Tumor Biomarkers

Signal Transduction and Targeted Therapy www.nature.
com/sigtrans
REVIEW ARTICLE OPEN
Tumor biomarkers for diagnosis, prognosis and targeted

therapy
Yue Zhou1, Lei Tao 1
, Jiahao Qiu1, Jing Xu1, Xinyu Yang2, Yu Zhang2,3, Xinyu Tian1, Xinqi Guan1, Xiaobo Cen 1,4
and
Yinglan Zhao 1 ✉
Tumor biomarkers, the substances which are produced by tumors or the body’s responses to tumors during tumorigenesis and
progression, have been demonstrated to possess critical and encouraging value in screening and early diagnosis, prognosis
prediction, recurrence detection, and therapeutic efficacy monitoring of cancers. Over the past decades, continuous progress has
been made in exploring and discovering novel, sensitive, specific, and accurate tumor biomarkers, which has significantly promoted
personalized medicine and improved the outcomes of cancer patients, especially advances in molecular biology technologies
developed for the detection of tumor biomarkers. Herein, we summarize the discovery and development of tumor biomarkers,
including the history of tumor biomarkers, the conventional and innovative technologies used for biomarker discovery and
detection, the classification of tumor biomarkers based on tissue origins, and the application of tumor biomarkers in clinical cancer
management. In particular, we highlight the recent advancements in biomarker-based anticancer-targeted therapies which are
emerging as breakthroughs and promising cancer therapeutic strategies. We also discuss limitations and challenges that need to be
addressed and provide insights and perspectives to turn challenges into opportunities in this field. Collectively, the discovery and
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application of multiple tumor biomarkers emphasized in this review may provide guidance on improved precision medicine,
broaden horizons in future research directions, and expedite the clinical classification of cancer patients according to their
molecular biomarkers rather than organs of origin.
Signal Transduction and Targeted Therapy (2024)9:132 ; https://doi.org/10.1038/s41392-024-01823-2
INTRODUCTION
Brief history of tumor biomarkers development immunoglobulin light chains from the urine of a patient with
Biomarkers are designated as “a biological molecule found in multiple myeloma, and named it BJP, the first biochemical tumor
blood, other body fluids, or tissues that is a sign of a normal or biomarker described in diagnostic laboratory medicine3 (Fig. 1).
abnormal process, or a condition or disease. A biomarker may be The monitoring of BJP in urine has become one of the parameters
used to see how well the body responds to a treatment for a related to the diagnosis and prognosis of multiple myeloma.4 The
disease or condition” according to the National Cancer Institute discovery of BJP marks the beginning of research on tumor
(http://www.cancer.gov/dictionary). Tumor biomarkers exist in biomarkers. Subsequently, hormones, isozymes, and other tumor
tumor tissues or body fluids such as blood, urine, stool, saliva, biomarkers that displayed abnormalities during the occurrence
and are produced by the tumor or the body’s response to the and development of tumors were discovered. In 1927, Selmar
tumor.1 The goal of the tumor biomarker field is to develop Ashheim and Bernhard Zondek found a gonadal stimulating
sensitive, specific, reliable, cost-effective, reproducible, and power- substance—human chorionic gonadotropin (HCG) from the blood
ful detection and monitoring strategies for tumor risk indication, and urine of pregnant women.5 Later on, HCG was identified as a
tumor monitoring, and tumor classification so that patients can tumor biomarker, which is frequently associated with gestational
receive the most appropriate treatment and doctors can monitor trophoblastic disease and testicular germ cell tumor.6 In 1959,
the progress, regression, and recurrence of the tumors.2 Since the lactate dehydrogenase (LDH), the first “isoenzyme”, was discov-
discovery of Bence-Jones protein (BJP), the first tumor biomarker, ered in the bovine heart by Clement L Markert at Johns Hopkins
in 1846, this field has been through many stages and has made University,7 and numerous clinical evidence subsequently demon-
significant and substantial progress with the joint efforts of strated that LDH was an essential prognostic factor for different
researchers, clinical staff, and patients. tumors.8 In 1962, Meador found that some tumors spontaneously
produced adrenocorticotropic hormone-like substances, which
hindered the normal secretion mechanism of adrenocorticotropic
Discovery and exploration stage (1847–1962). In 1847, Henry hormone and induced metabolic abnormalities dominated by
Bence-Jones described the findings of a large number of hypokalemia.9 Despite the aforementioned breakthroughs in
1
Department of Biotherapy, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China; 2West China School of
Pharmacy, Sichuan University, Chengdu 610041, China; 3School of Medicine, Tibet University, Lhasa 850000, China and 4National Chengdu Center for Safety Evaluation of Drugs,
State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China
Correspondence: Yinglan Zhao([email protected])
These authors contributed equally: Yue Zhou, Lei Tao, Jiahao Qiu
Received: 5 June 2023 Revised: 7 March 2024 Accepted: 2 April 2024
© The Author(s) 2024

Tumor biomarkers for diagnosis, prognosis and targeted therapy
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Fig. 1 Timeline of the history of tumor biomarker
knowledge about tumor biomarkers, these biomarkers did not inhibitor, sotorasib, which was approved by the United States
translate from bench to bedside for cancer diagnosis or Food and Drug Administration (FDA) in 2021 for non-small cell
monitoring. lung cancer (NSCLC) treatment.24 The first human oncogene,
retinoblastoma (Rb) gene, was successfully cloned in 198625
Clinical application stage (1963–1978). The next significant (Fig. 1). After that, large numbers of proto-oncogenes, oncogenes,
advances came from GI Abelev who is well known for his 1963 tumor suppressors, receptors, and kinases were discovered, and
discovery that mice inoculated with liver cancer cells can some of them were successfully used as tumor diagnostic,
synthesize alpha-fetoprotein (AFP)10 (Fig. 1). AFP has been used prognostic, and therapeutic biomarkers.
as a biomarker in clinical screening, diagnosis, prediction, and
treatment evaluation of hepatocellular carcinoma (HCC).11 At Innovation and development stage (2005-). The rapid develop-
around the same time (in 1965), Goldenberg and Freeman found ment of science and technology is driving the field of tumor
that carcinoembryonic antigen (CEA) in fetal colon mucosa12 biomarkers to an innovation and development stage. An
contributed a crucial part in tumor diagnosis and prognosis increasing number of methods and technologies have been
evaluation of lung cancer,13 breast cancer,14 ovarian cancer,15 developed and applied in tumor biomarker discovery and
colorectal cancer (CRC),16 etc. The discovery of AFP and CEA has detection. The molecular biological technologies, such as geno-
promoted the clinical application of tumor biomarkers. However, mics, transcriptomics, proteomics,26 metabolomics,27,28 the clus-
the application of CEA as a tumor biomarker was later challenged tered regularly interspaced short palindromic repeats (CRISPR)/
by Paul Lo Gerfo and his colleagues in 1971. They measured the CRISPR-associated protein 9 (Cas9) gene-editing technology,29,30
level of CEA in the serum or plasma of 674 hospitalized patients by and high-throughput sequencing,31 make it possible to obtain
radioimmunoassay (RIA). It was found that CEA expression level large-scale information on the diversity of tumor biomarkers. In
was elevated in the serum of patients with multitudinous diseases, 2005, the first high-throughput sequencer was launched, which
but not cancer-specific, which hampered the potential absolute contributed to the sequencing technology. In recent years, nucleic
benefit of CEA assessment independent of other surveillance tools. acid-based liquid biopsy for monitoring cancer has attracted much
attention.32 For example, cell-free DNA (cfDNA) in the plasma of
Exploration stage (1979–2004). James Watson and Francis Crick’s cancer patients contains tumor-derived DNA sequences, which
discovery of the DNA double helix structure in 1953 ushered in a can be used as biomarkers for the early detection of cancer,
new era in tumor biomarker research.17 After this discovery, guiding treatment, and monitoring drug resistance.33 In 2016,
modern molecular biology significantly promoted the research on ExoDx Lung (ALK), the world’s first exosomal oncology diagnostic
tumor biomarkers, with a large number of genes involved in test, was launched. It can be used for the diagnosis of NSCLC and
tumor occurrence and progression being discovered. In 1979, p53 the screening of NSCLC patients for targeted therapy with
was found by David Lane and confirmed by other independent anaplastic lymphoma kinase (ALK) inhibitors. Subsequently, the
groups.18,19 Although being considered as a cell tumor antigen at epidermal growth factor receptor (EGFR) gene mutation detection
the beginning, p53 was defined as a cancer suppressor gene in kit in plasma DNA samples was launched in 2018 and used to
198920 and more than 50% of p53 was mutant in cancer screen patients for EGFR-targeted drug therapy. China’s self-
patients.21 In 1981, Robert Weinberg and Geoffrey Cooper developed “CellRich” circulating tumor cell detection system was
discovered the small fragments of DNA in transgenic experiments approved by the National Medical Products Administration in
in which the transformation of mouse NIH/3T3 fibroblast cells 2018 and used to capture tumor circulating cells. Furthermore, the
transfected with DNA extracted from human tumors cell lines was emergence of “precision medicine” has pushed the field of tumor
successfully induced and soon followed the isolation of homo- biomarkers to a new stage, which requires the discovery of more
logous oncogenes HRAS and KRAS from human tumors.22,23 This effective tumor biomarkers and the integration of multiple tumor
discovery paved the way for the development of the first KRASG12C biomarkers to support personalized medicine.34
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Clinical application of tumor biomarkers of breast cancer associated with poor prognosis and limited
Early screening of tumors. Early screening of tumors is the most treatment options, thus improving the management and treat-
powerful public health tool that enables early detection, thus ment options with the ultimate goal of improving clinical
reducing annual cancer incidence, providing a higher chance of outcomes.52 Moreover, identifying curative predictive biomarkers
treatment, improving patient response to medical interventions, to distinguish patients who are most likely to respond to
and prolonging patient survival, especially for those cancers with anticancer therapy from all cancer patients enhances therapeutic
high mortality such as CRC.35 In addition to invasive or expensive efficiency, decreases treatment costs, and avoids adverse events.
screening methods such as endoscopy, low-dose computed For example, the implementation of patient selection prior to
tomography, and tissue biopsy, noninvasive and cost-effective programmed cell death 1 (PD-1)/programmed cell death ligand 1
screening based on biomarkers from body fluids including blood, (PD-L1) inhibitors therapy by the combination of biomarkers
stool, saliva, and urine has been gaining extensive attention. To reflecting tumor immune microenvironment and tumor cell-
date, thousands of these biomarkers including proteins, cytokines, intrinsic features, such as PD-L1, tumor-infiltrating lymphocyte,
metabolites, hormones, microRNA, and circulating DNA have been tumor mutational burden, mismatch-repair deficiency, and gut
explored, and several of them have been successfully developed microbiota, could enhance the treatment effect of anti-PD-1/anti-
and used in the early screening of cancers.2,34 For example, AFP was PD-L1 therapy in clinical practice.53
the first blood biomarker used for the screening of HCC in the
populations since 1964.36 After that, other biomarkers called Tumor recurrence monitoring. The level of tumor biomarkers is
“classical” tumor markers, such as CEA, carbohydrate antigen 19-9 valuable for indicating the disease recurrence of tumor patients.
(CA19-9),37 carbohydrate antigen 125 (CA125),38 prostate-specific Some classic biomarkers for tumor diagnosis and prognosis, such as
antigen (PSA),39 and LDH,40 have been used in the clinical screening PSA, CEA, CA19-9, and CA72-4, are used for indicating the recurrence
of various kinds of cancers. In addition to the “classical” tumor of cancers including prostate cancer, gastric cancer, breast cancer,
marker, a broad range of novel biomarkers have been explored in and liver cancer.54,55 The CEA is increased in most liver recurrence
recent years, which include microRNA41 and other RNAs,42 microbial cases of gastric cancer (90%), while the increase of CA19-9 after
proteins,43 circulating nucleosomes,44 circulating tumor DNA,45 and surgery in patients with gastric cancer could predict peritoneal
circulating tumor cells.46 Albeit currently undergoing clinical trials or recurrence more accurately (78.9%).56 In recent years, extensive
preclinical studies and unavailable in the market, they have great molecular and genetic characterization of disseminated tumor cells
potential for clinical screening. As some biomarkers from body and blood-based biomarkers have contributed significantly to
fluids may be difficult to detect because of fundamental biological monitoring cancer recurrence. Postoperative methylated septin 9 in
barriers such as short circulation times and very low density, plasma may represent a potential noninvasive biomarker for CRC
synthetic biomarkers including small-molecule, DNA-based, mam- recurrence monitoring in addition to CRC diagnosis and prognosis
malian cell-based, and bacterial cell-based sensors have been compared with CEA and CA19-9.57 The circulating tumor DNA
developed to amplify tumor signals, thus enhancing the sensitivity (ctDNA) minimal residual disease (MRD) following treatment in solid
and efficiency of early-stage tumor detection.47 New screening tests tumors predicts relapse and highlights the application of this
based on these novel techniques can be used in the clinic in the potentially transformative biomarker.58
near future. Collectively, for the detection of early-stage cancer, the Collectively, tumor biomarkers play an active role in all aspects of
noninvasive or minimally invasive test is ideal, and developing such clinical application, such as early screening, diagnosis, prognosis,
techniques is desirable in clinical applications. and relapse monitoring, and are of great value in helping patients
prolong their survival and improve their quality of life. To date,
Tumor auxiliary diagnosis. Due to the risk of false positive or excellent progress has been made in the discovery and application
negative results, relying solely on one biomarker level is not an of biomarkers. Besides classical biomarkers used in clinical practice,
accurate and reliable strategy for tumor diagnosis. Instead, the recent advances in molecular biology technologies have signifi-
combination of tumor biomarkers with other methods such as cantly improved the discovery of new candidates for cancer
tissue biopsy and endoscopy is a promising alternative to improve management, but most of them are still in the early stage of
the effectiveness of screening.48,49 For example, the combined development and validation. Great effort could be made to find
detection of AFP with cfDNA can improve the specificity of HCC new biomarkers with the right degree of specificity, sensitivity, and
diagnosis to 94.4%, which was superior to that of AFP alone in terms reliability, so as to provide evidence for individualized decision-
of higher sensitivity and better clinical correlation.50 The advantages making during the overall management of cancer patients. In this
of biomarker panels have been confirmed as compared with a review, we summarize the current progress that has been made in
single biomarker, especially a panel of biomarkers reflecting cancer biomarker development and discuss the promise, limitations,
changes in independent pathways. The combination of periodin and further challenges in biomarker development.
(POSTN) with CA15-3 and CEA for the diagnosis of breast cancer can
improve the diagnostic performance of CA15-3 and CEA.14 For CRC,
the detection of hemoglobin using fecal immunochemical testing in TECHNOLOGIES USED IN THE DETECTION OF TUMOR
combination with transferrin in stool improves the diagnostic BIOMARKERS
accuracy for CRC.51 The combination of various diagnosis strategies Multiple technologies have been developed for the detection of
with biomarkers could result in an easier, faster, more accurate, and tumor biomarkers as follows (Fig. 2). In the past decades, various
more specific diagnosis of cancer. immunoassay methods have played crucial roles in the discovery
of tumor biomarkers. Meanwhile, molecular hybridization technol-
Prediction of tumor prognosis and curative effect. Precision ogy and gene amplification detection technology further broaden
stratification of cancer patients based on prognosis and ther- the horizon of the application of tumor biomarkers in clinical
apeutic decision biomarkers has enabled the selection of practice. Immunohistochemistry (IHC) brings about the original
treatment strategies and more effective treatments for individual distribution of biomarkers in fixed tissue. Furthermore, rapidly
cancer patients. One successful example is to distinguish the type developed DNA sequencing and gene-editing technologies
of breast cancer by the expression of human epidermal growth accelerate the speed and numbers of digging out prognostic
factor receptor 2 (HER2), estrogen receptor (ER), and progesterone and predictive tumor biomarkers. Other technologies, such as
receptor (PR) in breast cancer tissues. These biomarkers help to liquid biopsy and different microscopy technologies, as well as
identify the triple-negative breast cancer (TNBC) lacking the single-cell sequencing analysis,59 also provide tremendous con-
expression of ER, PR, and HER2 which is the most aggressive type venience in cancer therapy.

