initiation. Moreover, the group also identified that the loss of epithelial
STAT3 leads to a reduced inflammatory cell infiltration as well as decreased
expression of inflammatory cytokines. These results indicate that STAT3 not
only has an influence on the proliferative, dedifferentiated state of the epithelial
cells but also contributes to inflammatory processes associated with metaplasia
(Fukuda et al. 2011). Lesina et al. (2011) observed the same events but addition-
ally identified the myeloid compartment to secrete the pro-inflammatory cytokine
IL-6 which leads to the activation of STAT3 in the pancreas and fosters the devel-
opment and progression of PanIN lesions. The identification of this mechanism
strengthens the role of the microenvironment in the development of PDAC and
was also shown to be valid for human PDAC by analyzing human PDAC speci-
men and patient data. Therefore, the results of these studies indicate STAT3 as a
potential therapeutic target for preventing inflammation-induced development of
PDAC at an early stage.
Inflammation has early been indicated to play a major role in pancreatic cancer
development. Similarities between the fibroinflammatory stroma (Fig. 6.1) com-
position in chronic pancreatitis and pancreatic cancer emphasize the pathogenetic
link between them (Chu et al. 2007). Inflammatory cells such as macrophages,
mast cells, neutrophils, dendritic cells, B and T lymphocytes as well as activated
PSC have all been described in the stroma of pancreatic cancer. However, only
6 The Role of Inflammation in Pancreatic Cancer 141
a few experimental studies exploited the functional role of immune cells in the
biology of PDAC. Most of these studies rely on correlation analysis which needs
further confirmation using functional analysis as well as animal experiments. For
example, studies on mast cells show that they foster neoangiogenesis (Esposito
et al. 2002, 2004) and that there is a positive correlation between the number
of mast cells in the pancreatic fibroinflammatory stroma and angiogenesis. To
such extent that the number of mast cells correlates positively with the occur-
rence of metastasis and negatively with survival of patients with PDAC (Esposito
et al. 2002, 2004). Epidemiologic studies show that inflammation significantly
increases the risk of pancreatic cancer development. Importantly, these studies
reveal that the elevated risk was independent of gender, ethnicity, and type of
pancreatitis (Lowenfels et al. 1993; Malka et al. 2002). A recently conducted
study identified inflammatory monocytes to play a role in survival of pancreatic
cancer patients. The study revealed that monocytes in the peripheral blood are
negatively correlated with the survival of pancreatic cancer patients, whereas a
low amount of peripheral monocytes showed an increased survival of pancreatic
cancer patients with a resected tumor (Sanford et al. 2013). It is also shown that
142 S. Hausmann et al.
Currently, there is not enough clinical evidence to support the routine usage of anti-
inflammatory drugs to improve outcome in pancreatic cancer patients. Some under-
powered studies show partial benefit when anti-inflammatory therapy (i.e., COX-2
inhibition) is added to conventional chemotherapy (Lipton et al. 2010). Similarly,
there is some circumstantial evidence that anti-inflammatory drugs reduce the risk of
malignant pancreatic lesions. Below, some experimental data are reported.
6.6.2 Cyclooxygenase-2 Inhibitors
Cyclooxygenase (COX) and 5-lipoxygenase are the main regulators of the arachi-
donic acid metabolism and have been shown to be dysregulated in pancreatic can-
cer (Hennig et al. 2002, 2005; Ding et al. 2001). COX-2, which is a prostaglandin
synthetase, catalyzes the conversion of arachidonic acid into prostaglandin G2. In
the pancreas, COX-2 expression is induced by inflammatory cytokines, growth fac-
tors, and mitogenic stimuli and was shown to be overexpressed in pancreatic cancer
144 S. Hausmann et al.
6.6.3 Inhibition of NFκB
The NFκB signaling pathway has been shown to play multitudes of roles in the
development of pancreatic cancer as well as in metastatic spread due to its role
in controlling proliferation, apoptosis, and angiogenesis. Therefore, inhibition
of NFκB expression is a promising therapeutic target to reduce tumor growth
and metastasis formation in pancreatic cancer patients. In vitro studies showed
that inhibition of NFκB signaling in combination with gemcitabine resulted in
6 The Role of Inflammation in Pancreatic Cancer 145
6.6.4 Anti-Fibrotic Therapies
Recently, the abundant fibrotic stroma, produced by the activated pancreatic stel-
late cells, has attracted attention as it might form a physical barrier for the effec-
tive delivery of therapeutic agents. There is a considerable amount of evidence
stemming from in vitro and animal experiment that PSC and various ECM com-
ponents support tumor growth by various mechanisms such as promoting tumor
growth, creating apoptosis resistance, creating a niche for cancer stem cells, ena-
bling immune escape of cancer cells, modulation of angiogenesis, facilitation of
metastatic spread, and increasing therapy resistance (Erkan 2013b). In line with
these observations, depletion of the desmoplastic stroma of the PDAC has led
to better chemotherapy delivery and drug response in Kras-based genetic mouse
models (Conroy et al. 2011; Erkan et al. 2012; Olive et al. 2009). Taken together,
anti-fibrotic therapy appears as a new hope in the treatment of PDAC. However,
as of today, data from clinical studies are largely missing. However, as a proof of
principle, Von Hoff et al. (2011) used in a phase I/II trial nanoparticle albumin-
bound (nab) paclitaxel (to deplete the stroma in PDAC) alone and in combination
with gemcitabine and showed that through depletion of the stroma, higher concen-
trations of gemcitabine can be delivered in the tumor.
Despite the initial hope mostly stemming from the success achieved in genetic
mouse models of PDAC, the clinical reality seems to be more complex. As men-
tioned above, the first trial using an inhibitor of sonic hedgehog signaling to
deplete the stroma of PDAC (IPI-926-03 trial, http://www.clinicaltrials.gov/) has
been stopped due to increased mortality in the treatment arm. Currently, several
other trials are recruiting patients where various forms of anti-fibrotic therapies
are applied concomitantly with conventional therapies. The results of these trials
146 S. Hausmann et al.
Pancreatic cancer is the fourth deadly cancer worldwide and has a 5-year sur-
vival rate of only 6 %. The cellular mechanisms contributing to pancreatic cancer
development and progression are still not completely identified. Inflammation has
emerged to be a key mediator of pancreatic cancer development. In a paper by
Guerra and colleagues, it could be shown that in adult mice the expression of the
mutant Kras is not sufficient to induce pancreatic cancer. However, when addition-
ally inflammation was induced, the mice developed pancreatic cancer stressing the
importance of inflammation in the development of pancreatic cancer (Guerra et al.
2007). Furthermore, many studies showed the impact of inflammatory molecules on
the development and progression of pancreatic cancer. So far, different approaches
have been made to inhibit the main inflammatory signaling pathways in pancreatic
cancer. Although, having shown promising results in vitro and in vivo experiments,
inhibitors of inflammation have not been successful in cancer prevention or cancer
progression in clinical trials. Therefore, further research is needed to elucidate the
mechanisms through which inflammation contributes to tumor initiation and pro-
gression. It is very likely that that there are several altered mechanisms on various
levels contributing to the aggressiveness of PDAC. Therefore, effective therapy of
PDAC should aim at overcoming various obstacles at several levels.
6 The Role of Inflammation in Pancreatic Cancer 147
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Fig. 7.2 Proposed progression model for prostate cancer development. Adapted from De Marzo
et al. (2007)
The cells of the innate immune system, including mast cells, phagocytes (mac-
rophages, neutrophils, and dendritic cells), basophils, eosinophils, natural killer
(NK) cells, and γδ T cells, are the human body’s first responders to invading path-
ogens. Interestingly, evidence suggests that these innate effector cells may also
serve as the first line of defense against cancer. Investigations in multiple forms
of human cancer as well as in animal models indicate that the innate immune sys-
tem, along with cells of the adaptive immune system, is actively involved in cancer
immune surveillance as part of a process that has been termed “extrinsic tumor
suppression” (Vesely et al. 2011; Swann and Smyth 2007). In essence, this pro-
cess presumes that along with malignant transformation, cells begin to produce
novel peptides or otherwise immunogenic molecules (i.e., “tumor antigens”) that
are recognized by the immune system. Indeed, recent genome-wide approaches
to sequencing human adult cancers have indicated that approximately 30–70 non-
silent somatic mutations are present in common solid tumors such as that of the
colon, breast, brain, or pancreas in coding regions of genes that result in altered
peptide sequences (reviewed in (Vogelstein et al. 2013)). Regardless of whether
these mutations confer a selective growth advantage on tumor cells (i.e., “driver
mutations”) or have no effect on tumorigenesis (i.e., “passenger mutations”), in
principle, these altered peptide sequences can serve as tumor-specific antigens
(Vogelstein et al. 2013). The elicited response, mediated by cells of both the
innate and the adaptive immune system, either eliminates the tumor cells before
they become clinically apparent or serves to prevent tumor outgrowth (Vesely
et al. 2011). Indeed, the concept of inducing innate immune responses for their
anti-tumorigenic properties has been harnessed by certain immunotherapy strat-
egies such as Bacillus Calmette–Guérin (BCG) treatment for bladder cancer. In
accordance with this model, the presence and number of innate immune cells, such
as mast cells and macrophages, in some types of human cancer have been shown
to serve as a prognostic factor, with larger numbers of tumor-infiltrating innate
immune cells or accumulation of innate immune cells at the tumor-invading front
conferring a better prognosis (Welsh et al. 2005; Fleischmann et al. 2009; Rajput
et al. 2008; Forssell et al. 2007; Zhou et al. 2010; Li et al. 2009a). However, as
inflammation in cancer is truly a “double-edged sword,” there are many impor-
tant exceptions to this rule (Hagemann et al. 2007). One notable exception is the
innate immune cells involved in dampening and/or regulating immune responses
such as the myeloid-derived suppressor cell (MDSC) lineage of human myeloid
progenitors (Ostrand-Rosenberg and Sinha 2009). Furthermore, innate immune
cells can produce pro-inflammatory cytokines, such as IL-6 and IL-1β, and factors
that enhance cell migration and invasiveness, such as matrix metalloproteinases
(MMPs) and CC family chemokines, which have been shown to promote tumor
initiation and/or progression (Kang et al. 2010; Li et al. 2009b; Allavena et al.
7 The Role of Inflammation in Prostate Cancer 157
2008; Kaler et al. 2009; Loberg et al. 2006; Mizutani et al. 2009). Therefore, the
influence of innate immune cells on cancer prevention versus cancer progression is
often very difficult to discern (Disis 2010; Zhang et al. 2012), and whether these
various cells provoke pro-growth versus growth-suppressive effects may relate to
the specific tissue type in question, the disease temporal state (e.g., initiation, pro-
motion, local extension/progression, metastasis), host genetic factors, and/or envi-
ronmental cofactors such as local (or systemic) coinfection and dietary exposures.
In regard to prostate cancer, a number of studies have been carried out to deter-
mine the prognostic significance of tumor-infiltrating innate immune cells. Herein,
although this research is still relatively early overall in terms of the scope of what
can be done, we will summarize these efforts and specifically focus on the prog-
nostic significance of prostate-infiltrating innate immune cells.
Mast cells are best known for their role in allergic reactions, where IgE–antigen
complex (IgE–Ag) binding to the mast cell receptor FceRI stimulates degranula-
tion, releasing effector molecules such as histamine, serotonin, leukotrienes, and
proteoglycans (Galinsky and Nechushtan 2008; Galli 2000). However, mast cells
have also been shown to play a significant role in defense against parasites, to be
key players in controlling bacterial infection, and to be very important producers
of immunoregulatory cytokines (Galinsky and Nechushtan 2008; Krishnaswamy
et al. 2001; Arock et al. 1998; Gordon and Galli 1990; Gordon et al. 1990). What
makes mast cells particularly interesting in cancer biology, however, lies in the
dynamic way in which these cells reside in and interact with the microenviron-
ment in their target tissues. Mast cell functions can have very potent effects on
their environment, effects that can be powerfully pro- or anti-tumorigenic, depend-
ing on the circumstances (Fig. 7.3). These functions are very dynamic and are sub-
ject to manipulation by outside forces, possibly even by the surrounding cancer
cells (Galinsky and Nechushtan 2008; Theoharides and Conti 2004). As such it
is likely that mast cells play different roles in different cancers and different can-
cer stages (Pittoni and Colombo 2012). This possibility is, in fact, reflected in the
literature. There have been a number of studies in many different cancers attempt-
ing to correlate mast cell density in or around the tumor, with the results varying
from associating mast cell density with good prognosis, poor prognosis, and hav-
ing no association with prognosis at all, even between studies in the same cancer
(Galinsky and Nechushtan 2008; Pittoni and Colombo 2012; Fisher et al. 1989;
Ribatti et al. 2003; Iamaroon et al. 2003; Ribatti et al. 1999; Molin et al. 2002;
Molin 2004; Ribatti et al. 2005; Tuna et al. 2006; Fisher et al. 1985; Aaltomaa
et al. 1993; Dabiri et al. 2004; Welsh et al. 2005; Chan et al. 2005; Johansson
et al. 2010; Sari et al. 1999; Fleischmann et al. 2009). In prostate cancer specif-
ically, higher numbers of “intratumoral” mast cells have been shown to be cor-
related with lower Gleason grade and better prognosis (Johansson et al. 2010;
158 K. S. Sfanos et al.
Fig. 7.3 Potential biological roles for mast cells in prostate cancer. Figure shows a photomi-
crograph from a region of human prostatic adenocarcinoma stained for mast cells (brown stain-
ing cells) by immunohistochemistry (H.A. Hempel, A.M. De Marzo, K.S. Sfanos, unpublished
observations). Original magnification, ×100
Fleischmann et al. 2009; Sari et al. 1999); however, the opposite finding has also
been reported (Nonomura et al. 2007). These and other studies on mast cells in
human cancers were performed using a variety of techniques and without a stand-
ardized definition of “intratumoral” versus “peritumoral” mast cells. Nevertheless,
the notion of mast cells having different effects in different circumstances is an
attractive one and is consistent with what is known about mast cell biology.
Mast cells, such as most immune cells, originate in the bone marrow. However,
mast cells travel to their targets before their final stages of development, and their
final differentiation into different subtypes is dictated in large part by the microen-
vironment of their resident tissues (Galinsky and Nechushtan 2008; Theoharides
and Conti 2004). The two main subtypes of mast cells are commonly known as
MCTC, which express chymase, tryptase, carboxypeptidase, and cathepsin G
and are usually found in mucosa, and MCT, which express mainly tryptase and
are usually localized to connective tissues (Krishnaswamy et al. 2001; Irani et al.
1986; Khazaie et al. 2011). These two subtypes also differ in number, type, and
content of secretory granules, as well as which stimuli to which they will respond
(Theoharides and Conti 2004). These subtypes are not exhaustive, however,
as mast cells have been discovered with phenotypes outside of these. In addi-
tion, there is significant evidence to suggest that mast cell differentiation is not
final, and phenotype and mast cell protease profile can be changed based on dif-
ferent conditions in their microenvironment, including exposure to different
cytokines, the presence of fibroblasts, and different host organ tissues (Galinsky
7 The Role of Inflammation in Prostate Cancer 159
and Nechushtan 2008; Lee et al. 1998; Gurish et al. 1995). As such, it is reason-
able to suggest that mast cell phenotype will be very different depending on differ-
ing conditions, such as in vitro versus in vivo, and on different cancers and cancer
stages. The best known method of mast cell effector function is via degranulation,
which is the release of the previously synthesized contents of intracellular granules
in response to IgE–Ag binding to the mast cell receptor. These contents include
effector molecules such as serotonin, histamine, heparin, tryptase, and chymase,
among others, many of which with pro-inflammatory, anti-tumorigenic properties.
However, there is also significant evidence for a method for “piecemeal degranu-
lation” of mast cells, allowing for the selective release of cytokines without the
release of the entire secretory granule (Crivellato et al. 2003). This alternative
activation model helps to fuel the mechanistic side of the argument for the pro-
tumorigenic potential of mast cells, as it provides the potential for the selective
release of pro-tumorigenic cytokines such as IL-1 and IL-6 (Lacy and Stow 2011)
and effector molecules such as MMP9 in the absence of anti-tumorigenic mast cell
granule components. The mast cell subtype also comes into play here, as differ-
ent subtypes will have different effector molecules available to them and thus can
have different effects on the tumor upon degranulation, piecemeal or otherwise.
In addition to these, mast cells are also potent producers of immune modulating
cytokines, chemokines, angiogenic factors, and proteases, all of which can cause
significant changes in the tumor microenvironment (Khazaie et al. 2011).
Mast cells are known to produce many different angiogenic molecules, includ-
ing VEGF, histamine, TNF-α, and Ang-1, and thus have been suspected for some
time to play a significant role in tumor angiogenesis and possibly even in the angi-
ogenic switch (Maltby et al. 2009). There have been several studies supporting this
idea, including a transplantation multiple myeloma mouse model, in which it was
found that transplanting mast cells with plasmacytoma tumors resulted in signifi-
cantly higher vascularization (Nakayama et al. 2004). In addition, one study in a
squamous carcinoma mouse model demonstrated a significant decrease in prema-
lignant angiogenesis in mast cell-deficient mice (Coussens et al. 1999). Evidence
in humans for a role for mast cells in tumor angiogenesis is also mounting, such as
in one study correlating melanoma progression with mast cell density and simulta-
neously increased vascular density (Ribatti et al. 2005). However, some have also
suggested a role for mast cells in suppressing angiogenesis by providing receptors
that can “soak up” angiogenic factors (Theoharides and Conti 2004). In addition,
there are studies correlating mast cell density with cancer progression without
finding a correlation with angiogenesis, suggesting that mast cells may also have a
role in tumor promotion aside from blood vessel development (Molin 2004; Molin
et al. 2002). In human prostate cancer, mast cell densities have not been correlated
with neovascularization; however, in an orthotopic rat model, implantation of AT-1
tumor cells resulted in peritumoral recruitment of mast cells and an increase in
peritumoral vascular density (Johansson et al. 2010). Furthermore, castration was
found to result in mast cell recruitment to the prostate both in men and in the AT-1
tumor model and the Dunning rat model, and this correlated with an increase in
vascular density in the Dunning model (Johansson et al. 2010).
160 K. S. Sfanos et al.
In addition to the possible roles for mast cells in angiogenesis, the role of
mast cells in tissue remodeling is of particular interest in tumor promotion and
includes a possible connection in prostate cancer. The idea that the extracellu-
lar matrix (ECM) plays a significant role in tumor promotion has gained impor-
tance in recent years (Bissell and Hines 2011; LaBarge et al. 2009; van Dijk et al.
2013). Mast cells produce potent proteases, including chymase, tryptase, colla-
genases, MMP9, and other gelatinases, and cysteinyl cathepsins. As such, research
into the role of mast cells in ECM modulation and tumor invasion is also gain-
ing ground (Khazaie et al. 2011). One study in prostate cancer explored the role
of mast cell MMP9 in early prostate tumor progression in transgenic adenocar-
cinoma of the mouse prostate (TRAMP) mice, arguing that lower-grade prostate
tumors would need mast cell-derived MMP9 for invasion, since well-differentiated
prostate tumor cells do not produce MMP9 (Pittoni et al. 2011). The results did in
fact suggest that mast cell MMP9 was necessary for early tumor invasion in mice.
Immunohistochemistry (IHC) studies in human prostate cancer tissues showed a
positive correlation between increased mast cell density, MMP9 production con-
fined almost entirely to tumor-infiltrating mast cells, and well-differentiated
tumors—supporting the observations in the TRAMP mouse studies (Pittoni et al.
2011). Whether mast cell-derived MMP9 plays a role in driving early invasion of
human prostate cancer is yet to be elucidated.
In addition to the direct effects mast cells might have on cancer cells and
their microenvironment, mast cells may also affect cancer through their roles as
potent immune modulators. Mast cells are capable of both suppressing and pro-
moting inflammatory responses, depending on the circumstances (Galinsky and
Nechushtan 2008). In fact, mast cells are known to produce a number of cytokines
and chemokines capable of recruiting, activating, suppressing, and driving the dif-
ferentiation of both innate and adaptive immune cells, including neutrophils, baso-
phils, macrophages, lymphocytes (such as B cells, TH2 T cells, and Treg cells), and
NK cells. As such, mast cells are also capable of anti- or pro-tumorigenic effects
through the suppression or activation of the inflammatory response and could also
have a significant effect on other inflammatory cells in different cancers.
Studies both in vivo in mice and in vitro in mouse and human cells have dem-
onstrated mast cell influences on cancer cells, with one in vivo study showing
decreased mitotic index and increased apoptosis in intestinal polyps and two in
vitro studies showing increased proliferation and invasion upon treatment with
mast cell-conditioned medium (Khazaie et al. 2011; Gounaris et al. 2007; Cheon
et al. 2011; Strouch et al. 2010). This activity of mast cells has been theorized to
be due to the production of tryptase, which is reported to promote proliferation of
colon cancer cells, fibroblasts, and other cell types (Yoshii et al. 2005; Cairns and
Walls 1996; Berger et al. 2001; Gruber et al. 1997; Levi-Schaffer and Piliponsky
2003; Frungieri et al. 2002). Mast cell histamine has also been suggested to play
a role in tumor cell proliferation; however, the literature is not in agreement with
whether it promotes or suppresses proliferation (Theoharides and Conti 2004).
In contrast, mast cell effector molecules such as IL-4 and TNF-α could result in
tumor cell death (Gooch et al. 1998; Gordon and Galli 1990).
7 The Role of Inflammation in Prostate Cancer 161
As mast cells and cancer cells evolve in the same microenvironment, the inter-
actions between the two could change significantly. With the myriad of mast cell
effector molecules, there is a delicate balance between the anti- and pro-tumori-
genic capabilities of mast cells that can conceivably change very dramatically with
time, even within the same tumor. It is possible that even if mast cells cannot serve
as an independent prognostic factor for all cancers, they might be an indicator of
cancer aggressiveness and invasiveness (Galinsky and Nechushtan 2008). In addi-
tion, it is very important that this ever-evolving relationship with cancer be better
understood before any mast cell-targeted therapies are attempted. Since mast cells
may have the potential to be pro-tumorigenic or anti-tumorigenic depending on the
microenvironment, even in the same cancer, certain therapies may be beneficial
at certain stages of cancer and detrimental in others (Pittoni and Colombo 2012;
Pittoni et al. 2011). As such, mast cells have great potential in cancer research, and
many possible roles of mast cells in prostate cancer continue to be an important
area of study.
Fig. 7.4 Inflammatory cells within tumor microenvironment and their potential biological roles.
Differentiation of monocytes into tumor-associated macrophages (TAM) with differing biological
roles may be mediated by secretion of cytokines and growth factors by tumor cells and stromal
cells within the tumor microenvironment. Reprinted from Hao et al. (2012)
for time to PSA progression has varied (Lissbrant et al. 2000; Wang et al. 2005;
Nonomura et al. 2011). In a study on disease progression after hormone therapy
for prostate cancer, TAM densities as assayed by IHC for CD68 on patient’s pros-
tate biopsy specimens prior to treatment were found to be positively correlated
with Gleason score and clinical stage (Nonomura et al. 2011). Furthermore, in
contrast to the studies by Shimura et al. (2000) and Yang et al. (2004), Nonomura
et al. reported that increased numbers of TAMs were significantly associated with
PSA progression and serve as a prognostic indicator for decreased time to progres-
sion-free survival (Nonomura et al. 2011).
Richardsen et al. (2008) also reported that the production of macrophage
colony-stimulating factor (M-CSF) and colony-stimulating factor-1 receptor
(CSF-1R) is significantly greater in tumor cells and in stromal areas in the pri-
mary tumors of patients with metastatic prostate cancer compared to those without
metastatic disease. The mechanism by which macrophages may promote prostate
cancer invasion may be mediated in part by secretion of proteases that act on the
ECM. Of interest is the serine protease urokinase plasminogen activator (uPA) that
stimulates the plasminogen activation system upon binding to urokinase receptor
(uPAR). Cleavage of the α6β1 integrin by uPA has been shown in in vivo stud-
ies to increase tumor cell motility, invasion, and prostate cancer metastasis (Ports
et al. 2009). Further in vitro studies indicated that this process may be mediated
by TAMs (Sroka et al. 2011). Likewise, the cathepsin proteases, and specifically
cathepsin K and cathepsin S, are also of interest in this regard. Cathepsins K and
S are both lysosomal cysteine proteases. Whereas cysteine cathepsins are gener-
ally thought to serve in intracellular lysosomal protein degradation and turnover,
these two cathepsins (K and S) can be secreted by macrophages (Punturieri et al.
2000) and may potentially play an important role in ECM remodeling to promote
tumor invasion and progression. Macrophage-secreted cathepsin-mediated tumor
invasion has been previously demonstrated in multiple forms of cancer (Vasiljeva
et al. 2006; Gocheva et al. 2010). In prostate cancer specifically, macrophage-
secreted cathepsin S was found in the TRAMP mouse model as a protein of inter-
est in advanced disease as assessed by differential protein-profiling studies of
normal prostate, primary tumors of differing histological grades, and metastatic
tumors (Lindahl et al. 2009). IHC for CD68 costained with cathepsin S demon-
strated that both in TRAMP tumors and in human prostate tumors, cathepsin S is
produced primarily by TAMs. Furthermore, the number of cathepsin S-secreting
macrophages was found to be significantly higher in castration-resistant prostate
cancer as opposed to hormone naïve prostate cancer in patients with high-grade
prostate tumors (Lindahl et al. 2009). Another recent study in prostate cancer
demonstrated that cathepsin K-deficient mice had a significant reduction in tumor
growth and bone resorption when PC3 cells were injected into the tibia compared
to wild-type mice (Herroon et al. 2013). This phenomenon was specific to bone,
as PC3 cells implanted subcutaneously had similar growth in both wild-type and
cathepsin K knockout animals. By IHC, cathepsin K was found to be restricted
to osteoclasts and macrophages in this model (Herroon et al. 2013). The role of
164 K. S. Sfanos et al.
TLRs are a class of molecules associated with innate immunity that are expressed
on cells such as macrophages and dendritic cells. TLRs recognize structurally con-
served pathogen-derived products such as lipopolysaccharide (LPS) contained in
Gram-negative bacteria. TLRs are also involved in tissue homeostasis and play
a role in tissue repair and regeneration. There are significant data to demonstrate
that TLRs may serve a role as negative regulators of cancer, as many antican-
cer therapeutic agents have been based on administration of TLR agonists (e.g.,
the historical use of Coley’s toxin as a cancer treatment and the currently used
strategy of BCG treatment for bladder cancer) (Rakoff-Nahoum and Medzhitov
2009). On the other hand, TLRs have also been implicated in the promotion of
tumorigenesis, which may involve recruitment of macrophages to sites of tissue
injury in the developing tumor (Rakoff-Nahoum and Medzhitov 2009). In prostate
cancer, early evidence of a role for TLRs in prostate cancer development came
from genetic studies of TLR polymorphisms associated with prostate cancer risk.
Specifically, multiple studies have shown that single nucleotide polymorphisms
(SNPs) in TLR4 and the TLR1-6-10 gene cluster are associated with prostate can-
cer risk (Chen et al. 2005; Zheng et al. 2004; Sun et al. 2005; Kim et al. 2012),
although results have varied among cohorts (Chen et al. 2007; Shui et al. 2012).
At the tissue level, TLRs have been reported to be up-regulated by prostate cancer
cells as assessed by IHC. For example, TLR3, TLR4, and TLR9 were shown to
be produced by prostate tumor cells as assayed by IHC, and interestingly, high
expression of these TLRs as assayed by real-time PCR was found to correlate
with biochemical recurrence (Gonzalez-Reyes et al. 2011). The mechanism by
which this relationship may arise, however, remains unclear, as, on the contrary,
TLR stimulation in cancer cells in prostate cancer models has also been consist-
ently shown to result in the upregulation of inflammatory cytokines and induction
of apoptosis and/or anti-tumor immune responses (Paone et al. 2008; Harashima
et al. 2012; Chin et al. 2010; Galli et al. 2010; Andreani et al. 2007).
evidence indicates that IL-6 may contribute to the progression of p rostate cancer,
and a high systemic level of IL-6 is considered to be a sign of a more aggressive
clinical course (Smith et al. 2001; Okamoto et al. 1997; Twillie et al. 1995). IL-6 is
secreted by a variety of cell types such as T cells, macrophages, endothelial cells,
and fibroblasts. In addition, studies have suggested that IL-6 is secreted by prostate
adenocarcinoma cells (Hobisch et al. 2000). IL-6 is responsible for skewing cellular
differentiation of multiple cell types including B cells, TH17 cells, and myeloid cells
(Kimura and Kishimoto 2010; Cheng et al. 2011; Tanner and Tosato 1992). As such,
both systemic and local production of IL-6 may drive accumulation of immune cell
subtypes. For example, IL-6, along with CCL2 (CC chemokine ligand 2), can induce
the differentiation of CD11b+ monocytes into M2-type macrophages, indicating that
systemic and/or local IL-6 production in prostate cancer patients may lead to differ-
entiation and accumulation of this immune-suppressive, pro-tumorigenic subset of
macrophages (Roca et al. 2009). There is additional evidence that CCL2 may assist
in recruitment of macrophages to the tumor site and promote tumor cell metasta-
sis to bone (Loberg et al. 2006; Mizutani et al. 2009). Furthermore, in experiments
using the TRAMP murine model, high circulating levels of IL-6 were associated
with recruitment of MDSCs (Wu et al. 2012); abrogation of IL-6 in this model inhib-
ited the recruitment of MDSCs, slowed tumor growth, and attenuated angiogenesis
(Wu et al. 2012).
A number of studies have been conducted to gain understanding of how IL-6
might act locally on tumor cells to contribute to prostate cancer development
and progression. One line of investigation has aimed to identify a role for IL-6
in progression of prostate cancer to androgen independence via direct regulation
of androgen receptor transactivation and/or androgen synthesis in prostate cancer
cells (Lee et al. 2003; Malinowska et al. 2009; Chun et al. 2009). Finally, it has
also been suggested that a positive feedback loop between IL-6 activation, STAT3
activation, and NF-κB activation in cancer maintains cells in an “epigenetic trans-
formed” state that might transform “non-stem cancer cells” into “cancer stem-like
cells” (Iliopoulos et al. 2011).
There are additional innate immune cells that potentially play a role in prostate can-
cer as indicated by studies in animal and/or in vitro models, but do not as of yet have
significant data in human prostate cancer tissues. One notable example of this is the
MDSC lineage of myeloid progenitors. MDSCs accumulate in the tumor microen-
vironment as well as the blood, lymph nodes, and bone marrow in association with
several forms of human cancer and may contribute to tumor immune escape due to
their general functional role in immune suppression (Ostrand-Rosenberg and Sinha
2009). In regard to prostate cancer, multiple murine models of prostate cancer dem-
onstrate accumulation of MDSCs in tumors along with a potential contribution to
tumor progression (Wu et al. 2012; Svensson et al. 2011; Rigamonti et al. 2011).
7 The Role of Inflammation in Prostate Cancer 167
The cells of the adaptive immune system (also known as the “acquired” immune
system) are more generally considered to be the “second-line” response against
invading pathogens, as they typically require costimulation from innate immune
cells (i.e., in the form of antigen presentation to cellular receptors) to drive cellular
proliferation and a pathogen antigen-specific response. Cells of the adaptive immune
system include T lymphocytes (i.e., CD4+ and CD8+ T cells) and B lymphocytes
(B cells). Like the cells of the innate immune system, cells involved in adaptive
immunity are known to play paradoxical roles in cancer development. For example,
whereas CD8+ T cells are thought to be major effector cells in anti-tumor immune
168 K. S. Sfanos et al.
with poor outcome in prostate cancer patients. One potential explanation for the
difference in results between studies may involve the heterogeneous nature of
prostate cancer (and prostate tumor-associated inflammation) that may not be ade-
quately represented on TMAs as opposed to whole tissue sections. Another poten-
tial explanation may involve the fact that many of these studies aimed to quantify
TIL as a whole and did not attempt to quantify different T-cell subtypes, such as
the different THELPER subsets of T cells (TH1, TH2, TH17) or regulatory T cells
(Treg), and it is known that the different subtypes can play varying and opposing
roles in the tumor microenvironment (Kennedy and Celis 2008). Treg, for exam-
ple, which are characterized by high expression of CD25 and FoxP3 and play a
suppressive role in immune responses, are known to actively suppress anti-tumor
immune responses (Mougiakakos et al. 2010).
The analysis and quantification of T-cell subsets in prostate tissue samples is
challenging, as these cells are typically differentiated by the different cytokines
that they secrete. IHC is often not a reliable assay for secreted cytokines, and the
isolation of immune cells from prostate tumor or tissue samples for use in flow
cytometry can be technically challenging. Nevertheless, one study did isolate
TIL from radical prostatectomy specimens using a fine-needle aspiration tech-
nique and quantitatively assayed for separate CD4+ T-cell subsets (TH1, TH2,
TH17, Treg) using flow cytometry (Sfanos et al. 2008). The results of this study
indicated that TH1 cells are quantitatively most abundant in the prostate of cancer
patients and that TH2 cells are almost completely absent. Furthermore, prostate-
infiltrating TH1, TH17, and Treg cells are significantly elevated when compared
to levels in the peripheral blood of the cancer patients, with the most significant
skewing toward the CD4+ TH17 and Treg phenotype (Sfanos et al. 2008). Finally,
although the sample size was limited (n = 20), greater numbers of CD4+ TH17
TIL were significantly associated with lower pathologic Gleason score (Sfanos
et al. 2008). Of interest, this finding of higher numbers of TH17 cells in lower-
grade tumors is inconsistent with literature in other types of cancer (Zhang et al.
2008; Grivennikov et al. 2012); however, there does not appear to be a consensus
as to whether TH17 cells are pro- or anti-tumorigenic (Wilke et al. 2011; Martin
et al. 2012; Zou and Restifo 2010), and they can possibly be both depending on
the inflammatory microenvironment and the stage of the tumor. One potential lim-
itation to the Sfanos et al. study was that the entire peripheral zone of the pros-
tate (where prostate cancer typically arises) was sampled and likely sampled areas
that contained both cancerous and benign regions. Therefore, localization of T-cell
subsets in prostate tumor versus benign prostate tissues was not conducted. This
type of analysis would likely need to be performed in frozen or formalin-fixed
paraffin-embedded (FFPE) tissues using in situ hybridization-based assays.
One study of note did utilize IHC for IL-17 to determine that IL-17-producing
macrophages accumulate in areas of PIA (Vykhovanets et al. 2011). Treg cells have
been examined in additional studies in prostate cancer patients and have consistently
been found to be elevated in tumor tissues (Miller et al. 2006; Kiniwa et al. 2007);
however, levels in peripheral blood vary (Miller et al. 2006; Yokokawa et al. 2008).
These studies also demonstrate the suppressive activity of CD4+ Treg (Miller et al.
170 K. S. Sfanos et al.
2006; Yokokawa et al. 2008; Kiniwa et al. 2007), as well as CD8+ Treg (Kiniwa
et al. 2007), isolated from peripheral blood and tumor tissues of prostate cancer
patients. CD8+ Treg cells are the newest class of T cells shown to exert suppressive
effects on CD4+ T cells (Leavy 2010). Another molecule of interest with respect to
its general suppressive function in regard to human prostate cancer is programmed
death 1 or PD-1. PD-1 is an inhibitory marker on T cells and is associated with a
non-functional or “exhausted” phenotype (Barber et al. 2006; Chen 2004; Freeman
et al. 2000). This molecule along with its ligand, PD-L1, may serve as a method of
immune escape in human tumors. As such, the PD-1 pathway has been targeted in
multiple immunotherapy strategies for different cancers, and preliminary studies in
human trials remain promising (Dotti 2009; Turnis et al. 2012). At least two stud-
ies have assayed for the presence of PD-1 in prostate tumors. One study identified
very high levels of PD-1 expression (close to 90 % of prostate-infiltrating CD8+
T cells in some patients) on prostate-infiltrating CD8+ lymphocytes isolated from
prostatectomy tissues and assayed by flow cytometry (Sfanos et al. 2009a). PD-1
was likewise found to be elevated in CD8+ T cells in the peripheral blood of pros-
tate cancer patients compared to controls (Sfanos et al. 2009a). In a separate study
that assayed for the presence of PD-1+ lymphocytes in prostate tissues via IHC,
clusters of PD-1+ T cells were found to surround most prostate tumors (Ebelt et al.
2009). Furthermore, overexpression of PD-1 in cancer patients may be associated
with poor disease outcome, and this pathway remains of interest in prostate cancer
immunotherapy strategies (Barach et al. 2011; Dulos et al. 2012).
7.4 Concluding Remarks
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Chapter 8
The Role of Inflammation in Bladder
Cancer
Georgios Gakis
Abstract The aim of this book chapter is to present the latest basic research
developments on the role of inflammation in bladder cancer and provide insights
into their future clinical significance in preventing bladder carcinogenesis and pro-
gression. Bladder cancer is a highly immunogenic malignancy. Urothelial cancer
cells aim to manipulate the immune system by inhibiting its cytotoxic function
while stimulating the secretion of growth promoting factors. Cytokine-induced
imbalances in the distribution and differentiation of tumor-infiltrating cytotoxic
cells can boost bladder cancer cell proliferation. Tumor-induced release of exces-
sive amount of cytokines causes an “inflammatory storm” which drives metastasis
formation via degradation of extracellular matrix proteins. Tumor-related selective
cyclooxygenase-2 (COX-2) upregulation suppresses the cell-mediated immune
response via aberrant prostaglandin metabolism resulting in failure of differentia-
tion of myeloid cell progenitors into mature antigen-presenting cells. T cells are
capable of increasing the oxidative stress on bladder cancer cells via induction of
COX-2 and STEAP expression. Some evidence also suggests that COX-2 activa-
tion may be also involved in inflammation-mediated cancer stem cell proliferation.
Antibodies against the VEGF-co-receptor neuropilin decrease the angiogenetic
potential of bladder cancer cells. Inflammation-based predictive bladder cancer
models have demonstrated to accurately predict response to treatment both in the
curative and palliative setting. While randomized trials do not support a clinical
benefit for the use of anti-inflammatory drugs (i.e., celecoxib, atorvastatin) in pre-
venting recurrence of low-grade bladder cancer, further investigations are war-
ranted in the setting of high-grade tumors since the immune response to cancer
stimuli is most probably more pronounced in advanced stages.
G. Gakis (*)
Department of Urology, University Hospital Tübingen, Eberhard-Karls University,
Hoppe-Seyler-Strasse 3, 72076 Tübingen, Germany
e-mail: [email protected]; [email protected]
8.1 Introduction
In Western countries, bladder cancer is the sixth most common cause of cancer
death in men and the eight most common cause in women. In the United States,
the incidence rate of bladder cancer has increased between the years 1973 and
2009 from 21 to 26/100,000 person-years. Stage-specific analyses have confirmed
an increase in the incidence of localized and distant tumor stages. Meanwhile,
improved 5-year survival rates were noted for all stages, except for distant disease
(Abdollah et al. 2013). Similarly, in Germany, a pronounced increase in the inci-
dence of approximately 35 % in men and 75 % in women has been noted between
the years 1980 and 2004, which is the second highest bladder cancer incidence
worldwide after Denmark. Despite this sharp increase in the incidence, the age-
standardized mortality has dropped around 20 % in men and 40 % in men (Robert-
Koch-Institut 2010).
Approximately 70–75 % of all bladder malignancies are diagnosed in
superficial tumor stages (Stenzl et al. 2011). Superficial bladder cancer is
characterized by a high recurrence rate and a relatively low progression rate
(Babjuk et al. 2011). Due to this, lifelong surveillance regimens are necessary
to detect recurrences at the earliest possible stage. This is the reason why blad-
der cancer causes the highest costs of all cancer entities in health care systems
(Stenzl et al. 2008). Despite the use of recently introduced sophisticated treat-
ment methods based on the combination of urine-based markers, fluorescence-
guided cystoscopy, and intravesical instillations (Gakis et al. 2010), 25–30 %
of all primary cases demonstrate histologically muscle invasion which is asso-
ciated with concurrent distant metastatic lesions in 20 % of the patients (Stenzl
et al. 2011).
Effective bladder cancer control requires an intact immune system (Balkwill
and Mantovani 2001). When bladder cancer cells invade into the subepithelial
tissue, further invasion can be arrested by intravesical instillations of Bacille-
Calmette-Guerin (BCG), which provides long-term recurrence-free survival in
about two-thirds of the patients (Kawai et al. 2013). However, the mechanisms
that mediate the antiproliferative effects of BCG on cancer cells are poorly
understood (Kawai et al. 2013). The clinical benefits seen in BCG patients sug-
gest that the immune system itself plays an important role in arresting bladder
cancer progression (Kawai et al. 2013). However, when tumor cells infiltrate into
the muscle layer of the bladder, the risk of micrometastatic disease increases
significantly. Therefore, at this stage, the chance of cure can be increased by
radical surgical treatment in conjunction with neoadjuvant cisplatinum-based
chemotherapy which, has shown to especially in patients with advanced tumor
stages (Gakis et al. 2013). In the last years, there is an increasing awareness
in the uro-oncological community that the immune system plays a critical role
in bladder cancer progression (Gakis et al. 2011). The aim of this review is to
provide molecular and clinical evidence for the role of inflammation in bladder
cancer development and progression.
8 The Role of Inflammation in Bladder Cancer 185
8.2 Evidence Acquisition
This review aims to describe the latest basic research developments on the role
of inflammation in bladder cancer and provide clinical insights into their future
role in preventing bladder carcinogenesis and progression. For this, a system-
atic Medline literature search was performed along with a free-text protocol,
using one or several combinations of the following terms: angiogenesis, blad-
der cancer, cytotoxic, immune system, inflammation, invasion, in vitro, in vivo,
macrophage, metastasis, molecule, pathway, proliferation, T-cell, urothelial
carcinoma.
receptor blockade, it did not affect cell viability (Dufresne et al. 2011). This sug-
gests that, besides activation of macrophages, cancer cell elimination requires
further action of the immune system. These two macrophage subtypes show
opposite effects in terms of their invasive potential.
Further molecular characterization of these subtypes helps to understand the
divergent effects on cancer cells. A recent study analyzed myeloid cells from
peripheral blood and tumor tissue collected from patients with urothelial carcino-
mas. Both blood and tissue analyses showed that two major CD11b(+) myeloid
cell subsets were present: granulocyte-type CD15(high) CD33(low) cells and
monocyte-type CD15(low) CD33(high) cells. Interestingly, the number of circulat-
ing granulocytic but not monocytic myeloid cells was markedly increased in can-
cer patients compared to healthy individuals. Both myeloid cell subsets produced
substantial amounts of proinflammatory chemokines. Granulocytic myeloid cells
were able to inhibit in vitro T-cell proliferation via induction of CD4(+) T regula-
tory cells. Further analysis revealed that tumor tissues were infiltrated with both
monocyte–macrophage CD11b(+)-HLA-DR(+) and granulocytic CD11b(+)-
CD15(+)-HLA-DR(−) myeloid cells (Eruslanov et al. 2012). Collectively, these
studies suggest that different subtypes of activated inflammatory myeloid cells not
only interfere with cancer cells but also with the T-cell system, thereby regulat-
ing the invasive potential of cancer cells and the functional efficiency of the local
immune response.
To effectively eliminate cancer cells, natural killer (NK) cells, CD4(+), and
CD8(+) T cells are of fundamental importance. In this respect, in vitro BCG mod-
els are ideally suited to elucidate the interactions between the immune and bladder
cancer cells. Human interferon-alpha 2B-secreting recombinant BCG augments
interferon-γ (IFN-γ) and interleukin-2 (IL-2) production by T helper cells (Liu et
al. 2009). This, in turn, potentiates cytotoxic effects of peripheral blood mononu-
clear cells (PBMCs) on bladder cancer cells. Blockage of IFN-α, IFN-γ, or IL-2
by neutralizing antibodies after rBCG-IFN-α stimulation of cancer cells reduced
the ability of PBMC to induce T-cell cytotoxicity. Conversely, NK and CD8(+)
T cells are also able to enhance PBMC cytotoxicity after exposure to BCG-IFN-α
(Liu et al. 2009).
An imbalance in the distribution of tumor-infiltrating Th17 cells and
regulatory T cells in the tumor area and peripheral blood seems to contribute to
the development or progression of bladder carcinoma. Upon interleukin stimu-
lation, T helper cells differentiate into Th17 cells which are capable of produc-
ing large amounts of cytokines. Using flow cytometric analyses, patients with
bladder cancer exhibited enriched Th17 cells in the bladder tumor and a higher
proportion of regulatory T cells in peripheral blood compared with healthy con-
trols. Exposure to IL-2 converted T regulatory cells into Th17 cells (Chi et al.
2010). Taken together, cytokine-induced imbalances in the distribution and
differentiation of tumor-infiltrating cytotoxic cells and macrophages provoke
a dysregulation of the immune system, thereby promoting bladder cancer cell
growth.
8 The Role of Inflammation in Bladder Cancer 187
Chronic inflammation may not only be the host’s response to bladder cancer devel-
opment but also actually elicit bladder carcinogenesis. Secreted protein acidic and
rich in cysteine (SPARC), a glycoprotein located in the extracellular matrix which
is increasingly expressed during tissue remodeling, has been recently implicated
with bladder carcinogenesis. SPARC-deficient mice and their wild-type littermates
were exposed to chemical bladder carcinogens. Loss of SPARC accelerated the
development of urothelial preneoplastic (such as atypia and dysplasia) and neoplas-
tic conditions. SPARC-deficient animals showed a stronger accumulation of reac-
tive oxygen species, increased urothelial cell proliferation, and carcinogen-induced
inflammation. Interestingly, loss of SPARC was associated with an increased acti-
vation of pro-inflammatory macrophages and NF-κB overexpression. In experi-
mental and spontaneous metastatic models, tumor- and stroma-derived SPARCs
reduced tumor cell growth and metastasis formation via inhibition of cancer-related
inflammation and lung colonization. These data indicate that SPARCs are produced
both in cancer- and non-cancer-related compartments of bladder carcinomas, where
they suppress bladder carcinogenesis and progression via modulation of the inflam-
matory response to cancer cells (Said et al. 2013).
The two isoforms of the enzyme cyclooxygenase catalyze the initial step in the
formation of prostaglandins (PGs). PGs are involved in various inflammatory cell
processes, i.e., inflammation, immune response, and carcinogenesis. Urothelial
cells predominantly express high levels of COX-1, while bladder cancer cells
show COX-2 overexpression (Boström et al. 2001). Therefore, the mechanisms
that modify the expression of COX isoforms may possibly contribute to the trans-
formation of normal urothelial cells to cancer cells. In in vitro studies, exposure to
IFN-α decreased significantly the expression of COX-1 in 5637 and T24 bladder
cancer cells, while an increased COX-2 expression was found in both cell lines
(Boström et al. 2001). These findings suggest that IFN-α plays a role in COX-2
upregulation in urothelial cancer cells.
Bacterial lipopolysaccharides have been found to exert tumorigenic influence on
the non-tumorigenic rat urothelial cell line MYP3 via cytokine-mediated increase in
oxidative stress (i.e., hydrogen oxide) (Okamoto et al. 1996). Hydrogen oxide is a
potent transforming agent which is released during inflammatory conditions of the
bladder mucosa. Besides IFN-α, an increase in TNF-α release during inflammation
has been causally related to the transformation of normal urothelial cells to malignant
cells. In a prior study, it was demonstrated that number of colonies of MYP3 cells,
which had been exposed to hydrogen oxide and subsequently to TNF-α, markedly
increased as compared to untreated controls. Conversely, exposure to TNF-α alone
188 G. Gakis
(mRNA) and protein was also present on the bladder cancer cell surface. In two
human urothelial cancer cell lines, T24 and BIU-87, and in tissues of 56 patients
with urothelial carcinoma, IgG mRNA and IgG proteins were detectable.
Increased cell apoptosis and inhibited cell growth via activation of the caspase
pathway was observed after blockade of tumor-derived IgG by either antihu-
man IgG antibody or antisense oligonucleotides. Furthermore, in xenotransplant
models, antihuman IgG antibody was able to suppress tumor growth. Moreover,
adding either antihuman IgG antibody or antisense oligonucleotides to the blad-
der cancer cell line T24 enhanced its sensitivity to mitomycin C (Liang et al.
2013). Besides immunoglobulines, tumor-derived exosomes exert antiprolif-
erative effects on bladder cancer cells. Exosomes are multi-protein complexes
which are capable of degrading RNA. Tumor-specific exosomes are promising
tumor vaccines antigens but show low antiproliferative activity. To enhance their
immunogenicity, melanoma-antigen-1 (MAGE-1)-expressing T24 cells were
transfected with a plasmid encoding the glycosyl-phosphatidylinositol-anchored
interleukin 2 (GPI-IL-2) gene. Hereafter, IL-2 was found on the cell surface in
the GPI-anchored form. The tumor-derived GPI-IL-2 exosome contained bio-
active GPI-IL-2 and tumor-associated antigen MAGE-1. The proliferation of T
cells and the antigen-specific cytotoxic T lymphocyte response was found to be
more pronounced in exosomes expressing GPI-IL-2-pulsed dendritic cells. In
future, these observations may pave the way for exosome-based tumor immuno-
therapeutic strategies as an alternative approach to traditional immunoglobulin-
based immunotherapy.
Inflammatory processes are not only necessary for carcinogenesis but also neces-
sary for metastasis formation. The Rho-GDP dissociation inhibitor (RhoGDI2)
suppresses the metastatic potential of various human bladder cancer cell lines
(Gildea et al. 2002). In patients with muscle-invasive bladder cancer, increased
RhoGDI2 expression has been associated with inferior survival (Theodorescu
et al. 2004). Versican, a complex and versatile extracellular matrix protein, is a
key regulatory molecule in cancer-related inflammation and is also associated
with invasive and metastatic cancer stages (Wight 2002). It acts as a substrate to
be depleted during invasion by cancer cells in order to facilitate metastasis forma-
tion. This metastasis-promoting effect depends on the recruitment of macrophages.
Thus, versican is an integral component in order to establish a highly inflamma-
tory microenvironment (Said and Theodorescu 2012). The excessive “crosstalks”
between the immune system and cancer cells cause an “inflammatory cytokine
storm” that drives cancer cell colonization. Targeting versican or the associated
excessive release of cytokines represents a promising strategy to delay the evolu-
tion of metastases (Said et al. 2012).
190 G. Gakis
of immune cells and limits their ability to eliminate cancer cells. Conversely,
blockade of adenosine A2 receptors activates cytotoxic T cells and stimulates
dendritic cells. Basically, there are two adenosine receptor subtypes [A(2A) and
A(2B)] which can be blocked in a selective and non-selective manner. Receptor
analyses have shown that the antitumoral effects of adenosine blockers are mainly
mediated by the selective adenosine A(2B) receptor. ATL801 is a selective A(2B)
receptor antagonist which induces the secretion of IFN-γ and IFN-inducible
chemokine CXCL10. CXCL10 is a ligand for CXCR3 expressed on tumor-infil-
trating cytotoxic T cells. Accordingly, administration of ATL801 in CXCR3-
deficient bladder tumor mice did not show to decelerate tumor growth (Cekic et al.
2012). Taken together, these data suggest that selective adenosine (2B) receptor
blockers activate dendritic cells and enhance CXCR3-mediated cytotoxic response
to bladder cancer growth. Whether a combined approach by a concurrent blockade
of A(2B) and COX-2 receptors might result in superior tumor growth inhibition
awaits further investigation.
Migration of macrophages into tumor tissues is essential for effective tumor cell
elimination. Increasing evidence suggests that the pro-inflammatory cytokine mac-
rophage migration inhibitory factor (MIF) serves as a link between inflammation
and carcinogenesis. Anti-thrombin III, an endogenous serine protease inhibitor,
which is known to inactivate several enzymes of the blood coagulation cascade,
also acts as an inhibitory binding protein for MIF. In the serum, an increased MIF
concentration was found in bladder cancer patients compared to healthy individu-
als, while the concentration of ATIII-MIF complexes was decreased in cancer
patients. These data suggest that increased circulating levels of bioactive MIF are
present in the sera of bladder cancer patients (Meyer-Siegler et al. 2010).
In recent years, besides increasing evidence for the role of inflammation in blad-
der cancer, the presence of cancer stem cells has been suggested to be causative for
the high risk of recurrence and progression (van der Horst et al. 2012). Therefore,
the question arises whether inflammation is capable of activating cancer stem cells.
Data from immunohistochemical analyses show that the immunoreactivity of dis-
tinct stemness markers (Oct3/4 and CD44v6) and COX-2 is significantly higher in
cystitis and cancer patients compared to healthy controls. Interestingly, the nuclear
localization of COX-2 was significantly associated with upregulation of Oct3/4
and CD44v6 in bladder cancer tissues irrespective of the degree of inflammation.
Therefore, COX-2 activation may be also involved in inflammation-mediated cancer
stem cell proliferation during bladder carcinogenesis (Thanan et al. 2012).
From a clinical point of view, as the degree of inflammation potentially reflects
tumor aggressiveness (Siemes et al. 2006), the use of serum markers for meas-
uring the degree of systemic inflammation may be useful in counseling patients
192 G. Gakis
for neoadjuvant and adjuvant treatment. Candidate markers which can be easily
assessed in daily clinical practice include interleukin-6, leukocyte levels, and
serum CRP (Siemes et al. 2006). In this respect, CRP is a highly sensitive marker
of acute and chronic inflammation (Ledue et al. 1998). After interleukin-6 medi-
ated release by hepatocytes, CRP acts an opsonizing agent for cancer cell detec-
tion and elimination. However, interleukin-6 can also be released by tumor cells
themselves, facilitating cancer cell survival by pleiotrophic effects (Trikha et al.
2003). Consequently, elevated serum CRP levels are not only an epiphenom-
enon of the tumor microenvironment but also a critical component of the host’s
response to the tumor.
A recent screening study among healthy individuals showed that elevated CRP
indicates a higher risk of developing bladder cancer (Trichopoulos et al. 2006). In
patients undergoing radical surgery for bladder cancer, preoperative serum CRP
levels have been shown to predict local tumor stage and prognosis. A novel pre-
diction model for cancer-specific survival after radical cystectomy, termed TNR-C
Score (Gakis et al. 2011), which accounts for critical determinants for survival
(Tumor-stage lymph Node density, Resection margin status, and CRP level),
yielded a considerably high predictive accuracy of 79 %. In the following, further
investigations have confirmed the clinical significance of pretreatment CRP levels
and kinetics in predicting response to first-line and second-line chemotherapy in
metastatic bladder cancer (Saito et al. 2012; Ishioka et al. 2012).
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196 G. Gakis
negatively (CXCL12, CXCR4, and MMP, neutrophils, and MDSC) and positively
(TH1 cells, IP-10, and MIG) with tumor progression and survival. Finally, there is
a discussion of different inhibitors of inflammation that may be useful in the treat-
ment of RCC.
9.1 Introduction
An estimated 65,150 new cases of kidney cancer will be diagnosed in the USA
in 2013 resulting in 13,680 deaths. Kidney cancer incidence has increased by
3.1 % per year from 2005 to 2009 while death rates have decreased by 5 % in the
same time period. The increased incidence is mainly explained by an increase in
early-stage diagnosis incidentally during abdominal imaging for unrelated issues;
there are, however, no current recommendations for a screening test for the gen-
eral population. Incidence appears to be reaching a plateau for the first time in
several decades. Renal cell carcinoma (RCC) are tumors arising from the renal
tubular epithelial cells and account for more than 90 % of primary kidney tumors.
RCC represents the eighth most common malignancy in adults in the USA and
more than 50 % of individuals present with metastatic disease (American Cancer
Society 2008).
Clear-cell RCC (CCRCC) represents the most common histological type of
RCC, accounting for up to 75 % of all renal cancer cases, followed by papillary
(15 %), oncocytoma (5 %), and chromophobe (5 %) (Devita et al. 2011). CCRCC
can affect all patient age groups but is most commonly found in their 60s or 70s,
predominantly in men (male-to-female ratio of 2:1) (Cheville et al. 2003).
The extent of the disease determines the treatment of RCC, and it greatly influ-
ences survival. Due to its location in the retroperitoneum, RCC tumors can fre-
quently grow unnoticed for many years until the development of metastasis.
Historically, less than 50 % of patients have localized disease at presentation,
20 % have local invasion, and 30–40 % of patients have metastatic disease at the
time of diagnosis (Golimbu et al. 1986; Zisman et al. 2002). However, recent data
(2003–2009) from the surveillance, epidemiology, and end results (SEER) pro-
gram reflect a pronounced increase in RCC with localized tumors at the time of
diagnosis, which brings the percentage of localized disease to a range of 61–71 %
depending on the demographic group.
Survival
The overall 5-year relative survival for kidney cancer has increased from 51 % in
the early 1980s to over 73 % in the past decade (Howlader et al. 2013). Tumors of
the renal pelvis carry a worse prognosis than RCC tumors (5-year survival of 50
vs. 72 %, respectively). Early diagnosis (i.e., at a local stage) increases the 5-year
survival rate to 91 %. Unfortunately, this percentage drops to 10 % in patients pre-
senting with metastatic disease (Cohen and McGovern 2005).
9 The Role of Inflammation in Kidney Cancer 199
Fig. 9.1 This figure provides a summary of the changes in the infiltrating immune cells that
promote immune suppression and angiogenesis resulting in tumor evasion (top left, purple).
Also detailed are the different signaling pathways associated with RCC development, growth,
and survival (lower right, pink). Included is a depiction of the central role VHL silencing has
on the constitutive activation of HIF transcription leading to gene expression of different mol-
ecules involved in tumor growth. The impact of proangiogenic protein production has on promo-
tion of the tumor vasculature is depicted (upper right). This figure also highlights the importance
of inflammatory proteins and their receptors in regulating RCC development and growth. Red
arrows represent inhibition or downregulation, while blue represents stimulation or upregulation;
green arrows indicate nuclear translocation of transcription factors
Chemotherapy
Chemotherapy has shown poor efficacy for advanced stage RCC in numerous
clinical trials. Single-agent regimens have shown response rates of 5–6 % in trials
involving over 4,000 cases and more than 30 individual agents (Motzer et al. 2000;
Yagoda et al. 1995). 5-fluorouracil and gemcitabine combinations have yielded
slightly better response rates (10–15 %) (Rini et al. 2005; Stadler et al. 2006).
Immunotherapy
Clear-cell RCCs are considered an immunogenic tumor based on several observa-
tions that include rare but documented cases of spontaneous regression sometimes
associated with cytoreductive nephrectomy, significant immune infiltrate in some
tumors, identification of tumor-associated antigens expressed by RCC, and their
sensitivity to immunotherapy.
9 The Role of Inflammation in Kidney Cancer 201
Cytokine therapy
Cytokines represent an active, non-specific immunotherapy for the treatment of
metastatic kidney cancer, and the most studied include interleukin-2 (IL-2) and
interferon-alpha.
IL-2
IL-2 was approved by the FDA for the treatment of advanced RCC, and mela-
noma over two decades ago and for years was the first-line agent for metastatic
RCC. Administration of recombinant IL-2 is associated with a 20–25 % objec-
tive response rate in patients with kidney cancer. In different clinical trials, the
5-year survival rate for these patients was almost 20 % of the responders (Clement
and McDermott 2009; Halama et al. 2010; Escudier 2010; Dillman et al. 2011).
High-dose IL-2, however, has profound side effects, in particular, one character-
ized as a “cytokine storm” or capillary leak syndrome, but the mechanism behind
it is not clearly understood. Clinically, this results in hypotension, cardiac, renal,
pulmonary, gastrointestinal, cerebral, and hepatic toxicity (Finkelstein et al.
2010). Strategies to improve efficacy and/or reduce the side effect profile have
been unsuccessful; agent combination such as TNF, iNOS, or VEGF inhibition,
IFN-α administration did not significantly improve outcome (Halama et al. 2010;
Escudier 2010). Efforts to identify which patients are more likely to respond to
IL-2 have identified possible biomarkers. Complete responders to IL-2 therapy
have unique protein and gene expression patterns, but clinical trials have failed to
identify markers that can be use prospectively (Clement and McDermott 2009).
The cloning and production of recombinant IL-2 allowed for the in vitro expan-
sion of lymphokine-activated killer cells (LAK). These cells were administered
to patients as a form of adoptive immunotherapy. The majority of LAK cells are
derived from precursor cells with the immunological marker spectrum CD3(−),
CD11(+), CD14(−), CD16(+), CD56(+). Following activation with IL-2, cells
express the markers CD2(+), CD3(−), CD56(+) and typically represent activated
NK cells (Fortis et al. 1991). However, IL2 combined with LAK cell infusion did
not improve the therapeutic activity of IL2 (Law et al. 1995).
IFN-α
IFN-α is a glycoprotein that has antitumor effects along with immunomodula-
tive, antiproliferative, and differentiation-inducing activities (Neidhart 1986;
McDermott and Rini 2007). Overall response rates range between 10 and 15 %
with different dose regimens and preparations. A meta-analysis involving 6,117
patients with metastatic RCC between 1995 and 2004 showed a median improve-
ment in survival of 3.8 months (Coppin et al. 2005). This small but significant
improved survival and a lower treatment-related toxicity (and cost) resulted in a
wide spread use of IFN-α in metastatic RCC. IFN-α has been tested in clinical
trials with multiple other agents including IL-2 at different doses, chemotherapeu-
tic drugs (5-fluorouracil, vinblastine, cis-retinoic acid), targeted therapy agents
202 A. R. de Vivar Chevez et al.
approach to the treatment of metastatic RCC and will likely to lead to interesting
combinational studies.
for cell proliferation, metabolism, protein synthesis, and angiogenesis, and its
activation was found in around 60 % of primary CCRCC (Robb et al. 2007);
furthermore, its activation worsens the prognosis of localized and metastatic
kidney cancer patients (Pantuck et al. 2007). Three analogs of rapamycin with
mTOR inhibition properties have been tested clinically: temsirolimus, everolimus,
and deforolimus. The first two have been extensively tested in RCC as second-
line agents in metastatic RCC that progressed after treatment with a TKI as first-
line drug treatment. Compared to IFNγ, IV infusion of temsirolimus improved
the overall survival of poor prognosis population with RCC (Hudes et al. 2007).
Everolimus can be taken orally as a second-line agent after progressive disease
following TKI treatment; its efficacy and safety in this patient population have
been established in a phase III clinical trial involving 416 patients. PFS was 4.9
and 1.9 months for the everolimus and placebo groups, respectively (Motzer
et al. 2008).
Combination therapy
There are a number of combinational studies that are being developed, and some
are underway. This includes combining anti-CTLA-4 antibodies with either anti-
PD1 or anti-PDL-1 antibodies as a strategy that will block two checkpoint path-
ways of T cell suppression. Other studies are combining checkpoint blockade
inhibitory antibodies with TKI since the latter are known to reduce angiogenesis
but can also reduce immune suppression by targeting myeloid-derived suppres-
sor cells and Treg cells (Ko et al. 2009; Ozao-Choy et al. 2009). Another focus is
to combine agents that reduce immune suppression mediated by different mecha-
nisms along with different immunotherapy approaches that are designed to stimu-
late the immune system. Clinical trials are underway testing whether sunitinib will
synergize with two different vaccine strategies to promote an antitumor immune
response and improve overall survival. This includes vaccination of RCC patients
with DCs expressing tumor-associated antigens following their transfection with
autologous tumor RNA (Argos Inc) or with a cocktail of peptides constituting
multiple proteins that are overexpressed on RCC (Immatics Biotechnology). It
also seems likely that additional studies will include combining these and other
vaccines with checkpoint blockade antibodies.
VHL pathway
Inflammation is mediated by certain environmental and pathogenic factors which
include upregulation of HIF-1α and the production of proinflammatory proteins
leading to the concept that hypoxia plays a role in inflammatory processes in
9 The Role of Inflammation in Kidney Cancer 205
RCC (Reuter et al. 2010; Fitzgerald et al. 2012). As mentioned earlier, the tran-
scription factors, HIF-1 and HIF-2, initiate transcription of a large set of genes
important in several biologic responses including angiogenesis, proliferation,
apoptosis, and metabolism (Fig. 9.1) (Kaelin 2009). The proteins produced as a
consequence of VHL silencing include those involved in angiogenesis [vascular
endothelial growth factor (VEGF), glycolysis (phosphoglycerate kinase), glucose
transport (Glut-1), and erythropoiesis (erythropoietin)]; the chemokine receptor
CXCR4 has also been identified as an HIF target (Staller et al. 2003), suggesting
that HIF activation may contribute to the metastatic potential of cancer cells.
Hypoxia
A recent study (Fitzgerald et al. 2012) suggests that the inflammatory cytokines
interleukin-6 and interleukin-8 (IL-6 and IL-8) are secreted from RCC cells after
exposure to hypoxia (VHL deficient RCC cells). Furthermore, the NADPH oxi-
dase isoform, Nox4, plays an important role in hypoxia-induced IL-6 and IL-8
production in RCC. Additionally, the AMP-activated protein kinase (AMPK) is a
key regulator of NOX oxidase protein expression. AMPK is a sensor of cellular
sensor status that has been shown to play a role in the regulation of cell inflamma-
tory processes. Ex vivo studies by Fitzgerald et al. showed that enhanced levels
of IL-6 and IL-8 result in RCC cell invasion and that activation of AMPK reduces
Nox4 expression, IL-6 and IL-8 production, and RCC cell invasion. These find-
ings shed light on a possible mechanism by which AMPK and Nox4 are linked to
inflammation-induced RCC metastasis and that activation of AMPK may represent
a relevant therapeutic strategy to reduce IL-6- and IL-8-induced inflammation and
cell invasion in RCC.
VEGF
Binding of VEGF to its receptor initiates a complex of signaling cascades that
promotes various cellular processes essential for new vessel formation including;
increased vascular permeability, endothelial cell growth, migration, and survival of
preexisting vasculature. VEGF also mobilizes endothelial progenitor cells from the
bone marrow to sites of neovascularization.
The exact mechanism by which VEGF changes vascular permeability is not
clearly understood but this change leads to macromolecule leak to the extravas-
cular space and followed by edema formation. This change in the extravascular
microenvironment makes it more proangiogenic compared to the stromal baseline
conditions (Dvorak 2002). Additionally, VEGF promotes antiapoptotic proteins
like bcl-2 and A1 which inactivates upstream caspases. This is mediated by activa-
tion of the PI3K-Akt pathway and promotes survival of endothelial cells. VEGF
signaling increases the expression of several genes in endothelial cells, includ-
ing several proteases, mitogens, and adhesion molecules that ultimately promote
endothelial cell changes in cytoskeleton, cell morphology, and migration and
invasion (Zachary 2001).
206 A. R. de Vivar Chevez et al.
mTOR pathways
Protein kinase B (Akt) and mTOR are key players in processes involved in onco-
genic transformation including cell survival, angiogenesis, and proliferation.
mTOR is part of two major signaling complexes, mTORC1 and mTORC2, with
two different functional roles. mTORC1 promotes cell proliferation and growth;
mTORC2 modulates cell polarity and cytoskeleton rearrangement. Signaling
of the mTORC1 pathway is initiated by growth factors binding PI3K on the cell
membrane, this in turn phosphorylates PIP2 to PIP3, and this step is negatively
regulated by PTEN. However, PIP3 activates Akt, which inhibits TSC (tuberous
sclerosis complex) results in an overall upregulation of mTORC1 (Lieberthal and
Levine 2009). In addition to its effects in cell growth and proliferation, mTOR
activation leads to HIF accumulation; this is mediated through downstream pro-
teins S6K1 and eIF-4E. PTEN is inactive in 20–30 % of RCC tumors (Brenner
et al. 2002); furthermore, these mTOR-related proteins measured by tissue micro-
array from 375 patients with RCC were found more active in tissue samples from
higher grade tumor and poor prognostic features (Pantuck et al. 2007), and some
of these biomarkers (pAkt and p-S6K1) could be predictive of response with
mTOR inhibition therapy (temsirolimus) (Cho et al. 2007).
TNF pathway
TNF has many different effects on tumor cells mostly dependent on ligation of
each of its receptors (TNFR1 and TNFR2). Ligation of TNFR1 activates apop-
totic signaling kinase and NF-κB promoting apoptosis. TNFR2 ligation leads to
activation of EtK and VEGFR2, stimulates the transcription of antiapoptotic pro-
teins, and promotes entry into the cell cycle acting as an autocrine growth factor
in ccRCC (Al-Lamki et al. 2010). Thus, strategies to reduce TNFR2 expression or
to selectively block signaling through TNFR2 may be more effective than global
TNF blockade to reduce tumor progression. Malignant transformation of tubuloen-
dothelial cells changes the profile of TNFR2 expression, in fact at both tissue level
and plasma levels of TNFR2 have been shown to be elevated in RCC, and this
elevation correlates with malignant grade of ccRCC (Elsasser-Beile et al. 2000;
Al-Lamki et al. 2010). Furthermore, TNF-α may play a role in tumorigenesis in
RCC, TNF-α-induced epithelial–mesenchymal transition and promotes tumor
invasion by repressing E-cadherin, upregulating vimentin, activating matrix metal-
loproteinase 9 (Ho et al. 2012; Chuang et al. 2008).
The persistent cytokine and growth factor signaling observed in cancer and
chronic inflammation leads to accumulation of activated STAT proteins, and this
persistent STAT activation has been observed in several types of cancer includ-
ing RCC (Buettner et al. 2002; Horiguchi et al. 2002). In the setting of constant
STAT activation, resulting in persistence of STAT in the nucleus and dysregulated
gene expression eventually will alter the genotype of the cell. Constitutive expres-
sion of STAT3 induces expression of BCL-XL, MCL1, and survivin, which have
antiapoptotic functions. Blocking STAT3 signaling can block the expression of
these proteins and makes cells more susceptible to apoptosis (Catlett-Falcone et al.
1999; Aoki et al. 2003). STAT3 also induces C-MYC expression which prevents
cells from reaching terminal differentiation and maintains mitotic cell capabilities
(Bowman et al. 2001).
Early in the formation of primary epithelial tumors, cells show excessive prolifera-
tion, angiogenesis, and invasiveness. This is thought to be initially characterized by
the invasion of the basement membrane which is the first step for tumor cells to
eventually disseminate and metastasize. The ability to advance through the base-
ment membrane is not exclusive of tumor cells, normal cells can do this as part of
the epithelial to mesenchymal transition (EMT) which is tightly regulated geneti-
cally and biochemically. Activation of this phenotype is postulated as a key step
in malignant transformation of epithelial cells and is characterized by altered mor-
phology, adhesion, migration, and cellular architecture (Thiery 2002). Expression
of vimentin and nuclear translocation of β-catenin are some of the molecular
208 A. R. de Vivar Chevez et al.
markers of EMT; cells also show resistance to apoptosis and increased migration
capacity. An invasion assay using CCRCC lines with mutated VHL showed that
tumor-derived IL-6, TNF-α, IL-1, and matrix metalloproteinase-2 (MMP2) pro-
moted tumor invasion. The most invasive cell lines showed higher levels of mRNA
of these proinflammatory cytokines and MMP2 and produced more TNF-α, which
was correlated with stronger invasive ability (Chuang et al. 2008). TNF-α has
shown to enhance migration of RCC cell lines via activation of PI3K/Akt p athway
which in turn inactivates GSK-3β pathway (Sourbier et al. 2006) which has been
reported to be involved in the regulation of EMT (Luo 2009). Moreover, inhibition
of PI3K/AKT reactivated the GSK-3β suppression of EMT in TNF-α-conditioned
RCC cells (Ho et al. 2012).
pVHL can play an HIF-independent role in tumor transformation. VHL muta-
tion results in dysregulation of HIF-1α which in turn activates a cascade of events
that favor tumor growth and proliferation. However, the mechanism by which a
normal kidney cell undergoes oncogenic transformation is poorly understood.
Some in vitro studies have shown that VHL has HIF-1α-independent effect on
RCC cells, and such effect could play an important role in tumor initiation. In fact,
overexpression of non-degradable HIF proteins in the absence of VHL mutation
leads to proliferation of normal-appearing blood vessels but no oncogenic trans-
formation (Elson et al. 2001). pVHL directly binds to fibronectin which interacts
with integrin to bridge cells to the extracellular matrix in vitro, and this assem-
bly is defective in mutant pVHL cells (Ohh et al. 1998). pVHL stabilizes tumor-
suppressor gene p53 which mediates cell cycle arrest and apoptosis. Additionally,
pVHL also suppresses the expression of the mitogen cyclin D1 which is required
for cells to exit the cell cycle upon serum starvation in vitro (Roe et al. 2006;
Zatyka et al. 2002; Pause et al. 1998). Functional VHL seems also to be required
for the formation of the primary cilium, which occurs in cells reaching quiescence,
and its dysfunction is associated with the formation of renal cyst which often pre-
cedes tumor formation (Esteban et al. 2006; Lutz and Burk 2006). Together, these
experiments suggest that there are independent functions of VHL separable from
its interactions with HIF-1α that likely play an important role in the oncogenic
transformation of kidney cells in RCC.
Transforming growth factor (TGF) β1 is a member of the TGF-β superfamily.
This cytokine plays a role in wound healing, fibrinogenesis, and tissue remode-
ling and can strongly influence the growth and phenotype of several types of cells
(Massague et al. 1992). Overexpression has been observed in different kinds of
cancer, including RCC. Primary RCC cells as well as different RCC cell lines
express TGF-β1, and a majority of cell lines are resistant to growth-suppressive
effect of exogenous TGF-β1 (Ramp et al. 1997). This suggests that transforma-
tion and/or progression of human RCC could be related to TGF-β1 resistance to
growth inhibition. Transfection of wild-type VHL gene into the human RCC line
786-O lacking WT pVHL suppressed TGF-β message mainly at the posttran-
scriptional level showing that TGF-β is a target for pVHL. However, this line was
unresponsive to TGF-β because it lacked the TGF-β type II receptor. While VHL
mutations appear early in RCC development, a second genetic event resulting in
9 The Role of Inflammation in Kidney Cancer 209
isoforms and targets them for ubiquitination and degradation (Okuda et al. 2001).
The nuclear fraction of RCC cell lines was analyzed by electrophoretic mobility
shift assay and showed higher basal level of NF-κB capable of binding its DNA
motifs compared with wild-type VHL reconstituted cells. Furthermore, treatment
with TNF-α showed a significant rise in NF-κB in the nucleus of WT VHL recon-
stituted cells compared to a lower response in non-reconstituted cells (Qi and
Ohh 2003). This suggests that in addition to downregulation of HIF proteins and
their target genes, VHL is a positive regulator of apoptosis and RCC cells with
a mutated VHL display resistant to NF-κB-mediated apoptosis. Functional VHL
protein may be required for TNF-α and TRAIL-induced apoptotic cell death.
CSF-1 is highly regulated in the kidney and plays an important role in renal
tubular injury by promoting repair and inhibiting apoptosis (Menke et al. 2009). It
also participates in progression of several epithelial tumors (Morandi et al. 2011).
RCC cell lines were found to co-express CSF-1, and its receptor CSF-1R stimula-
tion with TNF-α significantly upregulates this expression at the RNA and protein
level. Moreover, expression of CSF-1 and CSF-1R is induced in tubular epithe-
lial cells when treated with supernatant from an RCC cell culture in a concentra-
tion dependent fashion and with greater response when the supernatant was from
stimulated RCC cells. Treatment of RCC and TEC cell lines with CSF-1 showed
increased cell proliferation in a dose-dependent manner, and this response was
inhibited with a blocking antibody. RCC cell lines stimulated with TNF-α/LPS
showed an increased rate of apoptosis, and adding a blocking anti-CSF-1R Ab
further increased the response (Menke et al. 2012). CSF-1 may have an autocrine
and paracrine role in RCC promoting tumor cell proliferation, and decreasing
apoptosis.
RCC cell line resulted in significant downregulation of CXCR4 gene (Staller et al.
2003). Treatment with a recombinant ligand (rhCXCL12 alpha) induced changes
in intracellular calcium levels in A-498 RCC cells, proving functionality of this
receptor. Furthermore, a cDNA expression array showed increased stimulation
of cell cycle and apoptosis-related genes compared to unstimulated A-498 cells.
Upon binding of CXCL12 to CXCR4, a signaling cascade is initiated, the com-
plex is internalized and localizes into the nucleus (Wang et al. 2009). Silencing of
the CXCR4 receptor by RNA interference in A-498 cells induced apoptosis and
inhibited cell growth, migration, and invasiveness compared to control cells (Wang
et al. 2012b). Previous studies have shown that CXCR4-expressing cancer cells
commonly metastasize to organs that express abundant CXCL12 (Muller et al.
2001), and this chemotactic property could also play an important role in RCC
tumor migration. Moreover, while this HIF-dependent expression of CXCR4 takes
place in other solid tumors in response to hypoxia, RCC cells could manifest this
CXCR4 activation much earlier in the tumor progression. Thus, CXCR4 expres-
sion could provide kidney cancer cells with an increased ability to regulate the cell
cycle, invade tissue barriers, migrate to other organs, and inhibit apoptosis.
A-498 cells also express CCR3, CCR6, CXCR2, CXCR3, and CXCR4 on both
mRNA and protein levels (Johrer et al. 2005). CCR3 is a receptor for eotaxin-1
which has been characterized as a potent eosinophil chemoattractant but may also
play an important role in the proliferation of RCC cells. CCR3 and eotaxin-1 mRNA
expression is high in a wide range of organs including small intestine, colon, heart,
kidney, and pancreas (Levina et al. 2009). Proinflammatory cytokines including
TNF-α, IL-1, IFN-α, and IL-4 induce eotaxin-1 expression in vitro in a variety of
tissues (Garcia-Zepeda et al. 1996a, b; Schrader et al. 2002; Mochizuki et al. 1998).
Binding of eotaxin-1 to its receptors leads to activation of G proteins, increased
intracellular calcium, cytoskeletal rearrangements, activation of mitogen-activated
protein kinase pathway, and receptor internalization (Zimmermann et al. 1999).
When A-498 kidney cancer cells were treated with eotaxin-1, there was upregulation
of intracellular Ca2+, internalization of the receptor-ligand complex that coincided
with increased cell proliferation compared to control (Johrer et al. 2005).
tumor site. Inflammatory molecules play an important role in RCC that allows
these interactions to occur.
MMPs are metalloendopeptidases that have the ability to disrupt the extra-
cellular matrix continuity, thereby playing an important role in the inflamma-
tory response and in promoting histological processes such as tissue remodeling
and angiogenesis. MMPs also participate in pathological processes like cirrho-
sis, arthritis, and metastasis (Yoon et al. 2003). The loss of VHL function has
been linked with upregulation of gene and protein MMP expression through
HIF up-regulation (Petrella et al. 2005). Specifically, MT1-MMP gene is a tar-
get of HIF-2α, and MT1-MMP is thought to be a key mediator of invasion and
angiogenesis (Seiki and Yana 2003). An experiment using RCC cells either wild-
type (WT8) and null (pRc-9) for VHL looked at their invasive characteristics.
The pRc-9 cells had increased capacity to degrade and invade in a type I colla-
gen matrix transwell assay compared to WT8 cells. Expression of HIF-2α or
MT1-MMP in the WT8 cells, via transfection, promoted collagen degradation
and invasion of these cells comparable to levels seen in pRc-9 cells (Petrella and
Brinckerhoff 2006).
Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of
MMPs that protect tissues by regulating their function, and a high MMP to TIMP
ratio in a particular tumor has been found to correlate with malignant grade.
MMP-2 and MMP-9 are increased in RCC tissue (Kallakury et al. 2001; Kugler
et al. 1998) and overexpressed in RCC cell lines analyzed after oxidative stress.
However, the levels of their inhibitors TIMP-1 and TIMP-2 remained unchanged
compared to unstressed cells. Silencing MMP-9 reduced the expression of MMP-9
in a RCC cell line (Caki-2) as well as their invasiveness, but cell proliferation was
not affected (Ueno et al. 2009). The increase in MMP to TIMP ratio may repre-
sent a mechanism by which RCC cells acquire invasive capabilities in the pres-
ence of oxidative stress inducers such as tumor-associated macrophages (TAMs)
(Hemmerlein et al. 2004).
In addition to ROS, TAMs from RCC specimens have been shown to secrete
high amounts of IL1-β (Ikemoto et al. 2003). Treatment of serum starved human
786-0 VHL null RCC cell line with IL-1β resulted in induction of tumor cell inva-
sion in a type I collagen-coated transwell assay. Furthermore, pretreatment of
RCC cells with a pan-MMP inhibitor, blocked IL-1β-induced invasion thereby
demonstrating a MMP-dependent effect of IL-1β in promoting cell invasion.