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Fig. 2 Technologies for the detection of tumor biomarkers
RIA technology antigens as tracers, the principle of FIA is similar to enzyme-

RIA technology is an analytical method proposed in the late 1950s linked immunosorbent assay. The fluorescein is chemically bound
by the United States chemist Solomon A. Berson and Rosalyn S. to the antibody (or antigen) molecule, and after that, the latter is
Yalow.60 It integrates immunologic and radiolabeling techniques combined with the matching antigen (or antibody). The fluores-
to quantitate minute amounts of biological substances based cence is observed, or the fluorescence intensity is measured by a
upon the competition between labeled and unlabeled antigens fluorescence detector which determines the presence, distribu-
for specific antibody sites, forming antigen–antibody com- tion, and content of antigens (or antibodies) in samples. FIA has
plexes.60,61 RIA usually uses radionuclide 125I as a tracer, which the advantages of high specificity, high sensitivity, and good
has been widely used for its advantages of highly radioactive.62–64 practicability with cheap, stable, and safe reagents.69 Moreover,
In addition, RIA is advantageous in measuring a variety of FIA avoids the risk of handling radioactive materials. Thus, FIA is
immunoreactive substances for its high sensitivity, and specifi- widely used in the biomedical field in the measurement of drugs,
city.65,66 For example, RIA is utilized in the detection of early-stage hormones, and proteins; the identification of antibodies, and the
tumors, and is an effective method in combination with clinical quantification of antigens.70
pathological assay to provide comprehensive evaluations of The development of various fluorescent probes and instru-
tumors.67 However, the shortcomings of RIA are also prominent, ments also contributes to the continuing evolution of FIA.2,71,72
such as isotope contamination due to the radioactive wastes, the Multiple FIA-related technologies with high detection sensitivities
requirement of specific safety equipment, and the excessive and various measurable properties have been developed, includ-
radiation exposure of workers induced by the long incubation ing fluorescent excitation transfer immunoassay, fluorescence
time. which limits its wide use.65,68 RIA tends to be eliminated with polarization immunoassay, and time-resolved fluorescence immu-
the rapid update of other immunoassay methods, such as noassays.73–77 For example, the multicolor quantum dots based on
enzyme-linked immunosorbent assay and fluorescent immunoas- fluorescence polarization immunoassay have been applied in the
say (FIA), which use other substances such as fluorescent dye detection of tumor biomarkers such as α-AFP and CEA.78
instead of radioactive isotopes to label antigens.
Molecular hybridization technology
FIA technology Molecular hybridization technology is an important method for
FIA, combining the specificity of the immunological response with the investigation of gene expression and genome function by
the sensitivity of fluorescent technology, is a popular and fast- assessing chromosomal aberration using a fluorescent probe.79
growing nonisotopic immunoassay technology. As a new immu- The principle of molecular hybridization technology is to form
noassay technology using fluorescein-labeled antibodies or stable double-stranded hybrid molecules between DNA or RNA

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from different species, thereby detecting complementary intrinsic drawbacks of PCR that restrain its application have room
sequences or recognizing binding sites of transcription factors. for improvement, such as the requirement for instruments,
Common molecular hybridization techniques include fluorescence experienced operators, laboratory setting, and sophisticated
in situ hybridization (FISH) and in situ hybridization.79,80 In situ operations.90 Collectively, PCR is a valuable tool in tumor
hybridization uses labeled complementary DNA or RNA strands to biomarker detection, while novel PCR-based methods remain to
localize specific DNA or RNA sequences on chromosomes or tissue be explored to meet the needs of patient monitoring in clinic.87
sections fixed on slides (in situ), and the FISH technique helps to
localize genes to different chromosomal locations. They are all DNA sequencing technology
molecular tools in cancer diagnosis, treatment, and prognosis.79,81 DNA sequencing technology is a commonly used technology in
With the advantages of easy manipulation, fast hybridization molecular biology research, which is used to analyze the
process, possible automation of process and scoring, the FISH arrangement of the base sequence of specific DNA fragments.
technique is wildly utilized to detect the tumor biomarkers in The world’s first method of DNA sequencing was invented by
diagnosis and metastasis prognosis, such as the analysis of British biochemist Frederick Sanger, who performed the first
circulating tumor cells (CTCs) obtained from the patient blood complete DNA genome sequencing, bacteriophage ϕX174 in
sample82 in various of cancers, including lung cancer, glioma, 1977.91,92 Since that, DNA sequencing technology has witnessed
breast cancer, ovarian cancer, and soft tissue sarcomas. Extensive rapid development, which is now in its fourth generation of DNA
prognostic and predictive biomarkers, such as ALK, mesenchymal- sequencing technology.93 It not only opens up new perspectives
epithelial transition factor (c-MET), and ROS1, are identified by in traditional biology, medical research, and other fields, but also
FISH.82 Significantly, the timeless and costless FISH remains a gold promotes the further development of bioinformatics, molecular
standard in ALK-rearrangements NSCLC.83 In 2011, a novel genetics,94 genomics,95 precision medicine,96 and other disci-
anticancer drug crizotinib and its companion, the ALK FISH probe plines, which advances the progress of life science research.
detection kit, were simultaneously approved by the FDA, which DNA sequencing technology is not only the gold standard for
highlights the crucial position of the FISH assay in guiding ALK- microbial identification but can also be used to detect the
targeted therapy.84 existence of tumor biomarkers which indicate the occurrence and
In short, FISH is an increasingly demanded tool for biomarker development of tumors. At present, next-generation sequencing
research and personalized medicine despite the fact that the technology (NGS) is the most widely used among four DNA
process of FISH may be time-consuming and costly when sequencing technologies in clinical practice, which can detect
performed with standard chemicals and the retention of the multiple genomic alterations including nucleotide substitutions,
fluorescence is limited.82 small insertions, deletions, copy number variations, and chromo-
somal rearrangements. NGS promotes the identification of
Gene amplification detection technology somatic mutations associated with acute myeloid leukemia
Polymerase chain reaction (PCR), a molecular biological technol- (AML), melanoma, mesothelioma, small cell lung cancer (SCLC),
ogy used to amplify specific DNA fragments, is an invaluable tool breast cancer, and prostate cancer.97 For example, NGS was
for the assessment of nucleic acids in tissues and body fluids. It applied to detect mutations of many cancer-related genes, such as
can synthesize and amplify specific DNA into billions of copies in a TP53,98 phosphatase and tensin homolog (PTEN),99 KRAS,100 and
few hours by separating the DNA into two strands and incubating breast cancer type 1/2 susceptibility protein (BRCA1/2).101 These
it with oligonucleotide primers and DNA polymerase in vitro.85 detections are valuable for assessing the family history and the
PCR technology has developed to the third generation since the risk of tumorigenesis, and improving clinical diagnostics.97 Except
invention of Kary Mullis in 1985,86 and holds a pivotal position in for providing a high sensitivity in gene mutations, NGS is
biological research. The PCR technology includes three major dramatically cost-effective and less time cost compared with
steps: denaturation of double-stranded (ds) DNA template, current PCR-based tests. For example, the cost of using PCR to
annealing of primers (forward and reverse primers), and exten- detect RAS mutations is as high as several thousand dollars, while
sion/elongation of dsDNA molecules.85 The quantitative real-time NGS only costs one-third for detecting the same mutations.
PCR (qPCR)-based assay is considered to be the gold standard for Notably, NGS simultaneously sequences the remaining gene
prognostic and predictive biomarker analysis for the quantitative samples in the same pathway or multiple samples in a single
advantage.85 sequencing run with high speed and accuracy, which avoids
The application of PCR in diagnostic gene mutation analysis, incurring additional operating costs.97
such as the B-raf proto-oncogene (BRAF), EGFR, Kirsten rat Moreover, RNA sequencing has been utilized in multifarious
sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS aspects of cancer management, including prognostic and
viral oncogene homolog (NRAS), and phosphatidylinositol-4,5- predictive biomarker identification, the characterization of cancer
bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) genes from heterogeneity, and the monitoring of drug resistance. Some
the blood, is meaningful in initial cancer stratification and the special genomic biomarkers, including miRNA, lncRNA, and
monitoring of cancer progression. Moreover, several PCR assays circRNA, have been discovered by RNA sequencing.102 For
approved by the FDA are used for the diagnosis of KRAS mutation example, isocitrate dehydrogenase (IDH) mutation which is a
status in formalin-fixed paraffin-embedded tissue, thereby guiding good prognostic biomarker for glioma, and nuclear cyclooxygen-
anti-EGFR antibody treatment for metastatic CRC.87 Similarly, qPCR ase 2 combined with HER2 which serve as potential biomarkers for
assays are effective in the detection of MRD in leukemia, such as the diagnosis and prognosis of CRC, are identified by RNA
the quantification of BCR-ABL-positive cells post-induction che- sequencing.102
motherapy/transplantation in acute lymphoblastic leukemia In conclusion, the advent of sequencing technology sequences
(ALL).85 PCR technology is also widely used to detect abnormal individual cancer genomes, which opens a new chapter in
genes and abnormal mRNA amplification in tumors, such as MYCN precision cancer therapy. Novel sequencing technologies have
amplification in neuroblastoma.88 Ligand-targeted PCR is essential the potential to decode massive amounts of cancer genomes
for the detection of folate receptor-positive circulating tumor cells rapidly and cheaply to benefit cancer precision therapy.
as a potential diagnostic biomarker in pancreatic cancer.89
PCR methods are of great advantages in the detection of IHC technology
nucleic acid biomarkers, including relatively simple manipulation, IHC, a technology used to detect the distribution of antigens (or
providing rapid inexpensive diagnosis with good sensitivity, antibodies) on formalin-fixed paraffin-embedded tissue sec-
valuable for clinical molecular pathology.87 Nevertheless, several tions,103 identifies targets through antigen–antibody interactions,