Moreover, IL-1β potently induced the expression of MMP-1, MMP-3, and MMP-
10 (which have collagen I degradation activity) at the mRNA and protein levels in
a dose-dependent fashion (Ikemoto et al. 2003).
Urokinase-type plasminogen activator (uPA) has been known to mediate inva-
sion and metastasis of various tumor cells, its expression has been found elevated
in kidney tumors, and it possibly correlates with aggressive phenotype (Andreasen
et al. 2000; Swiercz et al. 1998). The protein C inhibitor (PCI) is an endogenous
inhibitor of several protease enzymes including protein C and uPA. In humans,
PCI is mainly produced by the liver and other tissues including the reproduc-
tive track, and the kidney also produces it in lesser amounts (Laurell et al. 1992;
9 The Role of Inflammation in Kidney Cancer 213
Francis and Thomas 1984). PCI is found in tissues in complex with uPA, which
inhibits it suggesting a role of PCI in protecting tissues from unopposed uPA; in
fact, PCI knockout mice have been reported to grow normally but are infertile, this
is thought to be due to disruption of the blood–testis barrier (Uhrin et al. 2000).
uPA has been known to mediate invasion and metastasis of various tumor cells.
An experiment using purified PCI to treat Caki-1, a kidney cancer cell line that
expresses uPA (but not PCI) showed that PCI inhibited cell invasion in a dose-
dependent fashion in gel matrix assay, heat inactivation of PCI, or addition of a
PCI antibody blocked this inhibition and treatment with an uPA antibody also
inhibited cell migration (Wakita et al. 2004).
RCC, especially the clear-cell type, develops a densely vascular architecture.
The VHL gene plays a crucial role in the cellular response to oxygen, and its abil-
ity regulates critical regulators of angiogenesis through the HIF transcription fac-
tors (Iliopoulos et al. 1996). In the renal tumor setting, HIF-2α is responsible for
activation of cyclin-D1, TGF-α, and VEGF pathways (Raval et al. 2005). VEGF
angiogenic activity is mediated through interaction with other proangiogenic fac-
tors many of which are also gene targets of HIF, some of these molecules, like
angioprotein-1, provide antiapoptotic properties and vessel stability; erythro-
poietin, also a target of HIF, promotes endothelial cell growth and migration
(Heeschen et al. 2003; Yamakawa et al. 2004).
MMPs can disrupt the extracellular matrix and promote tumor cell migra-
tion; this disruption of the tissue also allows for pericyte invasion and activation
through release of growth factors bound to the extracellular matrix; this step is
necessary for new vessel formation and is also facilitated by platelet-derived
growth factor receptor (PDGFR) activation on the pericyte (Yamakawa et al.
2004). PDGF-B and TGF-β1 participate in smooth muscle cell recruitment and
stabilization (Carmeliet and Jain 2000).
Animal models
The most widely used murine tumor model is the RENCA cell line that arose
spontaneously in Balb/c mice (Wigginton et al. 1996). More recently, streptozo-
tocin-induced renal cell tumor lines including SIRCC-1.15 (designated RCC#15)
have been developed and characterized. The Streptozotocin tumor develops spon-
taneous metastases to lung and mesenteric lymph nodes following into kidneys
(Gruys et al. 2001). Both of these tumor models are sensitive to different forms
of immunotherapy; however, neither have the molecular and cellular features of
human CCRCC as they lack the loss of the VHL gene function and the consti-
tutive expression of HIF and its targets. Conditional models of VHL inactivation
and VHL knockout mice have been produced but they do not develop spontaneous
RCC (Kleymenova et al. 2004; Rankin et al. 2006; Haase et al. 2001). A mouse
214 A. R. de Vivar Chevez et al.
TAMs
Tumor-associated macrophages or TAMs are cells that originate from recruited
myeloid cells such as monocytes and MDSC. These myeloid cells are highly
plastic and tumor-derived factors recruit and sustain them to support angiogen-
esis, tissue remodeling, and immune suppression (Sica and Bronte 2007). TAMs
can constitute a significant component of solid tumors, and depending on the
microenvironment, they can present different phenotypes (Sica and Bronte 2007;
Biswas and Mantovani 2010). In RCC microenvironment, there is an increased
metabolism of arachidonic acid partly due to the enzyme 15-lypoxygenase (LOX)
highly expressed in RCC TAMs. This, in turn, increases production of hydrox-
yeicosatetraenoic acids (HETE). An upregulated LOX-HETE pathway in RCCs
tumor microenvironment affects the immune phenotype of TAMs. For example,
RCC TAMs secrete immunosuppressive Interleukin-10 and the proinflammatory
chemokine CCL2. IL-10-producing macrophages are considered “regulatory mac-
rophages” in a recently proposed classification, and their presence could nega-
tively affect prognosis as well as efficacy of tumor vaccines and other kinds of
9 The Role of Inflammation in Kidney Cancer 215
immunotherapy (Mosser and Edwards 2008). RCC TAMs also induce u pregulation
of CTLA-4, IL-10-secreting TILs, and FoxP3+ T cells, a tolerogenic subset of
tumor infiltrating lymphocytes called Tregs (Daurkin et al. 2011). And, as dis-
cussed above, their ability to induce oxidative stress and produce IL1-β could
favor a more invasive and proangiogenic phenotype in RCC cells. RCC tumors
with high-infiltration TAMs were significantly associated with poor prognosis,
and inflammatory cytokines TAMs produce, including IL-1β, TNF-α, and IL-6,
are also independent factors of poor prognosis in RCC (Yoshida et al. 2002;
Komohara et al. 2011).
MDSC
Myeloid-derived suppressor cells constitute a heterogenous cell population with
immunosuppressive and angiogenic properties that originate from the bone mar-
row under pathologic conditions such as cancer. These cells have the morphol-
ogy of immature granulocytes, monocytes, and dendritic cells (DCs) (Gabrilovich
2004). These cells are functionally defined by their capacity to suppress T cell
immunity via different mechanisms (Gabrilovich 2004; Peranzoni et al. 2010).
MDSC express enzymes (e.g., arginase 1) that can deplete select amino acids in
the tumor microenvironment (L-arginine and L-cysteine), thereby limiting the
availability of these amino acid which are necessary for lymphocyte activation
(13) (Srivastava et al. 2010). Some MDSC produce reactive oxygen species (ROS)
and/or inducible nitric oxide synthase, resulting in reduced CTL activity and
IFN-γ production (Corzo et al. 2009; Kusmartsev et al. 2008; Cohen et al. 2012;
Ko et al. 2010). MDSC indirectly inhibit T cell immunity by stimulating expan-
sion of regulatory T cells (Treg) (Huang et al. 2006; Pan et al. 2010). MDSC can
also reduce L-selectin expression of naïve T cells, reducing their ability to enter
peripheral lymph nodes where DC presents antigen (Hanson et al. 2009). Besides
mediating immunosuppression, MDSC can stimulate angiogenesis. Injecting nude
mice with MDSC plus tumor cells compared to tumor alone increased vascular
density and maturation within the tumor that was dependent metallomatrix pro-
tein 9 (MMP9) production. Interestingly, a subset of MDSC can associate with
tumor endothelium followed by their differentiation into endothelial cells (Yang
et al. 2004). Furthermore, the production of VEGF and bFGF by MDSC is STAT3
dependent since MDSC- mediated angiogenesis can be blocked by STAT3 inhib-
itors (Kujawski et al. 2008). Similar to mouse models, granulocytic (G) MDSC
(CD33+HLADR−CD15+CD14−) dominate in the blood of patients with differ-
ent types of cancer including RCC, GBM, lung, and pancreatic cancer (Rodriguez
et al. 2009; Zea et al. 2005; Peggs et al. 2009; Ko et al. 2010; Youn et al. 2012;
Sippel et al. 2011). Monocytic (CD33+HLADR−CD15−CD14+) are also pre-
sent in modest numbers in RCC patients while a population of MDSC not typi-
cally seen in mouse models are prevalent in RCC, the linage negative subset
(CD33+HLADR−CD15−CD14−) (11, 23, 25). The granulocytic MDSC are
known to be suppressive. The impact-increased MDSC numbers in the blood has
on tumor progression were recently assessed in RCC patients. High pretreatment
216 A. R. de Vivar Chevez et al.
Neutrophils
Increased neutrophil numbers in the peripheral blood have been identified as a
predictor for shorter overall survival in metastatic RCC patients (Choueiri et al.
2007; Donskov and von der Maase 2006; Donskov 2013; Lopez-Lago et al. 2013;
Negrier et al. 2002). More recently it was reported that the presence of intratu-
moral CD66b+ neutrophils in RCC patients with localized disease was linked to
higher tumor size, lower recurrence-free survival, and reduced overall survival
(Jensen et al. 2009). It may be that the negative impact of neutrophils on RCC
patient outcome is linked to the evidence that neutrophils from RCC patients are
immunosuppressive. Peripheral blood from RCC patients contain already mature,
activated neutrophils with suppressive activity resulting from their expression of
arginase. Because of their similarities to granulocytic MDSC, they were identified
as G-MDSC (Rodriguez et al. 2009; Zea et al. 2005). These findings were similar
to the work of Schmielau and Finn who earlier showed that the presence of granu-
locytes from pancreatic, colon, and breast cancer patients had T cell suppressive
activity and their presence correlated with reduced T cell zeta-chain expression
and decreased cytokine production. They also showed that healthy donor resting
granulocytes could be converted to suppressive cells by exposure to the chemotac-
tic peptide N-formyl-L-methionyl–L-leucyl–L-phenylanine (fmlp) (Schmielau and
Finn 2001). However, other studies suggest that human G-MDSC constitute imma-
ture neutrophils (CD16−CD33+HLADR−CD66b+) because they display low to
absent expression of the neutrophil maturation marker CD16 (FcRγ1) (Brandau
et al. 2011). Additional functional and gene array studies are clearly needed to
define the interrelationship between G-MDSC, patient neutrophils, and activated
neutrophils from healthy donors.
from RCC patients with localized or metastatic RCC showed that primary human
RCC tumors are immunogenic and expresses high levels of HLA class I mRNA and
proinflammatory cytokines and chemokines. In contrast, metastatic RCC is charac-
terized by an immunosuppressive microenvironment, decreased expression of HLA
class I, reduced level of IFN-γ and suppression of IP-10, VEGF and SDF-1 at both
mRNA and protein level (Romero et al. 2006). This may indicate that a switch in the
tumor cells, its microenvironment, or both may provide an escape for tumor cells
from the cytotoxic response and favor tumor invasion and metastasis.
The macrophage migration inhibitory factor (MIF) is a cytokine with roles in
autoimmunity diseases, obesity, and cancer; it promotes inflammation and can pro-
long immune responses. This cytokine is regulated by hypoxia and is one of the
HIF target genes (Bernhagen et al. 1993; Welford et al. 2006). MIF is expressed
and correlated with poor prognosis in a number of solid tumors (Du et al. 2013).
In RCC, MIF was found in most tumor specimens, and it correlated with the pres-
ence of circulating MIF in RCC patients. shRNA inhibition of MIF expression in
RCC cell lines or the use of a direct antagonist reduced their proliferation rate.
Similarly, inhibition of MIF receptors CD74 or CD44 resulted in inhibition of
cell proliferation. Western blot analysis showed MIF signaling through CD74 and
CD44 increases Src expression and degradation of p27 which could represent a
mechanism for promotion of cell growth (Du et al. 2013).
evaluated showed CXCL12 expression which was correlated with poor overall
survival and DFS. Furthermore, RT-PCR analysis of 49 tumor samples showed
that the presence of CXCR4 and CXCR7 correlated with symptoms at the time
of diagnosis and lymph nodes status (D’Alterio et al. 2010b; Wang et al. 2012a;
D’Alterio et al. 2010a). Screening for the chemokine receptor CXCR3 (recep-
tor for eotaxin-1 discussed above) in 219 tumor specimens found this expres-
sion in almost a third of the samples, and its expression was found to correlate
with high tumor grade (Johrer et al. 2005). Two ligands for this receptor are CXC
chemokines, I-TAC and Mig, and they have antiangiogenic activities; however,
RCC samples showed high levels of both in vascular smooth muscle, pericytes of
tumor tissues compared to corresponding normal tissue. This paradoxical expres-
sion suggests a stronger opposing angiogenic stimuli or a possible dual role of
these chemokines generally considered angiostatic (Suyama et al. 2005).
In contrast with the above markers, Th1-associated cytokines could indicate
favorable prognosis. A total of 67 tumor specimens from sporadic RCC cases were
analyzed by rt-PCR for Th1- (IP-10, ITAC, MIG, MIP-1β, and RANTES) and
Th2- (MDC and eotaxin) associated genes. TH1 genes were higher compared to
normal kidney tissue, and it correlated with IFNγ expression. More importantly,
out of 59 patients who underwent curative surgery, 9 patients had recurrence, and
none of them presented high TH1-related cytokines IP-10, ITAC, MIP-1β, and
RANTES, and 1 patient with high MIG had recurrence after surgery (mean follow
up of 45.7 ± 29.3 months). Similarly, patients with high eotaxin expression had no
tumor recurrence after surgery (Kondo et al. 2006).
Matrix metalloproteinases (MMPs) and their regulators, TIMPs, are implicated
in RCCs invasive potential (see in vitro section). A total of 153 RCC sections
were analyzed for MMP2, MMP9, TIMP1, and TIMP2 by immune staining, and it
was found that their increased expression (including TIMPs) correlated with poor
prognostic characteristics including survival, and high tumor grade (Kallakury
et al. 2001). The paradoxical correlation of TIMPs expression with poor prognos-
tic features suggest a more complex biology of these inhibitors, in fact, when the
ratio of MMPs to TIMPs in RCC tumors was compared to normal kidney tissue,
it was found that tumors cells had a ratio of 2.4 MMP:TIMP (based on a ratio of
1 for normal kidney) for localized tumors and 4.86 for advanced disease suggest-
ing that a balance of these molecules has probably more prognostic value than the
absolute number (Kugler et al. 1998).
NSAIDs
Studies on the use of anti-inflammatory drugs and kidney cancer have been incon-
sistent. The overall risk of cancer in patients who use NSAIDs is reduced com-
pared to patients not taking this drugs (Bardia et al. 2007); this is thought to be
9 The Role of Inflammation in Kidney Cancer 219
COX-2 inhibitors
Cyclooxygenase-2 (COX-2) is an important enzyme involved in the synthesis of
prostaglandins. In cancer, COX-2 has been linked to several stages of develop-
ment of tumors including cell growth, antiapoptosis, and angiogenesis (Koga et al.
2004; Sawaoka et al. 1999; Masferrer et al. 2000). COX-2 inhibitors have been
used in preclinical and clinical studies to inhibit tumor growth and angiogenesis
and as a chemopreventive drug in different solid tumors (Rao et al. 2002; Wang
et al. 2013; Fujimura et al. 2007). RCC cell lines overexpress COX-2, and it has
a role in cell invasion capabilities (Chen et al. 2004a, b). Although in vivo stud-
ies showed COX-2 enhanced tumorigenesis and angiogenesis in a human RCC
xenograft, and human RCC specimens showed expression of COX-2, human clini-
cal trials using a selective COX-2 inhibitor in combination with IFN-α resulted in
no added clinical activity compared to IFN-α alone. Furthermore, selective treat-
ment of patients whose tumors showed high immunostaining for COX-2 did not
benefited from COX-2 inhibition when treated with IFN-α. Selecting high COX-2
expressing tumors could also represent a selection of highly immunosuppressive
tumors which could explain this lack of additional response. (Rini et al. 2006;
Schwandt et al. 2011).
levels (Farwell et al. 2008; Dale et al. 2006; Alsheikh-Ali et al. 2007). Khurana
et al. (2008) looked nearly 500,000 records of veterans who visited the VA Health
Care System over a period of 5.6 years, multivariate analysis adjusting for age,
race, sex, body mass index (BMI), and smoking showed an overall 44 % RCC risk
reduction in patients taking statins. Mechanistically, the effect of statins on renal
cancer cells has been shown to inhibit cell growth, proliferation, invasion, and
pro-apoptotic effects in vitro, it also leads to cell cycle arrest with upregulation
of p21 and p53 (Fang et al. 2013; Horiguchi et al. 2004). Protein analysis showed
elevated Bax and decreased Bcl-2; the balance of these two apoptosis-related
proteins favors apoptosis when it favors Bax. Looking downstream, cleaved cas-
pase-3 (an effector caspase) and cleaved PARP (a caspase-3 target) levels were
also elevated; furthermore, a statin (simvastatin) mediated these effects by target-
ing the AKT/mTOR pathway which is commonly activated in RCC as discussed
in the beginning of this chapter. Similarly, phosphorylation of ERK and IL-6
induced JAK2/STAT3 pathway that results in increased proliferation, migration,
and invasion of RCC cells was also inhibited by statin pretreatment. In vivo mod-
els showed that statins inhibited tumor growth, metastasis, and induced apoptosis
in the tumors and, consistent with in vitro experiments; AKT, ERK, and STAT3
phosphorylation were decreased (Fang et al. 2013; Horiguchi et al. 2004).
Reduction in number and function of suppressive MDSC
Because MDSC are immunosuppressive, and angiogenic multiple strategies are
being examined to reduce the number and/or function in tumor bearing mice and
humans (Gabrilovich 2004; Najjar and Finke 2013). Strategies tested in RCC
patients and mouse models includes the use of all-trans retinoic acid (ATRA) to
drive the differentiation of immature myeloid cells into mature cells without sup-
pressive activity (Kusmartsev et al. 2008). In RCC patients treated with ATRA,
the number of MDSC in the blood was significantly reduced, and this reduction
was associated with an increase in tetanus-toxoid-specific T cell responses (Mirza
et al. 2006). Others are testing whether MDSC can be converted into tumoricidal
macrophages by the use of CD40 ligand, TLR agonists, and/or T1-type cytokines
(Beatty et al. 2011; Shirota et al. 2012; Liscovsky et al. (2011); Zembala et al.
1994). Additional studies using a different approach demonstrated that blocking
reactive oxygen species production in MDSC with synthetic triterpenoid (CDDO,
Me) reduced the suppressive activity of MDSC isolated from patients with RCC
(Nagaraj et al. 2010). In a mouse model, this approach did not alter the number
of MDSC in the spleens but did reduce their suppressive activity and decreased
tumor growth. Select TKI that are used to treat metastatic RCC patients can reduce
the number of MDSC by promoting cell death. Sunitinib (front-line therapy) sig-
nificantly reduces the number of MDSC in the peripheral blood (Ko et al. 2009;
van Cruijsen et al. 2008) along with restoring Type-1 T cell IFN-γ responses
(Ko et al. 2009; Finke et al. 2008). Additionally, select chemotherapy agents can
also reduce MDSC levels in cancer patients (Gabrilovich 2004; Najjar and Finke
2013). In select mouse tumor models, sunitinib therapy when combined with vac-
cines and/or adoptive therapy can enhance tumor regression, improve survival, and
9 The Role of Inflammation in Kidney Cancer 221
The last couple of decades have seen exponentially growing evidence linking can-
cer and inflammation, and with this a shift to our approach to cancer from strictly
a disorder of cells with damaged or mutated genes that grow unregulated to one
that includes the interaction between tumor cells and the immune system where
tumor cells use host’s immune mediators to foster their growth and survival, in
turn, inflammation becomes chronic and tumors thrive and progress, a view that
Rudolph Virchow had over 150 years ago. RCC represents 90 % of primary kid-
ney tumors and is a prototypical tumor that interacts profoundly with the immune
system. The gene mutations that result in a loss of function of the VHL gene are a
common feature of sporadic and familial cases of RCC. Most solid tumors eventu-
ally outgrow their blood supply and enter a constant state of hypoxia; this activates
hypoxia-inducible genes via the HIF; in RCC, however, the loss of VHL function
results in a constant HIF expression and function early in the tumor formation
even in normoxic conditions. Early on there is upregulation of many pathways that
promote tumor growth, proliferation, invasion, angiogenesis, antiapoptosis, and
evasion of the immune system. These pathways are mediated by targets of HIF
which include VEGF, MMPs, chemokines, and chemokine receptors, cyclin-D1,
TGF-alpha, angioprotein-1, erythropoietin among others. The VHL protein itself
has HIF-independent functions that may favor malignant transformation and tumor
progression; it mediates cell–extracellular matrix interactions and assembly, stabi-
lizes p53, suppresses cycling D1, and helps cells reach quiescence.
Surgery remains the most effective treatment for RCC but is limited to local-
ized disease. Surgery for advanced RCC does not improve survival but anecdotal
cases of metastatic disease regression after nephrectomy has hints on the control
RCC has over immune-tumor interactions. Advanced RCC responds poorly to
chemotherapy and/or radiation although it is considered an immunogenic tumor
and is modestly responsive non-specific immunotherapy for RCC (IL-2 and IFN-α).
While a small percentage of patients achieved cures with this approach, signifi-
cant toxicity to high-dose IL-2 made way to more specific therapies. HIF target
genes and their pathways are now the main focus of the current and developing
222 A. R. de Vivar Chevez et al.
treatments of RCC (e.g., TKI targeting VEGF and mTOR inhibitors), termed
“targeted therapy,” and have proven to have greater survival benefit than IL-2 and
IFN-α treatment. Therapies targeting checkpoints of the immune system such
as CTLA-4 and PD-1 pathways have shown significant activity in some cancer
types such as RCC and providing renewed interest in immunotherapy approaches.
Additionally, growing evidence is emerging on inflammatory profiles on the
patient side that can help predict not only prognosis and survival but also predict
response to specific treatment. Inflammatory cytokines usually correlate poor
prognosis or advance disease, and immune infiltrating cell profiles can also predict
patient outcomes; even xenograft tumor model using fresh tissue tumor samples
implanted in nude mice are used in an attempt to predict best response to treat-
ment. Clearly, efforts are been made to characterize both patients and their tumor
inflammatory profiles and treatments that target a more specific biology. There are
many unanswered questions in the interactions of RCC with the immune system,
a better understanding of this bilateral dialog is vital to achieve clinical improve-
ments in patients with RCC.
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Chapter 10
The Role of Inflammation in Gastric Cancer
Kazım Şenol, Murat Bulut Özkan, Selahattin Vural and Mesut Tez
Abstract Gastric cancer, despite its declining incidence rate, is still the second
cause of cancer-related death worldwide, killing 750,000 people each year and
remaining the second common type of cancer. The best examples of inflammation-
associated cancer in human beings may be gastric cancer. Understanding the
molecular mechanism of the inflammation in gastric carcinogenesis is important
for developing new strategies against gastric cancer.
10.1 Introduction
Cancer is a major public health problem. Every year almost 1 million new cases of
gastric cancers are presented and there are about 750,000 deaths caused by g astric
cancer. It ranks second in terms of cancer-related deaths after lung and bronchus
cancer (Hussain and Harris 2007; Jemal et al. 2011). Although chemotherapy
improves life expectancy, complete resection of gastric cancer (R0) via gastrec-
tomy remains insufficient and more than 80 % of patients with advanced gastric
cancer die of the recurrent disease within 1 year after diagnosis (Group et al. 2010;
Gomceli et al. 2012).
The choice of treatment generally depends on the tumor’s size, tumor
location, stage of disease, and general health status of patient. Treatment of the
gastric cancer consists of surgery, chemotherapy, radiotherapy, and also targeted
therapy. Surgery is a common treatment of all stages of gastric cancer. The aim
of surgery is to remove as completely as possible all grossly visible tumor tissue
and to obtain histologically free surgical margins. Total and subtotal gastrectomy
are used for R0 resection. If the tumor is blocking the stomach but the cancer
Cell proliferation, differentiation, and function are principally arranged with a broad
signaling network mediated by stimulative/inhibitory hormones, neurotransmitters,
various cytokines, and growth factors. The interactions between cells are the most
important factors that keep the balance of this network which can influence cell
proliferation in positive or negative ways, as well as these interactions induce a
series of differentiated responses in appropriate target cells. When these networks
are inappropriately regulated, neoplastic cells may occur with its autonomy of
unrestrained growth and may harm the organism even the causes are disappeared
(Fedi et al. 2000).
Inflammation is one of the predominant manifestations of innate and adaptive
immune systems that different and also alternative inflammatory mechanisms play a
part in remodeling of tissue and re-establishment of tissue homeostasis in consequence
of infection or injury by exogenous or endogenous means. All pro-inflammatory
responses are accompanied by anti-inflammatory responses as a non-homogenous
result that depend on type of the pathogen or tissue damage, the genotype of the host,
and also discrepancies between the tissue involved. Any disturbance in tissue home-
ostasis activates the innate immune cells that are first line of defense which quickly
migrate into the injured tissue after vasodilatation and in response to chemokine gra-
dients, classically described as the inflammatory stage of wound healing (Velnar et al.
2009). The innate immune system cells are composed of macrophages, mast cells,
dendritic cells (DC), and natural killer cells (NK), etc., that regulates the inflammatory
response by releasing excessive growth hormones, cytokines, chemokines, matrix-
remodeling proteases, reactive oxygen, and nitrogen species on behalf of taking con-
trol of the inflammatory process (Coussens and Werb 2002; Nathan 2002). These cells
also promote healing by releasing cytokines such as tumor necrosis factor (TNF),
interleukin (IL)-1, interleukin (IL)-6 that achieves cell survival, activate stem cells
and promote epithelial proliferation. Also, the correlation between tumor-associated
macrophage abundance and poor prognosis has been shown (Nowicki et al. 1996).
Furthermore, macrophage-deficient mice display reduced progression of tumors to a
more malignant phenotype (Bromberg and Wang 2009). NK and DC also play a key
role in providing a link between adaptive and innate immune response, and both have
10 The Role of Inflammation in Gastric Cancer 237
crucial roles in maintaining the antigen-specific immunity (de Visser et al. 2006). Th1
response and its accompanying mediators interferon (IFN)-γ are not only necessary
for Helicobacter-induced inflammation but also for the development of atrophy or
metaplasia and spasmolytic polypeptide-expressing metaplasia (SPEM); however, a
Th2 response and its mediators (i.e., IL-4) appear to be protective. The presence of
a Th1, rather than a Th2, immune response is also associated with better survival in
gastric cancer patients (Marshall and Warren 1984).The host response to the inflam-
mation is a key factor takes role within inflammation process leading to carcinogen-
esis which includes the steps of initiation, promotion, and progression (Kinzler and
Vogelstein 1996).
In addition, inappropriate and steady regulation of the immune components may
be a cause of chronic inflammation by generating an initiative microenvironment
that alters normal cellular homeostasis and advances the stepwise accumulation of
genetic and epigenetic alterations of various proto-oncogenes and tumor suppres-
sor genes on behalf of cancer development. These genetic and epigenetic changes
include point mutations, deletions, duplications, recombinations, methylation of var-
ious tumor-related genes through various mechanisms (Chiba et al. 2012) and also
include altered microRNAs expression (Zhang et al. 2008). Although, multiple sign-
aling pathways including increased inflammatory cytokine production, abnormal
apoptosis, inappropriate cell proliferation/differentiation, and epithelial transfor-
mation are triggering causes of these alterations. The fact is that biology of cell
division, differentiation, and apoptosis is exceedingly similar in both normal and
cancer cells (Ooi et al. 2009).
Some of the earliest observations in cancer biology as well as recent advances
in molecular analyses contribute to our knowledge about the multistep process
of gastric carcinogenesis (Yokozaki et al. 1997; Balkwill and Mantovani 2001;
Gutierrez-Gonzalez and Wright 2008). Ooi et al. (2009) mentioned in a study that
among 70 % of GC patients, three oncogenic pathways are deregulated, which are
demonstrated as: proliferation/stem cell pathway (40 % of GCs), NF-κB pathway
(46 % of GCs), and Wnt/b-catenin pathway (39 % of GCs). Nuclear factor (NF)-κB
and STAT3 pathways have emerged as key regulators of the release of these pro-
inflammatory cytokines, and important mediators of both tumor proliferation and
persistence of chronic inflammation. The activation of these pathways results in fur-
ther cytokine release (Yang 2007; Rius et al. 2008; Guilford et al. 1998). Several
studies suggest that association between gastric carcinogenesis and cytokine overex-
pression, especially IL-1, IL-6, TNF which are also regulated by the NF-κB, showed
such a clinical correlation with NF-κB signaling pathway upregulation in gastric
cancer cells more than benign disorders such as gastritis (Yin et al. 2013). STAT3
drive in gastric cancer initiation and progression through its activation by cytokines,
IL-6 family ligands are expressed in the stomach that IL-6 and IL-11 provide a basis
in tumorigenesis (Giraud et al. 2012).
The gastrointestinal tract has rapid epithelial turnover and exposure to injury by
infections and dietary toxins. These conditions create very high cancer prevalence.
Intestinalization of gastric units, which is called “IM”; phenotypic antralization
of fundic units, which is called “spasmolytic polypeptide-expressing metaplasia
238 K. Şenol et al.
(SPEM)”; and the development directly from the stem/progenitor cell zone are
three pathways that have been described for gastric carcinogenesis (Fox and Wang
2007; Hoffmann 2008; Slack 1986).
Stem cell activation is a remarkable step in obtaining the tissue repair and self-
renewal. Despite the main disadvantages of addressing the response of human
immune system in the clinical researches remain obscure, these studies implicate
that only a subset of cancer stem cells (CSCs) propagate the tumor (Vries et al.
2010), while the frequency of CSCs greatly increases to possibly more than 25 %
of all tumor cells (Shmelkov et al. 2008; Quintana et al. 2008; Kelly et al. 2007).
Several signaling pathways such as, Wnt/β-catenin pathway, take role in assuring
the homeostasis and maintain the balance of gastric epithelia between progenitor and
stem cells (Lickert et al. 2001; Katoh 2007). Additionally, Wnt signaling pathway
is described as important steps of tissue repair and stem cell self-renewal in chronic
tissue injury-related carcinogenesis (Beachy et al. 2004). It has been shown in a study
that Wnt pathway in association with other inflammatory signaling pathways initiates
the gastric progenitor cells through the metaplasia-carcinoma sequence in rat gastric
mucosa (Oshima et al. 2006). In another mouse model, activated macrophages in the
inflamed or Helicobacter pylori-infected gastric mucosa express TNF-α, which stim-
ulate the surrounding cells to promote Wnt/β-catenin signaling activity in multistep
pathogenesis of inflammation leading to gastric cancer (Oguma et al. 2008).
Cyclooxygenases (COX) are the key enzymes that convert and array of fatty
acid substrates into pro-inflammatory prostanoids. There are 2 types of COX
genes, type 1 is a physiologic gene that constitutively expressed in many tissues
and responsible for the synthesis of prostanoids involved in protection of the gas-
trointestinal mucosa and for production of the pro-aggregatory prostanoid throm-
boxane by the platelets. In the contrary, type 2 COX gene is usually undetectable
in most tissues. COX-2 is an inducible gene and activated by several stimulus like
hormones, pro-inflammatory cytokines, growth factors, and tumor promoters.
Also COX-2 has been related to inflammation, reproduction, and carcinogenesis
(Taketo 1998; Dannenberg et al. 2001).
Although the subsequent pathways are different, chronic inflammation is the
first step in both the intestinal and the diffuse type of gastric cancer. While the
intestinal type has a sequence of multifocal atrophic gastritis, IM, and dysplasia,
which advances to carcinoma, the diffuse type tends to be primarily genetic in
origin (Correa 1995; Nardone et al. 2004). The progress from IM to gastric cancer
has a wide range of molecular alterations affecting transcription factors, such as
CDX1 and CDX2, telomerases, microsatellite instability, mutations of p53 protein,
overexpression of COX-2, cyclin D2, and decreased expression of p27 (Muller
et al. 2001). The next step is gastric dysplasia. During the progression of normal
tissue through the metaplasia-dysplasia sequence, there are mutations in genes
including p53, also loss of heterozygosity of the adenomatous polyposis coli gene,
overexpression of the anti-apoptotic gene bcl-2, and a mixture of polyploidy and
aneuploidy (Muller et al. 2001).
As described above, several signaling pathways take place in gastric carcino-
genesis, and detection of the form and complexity of interactions between these
10 The Role of Inflammation in Gastric Cancer 239
About 150 years ago Rudolph Virchow distinguished that inflammatory cells are
existed in tumor tissues suggesting that chronic inflammation played a role in
carcinogenesis. Since then it has been established that 25 % of all cancer types
related with chronic inflammation (Hussain and Harris 2007). After identifying
chronic atrophic gastritis and discover of H. pylori, gastric carcinoma has taken
place in one of the cancers caused by chronic inflammation.
Over 100 years several studies have been conducted on gastric cancer and its
relationship with atrophic gastritis and intestinal metaplasia. There has been a sig-
nificant progress through the understanding of the development of gastric cancers,
after in 1937, Magnus concluded that the presence of intestinal epithelium in the
stomach is the result of the faulty regeneration of surface epithelium in a mucosa
repeatedly damaged by gastritis and that it is, in fact, an example of metaplasia
resulting form chronic irritation, and in 1955, Morson suggested that gastric car-
cinoma has arose from the areas of intestinal metaplasia (Morson 1955; Magnus
1937). Interest in H. pylori as a cause of cancer began after the pioneering discov-
eries of Marshall and Warren in 1983. H. pylori infection is the most common bac-
terial infection worldwide, almost 80 % of the population in developing countries
are infected with H. pylori (Pounder and Ng 1995). H. pylori is a gram-negative
spiral-shaped rod that usually acquired in infancy. It has four to six flagella that
settle beneath the mucus layer of stomach. This is a defensive mechanism which
protects bacteria from low gastric pH. Another defensive mechanism is its highly
active urease enzyme which is capable of dividing urea into ammonia and bicar-
bonate, creating a non-acid microenvironment. H. pylori has various virulence
factors such as its screw-like shape, lipopolysaccharide, vacuolating cytotoxin
240 K. Şenol et al.
Inflammatory cytokines are the remarkable determinants cell survival and death.
IL-1 and IL-6 activate nuclear factor-кB (NF-κB) and STAT3 pro-survival tran-
scription factors to induce cell survival and tumor development, where as other
cytokines such as Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL)
induce apoptotic cell death (Kuraishy et al. 2011). It is now well accepted that if
the host-mediated anti-tumor activity is incapable of forming immune response
via several defending mechanisms, tumor cells undergo immune escape and grow
rapidly. Dunn et al. (2004) suggested as in “cancer immunosurveillance” theory
that cytokines have dual roles, while such cytokines especially TNF, TRAIL, FasL,
and TWEAK are inducing the apoptotic cell death, the other cytokines such as type
I interferon (IFN) and TGF-β limit the proliferation of epithelial cells. TNF-α, espe-
cially in combination with IFN-γ, were originally described for their anti-tumoral
activity, a cytotoxic action against tumor cells by regulating the immune response,
host defense and gene expression. It is demonstrated in a study that IFN-γ regu-
lates apoptosis by soluble TNF-R released by IFN-gamma in the injured gastric
epithelial cell line induced by TNF (Furuta et al. 2002). IL-12 and IL-18 both
242 K. Şenol et al.
allow proliferation of T cells and potent production of IFN-γ, which may lead to a
direct anti-proliferative and pro-apoptotic effect on the tumor cells as well as anti-
tumor activity (Ye et al. 2007). In a study, IL-18 enhance the proliferation of gastric
cancer lines via NF-κB signaling pathway in a dose-dependent manner, where as
L-18-pretreated gastric cancer cells, which were cultured with cytokine-activated
peripheral blood killer lymphocytes, showed less secretion of IFN-γ or perforin,
anti-tumor products of killer lymphocytes, resulting in a decreased susceptibility of
cancer cells to killer lymphocytes (Majima et al. 2006). Despite cytokines, several
in vitro studies have found that PPARγ activation results in cell cycle arrest and/
or apoptosis of gastric cancer cells (Takahashi et al. 1999). Cytokines produced
in response to injury have enormous effects on cell survival contributing to tumor
initiation, growth, progression, and metastasis, which is yet to be elucidated.
Chronic inflammation plays an important role in tumorigenesis and macrophages
are a key player in generating the chronic inflammation microenvironment by being
activated persistently until leading to continuous tissue damage (Macarthur et al.
2004). In the acute phase of inflammation, the release of endogenous reactive oxy-
gen (ROS) and nitrogen species (NOS) (O2−, H2O2, NO, OH, ONOO−, HOCl)
from such innate immune cells as macrophages together with other leukocytes con-
tributes a fight back to infection and pathogens (Maeda and Akaike 1998; Leach
et al. 1987). However, sustained generation of ROS and NOS may alter proliferating
cells via forming a tumorigenic microenvironment that generated in several path-
ways. Continuous deleterious ROS and NOS exposure triggers amplification of
inflammatory cytokine production that stimulates signal transductors, angiogenic
factors, and oncogene overexpression and post-translational modification of tumor
suppressor genes and also causes direct DNA damage by inhibition of DNA repair in
proliferating cells (Federico et al. 2007).
ROS and NOS secretion is under control of pro-inflammatory cytokines
through the activation of protein kinases signaling that accumulates the production
of free radicals such as hydroxyl radical (OH•), superoxide (O2−•), nitric oxide
(NO•), and peroxynitrite (ONOO−).
TNF-α induces ROS production in neutrophils, tumor cells, and also endothelial
cells via a ceramide-dependent signaling pathway (Corda et al. 2001), while TNF-
α, IL-1β, interferon-γ (IFN-γ) stimulates the expression of inducible nitric oxide
synthase in inflammatory and epithelial cells. In addition, in an increased cellular
oxidative stress process, TNF-α induced excessive production of reactive oxygen
species, influence its cytotoxic effects on tumor cells, and arrangement of gene
expression (Goossens et al. 1995; Schutze et al. 1992).
TNF-α and IL-1β also induce the formation of ONOO−, which formed by
a reaction of NO• with superoxide, is a constitutive producer of IL-8. IL-8 is a
potent pro-inflammatory chemokine derived from monocytes, macrophages, and
endothelial cells that promote adhesion, migration, invasion, and chemoresist-
ance of gastric cancer cells (Zouki et al. 2001; Kuai et al. 2012). When ROS levels
are significantly increased, oxidatively altered nucleic acids (Demple and Harrison
1994) cause DNA damage including strand breaks, intrastrand adducts, and DNA
protein cross-links (Valko et al. 2005). In addition, ROS mediates the formation of
10 The Role of Inflammation in Gastric Cancer 243
tissue remodeling and stromal development linking to tumor growth and spread as an
endogenous tumor promoter (Balkwill and Joffroy 2010). TNF-α can also promote the
angiogenesis by inducing a range of angiogenic factors, thymidine phosphorylase, and
matrix metalloproteinases (MMPs) (Balkwill and Mantovani 2001; Leek et al. 1998;
Balkwill and Joffroy 2010; Aggarwal 2003). In addition, TNF-α has a vital role in
DNA damage with the help of other inflammatory cytokines, IFN-γ, and IL-1β, by
upregulating iNOS and NO• production which causes direct damage of DNA and
inhibits the DNA repair (Jaiswal et al. 2000). Besides that, TNF-α implicates other
chemokines via regulating a tumorigenic signaling network, in inflammatory pro-
cesses leading to tumorigenesis (Balkwill and Joffroy 2010).
Thus, TNF-α secures its position as a major mediator of inflammation-associ-
ated carcinogenesis, by contributing a tumorigenic microenvironment via inducing
several cytokines, angiogenic factors, and MMPs that cause DNA damage and
promote tumor growth and tumor metastasis in the survival of tumor cells through
a tumorigenic signaling pathway, NF-кB (Balkwill and Joffroy 2010). NF-кB is
a transcription factor, and NF-кB-regulated genes provide several products that
inhibit apoptosis and enhance cell cycle progression, angiogenesis, and metas-
tasis, in consequence of forming the IKK complexes which pro-inflammatory
cytokines and microbial infections are being stimulated (Karin and Greten 2005;
Karin 2006; Luo et al. 2005). Soutto et al. (2011) demonstrated that Trefoil fac-
tor 1(TFF1, a tumor suppressor gene) knockout mice leads to activation of IKK
complex-regulated NF-κB transcription factors, and hence, NF-κB-mediated
inflammatory response causes a multistep carcinogenesis cascade in the progres-
sion of gastric carcinogenesis with the help of TNF-α-mediated NF-κB activation
through the TNF receptor 1 (TNFR1)/IκB kinase (IKK) pathway. In an another
study, Mochizuki et al., also showed that by using a green fluorescent protein
(GFP)-tagged human gastric cancer cell line, TNF-α-pretreated mice group exhib-
its an early progression of peritoneal metastasis which is more significant than the
non-pretreated group (Mochizuki et al. 2004). It is still a controversial manner that
gastric cancer patients show a significant increase in TNF-α levels.
TNF-α and IL-1β are essential in the initiation of chronic inflammation. Recent
works have shown that IL-1β overexpression, in the absence of H. pylori infection, is
sufficient to cause gastric cancer. In addition, IL-1β is one of the essential pro-inflam-
matory cytokines modulated during H. pylori infection that directs the mucosa toward
atrophy, metaplasia, and neoplastic transformation (El-Omar et al. 2000; El-Omar
2001; Pollard 2004). Beales (2002) demonstrated in a study that IL-1β stimulates the
proliferation of gastric cancer cell lines via tyrosine kinase-dependent signaling path-
way and autocrine stimulation of GM-CSF contributes to this stimulation in a dose-
dependent manner. Similar to findings for IL-1β, Uefuji et al. (2005) demonstrated
that IL-1α mRNA expression levels were relevant to COX-2 positive cancer cell
lines, that exogenous supplement of IL-1α enhances both IL-1α and COX-2 mRNA
expression levels which indicates IL-1α-COX-2 pathway might be involved in tumor
progression by regulating cancer cell proliferation. Several researchers have demon-
strated that IL-1α enhances angiogenesis and vascular endothelial cell proliferation in
gastric cancer cell lines (Ma et al. 2008; Furuya et al. 2000).
10 The Role of Inflammation in Gastric Cancer 245
IL-1β, IL-6, IL-8, and TNF-α mRNA expression levels were significantly elevated in
H. pylori-positive mucosa compared with H. pylori-negative mucosa. In H. pylori-
positive gastric mucosa, IL-1β, IL-6, and IL-8 mRNA expression levels correlated
significantly with activity and chronic inflammation scores, and TNF-α mRNA
246 K. Şenol et al.
expression levels correlated with chronic inflammation scores. There was a nega-
tive association between IL-6 and IL-8 mRNA expression and intestinal metapla-
sia scores IL-6 and TNF-α mRNA expression levels increased with the severity of
atrophic gastritis, while pro-inflammatory cytokine mRNA expression levels were
lower in the mucosa with intestinal metaplasia compared to mucosa with extended
atrophic gastritis (Isomoto et al. 2012).
Individual differences in the intensity of the inflammatory response (which
affects the maintenance, severity, and outcome of H. pylori infection) may
contribute to gastric mucosa transformation. Moreover, the impact of gene poly-
morphisms on the activity of key inflammatory molecules is relatively well known.
Previous studies on the association between IL-1 genetic polymorphisms and
the risk of gastric cancer have produced controversial results. In a meta-analysis,
authors observed that the IL-1B–511T carrier, as well as the IL-1RN*2 carriers,
are associated with an increased risk of developing of gastric cancer, markedly
the intestinal type. IL-1RN*2 carrier increased the risk of developing gastric can-
cer among Caucasian. However, the IL-1B–31C and +3954T genotypes are not
associated with an increased risk of developing gastric cancer (Wang et al. 2007).
In contrast, these polymorphisms are not consistently related to the risks of
esophageal or gastric cardia cancers (El-Omar et al. 2003).
A number of studies have shown that cyclooxygenase-2 (COX-2) gene poly-
morphisms were associated with gastric cancer. However, the results from different
research groups have not been consistent. At present, two polymorphisms in COX-2
have been reported. The promoter region polymorphic variant of −1195G>A and
−765G>C has been demonstrated to have a functional effect on COX-2 transcrip-
tion, which may cause gastric cancer (Pereira et al. 2009; Zhang et al. 2005).
Several studies have examined the association of polymorphisms in tumor
necrosis factor-A gene (TNF-A) with gastric cancer risk. However, the meta-
analysis of these studies have shown that TNF-A-308AA genotype was associated
with a increased risk of gastric cancer, whereas other polymorphisms are not
(Gorouhi et al. 2008).
Polymorphisms in the 5′-flanking region of IL-10 at positions −1082 A/G,
−819T/C, and −592A/C have been suggested to be associated with gastric can-
cer risk in different populations (El-Omar et al. 2003; de Oliveira et al. 2012).
IL-10-592 AA is a factor of protection against the development of this neoplasm
in Asians, but not among Caucasians and Latinos, indicating differences in the
genetic background of Asians and other ethnicities (Zhu et al. 2011).
IL-17A has a crucial role in the gastric inflammation and carcinogenesis.
Genetic polymorphisms of IL-17A may be involved in methylation-related carcino-
genesis in the stomach (Tahara et al. 2010). Similarly, it also indicates that IL8, and
maybe IL4R, variants may modify the risk for gastric cancer (Crusius et al. 2008).
Few studies have done combined analysis of different polymorphisms in gastric
cancer. El-Omar et al. (2003) analyzed 11 polymorphisms of the IL-1B, IL-1RN,
IL-4, IL-6, IL-10, and TNF-A cytokine genes and showed that the risk for non-
cardia gastric cancer increased progressively with the number of pro-inflammatory
genotypes to 27.3 for three or four polymorphisms. This finding is probably due to
10 The Role of Inflammation in Gastric Cancer 247
Wistar rats that underwent gastrojejunostomy stated that celecoxib has an inhibiting
effect on reflux-induced gastric carcinogenesis (Rocha et al. 2009).
In a human trial, patients with gastric preneoplastic lesion, who taken H. pylori
eradication therapy, received either celecoxib or placebo for 3 months, and a signif-
icant improvement in precancerous lesions was observed who received celecoxib
for placebo (Zhang et al. 2009). In an another study, etodolac was used as a selec-
tive COX-2 inhibitor to demonstrate the preventive effects on cancer development
in extensive metaplastic gastritis (Yanaoka et al. 2010). These results strongly
suggest that chemoprevention of cancer in the metaplastic stomach is possible by
controlling COX-2 expression.
In light of these findings, there is a high probability that in near future, gastric
cancer will be prevented by COX inhibition.
There are various studies about diet, nutrition, dietary supplements, and their
relation with gastric cancer and also its prevention. According to The World Cancer
Research Fund and the American Institute for Cancer Research, non-starchy veg-
etables and fruits probably protect against stomach cancer. Salt and also salt-pre-
served foods are probably the causes of this cancer (Wiseman 2008). A prospective
study with 10-year follow-up of the Japan Public Health Center study cohort sug-
gested that consumption of vegetables and fruits is associated with diminished
gastric cancer risk (Kobayashi et al. 2002). Current epidemiologic and human trial
evidence generally indicates that antioxidant foods or supplements provide little
protection against gastrointestinal cancers (Jayaprakash and Marshall 2011).
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Chapter 11
The Role of Inflammation in Sarcoma
Jürgen Radons
Abbreviations
ANG Angiogenin
AS Angiosarcoma
BGS Baller–Gerold syndrome
BLS Bloom syndrome
BS Bone sarcoma
CCS Clear cell sarcoma
J. Radons (*)
Department of Radiotherapy and Radiation Oncology, Klinikum rechts der Isar,
Technische Universität München, Ismaninger Straße 22, 81675 Munich, Germany
e-mail: [email protected]
CS Chondrosarcoma
DBA Diamond–Blackfan anemia
EC Endometrial cancer
ECS Endometrial carcinosarcoma
EFT Ewing's family tumors
ERK Extracellular signal-regulated kinase
ES Ewings’s sarcoma
FAK Focal adhesion kinase
FAP Familial adenomatous polyposis
FS Fibrosarcoma
GIST Gastrointestinal stromal sarcoma
HS Histiocytic sarcoma
HAS Hemangiosarcoma
Hh Hedgehog
HLRCC Hereditary leiomyomatosis and renal cell cancer
IRS Insulin receptor substrate
JNK c-Jun N-terminal kinase
KS Kaposi sarcoma
KSHV Kaposi sarcoma-associated herpesvirus
STS Soft tissue sarcoma
LFS Li–Fraumeni syndrome
LMS Leiomyosarcoma
LS Liposarcoma
MAPK Mitogen-activated protein kinase
MEK Mitogen-activated protein kinase kinase
MFH Malignant fibrous histiocytoma
MM Malignant mesothelioma
MPNST Malignant peripheral nerve sheath tumor
mTOR Mammalian target of rapamycin
NF1 Neurofibromatosis type 1
OPN Osteopontin
OS Osteosarcoma
PGHS-2 Prostaglandin H2 synthase 2
PI3K Phosphatidylinositol 3-kinase
RB Retinoblastoma
RMS Rhabdomyosarcoma
RTS Rothmund–Thomson syndrome
SS Synovial sarcoma
STAT Signal transducer and activator of transcription
TK Tyrosine kinase
WS Werner syndrome
11 The Role of Inflammation in Sarcoma 261
11.1 Introduction
11.1.1 Epidemiology
A recent population-based study from the SEER data base with >48,000 STS cases
clearly demonstrated that individuals over 50 years of age have an inferior survival
than younger patients (Ferrari et al. 2011), thus confirming results of the RARECARE
project. A similar pattern was observed for OS (Mirabello et al. 2009) and CS
(Giuffrida et al. 2009), demonstrating the lowest survival rates in the oldest age group.
The influence of gender on the incidence of sarcoma is controversially discussed in
the literature. According to RARECARE, STS overall had a slightly higher incidence
11 The Role of Inflammation in Sarcoma 263
in females than in males (Stiller et al. 2013), whereas SEER data revealed the direct
opposite (Toro et al. 2006; Ferrari et al. 2011).
Apart from RTS, WS and BLS are rare autosomal recessive cancer
p redisposition disorders caused by loss of function of the RecQ helicases Wrn or
Blm, respectively. BLS and WS are characterized by replication defects, hyperre-
combination events and chromosomal aberrations (Muftuoglu et al. 2008; Shen et
al. 2012) leading to genetic instability as one of the hallmarks of cancer (Colotta
et al. 2009). Genetic instability is associated with an increased predisposition to a
great variety of cancers including sarcoma, e.g., OS, CS, and spindle cell sarcoma
(Lahat et al. 2008). RECQL4 mutations can also lead to Baller–Gerold syndrome
(BGS), or RAPADILINO syndrome correlating with an increased risk for OS
(Suhasini and Brosh 2013). Diamond–Blackfan anemia (DBA) represents a fur-
ther inherited disorder which has been found as being associated with an enhanced
risk of OS (Lipton et al. 2001). DBA patients exhibit abnormal pre-rRNA matura-
tion patterns, and the majority bears mutations in one of several ribosomal protein
genes that encode structural components of the ribosome essential for the correct
assembly of the ribosomal subunits. Studies on the most frequently mutated gene,
RPS19, revealed that mutations prevent the assembly of the ribosomal protein into
forming preribosomal particles, thus triggering nucleolar stress pathways (Ellis
and Gleizes 2011). However, the role of ribosomal proteins in the pathogenesis of
OS remains to be elucidated.
Hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome is a
tumor predisposition syndrome caused by heterozygous germ line mutations in
the fumarate hydratase (FH) gene (Tomlinson et al. 2002). The condition is char-
acterized by predisposition to benign leiomyomas of the skin and the uterus,
renal cell carcinoma (RCC), and uterine LMS (Launonen et al. 2001; Lehtonen
et al. 2006). As shown by Pollard et al. (2005a, b), HLRCC tumors were found
to overexpress hypoxia-inducible factor-1α (HIF-1α) and hypoxia-responsive
genes encoding vascular endothelial growth factor (VEGF) and Bcl-2/adenovirus
E1B 19 kDa interacting protein 3 (BNIP3) accompanied by a higher microves-
sel density in comparison with the sporadic counterparts. From these findings, the
authors postulate that failure of the Krebs cycle in HLRCC tumors causes inap-
propriate signaling followed by a hypoxic cell state which may lead to angiogen-
esis as well as clonal expansion and tumor growth through some uncharacterized,
cell-autonomous effects.
Several heritable mutations in the genes encoding the stem cell factor recep-
tor c-Kit and platelet-derived growth factor receptor alpha (PDGFR-α) have been
identified in patients with familial GIST (Maeyama et al. 2001; Heinrich et al.
2003b). While about 70–80 % of GIST harbor mutations in the proto-oncogene
CKIT, approximately one–third of the remaining have mutations in PDGFRA
(Wardelmann et al. 2003; Heinrich et al. 2003a). Mutations in these genes result in
constitutive activation of the c-Kit and PDGFR-α signaling pathway culminating
in blockage of apoptosis and cell proliferation, respectively (Rubin et al. 2007).
On the other hand, genetic syndromes associated with Ewing’s sarcoma
(ES), a neoplasm of the undifferentiated small round cells generally affect-
ing the bone and deep soft tissues of children and adolescents, are extremely
rare. ES is associated with specific chromosomal translocations resulting
266 J. Radons
in oncogenic fusion transcripts and proteins (Ross et al. 2013). The chimeric
proteins commonly represent artificial transcription factors dysregulating gene
expression (Xia and Barr 2005) and consequently modifying growth and dif-
ferentiation processes leading to cell transformation due to their oncogenic
potential (May et al. 1993; Scheidler et al. 1996). A genome-wide association
study of 401 French individuals with ES recently identified candidate risk loci
upstream of TARDBP and EGR2 (Postel-Vinay et al. 2012). TARDBP encodes
the nuclear transactive response DNA-binding protein 43 (TDP-43) represent-
ing the dominant disease protein in amyotrophic lateral sclerosis and a subgroup
of frontotemporal lobar degeneration. TARDBP shares structural similarities
with EWSR1 and FUS, that encode RNA-binding proteins, and early growth
response gene 2 (EGR2) is a target gene of the EWSR1/ETS translocation.
Erythroblastosis virus E26 transforming sequence (ETS) family of proteins are
transcription factors that modulate the expression of genes involved in various
biological processes, including cellular proliferation, differentiation, devel-
opment, transformation, and apoptosis (Bassuk and Leiden 1997). The study
by Postel-Vinay et al. (2012) clearly demonstrated that variants at these loci
were associated with expression levels of TARDBP, ADO (encoding cysteam-
ine dioxygenase), and EGR2. Since our knowledge of the oncogenic pathways
underlying the pathogenesis of sarcoma is steadily increasing, one can assume
that fusion genes characteristic for a certain subtype could have the potential to
function as critical targets in diagnosis and therapy.
Moreover, specific molecular markers have been shown to impact STS progno-
sis. In addition to p53 and PDGFR-α that give rise to GIST, upregulated β-catenin
levels correlate with increased proliferative activity in high-grade STS (Kuhnen et
al. 2000). Apart from its membranous function as an effective molecule for cell
adhesion in sarcomas with epithelioid pattern, β-catenin may act as an oncoprotein
in sarcomas with intracytoplasmic and nuclear localization by binding to nuclear
DNA (Kuhnen et al. 2000). S-phase kinase-associated protein 2 (Skp-2) catalyzes
the ubiquitylation of the tumor suppressors p27Kip-1 and p21Waf-1 (Fasanaro et al.
2010). In myxofibrosarcomas, Skp-2 overexpression was found as being highly
representative of the biological aggressiveness, thus playing an important prognos-
tic role (Huang et al. 2006).
An intriguing novel aspect is presented by Savage and Mirabello (2011) who
discuss the putative impact of single nucleotide polymorphisms (SNPs) on the risk of
OS. SNPs are the most common type of genetic variation in the genome. Although
SNPs do not modify gene expression or protein functions, they can influence a wide
variety of biological effects such as upregulation of Mdm-2, a direct negative regula-
tor of p53, and inhibition of the p53 signaling pathway, thereby accelerating tumor
formation (Bond et al. 2004). SNPs have been found, among others, in the genes
encoding estrogen receptor (ESR1), collagen 1α1 (COL1A1), vitamin D receptor
(VDR), Mdm-2 (MDM2), insulin-like growth factor receptor 2 (IGFR2), Fas protein
(FAS), and TGFBR1*6A, a common hypomorphic variant of transforming growth
factor beta receptor 1 (TGFBR1). In these genes, genetic variations obviously appear
to be associated with an increased risk of OS (Savage and Mirabello 2011).
11 The Role of Inflammation in Sarcoma 267
11.1.3 Current Therapies
Results from the Meta-GIST meta-analysis showed that patients whose GIST
harbors a CKIT mutation in exon 9 garner a longer progression-free survival time
when treated initially with high-dose imatinib (800 mg daily) compared with those
patients harboring CKIT mutations in exon 11 or no mutation (Meta-analysis 2010;
Gronchi et al. 2010). Moreover, treatment interruption after three years resulted in
tumor progression in most patients with metastatic GIST (Le Cesne et al. 2010),
demonstrating that GIST patients benefit from an imatinib treatment. Imatinib
treatment has been approved by the Food and Drug Administration (FDA) to
decrease the risk of disease recurrence after resection of GIST with significant
relapse potential. Although this treatment strategy has improved the quality of life
and survival of patients with advanced GIST, several patients did not respond to
imatinib. Approaches to treat imatinib-resistant GIST include the use of alternative
kinase inhibitors such as sunitinib, dasatinib, nilotinib, AMG-706 (Amgen), the
mammalian target of rapamycin (mTOR) inhibitor everolimus (Afinitor, RAD001,
Novartis), and the Hsp90 inhibitor retaspimycin hydrochloride (IPI-504).
New drugs for the treatment of STS include eribulin (Halaven, Eisai), an analog
of the marine sponge natural product halichondrin B, which functions as a mechanis-
tically unique inhibitor of microtubule dynamics. Eribulin has been approved 2010
by the FDA for the treatment of patients with metastatic breast cancer. In a nonrand-
omized multicenter phase II study, patients were included if they had progressive or
high-grade STS and had received no more than one previous combination chemo-
therapy or up to two single drugs for advanced disease (Schoffski et al. 2011b). This
study clearly demonstrated that 32 % of patients with LMS, 47 % of patients with
adipocytic sarcoma, 21 % of patients with synovial sarcoma (SS), and 19 % of other
sarcomas were progression-free at 12 weeks after eribulin monotherapy.
Targeting the molecular pathways involved in sarcomagenesis represents a
promising novel approach in the treatment of sarcomas. Antagonistic antibodies, TK
inhibitors, and inhibitors of downstream molecules of the PI3K (phosphatidylinosi-
tol 3-kinase)/AKT/mTOR pathway demonstrated encouraging activities. Table 11.3
summarizes selected chemotherapeutics that have been used in clinical settings for
the treatment of different subtypes of sarcoma. Among them, monoclonal antibod-
ies against the insulin-like growth factor 1 receptor (IGF1-R) such as figitumumab,
cixutumumab, and ganitumab either alone or in combination with other agents are
currently under investigation for patients with sarcomas. The IGF signaling path-
way is constitutively activated in and drives cellular growth of a great variety of
sarcomas including ES, LS, LMS, RMS, and SS. Therefore, anti-IGF therapy rep-
resents a promising therapeutic option in the treatment of sarcoma because it also
affects mTOR, one of the downstream effector molecules of PI3K. The participation
of mTOR in sarcomagenesis is related to the primordial role of the IGF system in
these tumors. Therefore, mTOR inhibitors have been consequently tested for their
anti-tumor potential in sarcoma. Sirolimus, temsirolimus, everolimus, and ridaforoli-
mus are analogs of rapamycin, so-called rapalogs, and function as specific mTOR
inhibitors (Table 11.3). Clinical trials analyzing the clinical efficacy of rapalogs in
monotherapy or combination therapy in sarcomas are ongoing.
11 The Role of Inflammation in Sarcoma 269
Table 11.3 continued
Agents Sarcoma subtype References
Sunitinib mesylate, Imatinib-resistant GIST, Heinrich et al. (2008), George
sunitinib malate extraskeletal myxoid CS, et al. (2009), Demetri et al.
solitary fibrous tumor (2009b), Stacchiotti et al.
(2012a, b)
Tivantinib Clear cell sarcoma (CCS) Wagner et al. (2012)
mTOR inhibitors
Everolimus (RAD001) Advanced sarcoma, retroperito- Quek et al. (2011), Gennatas
neal perivascular epithelioid et al. (2012), Ho et al. (2012)
cell tumor (PEComa), SS
Ridaforolimus (AP23573) Advanced BS and STS Mita et al. (2013), Chawla et al.
(2012)
Sirolimus (rapamycin) KS, CS, pretreated STS, OS, Yaich et al. (2012), Bernstein-
ES Molho et al. (2012),
Schuetze et al. (2012)
Temsirolimus (CCI-779) BS, STS, Ewing's family Italiano et al. (2010), Naing
tumors (EFT), malignant et al. (2012), Schwartz et al.
PEComa (2012)
Insulin-like growth factor 6
receptor inhibitors
Cixutumumab EFT, refractory BS and STS Schoffski et al. (2011a), Naing
et al. (2012), Schwartz et al.
(2012), Malempati et al.
(2012), Chugh et al. (2012)
Figitumumab ES, STS Toretsky and Gorlick (2010),
Olmos et al. (2010), Quek
et al. (2011), Juergens et al.
(2011)
Ganitumab (AMG 479) EFT, desmoplastic small round Tolcher et al. (2009), Tap et al.
cell tumors (DSRCT), (2012)
advanced or metastatic RMS,
LMS, adipocytic sarcoma, SS
Rudolf Virchow (1821–1902) established cellular pathology and coined the phrase
Omnis cellula e cellula that means “each cell stems from another cell,” and he dis-
tinguished sarcomas from carcinomas (Virchow 1859). In 1863, Virchow noted
leukocytes in neoplastic tissues and postulated a connection between chronic inflam-
mation and development of cancer (Virchow 1863). 60 years later, Francis Harbitz
(1867–1950) demonstrated the significance of chronic inflammation with the for-
mation of sarcomas (Harbitz 1924). Epidemiological and experimental studies now
provide evidence that the development of cancer is indeed attributed to inflamma-
tion. Nowadays, it is generally accepted that up to 25 % of human malignancies are
related to chronic inflammation and to viral and bacterial as well as parasitic infec-
tions (Hussain and Harris 2007). Chronic inflammation increases the risk of cancer,
promotes tumor progression, and supports metastatic spread (Multhoff et al. 2012;
Kundu and Surh 2012). The connection between tumorigenesis and inflammation
is mediated via intrinsic and extrinsic pathways (Mantovani et al. 2008). The intrin-
sic pathway is activated by various epigenetic alterations causing inflammation and
malignant transformation. Epigenetic alterations comprise mutation-driven proto-
oncogene activation, chromosomal rearrangement/amplification, and inactivation
of tumor suppressor genes. Transformed cells secrete inflammatory mediators and
thus generate an inflammatory microenvironment. The extrinsic pathway is driven
by inflammation or infections consequently leading to malignant transformation and
further increasing the risk for cancer development. Both pathways converge in tumor
cells and induce the activation of several transcription factors such as NF-κB, STAT-
3, and HIF-1 culminating in the formation of numerous pro-inflammatory molecules
that recruit and activate various leukocyte populations into the tumor microenviron-
ment (for a review see Multhoff et al. 2012). These pro-inflammatory factors include
proangiogenic mediators (IL-8, VEGF), growth factors (IL-6, GM-CSF, osteopon-
tin), anti-apoptotic mediators (Bcl-xL, c-Flip, survivin), cell cycle mediators (cyclin
D1, c-Myc), adhesion molecules (ELAM-1, ICAM-1, VCAM-1), invasion-promoting
factors (MMP-2/-7/-9, uPA), inflammatory enzymes (lipoxygenase, prostaglandin H2
synthase 2: PGHS-2), prostaglandins, iNOS, as well as chemokines (CCL2/-20, IL-8,
osteopontin), and pro-inflammatory cytokines (IL-1, IL-6, IL-23, TNF, TGF-β, EGF),
promoting the malignant phenotype. The tumor cell-derived pro-inflammatory mol-
ecules now activate the same transcription factors within tumor cells and cells of the
microenvironment. This concerted action of tumor and micromilieu results in a more
pronounced generation of inflammatory mediators driving the progression of a posi-
tive amplification loop which further triggers tumor growth and invasiveness.
Emerging data suggest that genetic destabilization of tumor cells is regarded
as a further hallmark of most human cancers contributing to tumor initiation
and progression (Colotta et al. 2009). Apart from the production of cytokines,
chemokines, proteases, and prostanoids, inflammatory cells are able to produce
reactive oxygen (ROS) and nitrogen species (RNS) acting as chemical effectors in
272 J. Radons
inflammation-driven carcinogenesis (Kundu and Surh 2008). ROS and RNS have
been identified in tumor cells as inducers of the oxygen-dependent heterodimeric
transcription factor HIF-1 (Sandau et al. 2000) which plays a pivotal role in genetic
destabilization of these cells (Koshiji et al. 2005). All of these inflammatory media-
tors act together in perpetuating and amplifying the inflammatory cascade. On the
one hand, they suppress DNA repair mechanisms leading to microsatellite instability.
On the other hand, they can cause chromosomal instability culminating in abnormal
chromosomal segregation and aneuploidy (Multhoff and Radons 2012). Moreover,
the inflammatory factors induce DNA double-strand breaks, affect function of mitotic
checkpoint molecules, and dysregulate homologous recombination of DNA double-
strand break repair leading to random genetic diversification of tumor cells. Cancer
cells harboring the optimal combination of activated oncoproteins and inactivated
oncosuppressor proteins will develop the malignant phenotype (Colotta et al. 2009).
Inflammatory processes also play a critical role in sarcomagenesis. The classical
tumor with inflammatory etiology is the inflammatory malignant fibrous histiocy-
toma (MFH) predominantly occurring in adults (Sinkovics 2007a, b). The expres-
sion of cytokines in inflammatory MFH may account for local inflammatory cell
infiltration and the aggressive nature of the malignant cells (Melhem et al. 1993).
In this sarcoma subtype, the inflammatory process is driven by HIF-1 (Koga et al.
2005) which not only plays a crucial role in genetic destabilization of tumor cells
but also in tumor angiogenesis, invasion, survival, and growth (Multhoff et al. 2012).
As demonstrated by the group of Takaaki Akaike, ROS and RNS induce the forma-
tion of 8-nitroguanine, a product of nitrative DNA damage (Ohshima et al. 2006). In
MFH patients, 8-nitroguanine formation can be detected predominantly in the nuclei
of tumor cells and inflammatory cells in tumor tissues, while HIF-1α, the oxygen-
regulated subunit of HIF-1, is expressed in the cytoplasm and nuclei of tumor cells
(Hoki et al. 2007b). Apart from HIF-1α, iNOS, NF-κB, and PGHS-2 have been
found to colocalize with 8-nitroguanine in MFH tissues and to negatively correlate
with the survival indicating an NF-κB-driven sarcomagenesis (Hoki et al. 2007a, b).
Human herpesvirus 8 (HHV8) or Kaposi sarcoma-associated herpesvirus
(KSHV) is the known causative oncogenic virus for Kaposi sarcoma (KS), which
in general portends a poor prognosis (Mesri et al. 2010). KS is a chronic inflam-
mation-associated malignancy that arises from the initial infection of an appro-
priate endothelial or progenitor cell by KSHV/HHV8. Cellular hallmarks of KS
progression include both the hyperproliferation of KSHV-infected cells and the
infiltration of immune modulatory cells into KS lesions, which together result in
chronic inflammation, the induction of angiogenesis and tumor growth (Douglas
et al. 2010). Recent evidence has pointed to the involvement of the NF-κB path-
way in the biology of KSHV and in the pathogenesis of KS (Keller et al. 2006).
Many aspects of KS suggest that chronic inflammation associated with the lesion
and/or viral infection plays a role in tumor pathogenesis. A crucial role for inflam-
mation in the pathogenesis of KS is exemplified by the association of KS with the
Koebner (or isomorphic) phenomenon, a condition where lesions initiate or recur at
inflammatory sites of injury or trauma (Rubin and Stiller 2002), and the recrudes-
cent KS (KS flare) seen with the immune constitution inflammatory syndrome (IRIS;
Leidner and Aboulafia 2005). The inflammatory response generated is thought to
11 The Role of Inflammation in Sarcoma 273
attract infected cells to the site as well as exacerbate the oncogenic properties of the
viruses (Rubin and Stiller 2002). Progression of KS likely depends on a complex and
incompletely understood interplay between KSHV and the host immune system that
allows for the establishment of a tumor-promoting environment (Douglas et al. 2010).
In addition to the classical tumor-promoting molecules such as cytokines,
chemokines, ROS/RNS, matrix-degrading enzymes, the matricellular protein osteo-
pontin (OPN) has been identified as playing a crucial role in inflammation and tumor
progression. OPN mediates cell migration, adhesion, and survival in many cell types
(Lund et al. 2009). As reviewed by Kundu and Surh (2012), OPN signals via the cell
surface receptor RAGE and αvβ3-integrin. On the one hand, OPN/RAGE interaction
leads to upregulation of NADPH oxidase (NOX) and a concomitant raise in ROS levels
culminating in activation of AKT and MAPK (mitogen-activated protein kinase) as well
as NF-κB. On the other hand, OPN/αvβ3-integrin interactions lead to direct or indi-
rect activation of NF-κB via the mitogen-activated protein kinase kinase / extracellular
signal-regulated protein kinase (MEK/ERK) pathway, regulating the expression of sev-
eral genes involved in cell survival, angiogenesis, and metastasis and thereby promoting
tumor growth (Kundu and Surh 2012).
Several signaling pathways have been identified as playing a crucial role in sar-
comagenesis required for neoplastic transformation. These pathways include the
Ras/Raf/MAPK pathway, the PI3K/AKT/mTOR pathway as well as the recep-
tor tyrosine kinase c-Met and its ligand hepatocyte growth factor (HGF). Normally,
c-Met is expressed by epithelial and mesenchymal cells and regulates several cellular
responses such as cell proliferation, survival, motility, invasion, and morphogenesis
(Birchmeier et al. 2003). Activation via ligand binding and/or receptor overexpres-
sion, amplification, and mutation induces multiple downstream effector proteins and
cascades including Ras/Raf/MAPK, PI3K/AKT/mTOR, and STAT-3/-5 (for a review
see Liu et al. 2010). The c-Met/HGF pathway represents one of the most commonly
dysregulated pathways in human cancers with aberrant signaling found in most solid
tumors and hematological malignancies (Birchmeier et al. 2003; Liu et al. 2010).
Amplification or overexpression of c-Met has been demonstrated in many cancers
including OS and unclassified pleomorphic sarcoma/MFH (Lahat et al. 2011).
Induction of the PI3K/AKT/mTOR and Ras/Raf/MAPK cascades can also be
achieved via activation of TK receptors by either growth factors (IGF, HGF, VEGF,
bFGF) or upregulation/mutation. This triggers a number of mitogenic processes
that promote cell survival and proliferation, anti-apoptotic signals and upregulate
expression of cell cycle proteins such as cyclin D1 and CDK4 (Takebe et al. 2011).
Moreover, activation/upregulation of Ras culminates in the induction of ERK-1/-2
via Raf and MAPK/ERK kinases (MEK-1/-2) which regulate cell proliferation, sur-
vival, differentiation, and migration (Takebe et al. 2011). The possible contribution
of these pathways to sarcomagenesis is discussed in detail in Sect. 11.3.
One of the major pathways involved in sarcomagenesis is the insulin-like growth
factor (IGF) system. Signaling via the IGF receptor (IGF-1R) plays an impor-
tant role in normal cell growth and differentiation as well as key aspects of neopla-
sia such as transformation and anti-apoptotic signaling (Zha and Lackner 2010).
The ligands IGF-1 and IGF-2 are both capable of binding and stimulating IGF-1R.
Bioavailability of IGF-1 is modulated by circulating IGF-binding proteins (IGFBPs),
274 J. Radons
expressed at very low levels relative to GIST and LMS. The only experimentally
verified target for miR-143 is ERK5 (also known as MAPK7) whose role in sarcom-
agenesis is unclear. SSX1, a common 3′-fusion partner gene resulting from a t(X;18)
(p11.2;q11.2) translocation in SS (Sturgis and Potter 2003), is predicted to be a target
for miR-143, suggesting that underrepresentation of miR-143 in SS tumor cells ena-
bles the production of the SYT/SSX-1 oncoprotein (Subramanian et al. 2008; Hisaoka
et al. 2011). In alveolar RMS, high levels of miR-335 involved in mesoderm or muscle
differentiation can be found (Subramanian et al. 2008). From these data, the authors
speculate that the clearly distinct miRNA expression signatures among the tumor types
studied might implicate their role in tumorigenesis in these tumors and their potential
as diagnostic markers or even therapeutic targets. It is interesting to note that the induc-
tion of the cancer stem cell (CSC) phenotype in ES is the result of the combined effect
of suppression of miR-145 promoter activity and expression of EWS/FLI1 fusion gene
required for transformation (Riggi et al. 2010). SOX2, which participates in the devel-
opment of pluripotent stem cells, was identified as the target gene of miR-145 and
EWS/FLI1, thus providing insight how a single oncogene (EWS/FLI1) can reprogram
cells to display the CSC phenotype (Riggi et al. 2010).
Several groups demonstrated a strikingly decreased expression of miR-1 and
miR-133a in alveolar and embryonal RMS cell lines (Yan et al. 2009; Rao et al.
2010). Preclinical studies reported that forced re-expression of miR-206 leads
to cell cycle arrest and myogenic differentiation of RMS cells, preventing xeno-
graft growth in vivo by targeting the mRNA of the oncogenic c-Met receptor (Yan
et al. 2009; Taulli et al. 2009). miR-1 and miR-206 are downregulated in alveolar
and embryonal RMS compared to nonneoplastic skeletal muscle tissues and fail to
increase in RMS cell lines in response to differentiation-inducing treatment (Taulli
et al. 2009). Moreover, re-expression of miR-1 or miR-206 through lentiviral vec-
tors promotes cell differentiation in alveolar cell lines that are resistant to differen-
tiate cues, and blocks anchorage-independent growth and invasiveness in vitro and
in vivo (Taulli et al. 2009). Meanwhile, clusters of hundreds of genes up- (muscle
lineage) or downregulated (cell cycle) by miR-206 in RMS were identified, among
which c-Met was found as being a direct miR-206 target. Thus, the miR-206-de-
pendent post-transcriptional inhibition of c-Met expression markedly contributes
to the anti-tumor effects of this miRNA rendering tissue-specific miRNAs as hold-
ing great therapeutic potential.
The miR-155 has been shown to be the most overexpressed miRNA in LS cell
lines, and functional investigations assigned an important role in the growth of
dedifferentiated LS cells (Zhang et al. 2012). Knockdown of miR-155 retarded
tumor cell growth, decreased colony formation, and induced G1/S cell cycle arrest
in vitro and blocked tumor growth in murine xenografts in vivo. Casein kinase 1α
(CK1α) seems to be the direct target of miR-155 augmenting β-catenin signaling
and cyclin D1 expression and promoting tumor cell growth (Zhang et al. 2012).
In inflamed tissue, miR-155 inhibits the repair of DNA double-strand breaks or
allows mismatch repairs. According to Sinkovics (2012), these cells assume the
“mutator phenotype” and upregulate the expression of hypoxanthine phosphori-
bosyl-transferase as a consequence of the dramatically enhanced number of DNA
276 J. Radons
strand breaks and mutations. Inflammatory mediators such as TNF, IL-1β, IL-6,
IL-8, and LPS are able to upregulate miR-155 in cancer cells. miR-155 increases
the proliferation of adenocarcinoma cells and downregulates Wee-1, a recently
recognized tumor suppressor and the key inhibitor of cyclin-dependent kinase
1 (Cdk-1), enabling unlimited cell divisions to occur (Enders 2010). Apart from
being a regulator of mitotic entry, Wee-1 has been described to affect other cellular
processes, including regulation of mitotic spindle formation, positioning and integ-
rity, microtubule stabilization, and heat-shock protein 90 (Hsp90) phosphorylation
(Hashimoto et al. 2006; Garcia et al. 2009; Mollapour et al. 2010). Inactivation
of Wee-1 by miR-155 represents one of the hallmarks of inflammatory carcino-
genesis (Enders 2010; Butz et al. 2010; Tili et al. 2011). Whether downregulation
of Wee-1 as can be observed in human sporadic pituitary cancer cells (Butz et al.
2010) also occurs in sarcoma remains to be addressed.
Epithelial–mesenchymal transition (EMT) is the key process driving can-
cer metastasis characterized by the loss of the epithelial marker E-cadherin, an
increase in the mesenchymal markers vimentin and N-cadherin, and an increase
in the migratory and invasive behavior (Kraljevic Pavelic et al. 2011). Oncogene/
self-renewal factor Bmi-1 has been shown to induce EMT in cancer cells (Yang
et al. 2010b). Bmi-1 upregulation is associated with malignant transformation in
hepatocellular carcinoma (Sasaki et al. 2008). Recent studies suggest that miRNAs
function as crucial modulators for EMT. The group of Noriaki Sakuragi identified
Bmi-1 as an essential factor in EMT and in the development of an invasive pheno-
type in endometrial cancer (EC) cells (Dong et al. 2011). Furthermore, the expres-
sion of Bmi-1 could be suppressed by miR-194 via direct binding to the BMI1
3′-untranslated region. Ectopic expression of miR-194 in EC cells induced a mes-
enchymal to epithelial transition (MET) by restoring E-cadherin, reducing vimen-
tin expression, and inhibiting cell invasion in vitro. Based on these findings, it can
be concluded that targeting the oncoprotein Bmi-1 might provide a potential new
strategy to prevent EC progression.
Proto-oncogene activation represents a critical component in the intrinsic
pathway of cancer-related inflammation. In this context, mutations in RAS genes
play an important role in tumorigenesis of sarcoma. Overall, up to 30 % of all
human tumors harbor mutations in canonical RAS genes (KRAS, HRAS, NRAS).
Remarkably, these oncogenic mutations predominantly affect the KRAS locus,
with oncogenic KRAS mutations being detected in 25–30 % of all screened tumor
samples (Forbes et al. 2011). Activating RAS mutations are also present in up to
44 % of human STS (Yoo et al. 1999) and in up to 35 % of human embryonal
RMS (Martinelli et al. 2009). The high frequency of KRAS mutations and their
appearance in early tumor stages argue for a causative role of the K-Ras protein
in human tumorigenesis (Fernandez-Medarde and Santos 2011). Members of the
RAS family are crucial for the connection of upstream signals to downstream
effector pathways that are functionally related to cell cycle progression, growth,
migration, cytoskeletal changes, apoptosis, and senescence. In tumor cells, activa-
tion of mutated Ras is followed by the induction of several intracellular signal-
ing pathways including the Raf/MEK/ERK kinase cascade, the PI3K/AKT/mTOR
11 The Role of Inflammation in Sarcoma 277
pathway, and RalGDS proteins (Downward 2009), the latter belonging to the
family of nucleotide-exchange factors activating small GTPases such as RalB.
RalB stimulates the TANK-binding kinase 1 (TBK-1) resulting in NF-κB activa-
tion. NF-κB not only functions as crucial regulator of inflammatory and immune
responses as well as of cell survival, but it has also been implicated in cellular
transformation and tumorigenesis. In cancer cells, a constitutive activation of this
pathway, via chronic RalB activation, restricts the initiation of apoptosis after
oncogenic stress (Chien et al. 2006). Beside NF-κB activation, TBK-1 activates
the transcription factors IRF-3 and IRF-7 (Hacker and Karin 2006), leading to the
production of growth and inflammatory mediators. Previously, it has been shown
that oncogenic K-Ras is a direct inducer of pro-inflammatory IL-6 and pro-angi-
ogenic IL-8 in vitro required for the initiation of tumor-associated inflammation
and neovascularization promoting tumor growth (Sparmann and Bar-Sagi 2004;
Ancrile et al. 2007). Since TBK-1 and NF-κB signaling have been identified as
being essential in K-Ras mutant tumors (Barbie et al. 2009), it was assumed that
targeting the NF-κB signaling pathway might be effective in treating Ras-mutated
tumors (Downward 2009). Interestingly, NF-κB inhibition by dehydroxymeth-
ylepoxyquinomicin (DHMEQ) inhibited proliferation, decreased the mitotic
index, and triggered apoptosis of OS cells HOS and MG-63 (Castro-Gamero
et al. 2012) while NF-κB inhibition by the semisynthetic flavonoid 7-mono-O-
(β-hydroxyethyl)-rutoside (monoHER) potentiated the anti-tumor activity of doxo-
rubicin in the human LS cell line WLS-160 (Jacobs et al. 2011).