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and the antibody binding site is identified by direct labeling or EM has been widely used in investigating tumorigenesis-related
secondary labeling method. cellular and subcellular change133 and observing the ultrastructure
IHC is a gold standard and ubiquitously applied technology in changes in cancer cells,134 as well as in clinical applications for
cancer identification and diagnosis, especially in assessing cancer diagnosis and treatment.134–136 The ultrastructural features
biomarkers used for characterizing tumor subtypes, confirming of tumor cells by EM can provide vital clues such as evidence or
tissue origin, distinguishing metastasis from primary tumor, biomarkers of cytodifferentiation for correct diagnosis, which is
providing prognosis information, stratifying patients for treatment difficult for diagnosis of light microscopy.137,138 Especially, the
selection, and predicting therapy response in various can- ultrastructural examination provided by EM is necessary for the
cers.104–109 The American Society of Clinical Oncologists and precise categorization of biomarkers in apparently undifferen-
College of American Pathologists (ASCO/CAP) provides the tiated carcinoma.138 Thus, EM is useful in the differential diagnosis
HER2 scoring guidelines to determine breast cancer pathological of tumors, particularly in small-cell “undifferentiated” tumors, such
classification and clinical stage by using IHC-based staining as neuroblastoma, rhabdomyosarcoma, Ewing’s sarcoma, undiffer-
intensity and the percentage of HER2+ cells in cancer tissues.110 entiated squamous cell carcinoma (SCC) of the lymphoepithe-
IHC is used to detect p53 in cancer tissues.111,112 The detection of lioma type, and malignant lymphoma, amelanotic melanoma, and
the excision repair cross-complementation group 1 protein by IHC spindle-cell carcinoma.137 Scanning electron microscopy has been
has been approved for predicting the response of NSCLC patients used as an alternative to examine the morphology of exosomes
to chemotherapy.105 which is a diagnostic biomarker usually detected by liquid
As an indispensable technology, IHC holds the unique advantage biopsy.139 In conclusion, EM is a valuable complementary tool
of correlating the presence of protein with its location in tissues or for tumor diagnosis, especially providing valuable information on
cells compared with other protein detection methods, which is tumor differentiation which is difficult to define by light
essential for illustrating protein function in normal and pathological microscopies.134,140
tissues.113 Moreover, IHC can be operated by easy preparation and
automated manipulation.82 However, several limitations still exist in CRISPR/Cas9 technology
IHC, especially a lack of reproducibility. Conflicting results often occur The CRISPR/Cas9 technology is a gene-editing tool that is based
when different antibodies are used. The variables of the protocol on the bacterial immune system. The basic principle of CRISPR/
affect the reliability of IHC, including the fixation time of tissues, the Cas9 is to use a guide RNA molecule to direct a nuclease, Cas9,
absolute level of the antigen, the affinity and concentration of to a specific target gene. The nuclease then cleaves the DNA at
antibody, and the sensitivity of the detection system.111 Thus, high- the target site, allowing for precise modifications of
quality control of regents, standardized protocols, automated IHC, or genome.141,142
combined IHC with transcriptomics will improve the accuracy, By using CRISPR/Cas9 technology to precisely edit cancer-
reproducibility, and reliability of IHC and accelerate its application in related genes, researchers have created highly specific molecular
the discovery and validation of biomarkers.114 probes for the detection of cancer biomarkers in body fluids, such
as blood, urine, and saliva. CRISPR/Cas9 system is extensively used
Liquid biopsy technology for different kinds of cancer biomarkers including virus nucleic
Liquid biopsy, a minimally invasive methodology, is used to obtain acids, ctDNAs (i.e., EGFR mutation), miRNAs (i.e., miR-17, miR-31),
tumor-derived information from body fluids so as to facilitate proteins (i.e., TGF-β1, CEA, PSA, AFP), and extracellular vesicles.90
cancer diagnosis.115 Currently, liquid biopsy is used to detect CRISPR/Cas9 can combine with other assays for tumor biomarker
cfDNA, cell-free RNA, CTCs, extracellular vesicles,116,117 ctDNA,118 identification, such as qPCR, FISH, and nanotechnology, providing
circulating RNA, and exosomes119,120 in blood or other body an efficient way for tumor biomarker discovery. Moreover, CRISPR/
fluids.116,121 Liquid biopsy can enhance patient overall survival Cas9 has exerted significant effects in the treatment of cancers,
(OS) by improving early cancer detection and monitoring such as pancreatic cancer, prostate cancer, breast cancer, ovarian
treatment response continuously. Thus, liquid biopsies are widely cancer, liver cancer, and CRC.143
used in the clinical biomarker screening of tumors, such as The CRISPR/Cas9 system enjoys some advantages, including low
endometrial cancer,122 lung cancer,123 pancreatic cancer,124 cost, high efficiency, low application complexity, easy-to-operate,
CRC,125 melanoma,126 renal cell carcinoma (RCC),127 breast cancer, and time-saving.30 The exquisite specificity is also a character of
ovarian cancer, cervical cancer, and bladder cancer.128 In addition, the CRISPR/Cas9 system which could distinguish single base
liquid biopsy technology is also utilized to detect and monitor mismatch in target nucleic acid.90 Moreover, CRISPR/Cas9-based
KRAS, BRAF, and EGFR mutations in patients with lung cancer, nucleic acid amplification strategies exhibit high detection
CRC, and breast cancer.128 sensitivity comparable with PCR. However, several aspects of
Liquid biopsy can reduce the risk of biopsy by noninvasive CRISPR/Cas9-based diagnosis still need to be improved. CRISPR/
sampling,128 and it has the advantages of convenient sampling Cas9-based analysis requires the fluorescence spectrophotometer
and easy operation. Moreover, liquid biopsies have the potential and electrochemical workstation which is inconvenient for
to better detect heterogeneity across regions of the tumor.115 detection. Thus, the portable and quantitative detection strategy
Although there are still some challenges to overcome in terms of should be further explored to monitor cancer biomarkers. Cancer
assay sensitivity and specificity, liquid biopsy technology provides progression is influenced by the level of multiple biomarkers such
new opportunities for personalized cancer treatment and has the as various miRNAs, ctDNAs, and proteins, which makes the design
potential to revolutionize the field of oncology. of the high-throughput CRISPR/Cas9-based strategy for cancer
biomarkers detection promising and significant.90 In conclusion,
Electron microscopy technology CRISPR/Cas9 technology is a powerful gene-editing tool that holds
Electron microscopy (EM), a powerful imaging technique used to great promise and opportunities for the development of
visualize the ultrastructure of cells and tissues with high personalized cancer management.144
resolution, is applied based on a special type of microscope,
electron microscope.129,130 The first electron microscope was built
in 1931 by a German engineer and academic professor Ernst CLASSIFICATION OF TUMOR BIOMARKERS
Ruska.131 The electron microscope uses signals obtained from the Tumor biomarkers are diverse and can be classified by different
interaction between an electron beam and the sample to achieve standards. Here, we divide tumor biomarkers by tissue origin:
information about sample structure, morphology, and tumor biomarkers derived from blood, tumor tissues, and other
composition.132 biofluids such as feces, urine, and saliva (Fig. 3).

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Fig. 3 Overview of human tumor biomarkers
Tumor biomarkers derived from blood

Tumor biomarkers in the blood are highly significant for tumor antigens in cancer patients may be related to the activation of
diagnosis and treatment. They have vital reference values for early certain genes that have been turned off in adulthood when
tumor diagnosis, tumor stage assessment, anticancer strategy malignant cells transform, and these genes make embryonic
selection, treatment response monitoring, and prognosis.2,145 antigens. While there aren’t many embryonic antigen tumor
Here, we summarize the common tumor markers in blood and biomarkers, the ones that exist are crucial biomarkers for cancer
their roles in cancers. care in clinical practice.
AFP. AFP, first discovered in 1956 by Bergstrand Czar,146 is a 3–5%

Embryonic antigen tumor biomarkers. The 1960s saw the carbohydrate-containing single-chain glycoprotein.150 Encoded by
discovery of AFP and CEA, two tumor biomarkers that are still the AFP gene located in the q arm of chromosome 4 (4q25), AFP is a
widely employed as tumor biomarkers. AFP and CEA are member of the albuminoid gene superfamily.151 As the amino acid
embryonic antigen substances which are proteins that only sequences of AFP and albumin are very similar and highly
appear in the fetal period and gradually decline and disappear homologous, AFP is considered as an analog of serum albumin in
in adulthood.146–149 The reemergence of these embryonic the fetal period and is the main protein in fetal circulation. At 18