An intriguing aspect in cellular transformation is presented by the group of
Bharat Aggarwal, who discussed the potential role of oxidative stress in tumori-
genesis (Reuter et al. 2010). As stated by the authors, oxidative stress impacts any
stage of tumorigenesis. In the initial phase, oxidative stress induces genetic insta-
bility by enhancing the mutation rate of cells and consequently leading to onco-
genic transformation (Jackson and Loeb 2001). Apart from mediating genomic
destabilization, ROS have been found to activate several intracellular signaling
pathways promoting tumor growth and metastasis. In this context, the transcrip-
tion factor FoxM1, a member of the large evolutionarily conserved family of fork-
head box transcription factors involved in activating detoxifying enzymes (e.g.,
manganese superoxide dismutase), obviously plays a pivotal role in tumorigenesis
since it is overexpressed in various human malignancies (Pilarsky et al. 2004).
FoxM1 is also expressed in ES (Christensen et al. 2013), neuroblastoma (Wang
et al. 2011) and medullablastoma cell lines (Priller et al. 2011). In glioblastoma
cells, a high expression of FoxM1 was found to correlate with the tumorigenicity
of the tumor cells (van den Boom et al. 2003; Liu et al. 2006).
The glycophosphoprotein osteopontin (OPN) is implicated in several physiologi-
cal and pathophysiological processes including atherosclerosis, glomerulonephritis,
chronic inflammatory diseases, and cancer (Lund et al. 2009). OPN is involved in
proliferation, cell adhesion, migration, and invasion via interaction with its recep-
tor, αvβ3-integrin. It has been shown previously that OPN induction is required
for tumor promotor-mediated transformation of preneoplastic mouse epidermal
cells (Chang et al. 2003). Chen and coworkers convincingly demonstrated that
278 J. Radons
It has increasingly been recognized during the past years that malignancy not only
results from enhanced proliferation but also from decreased apoptosis, a funda-
mental process in tumor cell kinetics. Cancer cells are extremely dependent on
aberrations of the apoptotic pathways to survive. Critical regulators of this path-
way include the Bcl-2 family of proteins and p53. In cultured KS tumor cells,
flow cytometric and immunoblotting analyses revealed a predominant expres-
sion of anti-apoptotic Bcl-xL over Bcl-2 with no detectable pro-apoptotic Bax or
Bcl-xS (Foreman et al. 1996). Bcl-2 is also expressed in the SS cell line, SN-SY-1
(Noguchi et al. 1997). It has been shown previously that Bcl-2 expression can be
induced by VEGF in human dermal microvascular endothelial cells (HDMECs),
thereby enhancing endothelial survival and sustaining angiogenesis (Nör et al.
1999). These data suggest that the ability of VEGF to enhance endothelial cell sur-
vival might be attributed to its capacity to induce the expression of Bcl-2. Of note,
exposure of KS cells to pro-inflammatory IL-1 increased the expression of Bcl-2
and decreased that of Bax without affecting Bcl-xL expression providing a link
between KS cell escape from apoptosis and the immune dysregulation associated
with KS (Simonart and Van Vooren 2002). Bcl-2 overexpression has been recently
reported in SS in situ (Hirakawa et al. 1996).
Evidence for the role of inflammatory mediators in the survival of sarcoma cells
is given by several in vitro studies, revealing that cultured human astrocytes as
well as glioblastoma cell lines release GM-CSF that can be enhanced by addition
of TNF or IL-1, respectively (Frei et al. 1992; Curran et al. 2011). In human neu-
roblastoma and glioblastoma cell lines, exposure to GM-CSF showed cytoprotec-
tive effects on these cell lines by inhibiting staurosporine-induced apoptosis (Huang
et al. 2007; Choi et al. 2007). The same group found out that pretreatment of N2a
glioblastoma cells with GM-CSF inhibited staurosporine-induced expression of
pro-apoptotic p53 and Bax, while upregulated that of Bcl-2 (Huang et al. 2007).
These data imply that the GM-CSF-mediated modulation of pro- and anti-apoptotic
gene expression is crucially involved in inflammation-driven sarcomagenesis.
A wide range of pro-survival factors are activated by the essential embryonic
sonic hedgehog (Hh) signaling pathway which has been implied in tumor forma-
tion and progression of various cancers. Aberrant activation of the Hh signaling
pathway has been shown as playing a crucial role in RMS, OS, CS, ES as well
as medulloblastoma (Quesada and Amato 2012; Martin Liberal et al. 2012). Upon
Hh activation, upregulated expression of several marker genes, e.g., PTCH1,
GLI1, GLI3, and MYF5, can be observed in embryonal RMS and also in fusion
11 The Role of Inflammation in Sarcoma 279
gene-negative alveolar RMS (Zibat et al. 2010). Together with the finding of an
elevated Hh signaling in cancer stem cells and nonmalignant stromal cells sur-
rounding malignant tumors, the Hh pathway can be considered as key cofactor in
sarcomagenesis (Takebe et al. 2011).
Further critical signaling pathways in sarcomagenesis involve the PI3K/AKT/
mTOR as well as the Ras/Raf/MAPK cascade. The study by Sasaki and cowork-
ers detected RAF1 and MEK1/2 mRNA in OS and MFH cells (Sasaki et al. 2011).
Treatment with the MEK inhibitor U0126 decreased proliferation of OS and MFH
cells in a time- and dose-dependent manner. In human SS cells, inhibition of the
MAPK pathway by sorafenib led to downregulation of cyclin D1 and pRb, G1
arrest, and induction of apoptosis (Peng et al. 2009). Notably, the work by the
group of Hiroki Sakai convincingly demonstrated that the mTORC-2/AKT path-
way was constitutively activated in canine hemangiosarcoma (HAS) cell lines and
tumors (Murai et al. 2012a, b).
Angiogenin (ANG), a 14-kDa multifunctional pro-angiogenic growth factor, is
upregulated in several types of cancers including KSHV-associated cancers such
as KS (Sadagopan et al. 2009). ANG mediates its effects in multiple subcellular
compartments, including the nucleolus where it directly binds to DNA, thereby
inducing 45S rRNA transcription and cell proliferation (Moroianu and Riordan
1994). Studies by the group of Bala Chandran demonstrated that ANG plays a
crucial role in the anti-apoptotic state of KSHV-infected cells by suppressing p53
functions (Sadagopan et al. 2012; Paudel et al. 2012). Moreover, ANG expression
inhibited pro-apoptotic Bax and p21Waf-1 expression, induced anti-apoptotic Bcl-2
and blocked cell death. ANG was also found to colocalize with the p53 regulator
protein Mdm-2 and to increase p53/Mdm-2 interactions suggesting that ANG pro-
motes the inhibition of p53 functions to mediate anti-apoptosis and cell survival
(Sadagopan et al. 2012).
The crucial role of FoxM1 in tumorigenesis of sarcomas has already been men-
tioned. An altered expression and function of FoxM1B as can be found in sev-
eral human malignancies also has an impact on apoptosis, possibly by regulating
the cell cycle repressor protein p27Kip-1 (Liu et al. 2006). p27Kip-1 has been found
to modulate apoptosis in various cell types, including glioblastoma multiforme
cells (Hiromura et al. 1999; Lee and McCormick 2005). Knockdown of FoxM1 in
medulloblastoma cell lines significantly decreased cell viability which was caused
by a failure in mitotic spindle formation and caspase-dependent mitotic catastro-
phe (Priller et al. 2011).
Tumor cells have to evade various cellular stress factors for proliferation and
survival including a markedly increased production of ROS. In this context, the
serine/threonine kinase Mirk/Dyrk1B has been reported to be highly expressed
280 J. Radons
of human carcinomas. Epithelioid sarcoma cell lines have been shown to express
EMMPRIN and to upregulate MMP-2 in fibroblasts in coculture experiments criti-
cal for epithelioid sarcoma cell stromal invasion and vascular involvement (Koga
et al. 2007). These findings render tumor-associated EMMPRIN a potentially use-
ful target in the therapy of certain STS.
To analyze the functional and potential therapeutic relevance of IGF-1R signaling,
Friedrichs and collaborators treated SS cell lines with the IGF-1R inhibitor NVP-
AEW541 (Friedrichs et al. 2008). In this study, the IGF-1R antagonist was found
to inhibit cell growth through a reduction in phosphorylation of AKT and p44/42
MAPK. Moreover, inhibition of the receptor led to increased apoptosis and dimin-
ished mitotic activity. In a recent study, an upregulated expression of IGF-1R, c-Met,
HER-2, VEGFR-3, insulin receptor, and PDGFR-β was identified in OS cell lines
suggesting a contribution of these receptors to osteosarcomagenesis (Hassan et al.
2012). Further in vitro evidence for the contribution of the MAPK pathway to sar-
comagenesis is given by the work of Sasaki and colleagues who detected RAF1 and
MEK1/2 mRNA expression in several human sarcoma cell lines (Sasaki et al. 2011).
Treatment with the MEK inhibitor U0126 resulted in dose- and time-dependent
inhibition of cell proliferation and suppression of p-ERK expression (Sasaki et al.
2011). A similar observation was made with the SS cell lines SW982 and HS-SY-
II where the Raf inhibitor sorafenib effectively inhibited cell proliferation and phos-
phorylation of MEK and ERK, downregulated cyclin D1 and pRb levels, caused G1
arrest and S phase decrease, and induced apoptosis (Peng et al. 2009).
Lyn, a member of the SRC family of kinases, is a known regulator of tumor cell
proliferation, adhesion, motility, and invasion. Elevated Lyn kinase activity has been
found in ES cell lines (Rosen et al. 1986; Guan et al. 2008). Shor et al. (2007) also
reported high levels of phosphorylated Src in human OS and ES cell lines. In ES
cells, Lyn expression is regulated by EWS/Fli-1 which is known to transform cells
by acting as an aberrant transcriptional factor for specific target genes (Guan et al.
2008). In vitro, inhibition of Lyn kinase activity by the small-molecule Src fam-
ily tyrosine kinase (SFK) inhibitor AP23994 suppressed growth of ES tumor cells
TC71, while downregulation of Lyn reduced invasive capacity of the cells (Guan
et al. 2008). Activation of Lyn can be induced by the KSHV protein K1 in K1 lym-
phoma cells leading to production of VEGF and NF-κB activation, both strongly
implicated in the development of KSHV-derived disorders (Prakash et al. 2005).
As discussed earlier, FoxM1 is a central player in sarcomagenesis. FoxM1 is
exclusively expressed in proliferating cells and critically involved in cell cycle
progression (Laoukili et al. 2005; Wang et al. 2005a). FoxM1B overexpression
increased the growth of glioma cells both in vitro and in vivo, which was at least
partially caused by accelerated glioma cell cycle progression (Liu et al. 2006).
Consistent with previous studies (Kalinichenko et al. 2004); FoxM1 overexpression
was shown to diminish the expression of nuclear p27Kip-1 protein but increased the
expression of Skp-2 and cyclin D1 protein. From these observations, the authors con-
clude that FoxM1 probably regulates p27Kip-1 protein expression indirectly by induc-
ing Skp-2 expression, which mediates the degradation of p27Kip-1 protein. FoxM1
is also expressed in ES cell lines in which reduction of its expression resulted in
11 The Role of Inflammation in Sarcoma 283
neuroblastoma C1300 cells with OPN did not increase VEGF production and did not
affect gene expression of other proangiogenic factors confirmed by complementary
DNA microarray system suggesting a pro-angiogenic role independent of VEGF, at
least in this sarcoma subtype (Takahashi et al. 2002). Other pro-angiogenic factors
are differentially upregulated in STS cells such as angiopoietin-2, bFGF, MMP-2,
Notch-1/-4, and PDGF (Engin et al. 2009; Lee et al. 2010; Ye et al. 2012; Bai et al.
2012; Hönicke et al. 2012). Some chromosomal translocations and their gene prod-
ucts are able to upregulate the transcription of pro-angiogenic VEGF, HIF1A, MDK,
CMET, and TIMP2 as can be found in the alveolar soft part sarcoma cell line ASPS-1
(Quesada and Amato 2012). In this context, FoxM1 is required for invasion and angi-
ogenesis of glioma cells as VEGF was identified as being a direct transcriptional tar-
get of FoxM1B (Zhang et al. 2008). Furthermore, FoxM1 overexpression increased
and inhibition of FoxM1 expression suppressed the angiogenic ability of glioma
cells. According to Agulnik (2012), the PI3K/AKT/mTOR pathway has an important
role in the regulation of angiogenesis mediated by HIF-1α. Preclinical and clinical
studies provide further evidence for the anti-angiogenic effects of specific mTOR
inhibitors (rapalogs) in sarcoma (see also Table 11.3).
As already mentioned in Sect. 11.3.2, the multifunctional pro-angiogenic
growth factor ANG is upregulated in numerous cancers including KS (Sadagopan
et al. 2009). Apart from ANG, KSHV infection upregulates the transcription of
a broad range of host genes involved in angiogenesis such as VEGF and PGHS2
(Sivakumar et al. 2008; Sharma-Walia et al. 2010). The nuclear location of ANG is
a prerequisite not only for its pro-angiogenic and proliferative properties but also
for the pro-angiogenic potential of VEGF and bFGF (Kishimoto et al. 2005). In a
previous study, the group of Bala Chandran demonstrated robust PGHS2 expres-
sion and high levels of PGE2 secretion by KSHV during primary infection of
human microvascular endothelial cells (HMVEC-d) and human foreskin fibroblast
cells (Sharma-Walia et al. 2006). KSHV infection-induced PGHS-2/PGE2 expres-
sion also upregulated Rac1-GTPases in adhering endothelial cells, thereby acceler-
ating cell adhesion (Sharma-Walia et al. 2010). The same study also demonstrated
that KSHV infection-induced PGHS-2 potentially modulated survival, prolifera-
tion, and angiogenesis of latently infected endothelial cells by inducing secretion
of numerous growth (PDGF-BB, IGF-1, G-CSF, and IL-8), angiogenesis (VEGF,
ANG, oncostatin, IL-8, and MMP-2), inflammation (IL-1, TNF, RANTES, and
IL-8), and invasiveness-promoting factors (MMP-2/-9).
of mouse KRAS, INK4A/ARF−/− sarcoma, and human RMS cells in vitro and in
vivo (Hettmer et al. 2011). Consistent with a central role of Ras signaling in these
tumors, inhibition of the Ras/Raf/MEK/ERK signaling cascade diminished the
proliferation of mouse and human sarcoma cells (Marampon et al. 2009).
Several other candidate genes have been linked to mTOR signaling including
the actin filament-plasma membrane linker ezrin (encoded by VIL2) and the home-
odomain-containing transcription factor Six-1 (sine oculis-related homeobox-1
homolog) playing essential roles in determining the metastatic fate of RMS cells (Yu
et al. 2004). Similarly, phosphorylation of AKT, the upstream effector of mTOR, was
reported in approximately 50 % of human embryonal and alveolar RMS (Cen et al.
2007). The findings by Hettmer et al. (2011) are consistent with previous observa-
tions made with RMS cell lines and xenografts demonstrating that pharmacological
inhibition of mTOR signaling in mouse and human sarcoma cells impaired tumor
growth (Petricoin et al. 2007; Houghton et al. 2008). Beneficial effects were also
seen in some patients with advanced sarcomas including RMS (Yarber and Agulnik
2011; Agulnik 2012). All these data clearly document the important contribution of
aberrant Ras and mTOR signaling to the growth and malignancy of STS.
As already mentioned in Sect. 11.3.1, overexpression of oncogenic K-Ras was
found to induce the secretion of pro-inflammatory IL-6 and pro-angiogenic IL-8 in
different tumor cell types. The study by Ancrile et al. (2007) revealed that this Ras-
driven cytokine production is required for the initiation of tumor-associated inflam-
mation and neovascularization promoting tumor growth in vivo. In theses studies,
knock-down of IL6, genetic ablation of the IL6 gene, or treatment with a neutraliz-
ing IL-6 antibody retarded K-Ras-driven tumorigenesis in mice (Ancrile et al. 2007).
Ras-induced expression of several pro-inflammatory mediators including IL-1, IL-6,
and IL-11 as well as pro-angiogenic IL-8 has been demonstrated to promote tumor
growth and neovascularization in vivo (Sparmann and Bar-Sagi 2004). In an animal
model of human OS, increased IL-1 levels are also reported (Baamonde et al. 2007).
Further evidence for the impact of inflammation in sarcomagenesis comes
from a gene profiling approach in canine HAS (Tamburini et al. 2010). HAS, also
called malignant hemangioendothelioma or angiosarcoma, is a deadly cancer that
originates in the endothelium and invades the blood vessels. HAS is more com-
mon in dogs than any other species. The study by Tamburini et al. (2010) clearly
identified inflammation and angiogenesis as distinguishing features of canine
HAS. Six genes of the IL-8 signaling cascade were found as being enriched in
HAS. These genes included IL8, MMP9, VEGF, VCAM1, PGHS2, and cyclin D1
(CCND1) known as being involved in regulating pro-inflammatory and pro-angio-
genic responses via the IL-8 receptor-β (Tamburini et al. 2010). In a SCID mouse
xenograft model of human MM, Hillegass and colleagues identified an early and
sustained neutrophilia accompanied by early detection of cytokines that promote
inflammation (IL-6, IL-8, and IL-12), cell proliferation (IL-6, bFGF, IL-8, G-CSF,
and PDGF-BB), and angiogenesis (bFGF, IL-8, and VEGF) by human MM in per-
itoneal lavage fluid (Hillegass et al. 2010a)
Using a xenograft nude mouse model for human glioblastoma, Liu and cowork-
ers convincingly demonstrated that overexpression of FoxM1B directly promotes
11 The Role of Inflammation in Sarcoma 287
the growth of human glioma cells in the brain of nude mice, while inhibition of
FoxM1 by FoxM1-siRNA significantly suppressed glioma growth in these mice,
thus confirming in vitro results of the same study (Liu et al. 2006). Moreover,
depletion of FoxM1 inhibited anchorage-independent growth and tumorigenicity
of neuroblastoma xenografts (Wang et al. 2011).
The contribution of oxidative stress to sarcomagenesis is proven by investiga-
tions on a transgenic Rac-1 model for KS (Ma et al. 2009). The small GTPase
Rac-1, a member of the Rho family within the RAS superfamily, triggers ROS pro-
duction by members of the phagocytic as well as nonphagocytic NOX family (Abo
et al. 1991). Expression of a constitutively active Rac-1 (RacCA) in transgenic mice
is sufficient to cause KS-like tumors through mechanisms involving ROS-driven
proliferation, upregulation of AKT signaling, and HIF-1α-related angiogenesis (Ma
et al. 2009). Notably, the use of the ROS-scavenging agent N-acetylcysteine inhib-
ited angiogenesis and completely abrogated transgenic RacCA tumor formation,
indicative for the causal role of ROS in sarcomagenesis. These data clearly imply
the direct oncogenicity of Rac-1 and ROS and their contribution to a KS-like malig-
nant phenotype, further underscoring the carcinogenic potential of oxidative stress
in the context of chronic infection and inflammation (Ma et al. 2009).
In vivo evidence for the crucial role of OPN in sarcomagenesis is given by
the work of Liu and colleagues who successfully reduced the tumorigenecity of
human osteosarcoma cells OS-732 xenotransplanted into nude mice using an anti-
sense human OPN RNA expression vector (Liu et al. 2008). Takahashi and cow-
orkers recently generated a stable OPN transfectant from murine neuroblastoma
C1300 cells and demonstrated that culture medium with OPN-transfected C1300
cells significantly stimulates human umbilical vein endothelial cell (HUVEC)
migration and induces neovascularization in mice compared to control cells
(Takahashi et al. 2002). Further evidence for the pro-angiogenic role of OPN is
provided by the same group who found that OPN enhances tumorigenesis and
angiogenesis of murine neuroblastoma cells in mice rendering OPN a promising
target in sarcoma therapy (Hirama et al. 2003).
Experimental animal models suggest a contribution of the IGF system in tum-
origenesis of sarcomas. In addition as being linked to increased cancer risk and
certain neoplasias (see Sect. 11.3.1), dysregulated IGF expression has also been
demonstrated to have functional consequences. IGF-1 overexpression in basal
keratinocytes resulted in increased formation of squamous papillomas in trans-
genic mice (Wilker et al. 1999). Injection of ES cells carrying dominant nega-
tive IGF-IR into nude mice attenuated tumor formation and metastatic abilities
of ES cells and increased survival (Scotlandi et al. 2002). Furthermore, trans-
fected clones showed significantly higher sensitivity to doxorubicin, a major drug
in the treatment of ES. Ligand overexpression seems to be driven by pathologi-
cal alterations, particularly in sarcomas. The EWS/FLI1 translocation is a defin-
ing characteristic of ES and has been shown to upregulate IGF-1 expression and
downregulate IGFBP-3 expression, enabling an autocrine regulatory loop con-
sisting of IGF-1 and IGF-1R, which can be blocked by IGF-1R targeting agents
(Scotlandi et al. 2002; Prieur et al. 2004). In addition, EWS/FLI1 was shown to
288 J. Radons
upregulate expression of the Src kinase Lyn in athymic nude mice injected with
TC71 human ES tumor cells allowing increased bony lysis by creating space for
tumor growth, and providing simple access for tumor cells to the bone stroma
facilitating metastasis (Guan et al. 2008). Targeting Lyn using siRNA or the
pharmacological inhibitor AP23994 resulted in suppression of tumor growth,
decreased bony lysis due to tumor cells, and significantly fewer lung metastases in
this ES xenograft tumor model.
Current experimental data support the role of the PI3K/AKT/mTOR pathway in
sarcomagenesis. In an animal model of STS, intramuscular delivery of an adeno-
virus carrying Cre recombinase in mice with conditional mutations in KRAS and
TRP53 sufficiently initiated high-grade sarcomas with myofibroblastic differen-
tiation similar to RMS (Kirsch et al. 2007). The PI3K/AKT/mTOR pathway also
plays a substantial role in smooth muscle transformation and LMS genesis. As
demonstrated by the group of Carlos Cordon-Cardo, mice carrying homozygous
deletion of PTEN alleles developed widespread smooth muscle cell hyperplasia
and abdominal LMS (Hernando et al. 2007).
The role of inflammation in human sarcomagenesis has long been overlooked, but
emerging evidence suggests its contribution to the malignant process in humans.
The subsequent examples highlight the putative role of inflammatory mediators
in the pathogenesis of sarcomas. Among the inflammatory mediators present in
the tumor microenvironment, IL-1 acts as crucial player in inflammation-associ-
ated carcinogenesis (Lin and Karin 2007; Voronov et al. 2007). Elevated levels of
IL-1 have been identified in several human tumor entities. Overall, patients har-
boring IL-1-positive tumors have markedly worse prognoses (Lewis et al. 2006).
IL-1 is produced directly by cancer cells or by cells of the mircroenvironment and
stimulates other cell types to produce pro-angiogenic and pro-metastatic media-
tors, thus playing an important role in inflammation-associated carcinogenesis
(Lin and Karin 2007; Voronov et al. 2007). In this context, children with MFH
showed elevated serum levels of pro-inflammatory IL-1 and TNF (Ishii et al.
1991). Elevated serum levels of TNF and IL-6 were also found in a patient with
ovarian FS (Fukuda et al. 2001). Tumor cells from this patient revealed a focally
positive immunoreaction for vimentin, IL-6, TNF, and inhibin-α, a subunit of the
heterodimeric hormone inhibin produced in the ovary that antagonizes activin
signaling and FSH synthesis in the pituitary. Serum TNF levels were also sig-
nificantly higher in patients with aural cholesteatoma compared to controls cor-
relating with bone destruction (Sastry et al. 1999). Increased levels of IL-1 and
TNF were detected in acquired and congenital cholesteatoma tissues as com-
pared to normal skin (Akimoto et al. 2000). Tissue biopsy samples from chronic
otitis media patients with cholesteatoma also harbored significantly higher levels
11 The Role of Inflammation in Sarcoma 289
Leach et al. 1993; Shimada et al. 2006). In the latter, Mdm-2 overexpression occurs
with high frequency as a result of an upregulated MDM2 mRNA expression cor-
relating with disease recurrence and metastasis (Ladanyi et al. 1993; Gisselsson
et al. 2002). An upregulated expression of the FOXM1 mRNA could be detected
in neuroblastoma tissue samples compared to noncancerous ganglioneuroma or
less aggressive ganglioneuroblastoma (Koch et al. 2008; Hillegass et al. 2010a).
FoxM1 is a transcription factor critically involved in cell cycle progression (see
Sect. 11.3.1). As demonstrated by Liu et al. (2006), human glioma tissue specimens
apparently had a substantially higher level of FoxM1 expression than normal tissue
and this expression correlated directly with the grade of the glioma. Together with
the in vitro and in vivo findings from the same study, these data suggest the pivotal
role of FoxM1 in tumorigenesis of glioma. FoxM1 is also expressed at robust levels
in a variety of Ewing tumor specimens (Christensen et al. 2013). Previous stud-
ies have shown that nuclear expression of the cell cycle repressor protein p27Kip-1
decreases with malignancy in human astrocytic gliomas and that p27Kip-1 has inde-
pendent prognostic value in patients with astrocytomas (Piva et al. 1997; Alleyne
et al. 1999; Kirla et al. 2003). In human gliomas, the Skp-2 expression level
directly correlated with the tumor grade but inversely correlated with the p27Kip-1
level (Schiffer et al. 2002). FoxM1 has also been implicated in the pathogenesis
of human medulloblastoma, the most frequent malignant brain tumor in childhood
that can derive from cerebellar granule neuron precursors (Priller et al. 2011). As
documented in this study, FoxM1 was highly expressed in all subtypes of medul-
loblastoma tested. Importantly, expression levels of FoxM1 significantly correlated
with unfavorable clinical outcome and has been identified as an independent prog-
nostic marker (Priller et al. 2011). Overexpression of FoxM1 has been reported to
strongly correlate with metastasis in prostate cancer (Chandran et al. 2007).
Genetic evidence of the cooperative interactions of the PTEN and INK4A tumor
suppressor genes in the development of human histiocytic sarcoma (HS) is pro-
vided by Carrasco and coworkers who investigated the Pten and Ink-4A status in
human HS, a rare human neoplasm with only a limited number of cases reported
in the world literature (Carrasco et al. 2006). Immunohistochemical analyses of
a panel of ten cases of human HS revealed a loss of immunoreactivity for either
Pten or p16Ink-4A alone in four and five cases, respectively. Three of the ten cases
showed concomitant lack of immunostaining for both Pten and p16Ink-4A, while
four of the ten cases were positive for both proteins. Most human HS cases dem-
onstrated increased levels of pAKT in the histiocytic tumor cells compared to
normal cells. These results highlight the general role of AKT phosphorylation in
human HS pathogenesis suggesting that additional mechanisms besides Pten inac-
tivation can lead to AKT activation (Carrasco et al. 2006), including changes in
Src activity, Pten expression, PI3K activity, or receptor TK signaling (Lu et al.
2003; Nagata et al. 2004; Shekar et al. 2005; Wang et al. 2005b).
The implication of oxidative and nitrative stress in human sarcomagen-
esis is attributed, for instance, to the work of Fredika Robertson and colleagues
on AIDS-related KS, the most prevalent AIDS-related cancer arising under a
unique condition that is characterized by a combination of immunosuppres-
sion and immunostimulation (Shah et al. 2002; Restrepo and Ocazionez 2011).
11 The Role of Inflammation in Sarcoma 291
According to Mallery et al. (2004), nitrative stress occurred in situ within lesions
of AIDS-KS patients. Cultured AIDS-KS cells from these tumors were found to
harbor impaired functional activity of the putative tumor suppressor MnSOD as a
result of tyrosine nitration, implying a critical contribution of reactive oxygen and
nitrogen species to AIDS-KS pathogenesis. Because the fundamental functions of
MnSOD comprise, in addition to its function as tumor suppressor, its anti-oxidant
capacity, the loss of the cytoprotective activity of MnSOD might facilitate malig-
nant transformation (Mallery et al. 2004).
It is well known that osteopontin (OPN) plays an important role in tumor pro-
gression and that a high OPN expression level in several tumor entities correlates
with poor prognosis in cancer patients. In STS, elevated OPN protein in serum
as well as in tumor tissues correlates with clinical parameters and functions as an
important negative prognostic factor (Bache et al. 2010). In female STS patients
and those who received curative radiotherapy, high expression levels of OPN
splice variants were determined as negative prognostic and predictive markers
(Hahnel et al. 2012). An upregulation of certain OPN splice variants could also
be demonstrated in MM peritoneum specimens compared to healthy controls
(Ivanov et al. 2009). The putative role of OPN in human sarcomagenesis is further
given by the observation that in 90 % of patients with highly aggressive glioblas-
toma, OPN is upregulated at both, the mRNA and protein level (Atai et al. 2011).
Moreover, OPN protein expression was found to colocalize with neutrophils and
macrophages, suggesting that OPN promotes migration of cancer cells as well as
of leukocytes in tumors (Atai et al. 2011). Increased OPN serum levels were also
found in asbestos-induced MM (Pass et al. 2005).
Among other cancers, miR-155 is upregulated in endometrial carcinosarcoma
(ECS) which is known to undergo a true EMT. Castilla and collaborators ana-
lyzed the miRNA signatures associated to EMT in human ECS and determined
their relationships with EMT markers and repressors of E-cadherin transcription
(Castilla et al. 2011). This study clearly demonstrated that the loss of epithelial
characteristics, including cadherin switching and the acquisition of a mesenchy-
mal phenotype, was accompanied by changes in the profile of miRNA expression
and an upregulation of all the E-cadherin repressors analyzed. On the one hand,
members of the miR-200 family were downregulated in the mesenchymal part of
the ECS as well as miR-23b and miR-29c involved in the inhibition of mesen-
chymal markers, and miR-203, involved in the inhibition of cell stemness. On the
other hand, an upregulated expression of miR-155, miR-369-5p, miR-370, miR-
450a, and miR-542-5p has been noted, thus confirming, at least in part, previous
in vitro data on LS cell lines (Zhang et al. 2012). These data suggest that in human
ECS, the interplay between transcriptional repressors of E-cadherin and miRNAs
provides a link between EMT activation and the maintenance of stemness. In con-
trast, the group of Dirk Dittmer reported that multiple tumor suppressor miRNAs
(miR-155, miR-220/221, let-7 family) are downregulated in KSHV-associated
cancers, including KS and primary effusion lymphoma (PEL), whereas others
(miR-143/145) are upregulated exclusively in KS tumors highlighting the impact
of tumor suppressor miRNAs in oncogenic transformation and their clinical utility
for tumor classification (O’Hara et al. 2009).
292 J. Radons
The role of the IGF system as one of the central players in the tumorigenesis
of sarcomas has also been validated in humans. SS exhibits characteristic t(X;18)
(p11.2;q11.2) translocations that result in enhanced transcription of the IGF2
gene, hyperactivation of IGF-1R signaling, phosphorylation of AKT, and activa-
tion of p44/42 MAPK (Friedrichs et al. 2008). SS cell migration was found to
depend on signals transmitted by the IGF-1R, rendering the IGF-1R a promis-
ing therapeutic target in SS. Together with the in vitro data of the same group, it
can be postulated that IGF-1R obviously plays a central role in neoplastic trans-
formation and metastasis in a number of cancers, and pathological alterations in
the pathway may be particularly important in certain cancers including sarcoma.
There is a wealth of evidence illustrating the central role of the PI3K/AKT/mTOR
pathway in human sarcomagenesis. Abnormal activation of this pathway via
several growth factor receptors triggers the development of various sarcomas
(Vemulapalli et al. 2011). Hyperactivation of mTOR in humans can also result
from PTEN inactivation, lack of the tumor suppressor kinase Lkb-1, and loss of
inhibitory function of the tuberous sclerosis complex proteins (Hogendoorn et al.
2004; Wan and Helman 2007; Yang and Guan 2007).
It is well known that numerous TKs are crucially involved in sarcomagenesis.
Understanding the mode of their activation may help to develop new targeted
therapies. EWS/FLI1 is the most common translocation found in ES tumors. The
oncogene has been shown to transform cells by acting as a transcriptional factor to
modulate a cohort of target genes including the Src TK LYN (Guan et al. 2008).
Meanwhile, elevated Lyn kinase activity was demonstrated in numerous KS and
glioblastoma patient samples (Prakash et al. 2005; Stettner et al. 2005), suggesting
that Lyn activity plays a seminal role in promoting the malignant phenotype in these
cancers and further supporting the consideration of Lyn as being a potential thera-
peutic target for the treatment of patients with these sarcoma subtypes. A charac-
teristic translocation in alveolar soft part sarcoma (ASPS) results in a novel fusion
of the ASPSCR1 (previously designated ASPL) and TFE3 genes (ASPSCR1/TFE3),
leading to the formation of a functional transcription factor inducing unregulated
transcription of TFE3-regulated genes (Lazar et al. 2007). In this study, ASPS
biopsy specimen overexpressed a unique array of pro-angiogenic TFE3-regulated
genes and proteins including midkine, Jag-1, and ANG. Elevated serum levels
of ANG and VEGF can also be found in OS and ES patients (Kushlinskii et al.
2000). As KSHV infection upregulates ANG secretion in primary HMVEC-d cells
(Sadagopan et al. 2009), ANG upregulation can be considered as a crucial player in
inflammation-associated tumorigenesis of certain sarcomas.
in sarcomagenesis, little progress had been made to translate these findings into
effective clinical strategies. The identification of novel key effector molecules in
sarcomagenesis has resulted in the development of an increasing number of drugs
that need to be tested. As already discussed, aberrant activation of the Hh path-
way has been shown in certain sarcomas such as RMS, OS, CS, and ES. Moreover,
the aggressiveness of RMS and OS appears to be related to the Notch pathway
(Tanaka et al. 2009; Roma et al. 2011). Clinical trials of the Hh inhibitor GDC-
0449 and the Notch inhibitor RO4929097 as well as the histone deacetylase inhibi-
tor vorinostat are ongoing with no results up to date.
Another protein related to sarcomas is anaplastic lymphoma kinase (ALK)
upregulated in approximately half of inflammatory myofibroblastic tumor (IMT). A
phase I trial reported a sustained partial response to the ALK inhibitor crizotinib (PF-
02341066) in a patient with ALK-translocated IMT, suggesting a therapeutic strategy
for genomically identified patients with this aggressive STS (Butrynski et al. 2010).
It should be kept in mind that cancers including sarcomas are caused by dys-
regulation of multiple pathways due to cross talk between the pathways. It there-
fore seems reasonable that agents interfering with multiple pathways are likely as
being more effective. The best outcome might be achieved by combining agents
with distinct modes of action. Natural products such as nutraceuticals are a reason-
able choice due to their safety and ability to suppress multiple targets including
NF-κB, STAT-3, and Notch. The strategy of inhibiting multiple pathways simulta-
neously in sarcoma is currently under investigation. Ongoing studies explore, for
instance, the efficacy of a combination of targeted agents alone or together with
chemotherapy. An intriguing novel approach in sarcoma treatment relates to onc-
olytic virotherapy. Preclinical data revealed that oncolytic viruses exhibit potent
direct oncolytic effects against human sarcoma in vitro and in vivo (Li et al. 2011;
He et al. 2012). As the knowledge of the molecular pathways involved in sarcom-
agenesis is increasing, individualized targeted therapies aiming to cure sarcomas
are not illusive.
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Chapter 12
The Role of Inflammation in Lymphoma
12.1 Introduction
A. Carbone (*)
Department of Pathology, Centro di Riferimento Oncologico Aviano, Istituto Nazionale
Tumori, IRCCS, Via Franco Gallini, 2, 33081 Aviano, Italy
e-mail: [email protected]
C. Tripodo
Tumor Immunology Unit, Human Pathology Section, Department of Health Sciences,
University of Palermo, Palermo, Italy
C. Carlo-Stella · A. Santoro
Department of Oncology and Hematology, Humanitas Cancer Center,
Humanitas Clinical and Research Center, Rozzano, Milano, Italy
A. Gloghini
Department of Diagnostic Pathology and Laboratory Medicine,
Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy
and active molecular cross talk. Inflammatory cells and soluble mediators, i.e.,
cytokines and chemokines, are essential microenvironmental factors that sustain cell
growth and invasion, induce angiogenesis, and suppress antitumor immune functions
(Mantovani et al. 2008).
Lymphomas usually develop in specialized tissue microenvironments characterized
by different populations of accessory stromal and lymphoid cells that interact with
malignant cells. In multidimensional studies on hematolymphoid malignancies, a rel-
evant clinical role of the tumor microenvironment has recently emerged, bringing new
knowledge and suggesting new ideas and targets for treatment (Burger et al. 2009;
Dave et al. 2004; Lenz et al. 2007; Steidl et al. 2010).
The human lymph node is a complex tissue resulting from the microenvironmental
organization of different cell populations (lymphoid cells, accessory or non-lymphoid
cells, and stromal cells) linked by topographical and/or functional relationships. The
follicle is a structure made of B and T lymphoid cells within a network of follicular
dendritic cells (FDCs). Germinal centers (GCs) contain different microenvironmental
zones (i.e., the “dark” zone [DZ] and the “light” zone [LZ]). There is a sharp demar-
cation around the whole follicle center, which is highlighted by fibroblastic reticulum
cells (FRCs) (Gloghini et al. 1990). Tingible body macrophages (TBMs) are located
throughout the GCs (Gloghini and Carbone 1993; MacLennan 1994).
The GC in lymphoid organs is a dynamic and a complex cellular microenvironment
where B cells undergo repeated rounds of mutation and selection. Three major cellu-
lar components appear necessary for the GC reaction: The FDCs that define the locus
of GC formation and serve as antigen-retaining cells for GC B cells (GCBs), antigen-
specific T cells, and antigen-specific B cells. GCBs take up antigen from FDC, process
it, and present it to antigen-specific T cells. GC T cells that recognize the antigens pre-
sented by centrocytes deliver two types of stimuli that result in the proliferation and
differentiation of B cells: contact-mediated stimuli and activating cytokines.
12.5.1 Classic HL
12.5.1.2 Recruitment of HL Microenvironment
Numerous molecules (see below) (Aldinucci et al. 2010) are involved directly or
indirectly in the recruitment and/or proliferation of cells constituting the classic
HL microenvironment.
An abnormal network of cytokines and chemokines and/or their receptors in
RS cells are involved in the attraction of many of the microenvironmental cells
into the lymphoma background (see below). RS cells are surrounded by CD40L-
expressing rosetting T cells (Carbone et al. 1995) and are dependent on survival
signals received from other cells, such as CD40L-expressing T cells (Carbone
et al. 1995), CD30L+ mast cells and eosinophils, or by a proliferation-inducing
ligand (APRIL)-producing neutrophils (Kuppers 2009).
12 The Role of Inflammation in Lymphoma 319
12.5.3 Follicular Lymphoma
FL and the genetic events underlying the progression from in situ FL to overt
FL and to diffuse lymphoma are totally unknown. There is a wide spectrum of
genetic abnormalities identified in FL, such as genomic copy number changes and
somatic mutation of the histone modifying genes including EZH2 (7 %) (Morin et
al. 2010), MLL2 (89 %) (Morin et al. 2011), MEF2B (15 %) (Morin et al. 2010),
CREBBP (33 %) (Pasqualucci et al. 2011), and EP300 (8.7 %) (Pasqualucci et al.
2011), and inactivation of TNFRSF14 (18 %) (Cheung et al. 2010). It remains to
be investigated whether these genetic changes are driver mutations and cooper-
ate with t(14;18) in malignant transformation. In other words, in some cases, FL
“transforms” into an aggressive lymphoma resembling diffuse large B-cell lym-
phoma (DLBCL), and this transformation can be associated with a variety of
oncogenic changes (Lossos and Levy 2003). Accumulation of genomic alterations
and clonal selection account for subsequent FL progression and transformation.
However, a role for the immunological microenvironment of FL in determining
clinical behavior and prognosis of the disease has also recently been substantiated.
In addition to genetic events, microenvironment factors could underlie the FL
progression. The interaction between lymphoid tumor cells and their tissue micro-
environment may promote dissemination and progression from in situ lymphoma
to early or overt FL.
The molecular pathways of cross talk between the lymphoma cells and their
nursing stroma of the follicular mantle (Hopken and Rehm 2012; Skibinski et al.