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months after birth, albumin synthesis gradually increases, and AFP cancers.168,169 Furthermore, elevated serum SCCA is associated
concentration gradually decreases. The concentration of AFP in with the therapeutic effect of postoperative chemotherapy in
healthy adult serum is less than 10 μg/L.147 AFP is currently the most esophageal squamous cell carcinoma (ESCC),170 and with tumor-
widely used tumor biomarker for HCC and has been used for more node-metastasis stage in head and neck squamous cell carcinoma
than 60 years. Elevated AFP can be seen in ~80% of HCC (HNSCC).171 Peripheral SCCA has also been extensively utilized as
patients.152,153 Thus, AFP is currently applied for HCC screening, one of the tumor biomarkers for monitoring NSCLC and predicting
especially in China, Japan, Africa, and Alaska. The international patients’ response to platinum combination chemotherapy, and
academic community recommends limiting the reference value of serum SCCA level accurately reflects the survival status of
AFP to 20 μg/L. Moreover, early-stage HCC is frequently detected by patients.169 Despite its limited sensitivity in routine tests, SCCA is
AFP detection combined with ultrasound.154 Tumor prognosis and still a valuable diagnostic and prognostic biomarker in cancers.
treatment monitoring are additional applications for AFP. In patients
with HCC, a sharp increase in AFP indicates tumor recurrence or TPS. Tissue polypeptide-specific antigen (TPS) is an M3 antigen
metastasis. AFP >200 μg/L after surgery indicates incomplete determinant on the 18 fragments of cell keratin.172 TPS is
removal or metastasis of HCC.155 Nonetheless, AFP levels are not synthesized in the S and G2 phases of the cell cycle, and the
the perfect diagnostic criteria for HCC. Approximately 40% of patients level of TPS in serum specifically indicates the proliferative activity
with early-stage HCC express normal or acceptable AFP levels. The of cells.173 The levels of TPS mostly depend on the number of cells
elevation of AFP levels is observed in patients with chronic liver in the proliferative phase instead of the total number of tumor
diseases, including ~20% of patients with hepatitis and 40% of cells, which is different from other tumor biomarkers.173 The
patients with cirrhosis.156 serum levels of TPS are noticeably increased in multiple tumors,
such as endometrial cancer, bladder cancer, NSCLC, skin cancer,
CEA. CEA was first extracted from human CRC tissues and carcinoma of male urethra, prostate cancer, pancreatic cancer,
embryonic tissues in 1965, hence it was named for CEA.148 CEA CRC, gastric cancer, esophageal cancer, neuroblastoma, and
belongs to a family of glycoproteins on the cell surface, and its nephroblastoma.174–177 Thus, TPS has been employed as a serum
gene is located on chromosome 19q.157 The production of CEA in tumor biomarker. Due to its lack of sensitivity and organ
the digestive tract starts at the early fetal stage (week 9–13). In specificity, the prime application of TPS is monitoring treatment
addition to normal adult tissues such as the colon, stomach, cervix, efficiency, and predicting tumor progression and recurrence,
sweat glands, and prostate, CEA is highly expressed in various rather than diagnostic utility. In breast cancer patients, elevated
tumors.149 serum levels of TPS could predict distant metastasis after
As a broad-spectrum tumor biomarker, CEA is elevated in 70% treatment,178 and are recognized as an independent prognostic
of CRC, 55% of pancreatic cancer, 50% of gastric cancer, 45% of factor for disease-free survival (DFS) and OS of patients.179 In
lung cancer, 40% of breast cancer, 40% of urethral carcinoma, and gastric cancer, TPS is applied in monitoring the palliative
25% of ovarian cancer patients.149,158,159 Serum CEA levels are treatment response of patients with a 75% detection rate.180
proportional to tumor burden. Accordingly, CEA is applied to aid The potential clinical role of TPS in RCC prognosis has also been
the diagnosis, determine the prognosis, monitor recurrence, and demonstrated.181
evaluate the therapeutic efficacy in cancer patients.160 In patients Additionally, it is worth noting that TPS levels can alter in
with breast cancer, CEA is one of the most frequently used response to some pathological and physiological conditions, such
biomarkers in the diagnosis, prognosis, and prediction of survival as chronic pancreatitis, liver cirrhosis, ovulation, and menopausal
for different breast cancer molecular subtypes.161 In CRC patients, status. Thereby, TPS in combination with other prognostic factors
CEA level is a meaningful prognostic and diagnostic biomarker. is necessary to improve the clinical use of serum TPS levels in
The levels of CEA predict the 5-year survival rate of patients: 69% predicting patient prognosis and facilitating the individualization
of patients have a CEA level below 5 ng/mL, 44% have a level of of therapy for cancer patients.182 Further clinical studies are
5–200 ng/mL, and only 7% have a level equal or greater than required to fully determine the utility of TPS alone or in
200 ng/mL.162 The elevated CEA level also has a bearing on poor combination.182,183 In conclusion, TPS has a unique value in the
prognosis and progression of lung adenocarcinoma patients with prediction of recurrence and metastasis, treatment monitoring,
mutant EGFRs, and gastric cancer patients with lymph node and prognostic evaluation in cancer patients.177
metastasis.163 Additionally, CEA is also used for efficacy evaluation
and recurrence detection after tumor treatment.164 PSA. PSA, a serine protein kinase-releasing enzyme specifically
Nevertheless, CEA lacks good sensitivity and specificity, which secreted by the epithelial cells of prostate,184 is encoded by the
renders CEA inappropriate for tumor screening. A combination of prostate-specific gene kallikrein 3 which is a member of the tissue
CEA with other biomarkers could improve its actual significance in kallikrein family.185 PSA was first identified in the late 1970s.186
clinical practice.149,164 The elevated serum PSA levels represent prostate pathologies
including prostatitis, benign prostatic hyperplasia, and prostate
SCCA. Squamous cell carcinoma antigen (SCCA), a tumor-specific cancer.187,188 For the early diagnosis of prostate cancer, the
antigen, was first isolated from cervical SCC tissue by Kato and positive cut-off value of serum PSA is greater than 10 ng/mL. In
Torigoe in the 1970s.165 Initially, SCCA was used as a tumor 1986, PSA was approved by the FDA as an adjunctive test for the
biomarker for cervical cancer, and it has a high independent detection of prostate cancer in men over the age of 50.185,189
diagnostic value in cervical cancer.166 The serum level of SCCA Subsequently, in 1994, PSA was approved by the FDA as a
correlates with the stage, the degree of invasion, recurrence, and diagnostic biomarker.189 Later on, PSA became popular in prostate
the progression of cervical SCC.159 Cervical cancer patients with a cancer detection and patient management including screening,
high-level of pretreatment serum SCCA exhibit a higher risk for risk stratification for recurrence, surveillance following diagnosis,
death than patients with low serum SCCA. Pretreatment SCCA and monitoring therapy.186,188 Total PSA essentially consists of
cutoff ranging from 1.1 to 40.0 ng/mL is related to recurrence and free PSA and bound PSA, and the higher percentage of the free
death.166,167 Subsequent research has revealed that SCCA exists in PSA is connected to the lower the cancer risk. Studies have shown
tumors in the mouth, pharynx, esophagus, lung, and other tissues. that a free PSA percentage >25% indicates the cancer risk is <10%,
In particular, high levels of SCCA have been found in multiple SCCs but a free PSA percentage <10% means the cancer risk is ~50%.187
including lung cancer, esophageal cancer, and genitourinary However, PSA holds a poor specificity of 20–40% in prostate
system cancer in addition to cervical cancer, suggesting its cancer diagnosis. Some noncancerous pathologies such as
essential role in the diagnosis and prognosis of the above inflammation, trauma, or benign prostatic hyperplasia may also

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elevate the PSA level, which leads to a high rate of false positives. effective at converting lactic acid back to pyruvate than LDHA is at
Besides, PSA is unable to differentiate between indolent and converting pyruvate to lactic acid.211,212 Multiple factors, such as
aggressive forms of prostate cancer, which may ignore aggressive the oncogene c-Myc and hypoxia-inducible factor (HIF-1α),
prostate cancer with low initial serum PSA levels.187,190 All the stimulate the transcription of LDHA,213,214 which results in the
aforementioned factors make prostate cancer now an “over- overexpression of LDHA in most tumor tissues.215
diagnosed” and “overtreated” cancer.185 To sum up, PSA level is a High expression of LDHA provides cancer cells with many
promising biomarker in prostate cancer diagnosis and prediction. benefits, and multiple studies have proved that high levels of
serum LDH are associated with the proliferation of cancer-
NSE. Neuron-specific enolase (NSE), a member of the enolase initiating cells, enhanced aggressiveness and metastasis, the poor
gene superfamily in glycolysis, was originally identified by Moore prognosis of cancers, as well as radiation and chemotherapy
and McGregor in 1965 as an enzyme enriched in neurons and resistance.216–218 The serum LDH level is considered to be a
peripheral neuroendocrine cells.191,192 NSE consists of five dimeric primary predictor of prognosis in patients with adverse prognosis
isoenzymes with three different subunits, α, β, and γ, and is a sign and distant metastases in melanoma, RCC, and CRC.216 Accord-
of mature neural differentiation.193 Cell proliferation accelerates in ingly, an analysis of 76 studies comprising 22,882 patients with
response to oncogenic transformation in either central or solid tumors reveals that high serum LDH levels are linked to poor
peripheral neurons, accompanied by enhanced glycolysis and survival in patients with solid tumors, in particular in melanoma,
elevated NSE expression. Consequently, NSE plays pivotal roles in prostate cancer, and RCC, and is a valuable and affordable
diagnosis, prognosis, and treatment efficacy evaluation in cancers prognostic biomarker in metastatic cancers.40 Serum LDH levels
originating from neural and neuroendocrine.194,195 Moreover, are closely correlated with OS in an analysis of 2507 cancer
elevated NSE is also observed in SCLC which is with neuroendo- patients with brain metastasis216 and are a poor prognosticator for
crine properties. Serum NSE is currently believed to be a clinically OS and DFS in nasopharyngeal carcinoma (NPC) patients.
potential biomarker for staging, monitoring treatment, and Furthermore, the elevated serum LDH levels could be used to
predicting relapse of SCLC.196,197 Interestingly, NSE also exerts a develop individualized treatment strategies.219 A study of a total
significant function in NSCLC. An analysis of 363 patients with of 68 studies including 31,857 patients illustrates that LDH
advanced and metastatic NSCLC showed that patients with high overexpression is a predictor to guide individual therapy in solid
NSE level (≥26.1 ng/mL) have significantly shorter progression-free tumors,220 such as testicular cancer,221 SCLC,219 and gastrointest-
survival (PFS) (5.69 vs 8.09 months) and OS than patients with low inal cancer.222,223 In conclusion, LDH is a valuable indicator of
NSE level (11.41 vs 24.31 months).191 Besides, increased serum cancer diagnosis, efficacy evaluation, and recurrence and
NSE levels are found in 30–69% of patients with NSCLC,198,199 metastasis.
which is in accordance with a study of 621 NSCLC patients which
shows high NSE level (>12.5 ng/mL) is a prognosticate of poor CA72-4. Carbohydrate antigen 72-4 (CA72-4) is a mucin carcinoid
outcome.200 Thus, serum NSE level is a predictive biomarker of embryonic antigen found in liver metastases of breast cancer in
cancer treatment response and an independent prognostic 1981 and is highly expressed in human adenocarcinoma.224
factor.191 Enhanced serum CA72-4 levels are effective indicators for the
diagnosis of cancers, including gastric cancer, pancreatic cancer,
AFU. α-L-Fucosidase (AFU), consisting of two isoforms, AFU1 and CRC, breast cancer, ovarian cancer, lung cancer, cervical cancer,
AFU2, which are encoded by FUCA1 and FUCA2 genes, respec- and endometrial cancer.225,226 Notably, CA72-4 exerts diagnostic
tively, is a lysosomal enzyme that clears the terminal α-l-fucose value in patients with digestive system tumors, especially gastric
residues from glycoproteins.201 AFU is involved in the metabolism cancer, with superior sensitivity and specificity.227 Studies have
of glycoproteins, glycolipids, and oligosaccharides, and is widely demonstrated that the sensitivity and specificity of CA72-4 applied
distributed in human tissues and blood. The serum AFU level in the diagnosis of gastric cancer alone are 49 and 96%,
remains low under normal circumstances. While the serum AFU respectively, which outperforms other tumor biomarkers such as
level increases rapidly as long as tumors attack the body, its level CEA (sensitivity 41%, specificity 93%), CA19-9 (sensitivity 44%,
is closely related to the tumor stage and size.202 Multiple studies specificity 92%), and CA242 (sensitivity 38%, specificity 97%).228
have shown that AFU is one of the most valuable biomarkers for The serum level of CA72-4 is also correlated with the malignant
HCC detection, with 85% sensitivity and 91% specificity.203,204 85% grade of gastric cancer. Thus, CA72-4 is used as the best serum
of patients with HCC can be diagnosed with AFU detection six marker for gastric cancer diagnosis in China.229 However, CA72-4
months prior to the ultrasonography detection.205 Patients with a also has limitations. It has been uncovered that CA72-4 is highly
preoperative AFU >35 U/L have a lower recurrence-free survival expressed in normal tissues in addition to tumor tissues such as
(RFS) rate and OS rate than those with AFU ≤35 U/L, and they tend the endometrium and the colonic transitional mucosa, which
to form macrovascular invasion. Therefore, serum AFU is of great results in false positives in patients with atrophic gastritis.230 The
significance for judging the treatment effect, prognosis, and sensitivity of CA72-4 in the diagnosis of gastric cancer is far from
recurrence of HCC.205,206 Besides, the low AFU levels are satisfactory.231 The combination with other biomarkers may gain
significantly associated with longer OS in ESCC, which indicates increased sensitivity and specificity of CA72-4 in tumor applica-
that AFU is a potential prognostic biomarker for long-term survival tions. In conclusion, serum CA72-4 is a unique biomarker of gastric
in patients with early-stage ESCC.207 However, the serum levels of cancer for screening, diagnosis, the prediction of metastasis and
AFU are also mildly elevated in certain nonneoplastic conditions recurrence, and the evaluation of treatment efficiency.229
such as cirrhosis, chronic hepatitis, and gastrointestinal bleed-
ing.203,208 Presently, the combination of AFU and AFP biomarkers CA125. CA125, a highly glycosylated mucin, is originally dis-
is used in the diagnosis of HCC, which enhances the diagnostic covered in a monoclonal antibody OC125 screening against the
specificity, and makes the diagnosis more stable and reliable for ovarian cancer cell line OVCA433.232,233 Thus, CA125 has become
high-risk groups such as hepatitis and cirrhosis.155 one of the most important biomarkers for monitoring epithelial
ovarian cancer, and its sensitivity in the diagnosis of epithelial
LDH. LDH, an enzyme that catalyzes the reversible transfer of ovarian cancer reaches ~70%.234 The key role of CA125 in the
pyruvate to lactate and NADH to NAD + , consists of two different prognosis of ovarian cancer patients has also been recognized.
isoforms, lactate dehydrogenase A (LDHA) and LDHB.209,210 The The Gynecologic Cancer Group (GCIG) has shown that the serum
two isoforms can form five homotetramers or heterotetramers level of CA125 is associated with the progression and recurrence
with different functions.210 In the reverse reaction, LDHB is more of ovarian cancer. According to the criteria of GCIG, patients with

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serum CA125 levels within the reference range (<35 U/mL) after levels.256,257 Similar to other carbohydrate antigens, serum CA50
surgery or chemotherapy are considered fully effective. While the is also increased in patients with non-neoplasm diseases such as
CA125 level increased to twice of the minimum value (≥70 U/mL), chronic pancreatitis, colitis, cholecystitis, and pneumonia.256
the progression or recurrence is considered.235 Moreover, CA125 is
also a diagnostic and prognostic biomarker for other nonovarian CA19-9. CA19-9 is initially found in human CRC cell line SW1116
tumors, such as cervical cancer, endometrial carcinoma,236 and and belongs to the mucin glycoprotein antigen.258 It is extensively
gastric cancer.237 Of note, ~1% of healthy people and 5% of distributed on the cell membrane of Lewis antigen-positive
patients with menstrual or benign diseases such as endometriosis epithelial cells such as the pancreatic duct, gallbladder, and
and coronary artery disease have varying degrees of elevated gastrointestinal tract. CA19-9 is currently the most commonly used
serum CA125 levels.238–240 and gold-standard biomarker for pancreatic cancer,259,260 and
holds a median sensitivity of 79% for diagnosis of pancreatic
CA242. Carbohydrate antigen 242 (CA242), a sugar chain antigen cancer.261,262 In addition, CA19-9 has also been used as a
containing sialic acid, is obtained after the immunization of mice biomarker for other cancers, particularly digestive tract cancers.263
with a human CRC cell line COLO 205.241 An analysis of serum Other diseases such as liver damage, bile duct obstruction and
CA242 levels from 34,680 patients with 27 clinically defined inflammation, pancreatitis, acute diarrhea, stomach ulcer, and
diseases suggests that patients with pancreatic cancer, cervical pulmonary fibrosis have also been linked to increased CA19-9
cancer, and lymphoma have the highest level of serum CA242, levels.264–266 Notably, CA 19-9 is not expressed in cells from
which are followed by esophagus cancer, CRC, ovarian cancer, and patients with Lewis allele deficiencies, and it is necessary to
breast cancer.242 Hence, the primary application of CA242 is as a ascertain the patient’s Lewis gene type information when
biomarker for CRC and pancreatic cancer.243 Serum CA242 has a applying CA19-9 as a diagnostic biomarker.267,268
normal reference value of less than 17 U/mL. The sensitivity for
diagnosing pancreatic cancer and CRC is ~70 and 45%, HE4. Human epididymal protein 4 (HE4), an orotic acid protein, is
respectively, and the specificity is ~95 and 83%, respec- first identified in distal epididymal epithelial cells.269 HE4 is widely
tively.244–246 As CA242 exhibits a lower sensitivity for diagnosing expressed in the trachea, salivary gland, lung tissue, etc., and is
pancreatic cancer, the combination of CA242 with CEA is a highly expressed in ovarian cancer, endometrial cancer, and lung
promising strategy for improving diagnosis sensitivity in pancrea- cancer. Meanwhile, age and menopausal status are also momen-
tic cancer.247 In addition, CA242 is also used as a clinical indicator tous factors affecting HE4 levels.270,271 At present, serum HE4 is
of progression or recurrence during chemotherapy for pancreatic primarily used for the diagnosis and recurrence monitoring of
cancer.241,242 ovarian cancer with a sensitivity of 67%. HE4 is also used to
evaluate the treatment effect of ovarian cancer.270,272 In addition,
CA15-3. Carbohydrate antigen 15-3 (CA15-3, also known as HE4 is also overexpressed in other non-gynecologic malignancies,
mucin 1) is a large transmembrane glycoprotein derived from including NSCLC, pancreatic cancer, and transitional cell
the MUC1 gene.248 It is expressed in normal tissues including the carcinoma.273
breast, esophagus, stomach, duodenum, pancreas, uterus, pros-
tate, and lung.248,249 Notably, CA15-3 is overexpressed in the Ferritin. Ferritin is the leading protein that is essential for iron
majority of human cancers, and is thought to be a key biomarker storage and detoxification.274 Ferritin is present in numerous
for cancers, especially for indicating cancer metastasis.250 The normal tissues such as liver, spleen, bone marrow, and body
reference value for normal serum CA15-3 levels is less than 28 U/ fluids.274 Serum ferritin levels are linked to a broad range of
mL.243 In breast cancer, serum CA15-3 is used as an auxiliary conditions. The low serum ferritin concentration indicates iron
diagnostic index with a diagnosis sensitivity of 61.5–70% which is deficiency, e.g., anemia and diarrhea,275 and the high serum
higher than that of CEA.243 Thus, CA15-3 in combination with CEA ferritin concentration indicates iron overload, e.g., hemochroma-
is the most popular method for breast cancer diagnosis.251 tosis and hemolytic anemia, or infection and liver disease.276
Meanwhile, CA15-3 is also a crucial indicator for the evaluation of Moreover, ferritin is overexpressed in various cancers, such as HCC,
postoperative recovery, recurrence, and metastasis of breast lung cancer, lymphoma, melanoma, and CRC.277,278 As indicated
cancer.248 It is noteworthy that serum CA15-3 is also elevated to by its potential to promote tumor proliferation, angiogenesis,
varying degrees in benign diseases of the breast, liver, gastro- immunosuppression, and tumor drug resistance,276 ferritin is
intestinal tract, lung, and other organs, but the positive rate is valuable in evaluating the progression and prognosis of cancer
low.250 patients. Nevertheless, a number of factors influence ferritin levels,
and ferritin’s limited specificity for tumor detection means that it is
CA27-29. Similar to CA15-3, carbohydrate antigen 27-29 (CA27- not an ideal diagnostic marker for cancers.279
29) is a critical epitope for the MUC1 protein.243 With a sensitivity
of 84% for breast cancer detection, CA27-29 is primarily utilized in p2PSA. Prostate-specific antigen precursor (p2PSA) is a precursor
breast cancer patients for diagnosis, and efficacy evaluation.243 that is first secreted in the prostate gland ducts during the
Additionally, it also be used in combination with other markers to production of PSA.280 p2PSA is a relatively stable pro-PSA and has
increase the specificity of breast cancer diagnosis.252 The elevated certain clinical value in the diagnosis of early prostate cancer. The
CA27-29 is also observed in other cancers including CRC, stomach prostate health index, which forecasts the diagnosis of prostate
cancer, pancreatic cancer, ovarian cancer, and benign diseases of cancer, is calculated by PSA and p2PSA. Currently, the prostate
the breast and liver.253 health index is the strongest predictor of diagnosis at initial biopsy
when total PSA levels are between 2.0 and 10 ng/mL in prostate
CA50. Carbohydrate antigen 50 (CA50) was initially identified as cancer patients, and the prostate health index has been approved
a cancer-specific antigen screened from monoclonal antibodies by the FDA for early diagnosis and risk grading of prostate
against CRC cell line COLO 205 in 1983.254 It is generally absent in cancer.280,281
normal tissues, but elevated in multifarious cancers. Patients
suffering from pancreatic cancer, lung cancer, and colon cancer HCG. HCG is a polypeptide hormone composed of two
exhibit the highest levels of serum CA50. Serum CA50 is quite noncovalently linked subunits (α and β). The smaller α subunit is
effective in the diagnosis of pancreatic cancer, with a sensitivity of the part of follicle-stimulating hormone and luteinizing hormone,
more than 84%.255 Meanwhile, patients suffering from gastric while the larger β subunit is unique to HCG.282,283 Serum levels of
cancer and rectum cancer reveal comparable serum CA50 HCG in non-pregnant and menopause women maintain at a low