2001) might be mediated by factors expressed by mantle fibroblasts, also known
as FRCs (Gloghini et al. 1990). It is known that invasion requires active movement
on the part of the tumor cells. Movement of tumor cells through stromal tissues is
mediated in part by a “scatter factor” that is synthesized and secreted by fibroblasts
(Woolf 1998). Scatter factor, also known as hepatocyte growth factor (HGF), is a
multifunctional cytokine whose activities mainly include stimulation of epithelial
cell motility and invasiveness (reviewed in Skibinski et al. 2001). Its receptor is a
transmembrane tyrosine kinase encoded by the proto-oncogene, c-met (reviewed in
Skibinski et al. 2001), which can also be expressed, or induced, on normal B cells
(reviewed in Skibinski et al. 2001). Furthermore, B cells, when appropriately stimu-
lated, express the HGF receptor c-met, creating the potential for functional interac-
tion between mesenchymal and lymphoid cells (Skibinski et al. 2001; Weimar et al.
1997). We suggest that these functional interactions may influence lymphoid cell
motility and invasiveness. Microenvironmental factors should be further investi-
gated to clarify their role in the progression from in situ FL to early FL or overt FL.
DLBCL is the most common B-cell lymphoid neoplasm. DLBCL associated with
chronic inflammation, defined as DLBCL arising in the context of long-standing
chronic inflammation, is associated with Epstein–Barr virus (EBV) infection or
HCV infection.
322 A. Carbone et al.
Many studies have provided evidence that HCV infection is associated with
development of lymphoplasmacytoid lymphoma (immunocytoma) and with
other indolent and aggressive B-cell NHL (Mele et al. 2003; Germanidis et
al. 1999; Sansonno et al. 2007). However, a case–control study of patients
with various B-cell NHL subtypes indicated that HCV infection was detected
most frequently among those with DLBCL (Talamini et al. 2004). In con-
trast, the finding from several case–control studies did not support a notable
effect of HCV on T-cell lymphomas (reviewed in IARC). The similarities
shared by rearranged Ig genes present in B cells from patients with type II MC
and malignant B cells from HCV-positive patients with B-cell NHL support
the possibility that the antigens that promote type II MC and B-cell NHL in
HCV-positive patients are the same (De Vita et al. 1995; Sansonno et al. 1996).
These similarities also suggest that type II MC may be a precursor of B-cell
NHL (Dammacco et al.1998). Type II MC probably plays a central role in the
development of B-cell lymphoma in HCV-positive patients with Sjögren’s syn-
drome (SS) (Mariette 2001).
The liver is the main target of HCV infection and the major site of inflammatory
events, including recruitment of inflammatory cells. An emerging area of research
is directed to the definition of effective signals that enhance the survival of immu-
nocompetent cells (Taneda et al. 2001). Uncontrolled and inappropriate survival
signals are known to underlie many autoimmune disorders. The B-cell-activating
factor of the TNF family (BAFF), in particular, is a fundamental survival factor
(Mackay and Browing 2002; Schneider and Tschopp 2003).
Occurrence of HCV enrichment in intrahepatic inflammatory infiltrates
supports the notion that HCV is directly involved in the emergence and main-
tenance of these B-cell expansions (Sansonno et al. 2004). Intrahepatic B-cell
clonalities are invariably associated with extrahepatic manifestations of HCV
infection, frank B-cell NHL.
Molecular mechanisms of HCV-associated lymphoma development are still
poorly understood. Three general theories have emerged to understand the HCV-
induced lymphomagenesis: (1) continuous external stimulation of lymphocyte
receptors by viral antigens and consecutive proliferation; (2) direct role of HCV
replication and expression in infected B cells; (3) permanent B-cell damage, e.g.,
mutation of tumor suppressor genes, caused by a transiently intracellular virus
(“hit and run” theory) (IARC 2012; Peveling-Oberhag et al. 2013).
Other non-exclusive hypotheses have been proposed over the past two decades.
These hypotheses have variously emphasized the important role played by chro-
mosomal aberrations, cytokines, or microRNA molecules (Zignego et al. 2012).
However, the mechanisms by which B-cell lymphomas are induced by HCV
remain the subject of debate.
324 A. Carbone et al.
Our understanding of the biology of T-cell lymphomas is growing along with the
development of new tools for molecular profiling of T-cell clones. Lymphomas
of peripheral T cells (PTCLs) are commonly burdened by phenotypic aberrancies
implying antigenic losses, which impair specification of neoplastic T-cell differen-
tiation. Yet, gaining insight into the functional skewing of the neoplastic clone is
a fundamental step toward the correct interpretation of microenvironment biologi-
cal influence. It is conceivable that T cell clones with diverse Th or Tc polariza-
tion would differently prime the surrounding immunological and stromal milieu and
differently respond to the environment feedback. To date, we have limited but sig-
nificant evidence that PTCLs can originate from, or at least reproduce, functionally
differentiated T cells (Piccaluga et al. 2007; Iqbal et al. 2010). A notable example is
AITL, which has been demonstrated to derive from follicular helper T cells (Tfh),
a specific subset of T cells providing key help to B-cell responses under the fringes
of GC programs. Tfh cell differentiation and function are strictly reliant on IL-21/
IL-21R axis, and they express the stigmata of GC-associated lymphocytes such as
BCL-6, CD10, CXCL13, CXCR5, PD1, and ICOS expression. According to their
Tfh differentiation, AITL neoplastic cells display the Tfh phenotype and also syn-
thesize IL-21 and CXCL13. Signs of AITCl clone deregulated Tfh function can
be identified in the associated microenvironment such as the exuberant prolifera-
tion of FDC network and abundant B- and plasma cell infiltration. FDC expansion
can be directly induced by AITL cells through IL-21 and CXCL13 release and is
also sustained by the release of pro-inflammatory mediators by bystander myeloid
cells. Reactive B cells infiltrating the AITL microenvironment classically display
an activated phenotype and signs of EBV infection. These cells have been impli-
cated in the arousal of AITL-associated B-cell malignancies and in the orchestration
of autoimmune humoral responses. Abundant CXCL13 and IL-21 release by AITL
cells are effective stimuli favoring B-cell attraction and activation, which warn
about interpretation of the actual role of EBV in AITL-associated B-cell expan-
sion. Actually, EBV-infected B cells can be more susceptible to CXCL13 attraction
owing to the upregulation of CXCR5, and this event could underlie the enrichment
of EBV-infected B cells in the AITL milieu even in the absence of a trigger role for
EBV in B-cell expansion. The influence of AITL clone also extends to bystander T
cells via the activity of myeloid effectors. By CXCL13 release, AITL cells recruit
overly inflammatory mast cells eventually inducing Treg skewing toward Th17 dif-
ferentiation by IL-6 and OX40/OX40L interaction. The induction of a Th17-prone
background further contributes to magnifying myeloid cell accrual and fostering the
autoimmune diathesis of AITL cases (Tripodo et al. 2010).
The pressure exerted by the neoplastic clone over the mesenchymal components
of AITL-infiltrated lymphoid tissues also results in the induction of the characteris-
tic vascular proliferation. AITL angiogenic response is not mere expression of the
vascular remodeling associated with an expanding lymphoid clone, rather it reflects
the outcome of the uncontrolled release of pro-angiogenic factors such as the pro-
totypical VEGF-A, which is constitutively synthesized and released by neoplastic
12 The Role of Inflammation in Lymphoma 325
cells. Moreover, the angiogenic loop is further boosted through the engendering of
pro-inflammatory conditions to which mast cells, macrophages, neutrophils, and
eosinophils largely contribute. Notably, endothelial cells of AITL newly formed
vessels, which are characterized by BCL2 expression, give rise to a bidirectional
cross talk with neoplastic lymphocytes via the VEGF-A axis toward lymphoma
progression. Overall, the AITL model well explains the integrated effort of clonal
T cells and reactive lymphoid, myeloid, and mesenchymal elements to the orches-
tration of a vicious homeostasis and provides a precious insight into the influence
of T-cell clone polarization in the specification of the associated environment.
Recently, PTCLs other than AITL have been reported to be characterized by T cell
clones with Tfh phenotype. In these cases of PTCL, typical features of the AITL
microenvironment could be variably identified thus suggesting that the nature of
the neoplastic T-cell population and the quality of the microenvironment are both
determinant in the outcome of the lymphomagenesis. Accordingly, different polar-
izing environments can be associated with the establishment of diverse T-cell lym-
phoma histotypes. The induction of a Th-22 polarizing milieu has been reported to
favor CTCL development (Miyagaki T et al. 2011) while establishment of a Th-
17-skewed environment characterizes some anaplastic T-cell lymphoma (ALCL)
cases. The dynamics of induction of Th-17 skewing in ALCL is particularly inter-
esting being mediated by the NPM–ALK translocation via upregulation of miR-
135 and Th-2 program repression and thus indicating posttranscriptional regulation
of neoplastic T-cell fate as further element of complexity in PTCL biology.
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Chapter 13
The Role of Inflammation in Leukaemia
J. Krawczyk
Department of Haematology, Galway University Hospital, Galway, Ireland
M. O’Dwyer
Biosciences, National University of Ireland Galway, Upper Newcastle, Galway, Ireland
R. Swords
Sylvester Comprehensive Cancer Center, University of Miami, Miami, USA
C. Freeman
Department of Haematology, Barts and the Royal London NHS Trust, London, UK
F. J. Giles (*)
Northwestern Medicine Developmental Therapeutics Institute, Robert H. Lurie
Comprehensive Cancer Center of Northwestern University, Chicago, USA
e-mail: [email protected]
13.1 Introduction
Limiting factors in the treatment of AML include the development of drug resist-
ance, the relatively high treatment-related mortality and the long-term side effects.
Immunotherapy holds great promise to sustain AML remission once the disease has
been bulk-reduced with chemotherapy. Despite recent improvements in the treatment
of AML, the frequency of relapses and the difficulty to completely eradicate the dis-
ease warrant the search for innovative therapies.
The age-adjusted incidence of ALL is 1.7 per 100,000 persons, with a median
age at diagnosis of 13 years. ALL is the most common childhood acute leukaemia,
representing approximately 80 % of childhood leukaemia cases, although it repre-
sents only 20 % of adult leukaemias. The aetiology of ALL remains unknown in
most cases. Chromosomal translocations have been suggested as the primary cause
for paediatric ALL; some genetic disorders are associated with a higher risk of ALL
(trisomy 21, XXY). Some studies have suggested possible infectious aetiologies.
Therapy of ALL is one of the most complex types of anti-cancer programs. Multiple
drugs are combined into regimen-specific sequences in order to reconstitute normal
haematopoiesis, prevent resistance, provide adequate prophylaxis of sanctuary sites
and eliminate MRD through postremission consolidation and maintenance therapy.
CLL is a common monoclonal B-cell lymphoproliferative disease, derived from
antigen-experienced B lymphocytes. The CLL cells depend on external factors
for survival and proliferation. B-cell receptor stimulation and activation of a vari-
ety of signalling pathways, including PI3K/AKT, NF-κB, MAPK/ERK, WNT and
338 J. Krawczyk et al.
NOTCH, have also been associated with CLL cell survival, with the incidence of
three cases per 100,000 individuals, and it accounts for 35 % of all leukaemias diag-
nosed in the United States. Currently, several cytogenetic and molecular markers
have an established prognostic value, including among others, chromosomal abnor-
malities (especially deletions of 11q and 17p, beta-2-microglobulin, IgVH mutation
status, CD38 expression and ZAP70). The introduction of purine analogues, mono-
clonal antibodies and other targeted therapies has shifted the treatment paradigm for
CLL in recent years. These modern therapies commonly achieve CRs and even erad-
ication of MRD—endpoints that were essentially impossible in the past (NCI 2013).
Despite these advances, it remains an incurable disease.
CML is a clonal disorder of a pluripotent stem cell that affects myeloid,
erythroid, megakaryocytic lineages and lymphocytes. The age-adjusted annual
incidence of CML in the United States is 1.6/100,000 (NCI 2013). CML is charac-
terised by the presence of Philadelphia chromosome and the BCR-ABL oncogene.
The expression of the chimeric BCR-ABL gene in CML led to development of
agents specifically targeted at inhibiting the resulting tyrosine kinase that have sig-
nificantly changed the natural history of the disease.
13.3 Tumour-Associated Macrophages
only associated with increased levels of CCL5 and IL-2/5 and decreased levels
of CCL4 and CXCL8. A panel of 11 cyto- and chemokines (including CCL3 and
CCL5) allowed for the separation of patients into favourable, intermediate and unfa-
vourable remission groups with significantly different median survival rates (52 vs.
32 vs. 16 weeks, p = 0.003). The effect of chemo- and cytokines in leukaemia is
complex, as the level of receptor expression, the presence of receptor mutations,
para- and autocrine secretion and abnormalities in signalling pathways activation
modulate the effected cytokines (Kornblau et al. 2010).
One of the best-characterised chemokine is stromal-derived factor 1α, SDF-1α
(CXCL12). It is constitutively secreted by marrow stromal cells and binds to
C-X-C chemokine receptor type 4 (CXCR4), also known as CD184 (Koblas
et al. 2007). The main role of this axis is the homing of hematopoietic progenitors
and leukaemia cells within the bone marrow. CXCL12/CXCR4 mediates the adhe-
sion of leukaemic cells to marrow stromal cells, influences survival and prolifera-
tion, protects AML cells from the effects of chemotherapy in vitro and in vivo and
activates ERK and PI3K pathways (Tilton et al. 2000; Nebreda and Gavin 1999;
Datta et al. 1999). In primary AML samples, the increased CXCR4 expression
was found in 64 % of samples and was an independent poor prognostic factor for
relapse and survival (Spoo et al. 2007). In ALL, a high expression of CXCR4 was
strongly predictive for extramedullary organ involvement (Crazzolara et al. 2001).
The chemokine receptor CCR7 is an essential adhesion signal required for the
targeting of leukaemic T cells into the CNS. Ccr7 gene expression is controlled
by the activity of the Notch1 oncogene and is expressed in human tumours carry-
ing Notch1-activating mutations. The silencing of either CCR7 or its chemokine
ligand CCL19 in an animal model of T-ALL specifically inhibits CNS infiltration.
In a murine model, CNS-targeting by human T-ALL cells depended on the expres-
sion of CCR7 (Buonamici et al. 2009).
13.3.2 NF-κB in Leukaemia
Family of nuclear factor kappa B (NF-κB) transcription factors is the key signal-
ling pathway linking cancer and inflammation. NF-κB activates more than 200
genes including the expression of inflammatory cytokines, adhesion molecules, key
enzymes in the prostaglandin synthase pathway (COX-2), nitric oxide (NO) syn-
thase and angiogenic factors. In addition, by inducing anti-apoptotic genes (e.g.
Bcl2), it promotes survival in malignant cells. In resting cells, the majority of NF-
κB complexes are bound to the Inhibitor-κB (IκB) and remain sequestered in cyto-
plasm. The classical activation pathway of NF-κB in response to pro-inflammatory
cytokines and chemokines, DNA damaging agents, Toll-like receptors (TLRs)
ligands or viruses starts with the activation of inhibitor kappa B kinase (IKK) that
phosphorylates IκB and frees NF-κB complexes to enter the nucleus. The non-clas-
sical signalling responds to the subset of TNF receptors and depends on the pro-
cessing of a precursor protein p100 into a mature NF-κB subunit (p52) (Sun 2011).
13 The Role of Inflammation in Leukaemia 341
Fig. 13.1 The central role of NF-κB in signal crosstalk between inflammation and leukaemia;
only pathways with proven significance are shown (MMP matrix metalloproteases, TLR Toll-like
receptors, TRAIL tumour necrosis factor-related apoptosis-inducing ligand, TNF tumour necrosis
factor, VEGF vascular endothelial growth factor, NF-κB nuclear factor kappa B)
13.3.3 STAT3 in Leukaemia
In AML, STAT3 was constitutively activated in most of the cell lines and nearly
half of the primary paediatric samples (Redell et al. 2011). STAT proteins are
involved in the hematopoietic cytokine receptor signalling pathways that regu-
late cell proliferation, differentiation and survival. STATs are dysregulated in
AML; mechanisms of dysregulation include constitutive activation and trunca-
tion of the C-terminal transactivation domain; the latter results in a beta iso-
form that has a trans-dominant negative effect on gene induction, mediated by
the full-length STAT alpha form. A constitutive STAT3 activity in AML was
associated with poor prognosis (Benekli et al. 2002), possibly due to the resist-
ance to chemotherapy. Disease-free survival (DFS) was significantly shorter
in patients with constitutive STAT3 activity (median 8.7 vs. 20.6 months;
P = 0.01). The overall survival rate did not differ significantly. The subgroup
of patients with constitutive STAT3 activity and the STAT3 beta isoform had
the shortest DFS (P = 0.006) and the shorter overall survival rate (P = 0.049)
than all other patients. It is not clear whether adverse treatment outcomes are
attributable to constitutive STAT activity or to a process that leads to constitu-
tive STAT activity (Benekli et al. 2002). The constitutive serine phosphoryla-
tion of STAT1 and STAT3 is present, although the physiologic significance of
these modifications remains to be determined (Frank et al. 1997). CLL cells
have high levels of unphosphorylated STAT-3 (USTAT-3). It was confirmed
that USTAT-3 USTAT-3/NF-κB complexes bind to DNA and activate NF-κB-
regulated genes in CLL cells (Liu et al. 2011).
One of the newly discovered NF-κB activators is vascular endothelial growth fac-
tor (VEGF). In AML, the number of vessels in the bone marrow biopsies was sig-
nificantly increased at diagnosis, compared with normal bone marrow (P = 0.019)
and was restored to normal levels after achieving CR. The expression of VEGF
correlated with a degree of neoangiogenesis. These results suggest that malig-
nant cell proliferation, angiogenesis and VEGF expression are linked in AML
and might contribute to the growth advantage of the malignant clone (de Bont
et al. 2001). In CLL, VEGF mediates neovascularization in bone marrow. B-CLL
lymphocytes produced VEGF in vitro, and increased VEGF levels were found in
primary samples. Elevated VEGF receptor (VEGFR)-2 had a negative prognos-
tic impact on survival. Also VEGF stimulates NF-κB in malignant B-CLL cells.
The downstream transcriptional targets of NF-κB activation in CLL are also likely
to be diverse, but certainly include the inhibitor of apoptosis proteins (IAPs)
and anti-apoptotic members of the Bcl-2 family of proteins, for example, Bcl-2,
Bcl-XL and Bfl1/A1.
13 The Role of Inflammation in Leukaemia 345
13.3.4.4 Toll-Like Receptors
TLRs are pattern recognition receptors and take part, among others, in the
initiation of inflammation. They are involved in innate and adaptive immune
responses when activated by pathogen-associated molecular patterns (PAMPS),
and they mediate the secretion of cytokines. TLRs display both pro- and anti-
tumour properties. TLRs after the recognition of a specific ligand signal down
through adapter protein MyD88. MyD88 mediates the classical pathway of the
NF-κB activation. Pro-tumourigenic effects of endotoxin occur through TLR4-
mediated NF-κB activation. The focus of recent research has been aimed at acti-
vation of the immune system in order to inhibit cancer cell growth and induce
cancer cell apoptosis. TLRs, therefore, offer a unique target for cancer therapy.
TLR3 is an intracellular, type 1 trans-membrane receptor and is an important
“danger” signalling receptor that takes part in the control of the balance between
tolerance and inflammation on the one hand and inflammation and disease on
the other hand. In cytogenetically normal, but high-risk AML (based on FLT3–
ITD, a wild-type NPM1, or expression of both genes), the microRNA expression
13 The Role of Inflammation in Leukaemia 347
profiling revealed increased TLR2, TLR4 and TLR8 expression (Marcucci et al.
2008). On the contrary, the reduced expression of TLR4 was reported in both
CLL and AML in comparison with normal controls (Webb et al. 2009). Further
studies are needed to explain whether the decreased TLR4 expression contrib-
utes to the pathogenesis of leukaemia through impaired immune surveillance
and whether TLR4 agonists might serve to effectively strengthen the response of
the immune system in battling the leukaemic burden.
13.3.4.5 Matrix Metalloproteases
In the last few years, studies have also clearly demonstrated that leukaemia popu-
lations are highly heterogeneous and that the disease is propagated by a subpop-
ulation of LSC. LSCs, like normal hematopoietic stem cells, possess a range of
biological characteristics that enable for their long-term survival. Therefore, LSCs
reside in a mostly quiescent state, and as a consequence, the overall activity of
many chemotherapeutic agents that function by targeting cycling cells is possi-
bly reduced (Konopleva and Jordan 2011). Although some studies have indicated
a role for NF-κB in quiescent cells, the activation of NF-κB is an acquired phe-
nomenon. Unstimulated human CD34+ progenitor cells do not express NF-κB,
while primary AML cells display readily detectable NF-κB activity. NF-κB is
highly activated in leukaemic cells, which suggests that an intrinsic aspect of AML
biology resides in the constitutive activation of various pathways. Furthermore,
detailed analyses of enriched AML stem cells (CD341/CD382/CD1231) indi-
cate that NF-κB is also active in the LSC population. Interestingly, leukaemic
cells showed a rapid apoptotic response while stimulated by a NF-κB inhibitor
(MG-132), whereas normal CD341/CD382 cells showed a limited effect. Taken
together, these data indicate that primitive AML cells aberrantly express NF-κB
and that the presence of this factor may provide unique opportunities to preferen-
tially ablate LSCs (Guzman et al. 2001a).
T-cell acute lymphoblastic leukaemia (T-ALL) is associated with an increased
risk of central nervous system (CNS) relapse. Little is known about the mechanism
of leukaemic cell infiltration of the CNS. In an animal T-ALL model, chemokine
receptor, CCR7, was shown to be an essential adhesion signal required for the
targeting of leukaemic T cells into the CNS. The CCR7 gene expression is con-
trolled by the activity of the T-ALL oncogene Notch1 and is expressed in human
tumours carrying Notch1-activating mutations. The silencing of either CCR7 or its
chemokine ligand CCL19 (Bellosillo et al. 1998) in an animal model of T-ALL
specifically inhibits CNS infiltration. These studies identify a single chemokine-
receptor interaction as a CNS “entry” signal and open the way for future pharma-
cological targeting. The targeted inhibition of CNS involvement in T-ALL could
potentially decrease the intensity of CNS-targeted therapy, thus reducing its asso-
ciated short- and long-term complications (Buonamici et al. 2009).
As has been mentioned above, various inflammatory pathways are activated in leu-
kaemias; therefore, modulation of those pathways may have the potential for treat-
ment and possibly the prevention of leukaemias. Complete inhibition of a single
pathway is more likely to be toxic and less likely to be effective. Partial downregu-
lation of several pathways is more likely to inhibit the deregulated inflammatory
signalling and be less toxic and more efficient in therapy. The key agents modu-
lating inflammatory pathways include steroids, proteasome inhibitors, TNF inhibi-
tors, NF-κB inhibitors and COX2 inhibitors, TRAIL, chemokine modulators and
others. Another group of interesting agents is the naturally occurring modifiers of
NF-κB activation.
13.6.1 Cytokine-Based Interventions
stem cells into circulation and enhanced the sensitivity to chemotherapy or FLT3-
inhibitor-induced cell death. Other antagonists have also been investigated. One of
them, RCP168, had a strong antagonistic effect on chemotaxis of leukaemic cells.
Another candidate molecule, E-4031, a specific hERG1K(+) channel inhibitor and
CXCL12 blocker, inhibited the migration of leukaemic cell-induced G0/G1 arrest,
impaired proliferation and apoptosis of AML cells.
It was also found that ubiquitin is a natural ligand of CXCR4. Ubiquitin is a
small, highly conserved protein; it primarily targets intracellular proteins for deg-
radation via the ubiquitin proteasome system. Evidence in numerous animal mod-
els suggests that extracellular ubiquitin is an anti-inflammatory immune modulator
and an endogenous opponent of pro-inflammatory damage-associated molecular
pattern molecules. It is speculated this interaction may be through CXCR4 medi-
ated signalling pathways and regulatory effects on the growth of various leukae-
mia cell lines (Majetschak 2011).
recognised through well-controlled clinical trials. They are likely not to be utilised
as single agents, but rather as a part of multidrug protocols that can lead to a suc-
cessful therapy for different haematological malignancies
Further understanding of interactions between inflammation and leukaemia will
reveal novel targets for monitoring and novel therapies in combination with con-
ventional treatments. Therapeutic modifications of inflammatory pathways in leu-
kaemias will possibly improve the clinical efficacy. A better identification of bone
marrow and leukaemia-specific inflammatory mechanisms will allow personalisa-
tion of therapeutic strategies. Therapeutic manipulation of inflammatory pathways
is likely to change the inflammatory microenvironment into an anti-cancer micro-
environment. Taking into account the relevance of inflammatory networking in
leukaemia, it would be very important to incorporate inflammatory parameters into
traditional classification schemes to provide new prognostic tools. The challenges
for the future are to investigate the activation, function and prognostic value of
inflammatory pathways in leukaemia, as well as to evaluate the therapeutic poten-
tial of novel therapeutic strategies in clinical trials that interfere with inflammatory
signalling, including NF-κB. The novel agents interfering with inflammatory path-
ways look promising and will most likely be a useful addition to the treatments
that are currently available for many leukaemias.
In this chapter, we have provided conclusive evidence that inflammatory signal-
ling pathways play an important role in leukaemia. It is, therefore, evident that
anti-inflammatory agents should be explored for both the prevention and treatment
of leukaemia. Although numerous cell culture and animal studies have identified
several natural anti-inflammatory agents, their true potential will be recognised
only through well-controlled clinical trials.
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Chapter 14
The Role of Inflammatory Cells in Angiogenesis
in Multiple Myeloma
Abstract Both innate and adaptive immune cells are involved in the mechanisms
of endothelial cell proliferation, migration and activation, via production and
release of a large spectrum of pro-angiogenic mediators, thus creating the specific
microenvironment that favors increased rate of tissue vascularization. In this arti-
cle, we focus on the immune cell component of the angiogenic process occurring
during multiple myeloma progression. We also provide information on some anti-
angiogenic properties of immune cells that may be applied for a potential pharma-
cological use as anti-angiogenic agents in the disease treatment.
14.1 Introduction
D. Ribatti (*)
Department of Basic Medical Sciences, Neurosciences and Sensory Organs,
Section of Human Anatomy and Histology, University of Bari Medical School,
Piazza Giulio Cesare, 11, 70124 Bari, Italy
e-mail: [email protected]
A. Vacca
Department of Biomedical Sciences and Human Oncology (DIMO),
Section of Internal Medicine and Clinical Oncology, University of Bari Medical School,
Piazza Giulio Cesare, 11, 70124 Bari, Italy
e-mail: [email protected]
D. Ribatti
National Cancer Institute, Giovanni Paolo II, 70124 Bari, Italy
in tumor-bearing hosts, while inflammatory cells may produce growth factors that
suppress the anti-tumor immune response. The most aggressive human cancers are
associated with dramatic host inflammatory response, and inflammatory cells act
in concert with tumor cells, stromal cells, and endothelial cells to create a micro-
environment that is critical for the survival, development, and diffusion of the neo-
plastic mass. These interactions within the tumor microenvironment may represent
important mechanisms for tumor development and metastasis by providing an effi-
cient vascular supply and an easy escape pathway.
Inflammatory cells produce angiogenic cytokines, growth factors, and pro-
teases that contribute to new vessels formation at the site of tumor growth.
Conversely, microvascular endothelium activated by a number of cytokines and
angiogenic growth factors can express pro-inflammatory molecules involved in
leukocyte recruitment and activation (Pober and Sessa 2007). Various chemokines
may act both as leukocyte attractants and angiogenic inducers by acting directly
on endothelial cells. Several pro-inflammatory cytokines, including interleukin
(IL)-1α, IL-1β, IL-6, tumor necrosis factor alpha (TNF-α), and osteopon-
tin, induce vessel formation acting directly on endothelial cells or indirectly by
inducing leukocyte and/or endothelial cells to produce pro-angiogenic mediators.
Conversely, vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-
1) may elicit pro-inflammatory responses in endothelial cells by up-regulating the
expression of cell-adhesion molecules and inflammatory mediators.
Mast cells are bone marrow-derived tissue-homing leukocytes which were first
described by Paul Ehrlich in 1878. They appear as highly versatile cells playing an
important role in a large spectrum of biological settings, including inflammation,
14 The Role of Inflammatory Cells in Angiogenesis in Multiple Myeloma 365
angiogenesis, tissue repair and remodeling, and cancer. As concerns the role of
mast cells in tumor growth, although some evidence suggests that these cells can
promote tumorigenesis and tumor progression, there are some clinical data as
well as experimental tumor models in which mast cells seem to have functions
that favor the host (Ribatti and Crivellato 2009). Mast cells are attracted into the
microenvironment by stem cell factor (SCF) secreted by tumor cells and pro-
duce several angiogenic factors as well as MMPs, which promote, respectively,
tumor vascularization and invasiveness (Ribatti and Crivellato 2009). Mast cells
are capable of suppressing immune reactions by releasing histamine (which can
induce tumor cell proliferation through H1 receptors and suppress the immune
system through H2 receptors), TNF-α (Ullrich et al. 2007), and inhibitory
cytokines, such as IL-10, and are essential in promoting the immune tolerance
mediated by regulatory T cells (Treg) which, in their turn, stimulate immune toler-
ance and tumor promotion (Grimbaldenston et al. 2007). By contrast, mast cells
may promote inflammation, inhibition of tumor cell growth, and tumor cell apop-
tosis by releasing cytokines, such as IL-1, IL-4, IL-6, IL-8, MCP-3 and MCP-4,
transforming growth factor beta (TGF-β), TNF-α, and chymase. Mast cells also
produce chondroitin sulfate and tryptase: chondroitin sulfate may inhibit tumor
cells diffusion, while tryptase causes both tumor cell disruption and inflamma-
tion through activation of protease-activated receptors (PAR-1 and -2) (Ribatti and
Crivellato 2012).
Increased mast cell number has been correlated with a poor prognosis in sev-
eral human tumors, including melanoma (Ribatti et al. 2003a), oral squamous
carcinoma (Wanachantarak 2003), and squamous cell carcinoma of the lip
(Rojas et al. 2005). Mast cells produce several pro-angiogenic factors, includ-
ing FGF-2, VEGF, IL-8, TNF-α, TGF-β, and nerve growth factor (NGF) (Qu et
al. 1995, 1998a, b; Grützkau et al. 1998; Aoki et al. 2003; Abdel-Majid et al.
2004; Boesiger et al. 1998; Kanbe et al. 2000; Moller et al. 1993; Walsh et al.
1991; Nilsson et al. 1997). Mast cells migrate in vivo and in vitro in response to
VEGF and placental growth factor-1 (PlGF-1) (Detmar et al. 1998; Gruber et al.
1995;Detoraki et al. 2009). Human lung mast cells express VEGF-A, VEGF-B,
VEGF-C, and VEGF-D, and supernatants of prostaglandin E2 (PGE2)- and
5′-N-ethylcarboxamido-adenosine (NECA)-activated lung mast cells induced
angiogenic response in the chick embryo chorioallantoic membrane (CAM)
assay that was inhibited by an anti-VEGF-A antibody (Detoraki et al. 2009).
Mast cells store in their secretory granules pre-formed active serine proteases,
including tryptase and chymase (Metcalfe et al. 1997). Tryptase stimulates the
proliferation of endothelial cells, promotes vascular tube formation in vitro,
degrades connective tissue matrix, and activates MMPs and plasminogen activa-
tor (PA), which in turn degrade the extracellular matrix with consequent release
of VEGF or FGF-2 from their matrix-bound state (Blair et al. 1997). Histamine
and heparin stimulate proliferation of endothelial cells in vitro and are angio-
genic in the CAM assay (Sörbo et al. 1994; Ribatti et al. 1987). Mast cells
contain MMP-2 and MMP-9, and TIMPs, which intervene in regulation of extra-
cellular matrix degradation, allowing release of angiogenic factors. Granulated
366 D. Ribatti and A. Vacca
mast cells and their granules are able to stimulate an intense angiogenic r eaction
in the CAM assay, partly inhibited by anti-FGF-2 and anti-VEGF antibodies
(Ribatti et al. 2001). Moreover, intraperitoneal injection of compound 48/80
causes a vigorous angiogenic response in the rat mesentery window angiogenic
assay and in mice (Norrby et al. 1986, 1989).
Mast cells play a direct role in tumor angiogenesis. Mast cell-deficient
W/Wv mice exhibit a decreased rate of tumor angiogenesis (Starkey et al. 1988).
Heparin facilitates tumor vascularization by a direct pro-angiogenic effect and
its anti-clotting effect (Theoharides et al. 2004). An increased number of mast
cells have been demonstrated in angiogenesis associated with vascular tumors,
such as hemangioma and hemangioblastoma (Glowacki and Mulliken 1982),
as well as a number of hematological and solid tumors, including lymphomas
(Ribatti et al. 1998, 2000; Fukushima et al. 2001), multiple myeloma (MM)
(Ribatti et al. 1999), myelodysplastic syndrome (Ribatti et al. 2002), B-cell
chronic lymphocytic leukemia (Ribatti et al. 2003a; Molica et al. 2003), breast
cancer (Hartveit et al. 1981; Bowrey et al. 2000), colo-rectal cancer (Lachter et
al. 1995), uterine cervix cancer (Graham and Graham, 1996; Bentitez-Bribiesca
et al. 2001; Ribatti et al. 2005), and melanoma (Reed et al. 1996; Dvorak et al.
1980), in which mast cell accumulation correlates with increased neovasculari-
zation, mast cell VEGF and FGF-2 expression, tumor aggressiveness, and poor
prognosis (Tóth-Jakatics et al. 2000; Ribatti et al. 2003b, c). Indeed, a prognos-
tic significance has been attributed to mast cells and microvascular density also
in squamous cell cancer of the esophagus (Elpek et al. 2001). An association
between VEGF, mast cells, and angiogenesis has been demonstrated in laryngeal
carcinoma and in small lung carcinoma (Sawatsubashi et al. 2000; Imada et al.
2000; Takanami et al. 2000; Tomita et al. 2000).
The introduction of novel experimental procedures to induce carcinogenesis in
pre-clinical in vivo models contributed to our increased understanding on the role
of mast cells in tumor angiogenesis. Development of squamous cell carcinoma in
a human papilloma virus (HPV) 16 infected transgenic mouse model of epithelia
carcinogenesis provided experimental support for the early participation of mast
cells in tumor growth and angiogenesis (Coussens et al. 1999, 2000). Infiltration
of mast cells and activation of MMP-9 coincided with the angiogenic switch in
pre-malignant lesions through the release of pro-angiogenic molecules from the
extracellular matrix. Remarkably, pre-malignant angiogenesis was abrogated in a
mast cell-deficient HPV 16 transgenic mouse indicating that neoplastic progres-
sion in this model involved infiltration of mast cells in the skin (Coussens et al.
1999, 2000). By using the same in vivo transgenic mouse model, it has been dem-
onstrated that genetic elimination of mature T and B lymphocytes limits neoplas-
tic progression (de Visser et al. 2005; Andreu et al. 2010). Moreover, in prostate
tumors derived from both tumor transgenic adenocarcinoma of the mouse pros-
tate (TRAMP) mice and human patients, mast cells promote well-differentiated
adenocarcinoma growth (Pittoni et al. 2011).
14 The Role of Inflammatory Cells in Angiogenesis in Multiple Myeloma 367
Fig. 14.1 CD68 (red) and FVIII-RA (green) in (a) dual confocal laser microscopy, and (b, c)
immunofluorescence of bone marrow biopsies of (a, b) a patient with multiple myeloma (MM)
and (c) a patient with monoclonal gammopathy of undetermined significance (MGUS). In (a),
a microvessel lined by flattened FVIII-RA-positive endothelial cells (arrow) and FVIII-RA
positive macrophage (arrowhead) showing protrusions connected to endothelial cells; another
macrophage containing double-labeled CD68 (red arrowhead) and FVIII-RA (green arrowhead)
granules in the cytoplasm is connected to endothelial cells by a FVIII-RA-positive cytoplasmic
protrusion (double arrowhead). b Another microvessel formed by FVIII-RA-positive (green)
endothelial cells and CD68-positive (red, arrowheads) tracts belonging to the cytoplasmic pro-
trusions (double arrow) of macrophages, some of which are arrowed. c The MGUS microvessel
is formed only by FVIII-RA-positive endothelial cells: macrophages (arrows) are randomly scat-
tered in the tissue. [Reproduced from Scavelli et al. (2008)]
Fig. 14.2 Double FVIII-RA (green) and tryptase (red) confocal laser microscopy from bone
marrow biopsies of patients with (a) multiple myeloma (MM) and (b) monoclonal gammopathy
of undetermined significance (MGUS). In (a), a vessel is lined by both endothelial cells posi-
tive for FVIII-RA and by mast cells positive for tryptase (arrowheads). Mast cells containing
tryptase-positive granules (arrows) are also recognizable on the abluminal side of the vessel. In
(b), a MGUS vessel is lined only by endothelial cells positive for FVIII-RA and is surrounded by
tryptase-positive mast cells (arrows). [Reproduced from Nico et al. (2008)]
14.5.1 Immunomodulatory Drugs
apoptosis; decrease binding of tumor cells to bone marrow; inhibit constitutive and MM
cell binding-induced secretion of cytokines from bone marrow; and stimulate autolo-
gous NK and T-cell immunity to MM cells (Hideshima et al. 2007).
IMiDs inhibit TNF-α, IL-1β, IL-6, IL-12, and TGF-β production. These cytokines
enhance growth and survival of myeloma cells, drug resistance, cell migration,
and adhesive molecule expression. Moreover, IMiDs increase anti-inflammatory
cytokines (IL-10) production. Anti-inflammatory and anti-angiogenic properties of
thalidomide are partially controlled by NF-κB transcriptional factor. Moreover, tha-
lidomide significantly inhibits SDF-1α and CXCR4 receptor expression on MM cells
leading to decreased IL-6 and VEGF production supporting survival of MM cells.
14.5.2 Cytokines
In MM, both chains of IL-27 receptor are expressed in CD138 positive plasma
cells from patients (Cocco et al. 2011). IL-27 inhibits the angiogenic potential of
MM cells, down-regulates different angiogenic factors, and up-regulates the anti-
angiogenic chemokines CXCL9 and CXCL10 (Cocco et al. 2011). Pre-clinical
studies using highly immunodeficient non-obese diabetic/severe combined immu-
nodeficient mice injected with MM plasma cells demonstrated that IL-27 inhibits
MM plasma cell growth through inhibition of angiogenesis, thus supporting the
concept that IL-27 may represent a novel therapeutic agent for patients with MM.
Pentraxin 3 (PTX3) plays an important role in inhibiting the cross talk between
BMSCs and plasma cells in MM. Basile et al. (2013) demonstrated that PTX3 inhibits
FGF-2-induced angiogenesis of MM endothelial cells through its binding to FGF-2
and exerts a direct impact on FGF-2-induced biological activities on MM fibroblasts,
which also support MM cell growth. In particular, PTX3 is cytotoxic on MM cells by
inhibiting the activities of endothelial cells and fibroblasts, including cytokine produc-
tion, and causing loss of adhesive plasma cell capability to these cells.
14.5.4 Other Molecules
14.6 Concluding Remarks
The pathogenesis of most cancers includes complex and mutual interactions that affect
the number and phenotype of the tumor cells and host inflammatory cells. In this con-
text, angiogenesis in MM is the result of a complex balance between pro- and anti-
angiogenic stimuli generated in the tissue milieu. The evidences summarized highlight
the importance of the inflammatory microenvironment during angiogenesis in MM
and provide a novel perspective for the complex interplay between several inflamma-
tory and vascular components in the bone marrow microenvironment in MM.
Acknowledgments The research leading to these results has received funding from the
European Union Seventh Framework Programme (FP7/2007–2013) under Grant agreement
n°278570 to DR and 278706 to AV.
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Chapter 15
The Role of Inflammation in Cervical Cancer
Cancer is the major health problem and accounts for 14 % of the death worldwide.
Cervical cancer is the seventh most common cancer (including both sexes) and
accounts for about 4.2 % of the total cancers. It is also third most common among
the women and occupies 13 % of female cancer, which is next only to breast can-
cer. More than 85 % of the cases of cervical cancer are present in the develop-
ing countries. Incidence of cervical cancer is highest in Eastern, Western, Southern
Africa, and in south-central Asia. Incidence is lowest in Western Asia, Northern
America, Australia, and New Zealand, and Western Asia. Cervical cancer remains
the most common cancer among women only in Eastern Africa, south-central
Asia, and Melanesia. Cervical cancer is also the fourth most common cancer caus-
ing death next to breast, lung, and colorectal cancer. About 9 % of cancerous peo-
ple are diagnosed with cervical cancer.