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level of 5–10 U/L, and increase dramatically during pregnancy.284 related kinase (ERK) pathway, are activated in tumor cells, which
Increased serum HCG levels are observed in trophoblastic tumors, subsequently regulate tumor cell proliferation, migration and
ovarian cancer, testicular cancer, breast cancer, lung cancer, HCC, invasion, gene transcription, cellular metabolic reprogramming,
CRC, and kidney cancer. Although HCG level could be employed and tumor microenvironment (TME) remodeling.301–303
for monitoring the disease progression, it is too low to be
regarded as a diagnostic marker.282,285 RAS: RAS genes, named after the rat sarcoma,304 were identified
as the transformative factor in the Harvey and Kirsten strains of rat
CAM17.1. CAM17.1 is a mucin with high specificity for the sarcoma viruses305 and were identified in the human genome in
digestive system, such as the pancreas, colon, small intestine, and 1982.304,305 RAS proteins belong to a superfamily of GTPases, and
biliary tract.286 Several studies revealed that CAM17.1 is particu- three RAS genes (HRAS, NRAS, and KRAS) encode four highly
larly overexpressed in pancreatic cancer with a serum cut-off value homologous RAS proteins: HRAS, NRAS, KRAS4A, and KRAS4B, with
is 39 U/L.287 CAM17.1 has a sensitivity of 86% for the diagnosis of the latter two KRAS isoforms arising from alternative
pancreatic cancer, and a higher sensitivity of 89% in patients splicing.306,307
without jaundice.286 These findings suggested that CAM17.1 is a RAS proteins couple cell surface receptors to intracellular
potential biomarker for pancreatic cancer diagnosis, which effector pathways through binding to GTP or GDP, followed by
triggers the need for further study. a cycle between the GDP-bound inactive state (RAS-GDP) and the
GTP-bound active state (RAS-GTP). Under physiological conditions,
PIVKA-II. Protein induced by vitamin K absence or antagonist-II RAS proteins retain an inactive state, and are incapable of
(PIVKA-II) is an abnormal prothrombin elevated in the conditions interacting with downstream effectors. When activated by
of vitamin K reduction or the presence of vitamin K antagonists.288 upstream receptors, RAS is activated by guanine nucleotide
PIVKA-II is primarily used for the early detection of HCC, with a exchange factors (GEFs) which promote GDP to GTP exchange,
sensitivity and specificity of 97.5 and 90%, respectively.289,290 In thereby recruiting diverse downstream effectors such as the RAF-
other tumors such as gastric cancer and pancreatic cancer, PIVKA- MEK-ERK pathway and the PI3K-AKT-mTOR pathway.301,308 RAS
II is also elevated at varying degrees.291 In addition to being able activation has been linked to multiple tumor phenotypes,
to differentiate between other non-malignant conditions such including cell cycle progression, proliferation, metastasis, and
cirrhosis or chronic hepatitis, serum PIVKA-II is more accurate than apoptosis resistance.301 Furthermore, RAS is involved in diverse
AFP in the diagnosis of early-stage HCC.292,293 It is noteworthy that metabolic processes such as aerobic glycolysis, glutaminolysis,
certain patients with vitamin K deficiency also exhibit elevated redox homeostasis, and lipid metabolism in tumor cells to support
PIVKA-II levels.288 tumor growth.309 Importantly, RAS activation remodels the
TME,301 including the initiation and maintenance of proangiogen-
GRP. Gastrin-releasing peptide (GRP), first isolated from gastric esis,310 the production of proinflammatory factors,311 and immune
nerve fibers by McDonald in 1978, is a gastrointestinal hormone escape.301
that exits in the normal bronchial epithelial cells, pulmonary RAS mutation is a prominent factor that plays a vital role in
fibroblast, central nervous system cells, and neuroendocrine tumorigenesis and progression.312,313 Approximately 21% of all
cells.294 Significantly, GRP is overexpressed in multiple cancers, malignancies have RAS mutations,308 which include CRC,314
including 62% of CRC patients, 59% of pancreatic cancer patients, pancreatic ductal adenocarcinoma (PDAC),315 lung adenocarci-
60% of prostate cancer patients, 39% of breast cancer patients, noma,316 and melanoma.317
and 74% of SCLC patients.294 Since GRP has a short half-life and is Although the function of RAS in the physiological or
unstable, it is more appropriate to detect its precursor, pro-GRP.294 pathological states has been thoroughly elucidated in the past
With a sensitivity of 47 to 86%, serum pro-GRP detection is mainly decades, numerous unresolved concerns still need to be
utilized for the diagnosis, efficacy, and prognosis analysis of SCLC, investigated. For instance, the regulatory relationship between
outperforming NSE.294,295 The combined application of pro-GRP RAS and downstream effectors other than PI3K and MAPK.318 To
and NSE increases the sensitivity of SCLC detection.296 In addition, sum up, RAS is a crucial biomarker for tumor diagnosis, prognosis,
pro-GRP is also elevated in a few other diseases, such as gastritis and treatment.
and acute hepatitis, but the positive rate is generally low.297
KRAS: KRAS is by far the most frequently amplified and mutated
Tumor biomarkers derived from tumor tissues RAS isoform among the three RAS genes, accounting for 85% of all
Since the six hallmarks of cancer were proposed in 2000, tumor RAS mutations.319 KRAS mutations were first identified in 1984 in
characteristics are considered to be a set of functional capabilities patients with squamous cell lung cancer.320 Notably, KRAS
acquired by human cells during the transition from a normal to a mutations are present in 88% of pancreatic cancer, 50% of CRC,
tumor growth state.298 To date, tumors have possessed fourteen and 32% of lung cancer.319,321 The most common mutations in
major characteristics, including sustaining proliferative signaling, KRAS are G12D, G12V, G12C, G13D, and Q61R, which account for
evading growth suppressors, enabling replicative immortality, 70% of RAS mutations in cancer patients.321 KRASG12C mutation is
inducing angiogenesis, resisting cell death, activating invasion and the most frequent,321 and the G12C mutation alters KRAS
metastasis, genome instability, and mutation, tumor-promoting conformation and shape by forming binding pockets, leading to
inflammation, deregulating cellular metabolism, avoiding immune increased affinity for GTP and sustained activation of KRAS,
destruction, unlocking phenotypic plasticity, nonmutational epi- ultimately triggering the transduction of downstream oncogenic
genetic reprogramming, and polymorphic microbiomes, and signaling.319,321
senescent cells.298–300 Herein, we summarize the tumor biomar- KRAS mutations have emerged as biomarkers for the prognosis,
kers from tumor tissues divided by cancer hallmarks (Fig. 4). diagnosis, and treatment of some tumors, including PDAC,322
CRC,323 and lung cancer.324 A study in a pooled analysis has found
Sustaining proliferative signaling. Cancer cells are capable of that KRAS mutations are independently associated with shorter
multiple approaches to acquire the ability to sustain proliferation. time to recurrence, survival after relapse, and OS in patients with
Stimulated by growth factors and other proliferative signals, microsatellite-stable resected stage III CRC.313 Patients with the
proliferation-related signaling pathways, such as the RAS, the KRASG12C mutation are related to inferior PFS and OS compared
phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT)-mamma- with patients with non-mutated tumors, according to a prognosis
lian target of rapamycin (mTOR) pathway, and the RAF-mitogen- analysis in 1239 patients with metastatic CRC.323 Moreover, KRAS
activated protein kinase (MAPK) kinase (MEK)-extracellular signal- mutations link to the poor prognosis of patients with PDAC, and

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Fig. 4 The 14 cancer hallmarks-based biomarkers. Fourteen major characteristics of tumor cells have been proven so far, which have been
divided into acquired hallmarks including sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling
replicative immortality, inducing angiogenesis, activating invasion and metastasis, enabling hallmarks including genome instability and
mutation, tumor-promoting inflammation, nonmutational epigenetic reprogramming, and polymorphic microbiomes, and emerging
hallmarks including deregulating cellular metabolism, avoiding immune destruction, unlocking phenotypic plasticity, and senescent cells.
Each of the cancer hallmarks is involved in numerous essential biomarkers that play vital roles in tumor progression
KRAS mutation assays provide significant predictive information transcriptional upregulation of MYC and the nonoxidative pentose
on tumor progression and recurrence, which are of great value in phosphate pathway gene RPIA through activating MAPK, thereby
the diagnosis, prognosis, and treatment of PDAC.325 Consistently, enhancing nucleotide biosynthesis in PDAC cells.327
PDAC patients with KRASG12D mutation have shorter survival than In summary, KRAS mutations are among the most prevalent
all other PDAC patients.326 In lung adenocarcinoma patients, the drivers of tumorigenesis, and their activation is correlated with
patients with KRASG12C mutation have worse DFS than patients tumor progression and poor prognosis.334,335 The evidence
with nonG12C mutation KRAS or wild-type KRAS.324 presented above strongly suggests that KRAS is a crucial tumor
Mechanistically, KRAS drives tumor development and progres- biomarker.
sion through various signaling pathways. For example, the
extensive metabolic reprogramming induced by KRAS mutations, PI3K-AKT-mTOR: The PI3K-AKT-mTOR pathway plays valuable
such as glycolysis, glutamine metabolism, lipid metabolism, and roles in various cellular processes, such as cell proliferation,
nucleotide biosynthesis to facilitate tumorigenesis, has attracted angiogenesis, protein translation, and metabolic
much attention in recent years.327,328 KRAS-mutant cells exhibit reprogramming.302
the upregulation of glucose transporters329 and metabolic In normal cells, growth factor-stimulated PI3K activation leads
enzymes involved in the glycolysis,330,331 resulting in increased to the conversion of phosphatidylinositol-3,4-bisphosphate (PIP2)
glucose flux in the glycolytic pathway.329 KRASG12D stimulates to phosphatidylinositol-3,4,5-trisphosphate (PIP3), followed by the
hexosamine biosynthesis and the pentose phosphate pathway to recruitment of AKT and 3-phosphoinositide-dependent kinase 1 to
regulate glucose metabolism in PDAC.332 KRAS-mutant cells the plasma membrane. Following that, 3-phosphoinositide-
produce nicotinamide adenine dinucleotide phosphate (NADPH) dependent kinase 1 phosphorylates and activates AKT, thus
by promoting glutamine catabolism,329 and intracellular fatty acid phosphorylating the downstream mTOR complex, contributing to
uptake and oxidation.333 Furthermore, KRAS leads to the cell survival and proliferation.302,336,337 The atypical serine/

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threonine kinase mTOR consists of rapamycin-sensitive mTOR PTEN: PTEN was discovered in 1997 as a tumor suppressor,364
complex 1 (mTORC1) and rapamycin-insensitive mTORC2.338 AKT and it was the first phosphatase proven to have tumor suppressive
drives mTORC1 activation either directly by phosphorylating effects.365,366 As a phosphoinositide 3-phosphatase, PTEN nega-
mTORC1 at Ser2448 or indirectly by inhibiting TSC1/TSC2.302 tively regulates the PI3K-AKT-mTOR pathway by converting PIP3
mTOR supports cell growth and proliferation by promoting cell to PIP2, thereby hindering the proliferation and survival of tumor
cycle,302 sensing nutrient signaling339,340 by phosphorylating its cells.365,367 Furthermore, PTEN exerts both enzymatic and none-
downstream effectors such as S6K and 4EBP1.302,341 The tumor nzymatic effects in cellular epithelial-mesenchymal transition
suppressor PTEN is a critical negative regulator of the PI3K (EMT), migration and invasion, glucose and lipid metabolism, cell
signaling pathway.302 PETN rapidly metabolizes PIP3 by removing cycle, DNA repair, genomic stability, and gene transcription.365,368
the 3’-phosphate of PIP3, which in turn terminates PI3K PTEN function and expression are frequently altered in a variety
signaling.342 of cancers.369 Accordingly, PTEN acts as a prognostic and
In cancer cells, the PI3K-AKT-mTOR signaling pathway is predictive biomarker in various cancers including prostate cancer,
abnormally activated via the stimulation of tyrosine kinase growth RCC, PDAC, CRC, breast cancer, endometrial cancer, brain cancers,
factor receptors,343 the loss of PTEN functions, and the mutations skin cancers, and hematological malignancies.370 Aberration of
of PIK3CA, thereby promoting tumorigenesis in a wide variety of PTEN is associated with the mutations, downregulation or deletion
human cancers.302,342 The PI3K-AKT-mTOR pathway exerts sig- of the PTEN gene, and the abnormal subcellular localization of
nificant impacts on multiple cancers including lung cancer,344,345 PTEN protein.371,372 PTEN deletion modulates the downstream
ovarian cancer,302 breast cancer,346 and NPC.347,348 The PI3K-AKT- effector of mTORC1 by regulating 4EBP1 and p70S6 kinase to
mTOR has been proven to be crucial in ovarian tumorigenesis and increase protein synthesis.372 Significantly, PTEN deletion is
drug resistance.302 The level of pAKT is a diagnostic biomarker for strongly linked to a shorter OS and DFS of cancer patients.370
the treatment of SCLC involving the combination of clinically Taken together, PTEN is a significant biomarker for tumor
approved inhibitors against isoform-specific PI3K and mTOR.345 In prognosis. The mechanism studies of PTEN activation will be
addition, the class I isoform of PI3K, the most well-known PI3K beneficial for the development of antitumor strategies based on
protein, contains four distinct isoforms of catalytic structural the recovery of PTEN function.
domain: p110α (PIK3CA), p110β (PIK3CB), p110γ (PIK3CG), and
p110δ (PIK3CD).343 pIK3CA and PTEN aberrations lead to the Evading growth suppressors. In addition to inducing and main-
activation of the PI3K-AKT-mTOR pathway.349,350 The TCGA taining growth stimulus signals, tumor cells also eschew powerful
database has shown that PIK3CA gene mutations occur in a programs to evade growth restriction and blockade, which mainly
variety of cancers, including 53% of endometrial cancer, 35% of rely on the action of tumor suppressor genes.
breast cancer, 23% of cervical cancer, 21% of gastric cancer, 20%
of head and neck cancer, 20% of CRC, 15% of lung cancer, and Rb: Rb, the first tumor suppressor gene to be identified, was
10% of glioblastoma.343 PIK3CA mutation, PTEN loss, and pAKT originally discovered in retinoblastoma children.373,374 The altera-
activation are predictive biomarkers for the efficacy of tumor tion of the Rb gene or inactivation of the Rb protein is one of the
treatment.350,351 Moreover, PIK3CA mutations act as diagnostic most common events in cancers.375 Rb primarily restricts the
biomarkers for HR+ and HER2- metastatic breast cancer.352 In transcription of cell cycle genes by regulating E2F transcription
summary, the PI3K-AKT-mTOR pathway is an essential biomarker factors.376 Rb proteins are phosphorylated by cyclin-dependent
pathway for tumor diagnosis, prognosis, and treatment. kinases (CDKs),377 which lead to Rb functional inactivation,
followed by E2F transcriptional activation and cell cycle progres-
RAF-MEK-ERK: The RAS-RAF-MEK-ERK pathway participates in the sion.378 Inactivation of Rb causes abnormalities in cell division,
regulation of key processes such as cell proliferation, differentia- defects in cell cycle withdrawal, impaired induction of cellular
tion, migration, and apoptosis,303 which can be activated by senescence, and impaired cell cycle checkpoint control.379 The
growth factors, cytokines, integrins, and chemokine recep- function of Rb in tumor cells is disrupted in various ways including
tors.303,353 Active RAS binds to RAF kinase, which results in RAF Rb gene mutation, Rb protein hydrolysis, Rb-E2F interaction
dimerization and activation.354,355 RAF proteins possess three elimination, and the overactivation of CDK.375 Consequently, Rb
isoforms including BRAF, CRAF, and ARAF which share conserved dysregulation acts as a prognostic biomarker in cancers.380
regions of the regulatory domain and kinase domain,356 and
among them, ARAF exhibits the lowest kinase activities.357 The TP53: TP53, often referred to as the “guardian of the genome”, is
active RAF dimer phosphorylates and activates MEK1/2 which a gene encoding the p53 tumor suppressor protein.381 Numerous
subsequently phosphorylates and activates ERK1/2, followed by studies have shown that p53 plays an integral role in biological
the phosphorylation and activation of downstream effectors and processes such as cell cycle arrest, aging, DNA repair, and
the proliferation of cells.336 Various proteins, such as Hsp90,358 apoptosis.382–384
p50CDC37,359 and KSR,360 are engaged in the regulation of RAF In human tumors, TP53 is the most commonly mutated gene
activation. with ~50% of tumors carrying the mutations or deletion of
Abnormalities epically mutations in RAF-MEK-ERK signaling TP53.385,386 In addition to mutations or deletion, tumors may lose
lead to the aberrations of cell proliferation.361 A mutation the function of p53 due to other mechanisms. For example,
analysis of more than 3000 samples from 12 tumor types has overexpression of viral oncoproteins or MDM2 leads to the
shown that the mutations of RAF-MERK-ERK signaling occur in degradation of p53 protein.387 The expression and function loss or
~50% of cancers.362 In particular, BRAF mutations are widely gain function of TP53 are associated with poor prognosis, immune
investigated in cancers.362 Studies have revealed that the escape, and anticancer drug resistance. Thus, TP53 can serve as an
hyperactivity of the BRAF-MEK-ERK pathway is correlated with effective predictive biomarker to evaluate prognosis and monitor
worse survival in patients with ER-negative or progesterone therapeutic responses in various cancers.388 An analysis of over
receptor-negative breast cancers,363 suggesting that the altera- 29,000 cases from the International Agency for Research on
tions of the RAS-RAF-MEK-ERK pathway could serve as predictive Cancer database revealed that TP53 mutations are potential
and prognostic biomarkers for breast cancer.303 Meanwhile, the prognostic biomarkers, and can be used to bolster the predictive
aberrations of the RAS-RAF-MEK-ERK pathway can be predictive accuracy of the OS and DFS of cancer patients.389
biomarkers of drug sensitivity in cancer therapies.303 In conclu-
sion, the RAF-MEK-ERK signaling cascade functions as a Enabling replicative immortality. Unlimited proliferation is a
significant biomarker in tumor progression. critical characteristic of tumor cells.299 Normal cells undergo

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senescence due to their recurrent division cycle, whereas tumor referred to as VEGF), VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and
cells are capable of unlimited replication, a phenomenon known placental growth factor (PlGF).401,404 VEGF has been demonstrated
as immortalization. The protection of chromosome ends by to be a potent inducer of angiogenesis,399 and is widely expressed
telomeres is crucial for tumor immortalization.299 in normal adult organs along with two other related endothelial
growth factors VEGF-B and VEGF-C, suggesting their necessary
Telomerase: Telomere is a repetitive DNA–protein complex roles in tissue angiogenesis and homeostasis.399 VEGFs in tumor
located at the end of chromosome.390 Telomeres in normal cells tissues are extracted not only from tumor cells but also from host
gradually shorten with continuous cell division and eventually fail cells,405 and high levels of VEGFs are found in diverse tumor cells.
to protect the end of chromosomal DNA, thus triggering DNA Interestingly, tumor cells are able to produce VEGFs, but are
damage, cellular senescence, and apoptosis. Therefore, the length unable to respond to them due to the absence of VEGF receptors
of telomeres is closely related to the cellular lifespan.299,391 (VEGFRs) on the cell surface.406 Multiple factors such as genetic
Telomerase is a DNA polymerase that maintains telomere and epigenetic regulation influence the VEGF levels in tumor cells.
length by adding telomeric repeat fragments to telomeric DNA Among them, epigenetic factors include hypoxia-inducible
ends, thus compensating for the attrition of chromosomal ends in transcription factors 1α and 2α, low pH, inflammatory cytokines,
continuous cell division.390,392 Telomerase is encoded by the growth factors, androgens, estrogens, and chemokines.406,407
human telomerase reverse transcriptase (hTERT) gene which is the Genetic factors include the activation of oncogenes such as RAS,
catalytic subunit of telomerase holoenzyme.390,393 hTERT is EGFR, HER2, and the deletion and mutational inactivation of
silenced in almost all somatic cells and is significantly re- oncogenes such as p53, PTEN, and VHL.406,408
expressed in ~90% of human cancers by various approaches.390 VEGFs bind respectively to the three tyrosine kinase receptors
Thus, the large majority of normal human somatic cells lack the (RTKs) VEGFR1–3 with different specificities and affinities and
telomerase-maintenance mechanism, while a tremendous propor- VEGFR2 mediates the main VEGFR signals.409 VEGFR-1, the first
tion of cancer cells have a highly active telomerase-maintenance RTK to be identified as a VEGF receptor,410 binds VEGF with a
mechanism.392 The activation of the telomerase-maintenance binding affinity ten times higher than that of VEGFR-2, although its
mechanism is observed in numerous human cancers, such as ability of signal transduction is quite weak.406 VEGFR-1 serves as a
breast cancer, CRC, kidney cancer, cervical cancer, liver cancer, decoy receptor to chelate or trap VEGF under some circumstances,
lung cancer, pancreatic cancer, prostate cancer, thyroid cancer, thus negatively regulating VEGF activity by preventing VEGF
and bladder cancer,394 which ensure the replicative immortality of binding to VEGFR-2.411–414 The specific mechanism of VEGFR-1 in
cancer cells. In clinical practice, cancer patients with high hTERT VEGF-mediated angiogenesis needs to be explored in further
levels are along with worse survival than those with low hTERT detail.406,415 In contrast, VEGFR-2 is expressed in almost all
levels. Moreover, cancer patients with high hTERT levels have a endothelial cells, and exerts function through the activation of
higher risk of disease recurrence and death.395 Therefore, VEGF, VEGF-B, C, or D.416 The binding of VEGF to VEGFR-2 causes
telomerase is an independent prognostic biomarker of OS in receptor dimerization and subsequently activates the intracellular
cancer patients.395 Besides, TERT promoter mutations increase the signaling cascades, such as the PI3K-AKT and the RAF-MEK-ERK
expression of telomerase directly, which contributes to tumor- pathways, which generates neovascular branches required for
igenesis and is associated with poor OS of cancer patients, tumor growth, and ultimately promotes rapid tumor cell
suggesting that TERT promoter mutations are prognostic biomar- proliferation and migration.406 VEGFR-3 has similar functions to
kers for cancers.396 Moreover, the nonenzymatic functions of VEGFR-2, but its action site is mainly in the lymphatic blood
telomeres promote cancer cell proliferation and the resistance of vessels.401,415 VEGFR-3 is expressed in the lymphatic endothelial
apoptosis,299 regulate chromatin structure,397 impair DNA damage cells415 and mainly binds to VEGF-C and VEGF-D to induce
repair,397 and increase antioxidant protein expression,393 although lymphangiogenesis.417,418 In addition, VEGFs interact with the
the detailed mechanism remains to be elucidated.299 neuropilin receptor family.415
VEGF levels are associated with the aggressiveness of tumors.419
Inducing angiogenesis. In tumor initiation and progression, the The plasma VEGF levels in various cancer patients are elevated
new vascular system can transport nutrients and oxygen, and and negatively correlated with tumor prognosis.420 Moreover,
excrete metabolic waste, which is critical for tumor growth.299,398 VEGF levels are used to predict the efficacy of oral tyrosine kinase
The transition from prevascular hyperplasia to highly vascularized inhibitors (TKIs) in cancer patients.401 For example, VEGFR inhibitor
and progressively outgrowing tumors is known as the “angiogenic sorafenib has displayed better therapeutic efficacy against
switch”. In the early stage of tumor development, the angiogenic advanced clear cell renal cell carcinoma (ccRCC) patients with
switch is highly activated, which in turn sustains the continuous high levels of VEGF.401 In summary, circulating VEGF and VEGFR-2
generation of new blood vessels, and causes the transition from have been used as crucial biomarkers for the prediction of
dormant hyperplasia to outgrowing vascularized tumor, ultimately prognosis and antiangiogenic drug efficacy.405,421
promoting rapid proliferation of cancer cells.299,398,399 The
angiogenic switch, which favors a proangiogenic outcome during FGF: Fibroblast growth factor (FGF) is a secreted glycoprotein422
tumor angiogenesis, is controlled by the balance between that engages in the regulation of organogenesis,423 angiogenesis,
proangiogenic and antiangiogenic factors secreted by tumor cells and wound repair.422,424 FGF binds to the transmembrane FGF
or TME cells.398 Studies have ascertained that angiogenesis receptor (FGFR) on the surface of target cells with high
significantly contributes to the development of various cancers, affinity.422,425 The mammalian FGFR family consists of four highly
including CRC, breast cancer, bladder cancer, RCC, and conserved transmembrane RTK FGFR1-4, and FGFR5 which has no
NSCLC.400,401 A large number of angiogenic factors such as intracellular tyrosine kinase structural domain but has FGF binding
vascular endothelial growth factors (VEGFs) have been found to capacity.422 FGFR is widely expressed in a broad range of cells,
induce the proliferation and differentiation of endothelial cells especially endothelial cells.426
directly or indirectly. In tumors, FGF is essential for vascular endothelial integrity,
angiogenesis, tumor proliferation, survival, and metastasis.425,426
VEGF: VEGF, originally known as vascular permeability factor, Notably, abnormal FGF signaling accelerates tumor proliferation
was discovered as a tumor secretory factor in 1983 by Senger by promoting tumor angiogenesis.422 For example, the elevated
et al.402 In 1989, Ferrara isolated VEGF and renamed it vascular level of FGF2 in prostate cancer induces neovascularization to
endothelial growth factor.403 VEGFs are heparin-binding homo- boost tumor growth.427 The increased angiogenesis induced by
dimeric glycoproteins whose family includes VEGF-A (commonly FGF1 amplification in high-grade serous ovarian cancer leads to