Cervical cancer incidence varied between regions ranging from 5 per 100,000
in Western Asia to 35 per 100,000 in Eastern Africa in 2008. Highest incidence
rates were 56 and 53 per 100,000, respectively, in Guinea and Zambia. The UK
was 145th highest out of 184 countries worldwide. Cervical cancer is more prev-
alent in countries where Human Developmental Index is low. Cervical cancer is
the second most prevalent cancer in females (present in 37 countries in South and
Central America, Southern Africa, and Asia) (Ferlay et al. 2010b).
It was estimated that cervical cancer leads to 275,000 deaths worldwide in
2008, accounting for 8.2 % of all female cancer deaths (3.6 % of the total in men
and women). Cervical cancer is the tenth leading cancer in developed countries
(76,500 cases), and it shifts to second in case of developing countries (453,300). It
also ranks second (242,000) in causing mortality in developing countries which is
closely next to breast cancer (268,900 cases). Incidence of cervical cancer is 9.0 %
in developed countries, and it rises to 17.8 % in developing countries. Mortality
rate is 3.2 % in developed countries, and it increases to threefold in developing
countries (9.8 %). Mortality rates were less than 2 per 100,000 in Australia/New
Zealand and Northern America and 25 per 100,000 in Eastern Africa in 2008.
In Europe, the overall incidence rate of cervical cancer is 10.6 per 100,000.
Europe continent is diverse, and incidence of cervical cancer varies from region to
region. Incidence of cervical cancer in Western Europe is low (6.9/100,000), and
it increases to twofold in Central/Eastern Europe. Incidence in Northern Europe
and Southern Europe is almost similar with rate of 8.4/100,000 and 8.1/100,000,
respectively. Incidence is highest in Romania (23.9/100,000) and lowest in Malta
(2.1/100,000). Eastern Europe has increased 4–5 times risk of developing cervical
cancer when compared to western countries. The incidence of cervical cancer in
some countries in eastern part of Europe is 20 per 100,000. Incidence in Europe
has not changed, and it remains almost the same for the past few years (11.05 per
100,000 in 2002; 10.06 per 100,000 in 2008). For the past 10 years, the incidence
of cervical cancer in eastern European countries continues to increase. In Europe,
mortality due to cervical cancer decreased around 10 % from 5.0 per 100,000 to
4.5 per 100,000 (during the period 2002–2008) (Ferlay et al. 2010a).
in Nepal (32 per 100,000), followed by Mongolia (28.4 per 100,000) and India
(27 per 100,000). Incidence of cervical cancer is second highest in Asia–Oceania
region where the list is headed by breast cancer. Incidence is lowest in Australia,
Singapore, and Hong Kong. Mortality rate is around 7.9 per 100,000 in Asia–
Oceania region. Every year around 160,000 women dies because of cervical can-
cer. Mortality rate is highest in Nepal, Papua New Guinea, and India, and lowest in
Japan and Australia. Though the incidence of cervical cancer is second highest in
Asia–Oceania region, in mortality, it ranks fourth (Garland et al. 2012).
15.2 % of total cervical cancer burden is from Africa. The incidence of cervical
cancer varies from region to region. Incidence is high in Southern and Eastern
Africa (40 per 100,000). Incidence is high among Lesotho and Swaziland.
Mortality due to cervical cancer in Africa is 19.4 %.
Incidence of cervical cancer decreased to one-third from 15.0 to 9.8 (per 100,000
female populations) over the 20 years. United Kingdom has lower mortality rate
and ranked 157th among 184 countries worldwide. Mortality rates were higher in
the less developed regions of the world. It has been estimated that cervical cancer
contributes over 2.7 million years of life lost among women dying between the
ages of 25 and 64 years worldwide, some 2.4 million of which occur in the devel-
oping countries and only 0.3 million in the developed countries.
In USA, one-year survival rate of cervical patient is 87 %. Five-year survival rate
for all stages in cervical cancer is 68 %. Invasive cervical cancer, when detected at
early stage, can be successfully treated. In USA, the five-year relative survival rate
(measure of survival of cancer patients in comparison with the general population)
is 91 % and the rate decreases to 17 % when detected at a later stage. Cancer sur-
vival rate varies across the countries. Europe, North America, Australia, and New
Zealand have higher survival rate for cervical cancer. Survival data from African,
Asian, Caribbean, and Central American countries showed wide variation. Survival
rate was lower in Uganda (19 %) and Gambia (23 %), whereas it is higher in Seoul,
South Korea (76 %), and Hong Kong (77 %). Survival rate also varied drasti-
cally within the countries like India, where the 5-year survival rate for women in
Bhopal is 31 % and Chennai is 60 %. Lower cancer survival and higher mortality
in the developing countries is attributed to lack of screening facilities. In Singapore,
around 81 % of cervical cancer patients are diagnosed at the earlier stages, whereas
380 S. Deivendran et al.
in Chennai, India, it is only 7 %, similarly with the countries Costa Rica (33 %),
Manila, Philippines (35 %), and Cuba (53 %). Survival rate is higher in Singapore
when compared to other countries like USA. In China, Singapore, South Korea,
and Turkey, the median relative survival for cervical cancer is 63–79 %. One-,
three-, and five-year survival in invasive cervix cancer is 83, 61, and 54 %, respec-
tively in Costa Rica (Sankaranarayanan 2011; Coleman et al. 2008).
Inflammation is termed as the seventh hall mark of cancer, and chronic inflammation
is involved in cellular transformation, survival, proliferation, invasion, and metastasis.
Inflammation process involves tissue-remodeling events brought about by alterations
to epithelial, vascular, and immune cell function. These events occur by the involve-
ment of cytokines, chemokines, growth factors, and lipid mediators, and also by the
activation of transcription factors such as nuclear factor-κB, STAT3, and HIF-1.
Among the identified and categorized HPV’s, twelve HPVs namely 16, 18, 31,
33, 35, 39, 45, 51, 52, 56, 58, and 59 are defined as high risk by the World Health
Organization (WHO) along with types 68, 73 being as ‘possibly’ cancer causing.
HPV16 and HPV18 are found closely associated with high chances of cervical
cancer. Within the consolidated 8 early viral genes (E1–E8), E6 and E7 hold a lead
role in driving the HPV infection in the way to cancer. Recently, E5 is also added
to the same group considering its expression as a boon for the tumor progression
in HPV-infected conditions. Functional differences between high-risk type E6 and
E7 with low-risk type accounts for the potential of these viruses to be carcinogenic.
After infection, HPV amplifies the HPV E1/L1 genes through its tat protein which
leads to the viral replication and release of virions. Viral E6 protein binds to p53 and
degrades by ubiquitination. E7 protein binds to Rb protein and disrupts the Rb/E2F
complex leading to the increase in production of nitric oxide (NO), DNA damage
and the activation of the cyclooxygenase 2 (COX-2)/prostaglandin (PG)/PG G recep-
tor inflammatory pathways leading to increased inflammation and tumorigenesis.
Cyclooxygenase-Prostaglandin Pathway
Hypoxia-Inducing Factors
Inflammatory Cytokines
Inflammation is regarded as the seventh hallmark of cancer. Unlike the basic func-
tion of inflammation as in wound healing to destroy the infectious agents, during
cancer, inflammation persists to acquire a chronic condition and fails to undergo
healing process leading to a persistent infection. Occurrence of chronic inflamma-
tion is accompanied by release if certain agents from the immune response cell
that will support the better thriving of the pathogen in the host organism. This
enhances the betterment in incorporation of carcinogenic genetic material by the
virus to the host machinery. During an HPV infection, the inflammatory signal-
ing pathways mainly include pro-inflammatory cytokine pathway, interferon
pathway, TNF-α pathway, NF-κB, and COX-2 in order to link inflammation with
carcinogenesis. Thus, the key features of inflammation during a carcinogenesis
may include the infiltration of white blood cells, prominently tumor-associated
macrophages (TAMs); the presence of polypeptide messengers of inflammation
[cytokines such as TNF, IL-1, and IL-6, chemokines such as CCL2 and CXCL8,
and the occurrence of tissue remodeling and angiogenesis] (Colotta et al. 2009). A
better understanding of inflammation and tumor microenvironment is crucial for
the development of new therapeutic strategies based on the nature of tumor devel-
opment, progression, and metastasis.
Infectious agents have become a common cause for the development of tumor-
associated inflammation which triggers the inflammatory molecules in the micro-
environment to drive the persistence of tumor and therefore its progress. In
majority of the HPV infections, eradication of neoplastic lesions occurs in the ini-
tial stages, failure of which may impart the ability for HPV to evade the immune
barriers and integrate into the host genome. This whole process ultimately results
in a complex role for inflammation to occur during an HPV infection. The main
transmission of HPV pathogen to a cervix is via an infected partner’s semen dur-
ing coitus after which the virus resides in the epidermal mucosa with a total cut-
off from the connective tissue and paves the way to hide itself from host immune
system. Within this period, the virus attains the potential to induce mechanisms to
evade the host immune system by deregulating various pathways involving pat-
tern recognition receptor namely the toll-like receptor (TLR)-9 by the host. Mere
infection is incapable of inducing tumorigenesis in cervix. Under such instances,
oxidative stress induces the pathogen to drive the cell to attain oncogenic property.
Oxidative stress modifies the DNA and protein of the cell to provide a platform
for neoplastic development and its progress. This condition is further enhanced
384 S. Deivendran et al.
by binding of type I (IFNα/β) and type II (IFNγ) to the specific cell receptors
which in turn induces the transcription of IFN-induced genes via JAK-STAT path-
way (Chang and Laimins 2000). High-risk E6 and E7 proteins repress the STAT1
expression, which will inhibit its regulatory function on IFN response. It is also
found that high-risk E6 and E7 proteins downregulates type 1 IFN expression and
absence of influencing signals by these IFN during the process of antigen rec-
ognition leads to immune tolerance than expected immune responses (Sasagawa
et al. 2012). In vitro studies in support of these observation lead to clinical trials
of IFN to accomplish the eradication of viral infection on the onset of the disease.
Treatment of type I IFN to cells transfected with HPV31 episomes shows a loss of
viral episomes. This property of IFN is attributed to its clinical trials, but later on
reports indicated to explain the fact that HPV had gained resistance to overcome
the IFN therapy. It is now concluded that IFN response by HPV varies on the
type of IFN and also on the type of HPV infected and also cell specificity. Recent
reports came up with HPV16 oncoproteins E6 and E7 influencing the interferon
pathway. E7 oncoprotein physically interacts with the transcription factor and
interferon regulatory factor (IRF) 1, and hinders the binding of IRF1 on the IFNβ
promoter, thus inhibiting the transactivating function. This inhibition hinders the
heterodimerization of STAT1-STAT2, thus affecting the translocation to nucleus
(Park et al. 2000). Likewise, E6 oncoprotein interacts with IRF3 and repress the
transactivating function of IFNβ promoter by recruiting HDAC onto the promoter
(Ronco et al. 1998). Rincon-Orozco et al. (2009) came up with the regulatory
aspects of IFNκ by HPV16 E6 oncoprotein. Clinical sample revealed the informa-
tion that IFNκ was downregulated in cervical patients compared to that of normal
ones. This phenomenon was emphasized with the epigenetic silencing of IFNκ by
E6 protein.
Despite the antiviral properties of interferon, there are contradictory reports
regarding IFNβ. It is in limelight now that IFNβ facilitates the transcription of
HPV16 by promoting the binding of IRF1 to the viral promoters. Interferon also
plays a crucial role in proliferation. In vitro studies in HeLa cell lines, an HPV18
cervical cell line is shown to proliferate with the induction of IFNα2b. Evidences
from certain studies confirm that IFN-β is able to induce replication of HPV11,
HPV16, and HPV31 in human keratinocytes. Moreover, on continual IFN-β treat-
ments, it is reported that cells expressing high-risk type cutaneous HPV38 undergo
senescence by the induced expression of the tumor suppressor PML, known to be
downstream effector of IFN (Chiantore et al. 2012).
Cytokine production is a remarkable property of viral-induced human cancers.
As primary site of HPV infection, the keratinocytes are the immediate source of
cytokine. IL and TNF mainly contribute to this aspect. TNF has a definitive role on
any inflammation caused due to infection. Basically, TNFs are majorly involved
in the regulation of inflammation by binding to its cell-specific TNF receptors
(TNFR1 or TNFR2) in order to elicit signaling pathways against a viral infec-
tion, induce apoptosis, growth arrest, or aid in cell proliferation. TNF-α plays as
a growth stimulator for epidermal growth factor (EGF) or serum-depleted cervi-
cal cancer cells (Gaiotti et al. 2000). TNF-α acts as a potent keratinocyte inhibitor
386 S. Deivendran et al.
during the course of viral entry and onset of inflammation and elicit extrinsic
apoptotic pathway (Basile et al. 2001). Extrinsic pathway is initiated by the bind-
ing of these cytokines to receptors mainly TNFR1 or TRAIL. It is reported that E6
high-risk type proteins can bind on these TNFR1 or TRAIL and inhibit the down-
stream pathways (Filippova et al. 2002). More often, it is also found that E6 pro-
tein binds on to FADD and caspase8, thus blocking the FAS-mobilized apoptotic
response (Filippova et al. 2004; Garnett et al. 2006). In response to intrinsic path-
way, E6 plays a crucial role by interacting with Bax and Bak proteins of Bcl2 fam-
ily of pro-apoptotic proteins, which induces the upregulation of IAP and survivin
(Garnett and Duerksen-Hughes 2006). Thus, literature based on TNF gives out
the point that they are responsible for growth arrest in an HPV-infected keratino-
cyte. TNF activates NF-κB pathway to upregulate the expression of p21CIP1/WAF1,
which is a well-known cyclin-dependent kinase (Cdk) inhibitor. Moreover, TNF is
also having a critical function in downregulating certain cell cycle proteins such as
cyclinA, cyclinB, cdc2, and so on.
Polymorphism in inflammatory genes has become a common report, and it
stands as a potential therapeutic biomarker in cervical carcinomas. Polymorphism
detected in an anti-inflammatory gene IL-10 can lead to its underexpression,
thus allowing the cancer progression. PCR–RFLP-assay-based cohort stud-
ies implicated the potential effect of SNP on TNF-α-238 G/A in reducing the
risk of cervical cancer, whereas the multifunctional cytokine IL-10 is not, the
polymorphism of which are highly associated with the risk of cervical cancer
(Barbisan et al. 2012).
Persistence of HPV related to age old cervical cancer shows circulating inflam-
matory cytokines, namely TNF-α and also IL-8 (Kemp et al. 2010). An increasing
levels of resistin and Fas was noted in such a condition where an increased levels
of resistin, an adipokine, and sFas is observed in HPV-infected older women with
decreased immune function (Baker et al. 2011). Complexing of E7 with Rb leaves
a compliment by increasing the level of p53, a tumor suppressor gene, which hap-
pens to be the intrinsic mechanism of the cell to undergo apoptosis. To protect this
adverse situation the viral particle, E6 comes into play which aids in the protea-
somal degradation of p53 by recruiting E6AP (E6-associated protein), a HECT
domain containing E3 ubiquitin ligase (Huibregtse et al. 1991). In response to IFN
stimulus, p53 gains antiproliferative function which is highly tackled by high-risk
HPV proteins. Senescence is promoted in HPV16-expressing keratinocytes which
is mediated by acetylated p53.
Among the different strategies used by viruses in its survival, a colorful role
is engraved by the expression of class I MHC. HPV nonstructural viral proteins
like E5 protein have been shown to interact with several cellular processes in
15 The Role of Inflammation in Cervical Cancer 387
order to impair the antigen presenting function of MHC class I expression, thereby
suppressing the Th1 pathway to reduce the functional role of IFNγ. It is evident
that overexpression of HPV E5 attenuates MHC class I molecules to sequester
itself to the Golgi apparatus (Ashrafi et al. 2005). The viral protein, E7, may block
the ability of interferon-γ (IFN-γ) to induce IRF-1 expression, in turn inhibiting
the expression of downstream genes related to MHC class I antigen processing
and presentation. Recent study by Zhou et al. (2011) demonstrated that expression
of HPV16 E7 as a transgene product in epithelial cells does not directly impair,
but rather slightly increases MHC class I expression.
Direct role of inflammation to survival is evident from the participation of NF-κB
by bypassing apoptotic mechanism. NF-κB activation induces several pro-inflamma-
tory cytokines which are prominent in supporting the progression of HPV-induced can-
cer (Karin 2006). In cervical cancer cell lines, a constitutive activation of NF-κB was
studied which is usually seen to be accompanied with p50 and p65, the heterodimeric
complex of RelA family of proteins in the nucleus during the course of tumor progres-
sion. NF-κB acts in targeting apoptosis, cell cycle, and cell adhesion in the stage of
neoplasia. NF-κB competes with p53 for common transcriptional co-activators such as
p300 and CBP (Okamoto et al. 2007) and also for the binding to p21 promoter (Ma
et al. 2008). Effect of E6 proteins comes in act by degrading p53 via a tripartite com-
plex formation with p53 along with the acetylating agent p300, thus hindering the
acetylating property of p53 and suppression in the activation of p53-inducible genes for
senescence (Patel et al. 1999). Unlike the high-risk types, low-risk E6 can directly inter-
act with p53 which is an outstanding feature for survival mechanism in low-risk types.
From the development of macrophages from monocytes, there exist severe con-
ditions of hypoxia which is very evident in tumor microenvironment. At this point,
there is an upregulation of HIF1α which plays a crucial role in the survival of cells
during hypoxia. HIF1α is expressed immensely in the inflammation site leading to
support the survival by regulating several angiogenic genes like VEGF. Moreover,
studies show that in cervical cancer cell lines, HPV E7 enhances HIF1α regulating
the transcription activation of various pro-angiogenic genes by inhibiting HDAC
activity (Bodily et al. 2011). Thus, we can see that HPV protein is facilitating the
existing microenvironment rendering it favorable for its own survival and prolif-
eration through their epigenetic control.
During a persistent infection with HPV, there occurs tissue injury which paves
the way for epigenetic alterations and remodeling of epithelial cellular events
promoting angiogenesis, invasion, finally leading to metastasis. Angiogenesis,
invasion, and metastasis are a complex network underwent by tumor for the well
progression and spreading of the own. In such a condition, an overall remodeling
occurs causing the degradation of extracellular matrix (ECM), cell migration,
proliferation, and also generation of vasculature. Angiogenesis and inflammation
are two mutually dependent processes for which inflammatory molecules come
in contact with cells such as the endothelial cells, fibroblast cells, and also the
ECM to promote angiogenesis. In cancer cells, angiogenesis occurs as an abnor-
mal process where there is an imbalance between pro- and anti-angiogenic fac-
tors requiring the activation of several receptors by growth factors coupled with
the triggering of inflammation. Attack of an HPV fires inflammation and lead to
the production of monocytes. It is noted that there are two types of macrophages
produced during such a condition in which one acts as tumor supportive and the
other as tumor suppressive. Tumor-supportive macrophages are greatly involved
in angiogenesis. These macrophages are often referred as TAMs. TAM produces
a cytokine MCP-1, which is an indication of macrophage accumulation at the
tumor site, in turn giving a feedback regulation.
15 The Role of Inflammation in Cervical Cancer 389
Even though the direct relationship between HPV and inflammation remains
undecided and contrary, experiments in animal model support to the fact that HPV
controls inflammatory pathways in the host. Direct evidences exist describing the
role of HPV16 early genes expressed under human keratin 14 promoter which
facilitate the induction of the chemokine CCL2 from the neoplastic lesion to recruit
macrophage in the tumor microenvironment. In the development of cancer COX-2-
derived PGE2 has a direct effect on inflammation. HIV1 infection augmented with
HPV infection is associated with increased cervical COX-2 and increased systemic
PGE2 levels (Fitzgerald et al. 2012). Findings in human cervical epithelial cells of
neoplasia explain that late genes of HPV16 namely E6 and E7 upregulate COX-2
activate COX/PG pathways. The expression of pro-inflammatory cytokine inter-
leukin-32 (IL-32) is very evident in high-risk HPV-positive cervical cancer which
is mediated by COX-2 stimulation controlled by E7. There also exist an E7 and
COX-2 downregulation in the IL-32γ overexpressing cells suggesting a feedback
390 S. Deivendran et al.
mechanism by IL-32γ on E7 and COX-2 cervical cancer cells (Lee et al. 2011). In a
study related to mouse corneal angiogenesis induced by IL-1β, inhibition of COX-2
almost completely attenuated angiogenesis (Kuwano et al. 2004).
IL-17 is a pro-inflammatory cytokine produced by Th17 cells, found to play a
main role in inflammation-triggered oncogenesis. This observation is further sup-
ported by studies in vivo that IL-17 overexpressing human cervical cancer shows
high oncogenic growth and the functional status of IL-17 is determined to be pro-
angiogenic (Tartour et al. 1999). Studies in transgenic mice with defective E6 pro-
tein ensures that there is no development of potent tumor in these mice models
due to mutated PDZ binding domain which is associated with PDZ protein and in
turn a direct target of p53. Mutations linked to the mentioned PDZ binding domain
of the E6 protein may lead to ultimate loss of function of the protein in terms of
pathogen survival.
Macrophages secretes (TNF-α), and their role in risk of cervical cancer remains
controversial until meta-analysis was performed by Ding et al. where polymor-
phism status is studied in 2298 cases and 1903 controls. They found that poly-
morphism in TNF-α-308 G > A is a risk factor for developing cervical cancer in
Asians and whites but not in Africans (Ding et al. 2012). Recently, TNF-α pol-
ymorphism rs1800629 was studied in Chinese population, which included three
groups with Group 1 consisted of 285 high-risk HPV-positive cervical cancer
patients, Group 2 with 225 high-risk HPV-positive patients without cervical can-
cer, and Group 3 with 318 HPV-negative women with no cervical cancer. They
found that TNF-α polymorphism rs1800629 has no association with HPV infec-
tion or risk of developing cervical cancer (Wang et al. 2012). Polymorphisms
TNFA-308G/A (rs1800629) and -238G/A (rs361525) are associated with the
increase in risk in cervical cancer. Association of polymorphisms in pro- and
anti-inflammatory cytokines (TNF-α, TNFA, and interleukin, IL-10) with cervi-
cal cancer was studied in 2009 (Singh et al. 2009). The study included 150 histo-
pathologically confirmed cervical cancer patients and 162 age, ethnically matched
cervical cytology-negative controls. They found that polymorphism in TNFA
(-308 G > A) is associated with developing cervical cancer stages early (IB) and
advanced stages (III). They have also concluded that polymorphism IL-10 (-819
C > T) is not associated with cervical cancer (Singh et al. 2009). Recently, pol-
ymorphisms were analyzed in North Indian population for IL-6 and IL-10, and
contradictorily, they found that polymorphism IL-4 Rp1/Rp2 and IL-10 (AC)
genotype is associated with risk in cervical cancer (Shekari et al. 2012) which
supported the previous results where promoter polymorphism in IL-6 promoter,
15 The Role of Inflammation in Cervical Cancer 391
which regulates inflammation has the association with increase in cervical cancer
(Gangwar et al. 2009).
Hydralazine/valproate Neolpharma S.A. de Repositioned CC, myelodysplastic Phase III DNA methylation and HDAC
C.V. Mexico small syndrome (MDS) inhibitors, respectively
molecules and cutaneous T-cell
lymphoma (CTCL)
Sorafenib Bayer Small molecule Metastatic renal Phase II Inhibit multiple intracellular (CRAF,
cell carcinoma, BRAF, and mutant BRAF) and cell
hepatocellular surface kinases (KIT, FLT-3, RET,
carcinoma VEGFR-1, VEGFR-2, VEGFR-3,
and PDGFR-b)
Sunitinib Pfizer Small molecule Renal cell carcinoma, Phase II Inhibits (PDGFRa and PDGFRb)
GISTs after disease (VEGFR1, VEGFR2, and
progression on or VEGFR3), stem cell factor
intolerance to imatinib receptor (KIT), Fms-like tyrosine
kinase-3 (FLT3)
(continued)
393
Table 15.1 (continued)
394
DPD dihydropyrimidine dehydrogenase, EGF epidermal growth factor, EGFR epidermal growth factor receptor, (FGFR)-1 and -3 fibroblast growth factor receptor,
cytokine receptor (Kit), mTOR mammalian target of rapamycin, NSCLC non-small-cell lung cancer, PDGFR platelet-derived growth factor receptor, VEGF vascular
15 The Role of Inflammation in Cervical Cancer
15.5 Conclusion
HPV infection is the one of the most common causes of sexually transmitted viral
disease. Preceded by breast cancer, cervical cancer holds a second position among
the most common cancer prevalent in women across the world. The HPV–cervical
cancer bond is so strong that among the 30 HPV types identified that can spread
through sexual contact to infect primarily the cervix, vagina, vulva and penis; four
are most often associated with cervical cancer malignancies. HPV-related cervi-
cal cancer is one of the typical examples for the role of viral infection in devel-
oping a malignancy. The primary viral particles responsible for this malignancy
by altering the cellular intrinsic pathways are E5, E6, and E7 proteins. The com-
plex interplay between the HPV particles and inflammation is well deciphered and
their functional and regulatory aspect in the development of malignancy is also
highly focused. The molecular studies in HPV-related cervical cancer had solidi-
fied its epidemiological side also. This helped in decreasing the incidence of mor-
tality related to cervical cancer and the emergence of Pap smear tests had greatly
added to the importance in detecting the cancer at a very early stage which has
a significant influence on the morbidity of cervical cancer. Although Pap smear
remains the major screening method, HPV-based testing has been more effective
than cytology for the detection of cervical cancer precursors and prevention of cer-
vical cancer. Successful designing of effective therapeutics and vaccines has also
greatly helped to eradicate the prevalence of HPV infection at least in developed
countries and contributed to the control of cervical cancer.
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Chapter 16
The Role of Inflammation in Liver Cancer
Anupam Bishayee
A. Bishayee (*)
Department of Pharmaceutical Sciences, School of Pharmacy,
American University of Health Sciences, 1600 East Hill Street,
Signal Hill, CA 90755, USA
e-mail: [email protected]
16.1 Introduction
factor for HCC as men are more susceptible than women with a male-to-female
ratio of 2:1–4:1 (El-Serag and Rudolph 2007; Ruggieri et al. 2010).
Current treatment options for patients with HCC are limited. Surgical resection
is the treatment of choice for patients with well-preserved hepatic functions.
Unfortunately, it involves a high risk of postoperative complications and tumor
recurrence. At the present time, liver transplantation is the most effective way to
improve the survival of HCC patients (Dutkowski et al. 2010). However, this option
has limitation due to inadequate number of qualified donors as well as occurrence
of the disease in the transplanted liver. Although various strategies, such as cry-
oablation, microwave ablation, radio-frequency ablation, trans-catheter arterial
chemoembolization, percutaneous ethanol injection, and yttrium-90 microspheres,
are available for inoperable patients, difficulty in the management of patients and
tumor recurrence remain two significant limitations for these treatments (Senthil
et al. 2010). Currently, sorafenib [N-(3-trifluoromethyl-4-chlorophenyl)-N′-(4-(2-
methylcarbamoyl pyridin-4-yl)oxyphenyl)urea, Nexavar®, Bayer] is the only drug
approved by the United States Food and Drug Administration for the treatment of
advanced HCC based on two large phase III clinical trials (Llovet et al. 2008; Cheng
et al. 2009). Nevertheless, only moderate improvement of survival, a number of
adverse side effects, and high costs underscore the need for other novel therapeutic
as well as preventive approaches for HCC (Je et al. 2009; Lu 2010; Bishayee et al.
2010a; Bishayee 2012).
16.4.1 Cytokines
Cytokines are synthesized by various cell types in the liver. Hepatocytes also
express cell surface receptors for cytokines. Various inflammatory cytokines, such
as interleukin-1α (IL-1α), IL-1β, IL-6, IL-8 and tumor necrosis factor-α (TNF-α),
participate in chronic hepatic inflammation, and IL-6 is probably the most impor-
tant and studied one (Budhu and Wang 2006; Naugler and Karin 2008). During
chronic hepatitis, activated Kupffer cells produce IL-6 which enhances local inflam-
matory responses and induce compensatory hepatocyte proliferation resulting in
neoplastic transformation of hepatocytes (Naugler and Karin 2008). Elevated serum
levels of IL-6 have been found in patients with chronic liver ailments, including
alcoholic hepatitis, NASH, and hepatic infections with HBV and HCV (Deviere
et al. 1989; Lee et al. 1998; Wieckowska et al. 2008). Moreover, higher serum levels
of IL-6 have been found to be associated with increased risk of HCC development
in patients with chronic hepatitis B and C infections (Nakagawa et al. 2009; Wong
et al. 2009). All these reports underscore the pivotal role of IL-6 in chronic inflam-
mation-mediated hepatocarcinogenesis in humans.
An elegant study conducted by Naugler et al. (2007) investigated the role of
IL-6 in liver cancer using IL-6 knockout mouse model. IL-6 knockout mice
exhibited a significant reduction of diethylnitrosamine (DENA)-initiated HCC
development, suggesting direct involvement of IL-6 signaling in experimental
hepatocarcinogenesis. The results from this study also showed the critical role
played by the toll-like receptor (TLR) adapter protein MyD88 in the production of
IL-6 by Kupffer cells during DENA-induced HCC development. The production
of IL-6 by necrotic hepatocytes was reduced considerably in Kupffer cells defi-
cient for MyD88. The ablation of MyD88 also suppressed DENA-induced hepatic
tumorigenesis, indicating that IL-6 production by the TLR/MyD88/nuclear factor-
kappaB (NF-κB) pathway is critical for HCC development. Another study from
the same laboratory found that DENA-induced acute inflammatory response is
triggered by IL-1α release from necrotic hepatocytes, and IL-1α stimulates IL-6
production by Kupffer cells (Sakurai et al. 2008). Moreover, the investigators have
found that IL-1α released by damaged hepatocytes is essential for the compensa-
tory proliferation which is essential for DENA-initiated hepatocellular carcinogen-
esis. Rogers et al. (2007) proposed a multistep model linking chronic hepatitis to
406 A. Bishayee
16.4.2 NF-κB Pathway
and suppresses tumor formation in other models. During early stages of liver tumor
development, the cytoprotective role of NF-κB prevails as it prevents hepatocyte
death. In late stages, NF-κB promotes tumor cell survival and proliferation.
The influence of NF-κB signaling in myeloid cells has also been investigated
utilizing DENA-induced HCC in mice. Ablation of IKK-β in both hepatocytes and
myeloid cells (including Kupffer cells) has been found to inhibit DENA-induced
HCC development (Maeda et al. 2005). This effect was accompanied by dimin-
ished production of proinflammatory cytokines, such as IL-6, TNF-α and hepato-
cyte growth factor, which are secreted by non-parenchymal cells in response to
dying hepatocytes to stimulate compensatory proliferation of remaining hepat-
ocytes (Maeda et al. 2005). Another study showed that IKK-β in myeloid cells,
especially in Kupffer cells, has also been involved in the development of meta-
static liver malignancy through IL-6 production (Maeda et al. 2009).
Several reports support the notion that NF-κB plays an indispensable role in
the promotion of obesity-associated HCC. The effects of obesity-induced activa-
tion of NF-κB are believed to be mediated through the synthesis of NF-κB target
genes, including IL-1β, IL-6, and TNF-α (Shoelson et al. 2006). Experimental
results showed that high-fat diet increased NF-κB activation, resulting in sustained
elevation of IKK-related kinase IKK-ε in the liver, adipocytes, and adipose tissue
macrophages. Interestingly, IKK-ε ablation was found to reduce the expression
of inflammatory cytokines and protected mice from high-fat diet-induced obesity,
chronic hepatic inflammation, and hepatic steatosis (Chiang et al. 2009). Wang
et al. (2009a) reported that administration of DENA enhanced the development of
preneoplastic lesions in the livers of rats fed with a high-fat diet with simultaneous
increase in TNF-α/NF-κB signaling and ERK-related hepatocyte proliferation. The
role of hepatic NF-κB in obesity-associated liver tumorigenesis has been inves-
tigated in mice with liver-specific inactivation of the NF-κB essential modulator
gene NEMO exposed to a high-fat diet. Hepatic NEMO deficiency has been found
to synergize with high-fat diet in the development of liver steatosis, increased
inflammation, and aggravated liver tumorigenesis (Wunderlich et al. 2008).
16.4.3 JAK-STAT Signaling
STAT proteins are known to play vital roles in cytokine signaling pathways
involved in cell growth and differentiation in various species, including mammals
(Darnell et al. 1994). The STAT family consists of seven members, such as STAT1,
STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6. Among STAT family pro-
teins, STAT3 has gained substantial attention as a convergent point for a number of
oncogenic signaling pathways as well as regulator of signal transduction pathways
of several proinflammatory cytokines and growth factors involved in hepatic dam-
age and repair mechanisms (Taub 2003; Costa et al. 2003). Following phosphoryl-
ation and activation by JAKs, especially by JAK2, STAT3 undergoes dimerization
before entry to nucleus for DNA binding (Yoshimura et al. 2007).
16 The Role of Inflammation in Liver Cancer 409
16.4.5 Cyclooxygenase-Prostaglandin Pathway
in early as well as late states of hepatic cancer (Sung et al. 2004; Yildirim et al.
2008). COX-2-derived PG signaling has also been shown to be involved in chol-
angiocarcinoma, a highly malignant epithelial tumor arising within the biliary tract
(Wu 2005).
Experimental evidence supports a close interaction between COX-2 and
EGFR signaling pathways. The activation of EGFR in human HCC cells has been
found to upregulate COX-2 expression and PGE2 synthesis (Dajani et al. 2008).
Likewise, COX-2-derived PGE2 is known to transactivate the EGFR receptor (Wu
2005; Han et al. 2006). Moreover, COX-2-derived prostanoids may be one key sig-
nal in the activation of EGFR involved in the early stages of hepatic inflammation
and neoplasia (Berasain et al. 2005; Castillo et al. 2006).
16.4.7 Inhibitor of Apoptosis
16.4.8 Chemokines
16.4.9 MicroRNAs
Numerous in vitro, in vivo, and clinical studies as described above have validated
the critical role of chronic inflammation in the development and progression of
liver cancer. Identification of cellular pathways necessary for the initiation and
propagation of inflammatory cascade in HCC not only aids in understanding the
pathophysiology, progression, and diagnosis but also provides a valuable tool in
designing effective prevention and treatment of this disease. Hence, interfering
with various inflammatory signaling molecules and pathways may offer poten-
tial opportunities for the development of novel drugs for the prevention as well as
therapy of HCC. The following section highlights preclinical animal studies show-
ing innovative approaches of targeting inflammatory mediators and signaling by a
variety of natural compounds as well as synthetic agents.
414 A. Bishayee
16.5.1 Natural Compounds
(continued)
Table 16.1 (continued)
416
↑IκB-α
cinogenesis in male
Sprague-Dawley rats
Etodolac Spontaneously developed ↓HCC nodules ↓PGE2 Liu et al. (2006)
HCC in male fatty liver
Shionogi mice
Fenretinide DENA-initiated and 2-AAF- ↓GST-P-positive foci; ↓iNOS; ↑IκB; ↓NF-κB Simile et al. (2005)
promoted hepatocarcino- ↓hepatic nodules and
genesis in male F344 rats HCCs
JTE-522 CDAA-induced hepato- ↓GST-P-positive foci; ↓HCC ↓COX-2; ↓PGE2 Yamamoto et al. (2003)
carcinogenesis in male
Wistar rats
Nimesulide CDAA-induced hepatocar- ↓GST-P-positive foci; ↓COX-2 Denda et al. 2002
cinogenesis in male F344 ↓hepatic nodules; ↓HCC
rats
(continued)
417
Table 16.1 (continued)
418
Nodule
Foci
COX-2
iNOS
NF- B
been studied for its chemopreventive activity during DENA-initiated early hepa-
tocarcinogenesis in rats. Chrysin administration significantly reduced the number
and size of hepatic nodules with an inhibition of hepatic expression of COX-2 and
NF-κB (Khan et al. 2011).
Curcumin (diferuloylmethane) is the predominant active component present in
the roots of perennial plant turmeric (Curcuma longa). Curcumin has been shown
to prevent DENA-induced hepatic hyperplasia in rats with reduced hepatic NF-
κB expression (Chuang et al. 2000). Yoysungnoen et al. (2006) reported antian-
giogenic potential of curcumin in nude mice xenografted with HepG2 cells.
Additional studies showed suppression of intratumor COX-2 expression.
Epigallocatechin-3-gallate (EGCG) is the primary catechin present in green tea.
Shimizu et al. (2011a) have investigated the effects of EGCG on the development
of DENA-induced liver tumorigenesis in obese and diabetic mice. EGCG in drink-
ing water has been found to inhibit the phosphorylation of the IGF-IR, ERK, Akt,
STAT3, and JNK proteins in the livers of experimental mice. The serum levels of
insulin, IGF-I, IGF-II, free fatty acid, and TNF-α were all decreased by drinking
EGCG, which also lowered the expression of TNF-α, IL-6, IL-1β, and IL-18
mRNAs in the livers.
Genistein, a phytoestrogen, can be found in soybeans and other legumes, such
as chickpeas. Genistein retarded the growth of established tumors generated by
injecting HepG2 cells in nude mice. Mechanistic results showed suppression of
Akt activation, NF-κB activity, and downregulation of NF-κB regulated gene
COX-2 (Ma et al. 2011).
Geranylgeraniol, a dietary diterpene, showed reduction in the number and size
of GST-P hepatic foci and nodules during the pre- and post-initiation stages of
DENA-induced hepatocarcinogenesis in rats. This study also revealed decreased
cell proliferation, DNA damage, and NF-κB p65 expression following the treatment
with geranylgeraniol (Espindola et al. 2005).
Lycopene, a bright red carotenoid pigment, is mostly found in tomatoes along
with other red fruits and vegetables, including red bell peppers, red carrots, water-
melons, and papayas. Lycopene as well as tomato extract curtailed the development
of GST-P foci in DENA-initiated NASH-promoted hepatocarcinogenesis in rats.
Additional results showed reduced activation of ERK and NF-κB and decrease in
mRNA expression of proinflammatory cytokines, such as TNF-α, IL-1β, and IL-12
(Wang et al. 2010).
Morin (3,5,7,2′,4′-pentahydroxyflavone), a bioflavonoid, is found in red wine,
almonds, figs, and Osage orange. Sivaramakrishnan and Devaraj (2009, 2010)
provided evidence for morin-mediated reversal of hepatic ultra-structural changes
in DENA-exposed animals via apoptosis induction through modulation of the
PI3K/Akt and NF-κB signaling pathways.
Perillyl alcohol, a monoterpene, is found in lavender oil, sage, cherries, orange
peel, and peppermint. Mills et al. (1995) showed that dietary perillyl alcohol treat-
ment in rats exposed to DENA inhibited hepatic tumor growth. The mRNA levels
of mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF-IIR),
16 The Role of Inflammation in Liver Cancer 421
and TGF β type I, II, and III receptors (TGF-β I, II, III R) were also significantly
increased in the liver tumors of perillyl alcohol-treated rats.
Our laboratory has demonstrated that a pomegranate-based formulation containing
various phytochemicals, including caffeic acid, gallic acid, and ellagic acid, exerts
a striking chemopreventive activity in rats subjected to DENA-PB hepatocar-
cinogenesis as evidenced by reduced incidence, number, multiplicity, size, and
volume of hepatocyte nodules as well as GGT-positive focal number and area
(Bishayee et al. 2011b). We have also reported that pomegranate bioactive phyto-
constituents are capable of suppressing DENA-induced inflammatory cascade by
reversing the elevated expression of iNOS, COX-2, and NF-κB during experimental
hepatocellular carcinogenesis in rats (Bishayee et al. 2013b).
Resveratrol (3,4′,5-trihydroxy-trans-stilbene), a naturally occurring antioxidant
and anti-inflammatory agent found in grapes, berries, peanuts, plums as well as
red wine, has been shown to prevent the development or reduce the growth of
tumors in multiple organs (Bishayee 2009). According to our study, dietary resver-
atrol reduced the incidence, total number, and multiplicity of hepatocyte nodules
(Bishayee and Dhir 2009). Ancillary studies showed that resveratrol dose-depend-
ently suppressed DENA-induced elevated expressions of hepatic inflammatory
markers, such as iNOS, COX-2, and NF-κB, and attenuated the translocation of
NF-κB to the nucleus by stabilizing IκB (Bishayee et al. 2010b, c). Additionally,
we have also observed that resveratrol treatment reversed the DENA-induced
alteration in the level and expression of hepatic TNF-α, IL-1β, and IL-6 (Mbimba
et al. 2012).
Saikosaponin-d, a triterpene saponin, is extracted from Bupleurum falcatum L.
(Umbelliferae). A recent study has investigated the chemopreventive potential of
Saikosaponin-d against hepatocarcinogenesis and its possible molecular mechanism
in vivo. The liver nodule formation, tumorous invasion to surrounding organs, and
increased cellular atypia induced by DENA were markedly reduced by intraperito-
neally injected saikosaponin-d. The immunohistochemical staining demonstrated
that the expression of COX-2 and C/EBPβ (a protein involved in inflammation and
carcinogenesis) was significantly increased in tumor cells and macrophages of liver
tissue from DENA-treated rats, whereas the expression of these two proteins was
markedly lowered in the saikosaponin-d plus DENA group (Lu et al. 2012).
Silymarin is a complex mixture of polyphenolic flavonoids present in the
seeds of milk thistle (Silybum marianum L. Gaertner). Silibinin (also known as
silybin) represents the major active component of silymarin. Silibinin reduced the
growth transplanted HuH7 tumor through the inhibition of phosphatase and ten-
sin homolog (PTEN)/p-Akt and ERK signaling and reduced the level of NF-κB
(Cui et al. 2009). It has been showed time that both pre- and post-treatment of
DENA-initiated rats with silymarin significantly inhibited the multiplicity and size
of hepatic nodules (Ramakrishnan et al. 2006). A separate study from the same
laboratory documented that dietary silymarin supplementation downregulated
the hepatic expression of COX-2 during DENA-induced hepatic carcinogenesis
(Ramakrishnan et al. 2008).
422 A. Bishayee
16.5.2 Synthetic Agents
Shimizu et al. (2011b) examined the effects of acyclic retinoid on the development
of DENA-induced liver tumorigenesis in C57BLKS/J- +Leprdb/+Leprdb obese
mice. The development of liver cell adenoma was significantly inhibited by acyclic
retinoid which also markedly reduced the phosphorylation of ERK. The serum levels
of TNF-α and the expression of levels of TNF-α, IL-1β, and IL-6 mRNA in the liv-
ers of DENA-treated mice were decreased by acyclic retinoid treatment, indicating
attenuation of the chronic inflammation induced by excessive fatty deposits.
Aspirin (acetyl salicylic acid) significantly reduced the degree of highly
metastatic HCC developed in rats by sequential treatment with DENA and
N-nitrosomorpholine (NMOR). This effect was associated with aspirin-mediated
downregulation of COX-2 in primary HCC (Futakuchi et al. 2002).
The chemopreventive effect of celecoxib, a specific COX-2 inhibitor, on the
development of liver preneoplastic lesions in rats has been evaluated using a
medium-term experimental hepatocarcinogenesis protocol. A reduction by 80
and 90 % both in the number and size of altered hepatic foci was observed in the
group treated with celecoxib following carcinogen treatment, respectively. Neither
COX-2 expression nor PGE2 production has been altered by the hepatocarcino-
genic exposure or celecoxib treatment. Interestingly, celecoxib inhibited the trans-
location of Rel A/p65 to the nucleus from the cytoplasm with significant effect on
stability of the repressor IκB-α (Márquez-Rosado et al. 2005).
The effect of etodolac ([±]-1,8-diethyl-1,3,4,9-tetrahydropyrano-[3,4-b] indole-
1-acetic acid), a specific COX-2 inhibitor, on spontaneous development of HCC in
fatty liver Shionogi mice has been evaluated. The development of HCC has been
suppressed slightly in the high-dose group and suppressed markedly in the low-
dose group, although the development of fatty liver has not been inhibited in either
group. Plasma PGE2 levels were also decreased significantly in the low-dose
group, consistent with the suppression of HCC (Liu et al. 2006).
Simile et al. (2005) have investigated the chemopreventive potential and pos-
sible mechanisms of action of fenretinide [N-(4-hydroxyphenyl)retinamide], a
synthetic retinoid, using rats subjected to the “resistant hepatocyte” protocol that
included initiation by DENA followed by 2-acetylaminofluorene (2-AAF) treat-
ment and partial hepatectomy. Fenretinide suppressed the development of GST-P-
positive foci, nodules, and HCC through inhibition of iNOS and inactivation of
NF-κB.
Yamamoto et al. (2003) have investigated the inhibitory effects of JTE-522
[(4-(4-cyclohexyl-2-methyloxazol-5-yl)-2-fluorobenzenesulfonamide], a selective
COX-2 inhibitor, on liver fibrosis and carcinogenesis induced by CDAA. JTE-
522 significantly inhibited fibrosis and development of preneoplastic lesions in a
dose-dependent manner and completely inhibited generation of cirrhosis and HCC
at both low and high doses. Mechanistic studies indicated that the CDAA model
displayed upregulation of several biomarkers, including COX-2 and PGE2, and
increased the proportion of activated hepatic stellate cells, proliferating cell nuclear
16 The Role of Inflammation in Liver Cancer 423
antigen index, and CD45-positive inflammatory cells in the liver. JTE-522 effec-
tively reversed all these changes.
The chemopreventive efficacy of nimesulide, a specific COX-2 inhibitor, has been
tested in CDAA-induced rat hepatocarcinogenesis. Administration of nimesulide
through diet decreased the number and size of preneoplastic, enzyme-altered liver
foci, multiplicity of neoplastic nodules and hepatocellular carcinomas, and prevented
the development of cirrhosis with reduced expression of COX-2 (Denda et al. 2002).
The effects of pitavastatin, a drug used for the treatment of hyperlipidemia,
on the development of DENA-induced liver preneoplastic lesions have been
examined in C57BL/KsJ-db/db (db/db) obese mice. Feeding of animals with
10 ppm pitavastatin significantly inhibited the development of hepatic prema-
lignant lesions (foci of cellular alteration) as compared to the untreated group
through inhibition of cell proliferation and induction of apoptosis. Pitavastatin
improved liver steatosis, decreased free fatty acid and aminotransferases levels,
while increasing adiponectin levels in the serum. Additionally, the serum levels of
TNF-α and the expression of TNF-α and IL-6 mRNAs in the liver were decreased
by pitavastatin treatment (Shimizu et al. 2011c).
Roxithromycin, a macrolide antibiotic, inhibited oxidative stress as measured
by the level of thiobarbituric acid-reactive substances, NO production, and acti-
vation of NF-κB during DENA-induced hepatic carcinogenesis in rats. All these
results were associated with a dose-dependent inhibition of hepatic tumor volume
in experimental animals (Ueno et al. 2005).
SC-236, a selective COX-2 inhibitor, has been tested for its antihepatocarci-
nogenic potential using a choline-deficient, ethionine-supplemented (CDE) diet-
induced rodent model of HCC. The test compound not only suppressed hepatocyte
peri-cellular fibrosis and steatosis, but also inhibited the early stages of HCC
(Davies et al. 2006).
Sodium selenite has been found to exert chemoprevention of DENA-initiated
and 2-AAF-promoted hepatocarcinogenesis in rats as evidenced from histopatho-
logical observations with simultaneous inhibition of hepatic NF-κB expression
(Alwahaibi et al. 2010).
Activation of Ras and its downstream signaling pathways are likely to contrib-
ute to the development of hepatocarcinoma. It has been shown that intraperitoneal
injections of the S-trans- trans-farnesylthiosalicylic acid (FTS), a Ras inhibitor,
blocks Ras activation and prevents heptocarcinoma development in rats challenged
with DENA (Schneider-Merck et al. 2009). A follow-up study from the same
laboratory showed that DENA-induced activation of NF-κB and STAT3 has been
abrogated by FTS treatment. Although FTS treatment showed no effect on DENA-
induced elevation of TNF-α, IL-6, and TLR4, it significantly reduced phosphoryla-
tion of the MAPK p38 and of the p70S6 kinase, a surrogate marker for mTOR
activation, without affecting ERK and Akt phosphorylation (Stärkel et al. 2012).
TNP-470 (O-chloroacetyl-carbamoyl-fumagillol) is a synthetic derivative
of fumagillin, a naturally secreted antibiotic from Aspergillus fumigatus. The
expression of GST-P was significantly reduced in rats with hepatocarcinogenesis
424 A. Bishayee
Emerging in vitro, in vivo, and clinical studies carried out during the last decade
provide substantial evidence that activation of inflammatory signaling pathways
plays a vital role in the pathogenesis and progression of liver cancer. It is also
apparent that there are several mechanisms which contribute to the activation of
inflammatory cascade in the liver in response to various etiological factors of
liver cancer. There also exists the possibility of cross talk between inflamma-
tory pathways and other signaling events in liver cancer. Since various inflam-
matory pathways are closely regulated at multiple cellular and subcellular levels,
these pathways provide opportunities to develop novel preventive and therapeu-
tic strategies for management of liver cancer. Based on current interest in inflam-
matory signaling pathways in liver cancer, it is conceivable that new signaling
molecules and pathways of inflammation-linked HCC would be identified in the
near future.
Several animal studies as presented in this chapter clearly demonstrate that var-
ious natural and synthetic compounds are capable of disrupting activated inflam-
matory signaling to halt or reverse the growth of a variety of transplanted HCC
cells in vivo and inhibit the development and progression of chemically initiated,
dietary-induced, or spontaneously occurring liver tumors in rodents. All these anti-
hepatocarcinogenic effects could be possible due to inhibition of upstream acti-
vators of key inflammatory regulators, subunits of lead inflammatory mediators,
activating kinases or target genes. The unique inflammation-hepatocarcinogenesis
sequence in liver cancer clearly indicates that specific inhibitors of inflammatory
pathways have the potential to block or disrupt the continuous transition from
chronic liver injury to liver neoplasia. It is, indeed, noteworthy that all these struc-
turally dissimilar compounds target nearly all known proinflammatory factors and
signaling pathways in hepatic carcinogenesis. Since activation of inflammatory
insult occurs during the early as well as late phases of multistage hepatocarcino-
genesis, naturally occurring or synthetic anti-inflammatory agents could be effec-
tive in both chemoprevention and therapy of liver cancer.
Although a large number of preclinical and clinical studies underscore the
importance of inflammation in liver cancer, the direct clinical application of this
knowledge has not been fully realized. Similarly, despite the identification of a
large number of natural as well as synthetic agents targeting inflammatory path-
ways during hepatocellular carcinogenesis, there remains a gap in the transition of
these impressive results into clinical practice. Hence, future well-controlled clini-
cal studies are needed to validate the promising preclinical results of blocking or
diminishing liver cancer by interference with the inflammatory signaling by vari-
ous natural and synthetic compounds. The safety of these agents also needs to be
16 The Role of Inflammation in Liver Cancer 425
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Chapter 17
The Role of Inflammation in Skin Cancer
17.1 Introduction
in the air, water, food, and workplace. The chance that an individual will develop
cancer in response to exposure to a specific environmental agent depends on com-
plex interactions between environmental and host factors (genetic/acquired sus-
ceptibility/protective), how long and how often a person is exposed to a particular
substance, exposure to other agents, genetic factors, diet, lifestyle, health, age and
gender, etc.
The environmental agent(s)-mediated cancers have been observed to share
ten common traits that govern the transformation of normal cells to cancer cells
(Hanahan and Weinberg 2011). Accumulating evidence has resulted in the accept-
ance of “chronic inflammation” to be one of the ten hallmarks of cancer. Cancers
caused by environmental agents frequently occur in tissues with the greatest sur-
face exposure to the agent(s), e.g., lungs, gastrointestinal tract, and skin (Loeb
and Harris 2008). Skin cancer is the most common of all cancers. Therefore, this
review focuses mainly on compiling available evidence and understanding the role
of chronic inflammation in the development of skin cancer. Before presenting the
available evidence on (a) the role of inflammatory molecules in the development
of skin cancer in vitro and in vivo, and (b) observations on inhibitors of inflam-
mation for the prevention and treatment of skin cancer, brief description of skin
structure, function, types and prevalence of skin cancer, causative agents and risk
factors, treatment modalities and survival, etc., has been included for enhancing
the understanding and clarity of the presentation.
The skin is the largest and dynamic organ of the body, making up 16 % of body
weight, with a surface area of 1.8 m2 and situated at the interface between the body
and environment. Skin serves as the armors for the body against mechanical, thermal,
and physical injury and hazardous substances (Proksch et al. 2008). There are three
structural layers of the skin: epidermis, dermis, and subcutaneous layer (Fig. 17.1).
Epidermis is an external and continually regenerative, stratified epithelium
devoid of blood or nerve supplies of approximately 5–100 μm thickness. It is
composed of several distinct cell populations, keratinocytes and melanocytes
being the main constituents. Keratinocytes, which comprise 95 % of the epider-
mis, are arranged in four layers. The inner layer is the stratum germinativum
(stratum basale, basal layer), from which columnar-shaped keratinocytes divide
to migrate to the next layer. The stratum spinosum (spinous layer) is composed
of polygonal keratinocytes that become eventually more condensed. Further dif-
ferentiation of the cells leads to the stratum granulosum (granular layer), which
contains basophilic granules. In thick skin areas, such as the soles of feet or the
palms of hands, there is a clear layer of flattened cells called the stratum lucidum.
The outermost layer is the stratum corneum (horny layer), which contains keratin
and dead cells that confer to the skin its barrier function. Melanocytes are cells of
neural crest embryogenic origin whose primary function is to produce melanin,
17 The Role of Inflammation in Skin Cancer 439
Fig. 17.1 Schematic presentation of skin structure, cell types, causes, and risk factors associated
with skin cancer
the pigment that determines skin and hair color. They are located in the basal layer
of the epidermis, which comprise from 5 to 10 % of the cells and in hair follicles,
and are also found in other anatomical areas such as the inner ear, the eye, and the
meninges (Costin and Hearing 2007).
Dermis, the middle layer of the skin found below the epidermis, is composed
of a tough, supportive cell matrix. The dermis contains fibroblasts, which produce
collagen, elastin, and structural proteoglycans, together with immune-competent
mast cells and macrophages. Collagen fibers, which make up 70 % of the dermis,
give skin its strength, elasticity, and toughness. Dermis contains hair follicles,
sweat glands, blood vessels, and nerves that are held in place by collagen.
Subcutis or subcutaneous layer consists of loose connective tissue and fat. It
helps the body conserve heat and has a shock-absorbing effect that helps protect
the body’s organs from injury.
Skin has several functions, the most important being to form a physical bar-
rier to the environment, allowing and limiting the inward and outward passage
of water, electrolytes, and various substances while providing protection against
microorganisms, ultraviolet radiation (UVR), toxic agents, and mechanical insults.
Its other functions are insulation, temperature regulation, sensation, and synthesis
of vitamin D and the protection of vitamin B folates.
There are four different types of skin cancer: basal cell carcinoma (BCC) and squa-
mous cell carcinoma (SCC) collectively referred to as non-melanoma skin cancers
(NMSC) or keratinocyte carcinoma; melanoma and other non-epithelial skin cancer.
BCC and SCC account for approximately 80 and 16 % of all NMSC, respectively.
440 G. B. Maru et al.
Melanoma accounts for only 10 % of skin cancer cases, but it is the most serious
type, which can also occur in other body organs (Ibanez et al. 2011). Along with
melanoma and keratinocyte cancers, there are some other less common types of
skin cancer, e.g., Merkel cell carcinoma, Kaposi’s sarcoma, cutaneous (skin) lym-
phoma, skin adnexal tumors, dermatofibrosarcoma protuberans, and angiosarcoma.
17.1.3 Prevalence
Skin cancer is the most common malignancy in the USA, Australia, and
New Zealand with substantially associated morbidity and cost, as well as relatively
smaller but significant mortality (Rogers et al. 2010). Australia and New Zealand
have the highest rates of skin cancer incidence in the world, almost four times the
rates registered in the USA, the UK, and Canada. Skin cancer is 10 times more
common in whites than in African Americans. It is estimated that one American
dies every hour from skin cancer, whereas the incidence of UV-induced NMSC
has increased dramatically worldwide accounting for more than 40 % of all human
cancers in the USA, with about 1.3 million new cases being diagnosed annually of
which roughly 20–30 % is of SCC (Madan et al. 2010).
UVR from sun exposure is the main cause of skin cancer, accounting for at least
65 % of melanomas worldwide. The geographic variation and risk of development
of NMSC are associated with ambient sun irradiance, genotypic, phenotypic, and
environmental factors. Risk is greatest in residents of high ambient solar irradiance
who have markers of UV susceptibility, such as light skin, eye and hair color, or an
inability to tan, and those with benign sunlight-related skin disorders, e.g., actinic
keratosis (AK) and solar lentigines. Incidence within countries is associated with
increasing proximity to the equator. The thinner ozone layer and shorter distance
traversed by UVB at lower latitudes than at high latitudes make residents of these
regions most vulnerable to the effects of this radiation (Madan et al. 2010).
Individuals with familial genetic syndromes, viral infections such as human
immunodeficiency virus (HIV), human herpesvirus 8 (HHV8), and human papil-
loma virus (HPV) or exposed to artificial UVR (tanning beds and lamps), aging,
diet, and smoking are attributed risks. Some treatment modalities, including radi-
otherapy, phototherapy, psoralen, long-wave ultraviolet radiation (PUVA), and
immunosuppressant drugs (cyclosporin A, methotrexate) besides work-related
exposures such as arsenic, tar product, and chemical carcinogens (petroleum refin-
ing, pesticide manufacturing, etc.), also predispose individuals to skin cancers
(Fig. 17.1) (IARC 1987; Boffetta et al. 2001). Skin cancers are also attributed to
chronically injured or non-healing wounds and scars or ulcers that occur at sites of
17 The Role of Inflammation in Skin Cancer 441
previous burns, sinuses, trauma, osteomyelitis, prolonged heat (Kangri cancer) and
chronic friction (Saree/Dhoti cancer) (Aziz et al. 1998; Patil et al. 2005; Saladi
and Persaud 2005). The incidence of malignancy in scar tissues is 0.1–2.5 %.
A variety of modalities for the treatment of skin cancer is available, including sur-
gery, radiation therapy, chemotherapy, photodynamic therapy (PDT), and biologi-
cal therapy. Surgical options, including curettage with electrodessication, Mohs
micrographic surgery, and surgical excision, are the most frequently used treat-
ments, providing a high control rate and satisfactory cosmetic results. Radiation
therapy, including brachytherapy techniques and external beam radiations such as
superficial/orthovoltage X-rays, megavoltage photons, and electron beam radia-
tion, has been used as primary and post-surgical adjuvant therapy for skin cancers.
Chemotherapy includes the topical agents (in the form of ointment) such as fluo-
rouracil, diclofenac sodium, and imiquimod. In PDT, a photosensitive drug and
a certain type of laser light are used to kill cancer cells. However, in biological
therapy (biotherapy or immunotherapy), substances such as interferon and imiqui-
mod (made by the body or in a laboratory) are used to boost, direct, or restore the
body’s natural defenses against cancer.
17.1.6 Survival
Table 17.1 Characteristics of inflammation
Characteristics Acute inflammation Chronic inflammation
Duration Short Relatively long
Pattern Stereotyped Varied
Predominant cell Neutrophils, leukocytes Lymphocytes, macrophages,
plasma cells, giant cells, fibroblasts
Tissue destruction Mild to moderate Marked
Fibrosis Absent Present
Inflammatory reaction Exudative Productive
IL-22), and various peptides (Zhu et al. 2011). STAT3 regulates the expression of
genes that mediate survival (survivin, Bcl-xl, myeloid cell leukemia sequence 1
[mcl-1], c-FLIP), proliferation (c-fos, c-myc, cyclin D1), invasion (MMP-2), and
angiogenesis (VEGF) (Aggarwal et al. 2009). Along with NF-κB, STAT3 is a point
of convergence for numerous oncogenic signaling pathways. Maintenance of NF-κB
activation in tumors requires STAT3 which is constitutively activated both in tumors
and in immune cells and plays a role in carcinogenesis (Colotta et al. 2009).
Several clinical observations and experimental findings indicate that the process of
metastasis is non-random and involves a sequence of multistep events targeted for
therapy. Metastatic cancer cells exploit the mechanisms of the inflammation pro-
cess, which successfully migrate into distant organs. This implies a pivotal role for
specific adhesive interactions between cancer cells and vascular endothelial cells
and activation of migratory pathways in the cancer cells (Laferriere et al. 2002).
The tumor cells follow the extravasation strategy of leukocytes in their migration
toward inflammatory sites (Witz 2006). For instance, VCAM-1, an integrin recep-
tor located on an endothelial cell, binds to the integrin α4 β1 (VLA-4—very late
antigen-4), which are normally expressed on leukocyte plasma membranes, but
they do not adhere to their appropriate ligands until the leukocytes are activated by
chemotactic agents or other stimuli (Schadendorf et al. 1995).
Selectins have been involved in the progression of cancer. In fact, several
types of tumor cells express functional ligands of selectins and contact selec-
tins expressed on blood vessel walls (Laferriere et al. 2002; Witz 2006; Barthel
et al. 2007). Keratinocyte cell lines—A431, HaCaT, SVK14—express selectin
17 The Role of Inflammation in Skin Cancer 451
ligands including sialyl Lewis X and S-Le(a) expression, whereas normal human
keratinocytes do not. These findings suggest a potential role for selectin-mediated
events in the early and late metastasis (Groves et al. 1993). To this end, the study
of the role of selectins in leukocyte and tumor cell extravasation merits particu-
lar attention in understanding the pathophysiology of inflammation and cancer and
is substantiated by a number of recent studies (Witz 2006; Barthel et al. 2007).
There is also growing awareness that platelets and leukocytes may potentiate
and even enhance the hematogenous dissemination of cancer cells, suggesting a
link between inflammation and cancer progression. Indeed, the tumor microenvi-
ronment often contains infiltrates of platelets, macrophages, dendritic cells, and
lymphocytes (Mantovani et al. 2008). These cells may be critical sources of pro-
inflammatory cytokines, including TGF-β, TNF-α, IL-1β, and IL-6, all of which
may promote the up-regulation of selectin expression on the vascular wall and
synergize with chemokines, such as IL-8, secreted by tumor cells.
Tumor invasion and metastasis represent a multistep process that depends on
the activity of many proteins (Hua et al. 2011; Shanmugam et al. 2012; Ravi and
Piva 2013). Several classes of proteases, including MMPs, serine proteases like
furin, and cysteine proteases such as cathepsin have been implicated in the tumor
cell-invasive process. Of these, MMPs appear to be primarily responsible for
extracellular matrix (ECM) degradation observed during invasive processes (Hua
et al. 2011; Pytliak et al. 2012; Ravi and Piva 2013). They contribute to tumor
growth by degradation of the ECM as well as by the release of sequestered growth
factors such as VEGF, b-fibroblast growth factor (b-FGF), or TGFβ, the suppres-
sion of tumor cell apoptosis and the destruction of immune-modulating chemokine
gradients (Ravi and Piva 2013). In normal skin, MMPs are not constitutively
expressed but can be induced temporarily in response to exogenous signals such
as UVR. UVR is known to elevate the expression of MMP-1, MMP-3 (stromely-
sin-1), and MMP-9 in human skin. MMP-2 and MMP-9 have been frequently
associated with the invasive and metastatic potential of tumor cells (Ramos
et al. 2004; Dong et al. 2008; Ravi and Piva 2013).
Furin is a serine protease that is frequently over-expressed in several cancer cell
lines and malignancies, including several murine cell lines derived from chemically
induced skin tumors (Fu et al. 2012). Its activity results in proteolytic cleavage
of substrates, leading to the activation of many cancer-related proteins including
important growth factors and receptors such as insulin growth factor-1 (IGF-1) and
its receptor IGF-1R, TGF-β, and VEGF. (Siegfried et al. 2003; Ravi and Piva 2013).
Furin is also involved in the maturation of both TNF-alpha-converting enzyme
(TACE) and MMP within skin cells and mainly activate MT1-MMP, which directly
contributes to the motility and invasiveness of the tumor cell, thereby indicating
that furin activity has an influence on the inflammation seen in the skin, follow-
ing exposure to UVR (Ravi and Piva 2013). It has been shown that furin mRNA,
protein, and enzyme activity increase immediately after UVA and UVB treatment
in human epidermal keratinocytes (HaCaT cells). Furin/PC processing of substrates
has been shown to contribute to tumor progression, aggressiveness, metastasis, and
angiogenesis (Arsenault et al. 2012; Fu et al. 2012; Ravi and Piva 2013).
452 G. B. Maru et al.
Various skin tumor promoters induce IL-1α mRNA and protein expression in the
epidermis in vivo (Oberyszyn et al. 1993; Lee et al. 1994). Blocking the activity of
IL-1α with intradermal injections of a neutralizing antibody inhibits TPA-induced
vascular permeability, inflammatory cell infiltration, and epidermal hyperplasia,
which demonstrates the central role of IL-1α in mediating these tumor promoter-
related events (Lee et al. 1994). Transgenic mice over-expressing IL-1α in basal
keratinocytes (K14 promoter) develop spontaneous inflammatory skin lesions, as
well as dermal neutrophil infiltration even in non-lesional skin (Groves et al. 1995).
Moreover, stable over-expression of antagonist of IL-1 (IL-1Ra) in mouse skin car-
cinoma cell line results in down-regulated COX-2 expression and slower in vitro
and in vivo growth. These results indicate that IL-1 is contributing to malignant
cell proliferation (Rundhaug and Fischer 2010). IL-12 and IL-23 also play role in
skin tumorigenesis, wherein IL-12 acts as a tumor suppressor by inducing immune
surveillance and IL-23 promotes skin tumorigenesis by driving inflammation and
reducing immune surveillance. While IL-12p35-null mice develop papillomas ear-
lier and more frequently than wild-type mice, IL-23p19-null mice, as well as p40-
null mice, are resistant to DMBA/TPA induction of skin tumorigenesis (Langowski
et al. 2006). In addition, IL-12p35- and IL-12p40-null mice are more sensitive to
UV-induced skin carcinogenesis, with reduced repair of UV-induced DNA damage,
increased number of tumors per mouse, more rapid growth, and greater malignant
potential than wild-type mice. UV-induced tumors from IL-12p35-null mice also
have increased angiogenesis and up-regulated expression of pro-inflammatory IL-6
and IL-23 (Meeran et al. 2007). Thus, IL-12 counteracts UV-induced immunosup-
pression, inflammation, and skin carcinogenesis.
Proteinases such as MMP-2 and MMP-9, provided by mast cells as well as
granulocyte neutrophils in inflammatory microenvironment, play important roles
as regulators of development, angiogenesis, and tumor progression. The essential
role of stromal MMP-9 for tumor development in K14-HPV16 transgenic mice
showed that mice deficient for MMP-9 resulted in decreased tumor incidence
(Coussens et al. 1996). In addition, lack of MMP-9 was associated with delayed
activation of angiogenesis in the stroma of the lesions (Mueller 2006). In another
study, it has been shown that MMP-9 expressed by inflammatory cells is function-
ally involved in distinct processes of epithelial carcinogenesis such as regulation
of oncogene-induced keratinocyte hyperproliferation, progression to invasive can-
cer, and end-stage malignancy (Coussens et al. 2000).
however, these studies do not allow any insight in the cellular and molecular
mechanisms that lie at the basis of the tumor and progression-promoting effect of
inflammation in epithelial skin cancers.
Dietary polyphenols which are widely present in fruits, vegetables, dry legumes,
and beverages (such as tea, coffee, juice, wine, beer) have gained considerable
attention for the prevention of UV-induced skin photodamage including the risk of
skin cancer. Experimental and epidemiologic studies have suggested that polyphe-
nols protect the skin from the adverse effects of UV radiation. Polyphenols have
been shown to (a) scavenge radical species such as ROS/RNS, e.g., O2−, H2O2,
OH•, ONOO−; (b) suppress ROS/RNS formation by inhibiting some enzymes or
chelating trace metals involved in free radical production; and (c) up-regulate or
protect antioxidant defense (Patel et al. 2007).
Oral administration of green tea polyphenols (GTPs) to SKH-1 hairless mice
resulted in significant inhibition of UVR-induced cutaneous edema, erythema,
and bifold skin thickness (a biomarker of inflammation). Administration of GTPs
in drinking water decreased COX-2, PGE2, PCNA, and cyclin D1 and also sig-
nificantly reduced the levels of various pro-inflammatory cytokines in chronically
UVB-exposed skin/skin tumors of mice (Meeran et al. 2009). Topical treatment
with GTPs prior to UV exposure reduced the UV-induced hyperplastic response,
myeloperoxidase (MPO) activity, and the numbers of infiltrating inflammatory
leukocytes in the skin (Afaq et al. 2003). Moreover, similar administration of
both agents in the untanned backs of humans resulted in significantly less devel-
opment of erythema as compared to the UV-irradiated skin that was not treated
with GTPs (Katiyar et al. 2001). Topical application of EGCG, an active constitu-
ent of green tea, in mice and humans, resulted in the inhibition of UVB-induced
production of PG metabolites (PGE2, PGF2-α, and PGD2), which play a critical
role in inflammatory disorders, free radical generation, proliferative skin diseases,
and skin tumor promotion (Katiyar et al. 2001; Katiyar and Mukhtar 2001). The
inhibitory effects of GTPs on these biomarkers of inflammation in UV-exposed
skin provide mechanistic evidence of the anti-carcinogenic effects of GTPs.
Studies have also shown that topical pretreatment with polymeric black tea poly-
phenols in Swiss bare mouse skin decreased TPA-induced inflammatory pro-
tein (COX-2) and cellular proliferation through decreasing activation of cellular
kinases (JNK, ERK, p38, and Akt) and transcription factors (AP-1 and NF-κB)
as well as apoptosis (Patel et al. 2008). The above in vivo observations generated
using both animal and human systems provide insights into the possible protec-
tive mechanisms involved in the anti-initiating and/or anti-inflammatory effects
of tea polyphenols.
460 G. B. Maru et al.
Melanoma and non-melanoma skin cancers are among the most prevalent cancers
in human. Epidemiological and experimental evidence suggests “chronic inflam-
mation” to be one of the hallmarks in solar UVR and several other environmen-
tal agent-mediated skin cancers. The identification of transcription factors, i.e.,
NF-κB, STAT3, and HIF-1α, and their gene products, i.e., COX-2, cytokines,
chemokines, and chemokine receptors, suggests critical role of inflammation
in skin carcinogenesis. Considering the potential role of inflammation in initia-
tion and its major as well as convincing role in promotion, progression as well as
tumor angiogenesis and metastasis, inflammatory pathways may become attractive
targets for skin cancer prevention. Efforts to prevent or minimize the exposure to
known skin carcinogens and ongoing studies on evaluating the role of various pro-
inflammatory mediators in carcinogenesis and assessing them as potential targets
for chemoprevention of skin cancers need to be enhanced/encouraged.
Acknowledgments We thank Dr. Vikram Gota for useful discussion and Council of Scientific
and Industrial Research (CSIR) and ACTREC, Government of India, for awarding fellowship to
Mr. Gaurav Kumar.
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Erratum to:
The Role of Inflammation in Leukaemia
Erratum to:
Chapter 13 in: B. B. Aggarwal et al. (eds.), Inflammation
and Cancer, DOI 10.1007/978-3-0348-0837-8_13
Please note that the sequence of authors’ names was incorrect in the original
version. The correct sequence should read as:
Janusz Krawczyk, Michael O’Dwyer, Ronan Swords, Ciara Freeman, and
Francis J Giles
The online version of the original chapter can be found under DOI 10.1007/978-3-0348-0837-8_13
J. Krawczyk
Department of Haematology, Galway University Hospital, Galway, Ireland
M. O’Dwyer
Biosciences, National University of Ireland Galway, Upper Newcastle, Galway, Ireland
R. Swords
Sylvester Comprehensive Cancer Center, University of Miami, Miami, USA
C. Freeman
Department of Haematology, Barts and the Royal London NHS Trust, London, UK
F. J. Giles (*)
Northwestern Medicine Developmental Therapeutics Institute, Robert H. Lurie
Comprehensive Cancer Center of Northwestern University, Chicago, USA
e-mail: [email protected]
0–1 A
1-methyl L-tryptophan, 91 A431, 450
12-LOX pathway, 31 Aberrant crypt foci (ACF), 29
12-O-tetradecanoylphorbol-13-acetate Acinarto-ductal metaplasia (ADM), 132, 140
(TPA), 453, 454, 455, 461 Acquired immune systems. See Adaptive
induced inflammatory protein, 459, 460 immune systems
induced skin tumorigenesis, 455, 457, 458 Actinic keratosis (AK), 440
15-hydroxyprostaglandin dehydrogenase Acute bacterial prostatitis, 154
(15-PGDH), 112 Acute lymphoid leukaemia (ALL), 336, 337,
15-lypoxygenase (LOX), 29, 214 340, 343, 346, 347, 348
lipid molecules derived from, 31 age-adjusted incidence of, 337
metabolites, 30 Acute myeloid leukaemia (AML), 336, 338,
15-lipoxygenase-2 (15-LOX2), 221 339, 340, 346, 351, 352
2, 3,7, 8 tetrachlorobenzo-p-dioxin cytogenetic risk groups, 337t
(TCDD), 91 high serum TNF level, 345
3-hydroxyanthranilate 3,4-dioxygenase murine model data, 350
(3HAO), 91 NF-κB activation in, 342
4-aminobiphenyl exposure, 402 primary AML cells, 348
5-aminosalicylic acid (5-ASA), 28 prognostic factors in, 337t
5-bromocytosine (5BrC), 86 smoking, 349
5-carboxylcytosine (5caC), 82 Stat3, 343–344
5-chlorocytosine (5ClC), 86 TLR4 expression in, 347
5-fluorouracil, 65, 130, 200, 236 Acyclic retinoid, 417t, 422
5-formylcytosine (5foC), 82 Adaptive immune systems, 9, 156, 167, 168,
5-hydroxymethylcytosine (5hmC), 82, 83, 96 236
5-hydroxymethyluracil (5hmU), 82 B lymphocytes, 167
5 LOX pathway, 31, 34f NK and DCs, 236–237
5-LOX-activating protein (FLAP), 31 T lymphocytes, 167
5-methylcytosine (5mC) patterns, 80, 86 Adenocarcinomas, 3, 14, 32, 39
7, 12-dimethylbenz(a)anthracene (DMBA), bronchoalveolar subtype, 8
453, 454, 457, 458, 460, 461 of colon, 37, 38
8-oxo-2’-deoxyguanosine (8-oxodG), 85 in CRC, 29, 30
1 Page numbers followed by “f” and “t” indicate figures and tables respectively
IFN-γ, 7, 186, 216–217, 237, 363 John Cunningham virus (JCV), 240
regulatory factor (IRF) 1, 385 JTE-522, 417t, 422–423
stimulated genes (ISG), 384
Interleukins (ILs), 6
IL-1, 288, 289 K
genetic polymorphisms, 246 Kainate receptors, 92
in skin cancer, 454–455 Kaposi’s sarcoma (KS), 291, 440
IL-1β, 59, 118 associated herpesvirus (KSHV), 272, 273
IL-10, 10–11, 14, 29, 185, 370 Keratinocyte carcinoma, 439
polymorphisms, 245 Kidney cancer, 198.See also Renal cell
IL-2, 186 carcinoma (RCC)chemotherapy, 200
IL-21, 320, 324 infiltration cells, changes in, 200f
IL-32, 114, 389 inflammatory molecules, 207
IL-4, 216–217, 320 in cancer cell survival, 209–210
IL-6, 12, 14, 29, 57, 113, 165–166, in cancer cell transformation, 207–209
216–217, 281, 286, 408, 456 chemokines in cell proliferation,
oral keratinocytes, 113 210–211
in prostate cancer development, 166 in invasion and metastasis, 211–213
in skin cancer, 452 as prognostic tools, 217–218
TRAMP murine model, 166 inflammatory signaling pathway
IL-7, 390 mTOR pathways, 206
IL-8, 29, 61 STAT signaling pathways, 206–207
signaling cascade, 286 TNF pathway, 206
Intestinal metaplasia, 239 VHL pathway, 204–205
Invasion major gene products associated with, 199
of bladder cancer calls, 189–190 surgery, 199
of cervical cancer cells, 388–389 radical nephrectomy, 199
of head and neck cancer cells, 112–118 survival, 198
of IBC cells, 60–61 transforming growth factor (TGF) β1,
of kidney cancer cells, 211–213, 216–217 208–209
of lung cancer cells, 12–13 Kidney cancer cells
of pancreatic cancer cells, 137–139 animal models, 213–214
of sarcoma cells, 283–284 chemokines in proliferation of
of skin cancer cells, 450–452 A-498 cells, 211
IRF4 gene, 317 CXCL12, 210
Irinotecan, 130, 382 CXCR4, 210
Ixabepilone, 269t, 394t rhCXCL12 alpha, 211
evidence from patients, 214
chemokine receptors and ligands,
J 216–218
JAK/STAT, 403 cytokines, 216–218
pathway, 209, 385 immune inflammatory cells, 214–216
JAK-STAT signaling, 56–57 inflammation inhibitors
and liver cancer COX2 inhibitors, 219
mitogen-activated protein kinase NSAIDs, 218–219
(MAPK) signaling, 409 statins, 219–220
STAT family, 408, 409 suppressive MDSC reduction, 220
suppressor of cytokine signaling 3 tumor-promoting TAM reduction, 221
(SOCS3), 409 invasion, metastasis, and angiogenesis
signal transducers, 403 MMPs, 212, 213
Janus kinase/signal transducer and activator of protein C inhibitor (PCI), 212, 213
transcription. See JAK/STAT TAMs, 212
Janus-activated kinase (JAK) family, 133 tissue inhibitors of metalloproteinases
JCV T-Ag (oncogenic transforming antigen), 240 (TIMPs), 212
482 Index
R S
Raf, 403 S,S-1, 4-phenylene-bis(1, 2-ethanediyl)bis-
RANK/RANKL pathway, 280 isothiourea (PBIT), 37
RANTES (Regulated on Activation, Normal S1, 394t
T cell Expressed and Secreted), 14, 319 Saikosaponin-d, 416t, 421
RAPADILINO syndrome, 264, 265 Sarcoma, 261
Rapamycin, 203 current therapies, 267–270
Ras, 30, 243, 443 chemotherapeutics for, 269–270t
chemical inhibition of, 285 epidemiology, 261–263, 262t
Ras/Raf/MAPK pathway, 273 inflammatory molecules
Ras/Raf/MEK/ERK, 403 in cancer cell proliferation, 279–283
Reactive nitrogen intermediates (RNI), 362 in cancer cell survival, 278–279
Reactive nitrogen species (RNS), 31–32 in cancer cell transformation, 274–278
Reactive oxygen intermediates (ROI), 362 in development, in vivo studies,
Reactive oxygen species (ROS), 5–6, 31–32, 284–288
215, 442 evidence from patients, 288–292
Receptor tyrosine kinase (RTK) signaling in invasion, metastasis, and angiogen-
pathway, 77 esis of, 283–284
in glioblastoma, 78f inhibitors, 292–294
Receptor tyrosine kinase c-Met, 273 inflammatory signaling pathways, 271–274
RECQL4 mutations, 265 epigenetic alterations, 271
Reed-Sternberg (RS) cells, 317 IGF-1 system, 273, 274
Regulatory macrophages, 214 malignant fibrous histiocytoma (MFH),
Regulatory T cells (Tregs), 29, 91, 186, 456 272
relA, 341 pro-inflammatory factors, 271
Renal cell carcinoma (RCC), 198.See also ROS and RNS, 272
Clear-cell RCC (CCRCC)checkpoint sarcomagenesis, 272
inhibitors, 202–203 inherited disorders, 263t
IFN-α, 201–202 major gene products, 263–266
488 Index
U
U-2 OS, 281 X
Ubiquitin, 351 Xenograft model, 284, 286
Ulcerative colitis, 129 Xeroderma pigmentosum, 443
Ultraviolet radiation (UVR), 439, 440, 441 XIAP, 411, 445
chronic inflammation, 462 survivin-XIAP complex, 412
induced skin cancer, 443, 447, 457
IL-6 role in, 452
Unspecified lymphomas, 324–325 Z
Urokinase plasminogen activator (uPA), 163, ZAP70, 338
445 Zeta-chain expression, 216