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reduced OS in patients, suggesting that FGF1 is a prognostic mitochondrial outer membrane permeabilization and the release
biomarker for ovarian cancer.428 of multiple proapoptotic factors, followed by the release of
Furthermore, FGFR is strongly associated with the development cytochrome c from mitochondria to the cytoplasm. Subsequently,
of various tumors,422,429 such as prostate cancer,430,431 lung the apoptotic peptidase activating factor 1 interacts with
cancer,432 breast cancer,433 and pancreatic cancer.434 In particular, cytochrome c, and form the apoptosome that induces the
studies have revealed that FGFR with mutations or amplification activation of the initiator caspase pro-caspase 9. Later on, the
functions as driving oncogene to aberrantly activate downstream caspase 9 binds to the apoptosome and is cleaved and activated,
pathways, resulting in mitogenic, mesenchymal, and antiapoptotic which subsequently stimulates the activation of initiator caspase
responses in cells.422 Somatic mutations of FGFR3 have been 3.465,466 This process in which cytochrome c is released from the
observed in more than 30% of bladder cancers.435,436 The somatic mitochondria is negatively regulated by antiapoptotic BCL-2
mutations of FGFR2 have been discovered in 12% of endometrial family members such as BCL-2, B-cell lymphoma-extra large
cancers, and mutant endometrial cancer cell lines are highly (BCL-XL), BCL-W, BCL-2-A1, and MCL1.463 The membrane permea-
sensitive to FGFR TKIs.437 Besides, FGFR amplification is also tightly bilization and the release of cytochrome c into cytoplasm are key
linked to the progression of numerous cancers.438,439 Approxi- processes for triggering apoptosis.463,467
mately 10% of gastric cancers have shown FGFR2 amplification, The extrinsic apoptotic pathway is initiated through the
which is associated with the poor prognosis of gastric cancer proapoptotic death receptors which include Fas, the tumor
patients.440 Amplification of FGFR1 occurs in approximately 10% necrosis factor receptor (TNFR) family such as TNFR1, TNFR2,
of breast cancers, especially ER+ type.441,442 In brief, FGF and FGFR and theTRAIL receptors DR4 and DR5. The proapoptotic death
are vital biomarkers of tumor prognosis and treatment. receptors bind to ligands and then trimerize and aggregate within
the cell membrane, subsequently recruiting adapter proteins such
PDGF: Platelet-derived growth factors (PDGFs), an α-granule as FADD, caspase 8 and/or caspase 10 to form the death-inducing
component secreted in an autocrine manner during platelet signaling complex, which activates the initiator caspase 8, which
activation,443 are critical proangiogenic factors for tumor angio- in turn induces the activation of the effector caspases such as
genesis.443,444 The PDGF family contains four different monomeric caspase 3, 6, and 7, and apoptosis.463,467 Consequently, the
polypeptide chains: PDGF-A, -B, -C, and -D, which form four potential strategy for cancer therapy is targeting the proapoptotic
homodimers through disulfide bonds (PDGF-AA, -BB, -CC, -DD) and antiapoptotic proteins to induce apoptosis.468
and a PDGF-AB heterodimer.443,445 The PDGF receptor (PDGFR)
consists of RTKs PDGFRα and PDGFRβ. PDGF isoforms trigger BCL-2/BCL-XL: The BCL-2 family proteins have four conserved
different receptor dimerization and phosphorylation by binding to BCL-2 homology (BH) structural domains (BH1, 2, 3, and 4) which
the corresponding PDGFRs, thus activating multiple downstream can be divided into three subfamilies based on the homology and
growth signaling pathways, such as PI3K, MAPK, and JAK/STAT function of proteins: the antiapoptotic BCL-2 family members
pathways, to promote cancer cell proliferation, migration and (such as BCL-2 and BCL-XL), the multi-BH-domain proapoptotic
invasion, angiogenesis, and drug resistance.443,446,447 members, such as the BCL-2-associated X protein (BAX) and the
PDGFs and their receptors are extensively expressed in a BCL-2 antagonist/killer (BAK), and the proapoptotic “BH3-only”
number of cancers, such as oral squamous cell carcinoma proteins, such as the BCL-2 interacting mediator of cell death
(OSCC),448 skin SCC,449 soft tissue sarcomas,450 ccRCC,451 derma- (BIM), and PUMA.469
tofibrosarcoma protuberans, gastrointestinal stromal tumors BCL-2 was the first identified apoptosis regulator, which was
(GIST),452 CRC,453 breast cancer,447 pancreatic cancer,454 gastric activated by chromosome translocation in human follicular
cancer,455 neuroendocrine tumors,456 NSCLC, ovarian cancer, and lymphoma oncoprotein.470 The BCL-X gene was cloned in
HCC.443 High PDGF-A levels correlated independently and 1993,471 and the BCL-XL protein, which is localized in the
inversely with the risk of metastatic relapse in cancer patients.450 mitochondrion, is the first protein whose three-dimensional
The level of PDGF-D is associated with advanced tumor stages and structure has been identified in the BCL-2 protein family.472,473
the development of bone metastasis.457,458 High expression of The “BH3-only” proteins can be divided into activators and
PDGFR-β is independently linked to prostate cancer recurrence.445 sensitizers.474,475 Activators of BH3 proteins, such as BIM, BID,
In conclusion, PDGFs and PDGFRs are meaningful diagnostic initiate apoptosis by directly inducing BAX and BAK oligomeriza-
biomarkers. tion and cytochrome c release. However, sensitizer BH3 proteins,
such as BAD, and BIK, exert proapoptotic functions by binding to
Resisting cell death. Resisting cell death is a significant tumor antiapoptotic BCL-2 family members, rather than directly activat-
hallmark that contributes to tumor progression and therapeutic ing BAX or BAK.475–477 The interaction of one protein’s BH3 α-helix
resistance.299 Apoptosis that leads to programmed cell death with a sizable hydrophobic pocket on binding partners regulates
hinders tumorigenesis, and the apoptotic program is considerably the activity of BH3-only proteins,475 which initiates apoptosis by
reduced in highly aggressive and therapy-resistant tumor cells.299 activating proapoptotic proteins or by inhibiting antiapoptotic
Increasing autophagy activation might inhibit tumorigenesis in proteins.478
parallel with or in concert with apoptosis.459,460 Moreover, necrosis BCL-2 can drive oncogenic transformation, hinder apoptosis,
also significantly contributes to tumor cell death.461 The identifi- and increase tumor cell survival.479,480 The high expression of BCL-
cation of biomarkers in these processes is useful for tumor XL is involved in tumor cell invasion, the maintenance of tumor
diagnosis or prognosis. stem cell phenotype, angiogenesis, and metastasis through
inducing apoptosis resistance.480 The overexpression of BCL-2
Apoptosis: Sydney Brenner, Robert Horvitz, and John Sulston and/or BCL-XL may contribute to tumor progression and the
shared the 2002 Nobel Prize in Physiology or Medicine for their resistance of chemotherapeutic agents in various tumors,467,475
contributions to the discovery of apoptosis procedure.462 Cellular including pancreatic cancer,481 ovarian cancer,481 lung cancer,481
stress, DNA damage, and immune surveillance systems frequently prostate cancer,481 breast cancer,482 neuroblastoma,483 CRC,484
cause apoptosis, a type of cell death that is initiated by the gastric cancer,485 HCC,469 chronic lymphocytic leukemia (CLL),469
proteolytic cleavage of numerous proteins and the regulation of lymphoma,481 and multiple myeloma.481 Furthermore, BCL-XL can
caspase protease activity.463 Apoptosis can be triggered through be used as an independent biomarker for the prognosis prediction
the intrinsic or mitochondrial pathway and the extrinsic path- of CRC patients.484 BCL-2 is a prognostic biomarker in TNBC
way.464 The intrinsic pathway is controlled by the B-cell leukemia patients. Lower BCL-2 expression level is associated with better
or lymphoma gene number 2 (BCL-2) family. BCL-2 induces outcomes of TNBC patients treated with both adjuvant and

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neoadjuvant chemotherapy.486 In summary, BCL-2 and BCL-XL are which includes VPS15, VPS34, ATG14, Beclin1, UVRAG, and
essential biomarkers in tumor prognosis and treatment. AMBRA1, which mediates vesicle nucleation.460 Then, the ATG5-
ATG12 complex binds to ATG16 to extend the autophagosomal
BAX/BAK: BAX, a cytosolic membrane protein that works as a membrane, and members of the LC3 and GABARAP protein
critical regulator of the apoptotic process, was identified by families conjugate with lipid phosphatidylethanolamine (PE) and
immunoprecipitation and yeast two-hybrid screening.480,487 BAX recruit PE to the membrane. ATG4B binds to ATG7 and then
protein has BH1, BH2, and BH3 structural domains,480,488 which is couples with LC3-I and PE to form LC3-II. Eventually, autophago-
highly homologous with BCL-2.480 BAX stimulates apoptosis either somes fuse with lysosomes to degrade macromolecules and reuse
by inhibiting BCL-2 and BCL-XL or by directly triggering the them. The adapter protein sequestosome-1 (also known as p62)
apoptotic pathway.480 BAX moves from cytoplasm to mitochon- targets autophagosome-specific substrates and LC3-II which are
dria during apoptosis, followed by oligomerization and the simultaneously degraded.460,510
formation of pores in the outer mitochondrial membrane, thus Autophagy is a double-edged sword in cancer. The enhanced
facilitating the release of cytochrome c which activates the autophagic flow in tumor cells accelerates tumor cell growth,
downstream effector caspases and leads to cell death.469,489,490 while the induction of autophagy can prevent the development of
Downregulation and mutations of BAX are essential for apoptosis cancer.459,460 Therefore, autophagy inhibition and promotion are
resistance,491 and BAX acts as a potential prognostic and both promising strategies for cancer therapy, and their application
predictive biomarker in various cancers including gastric cancer, depends on the actual situation.511
esophageal cancer, and CRC.492 The somatic frameshift mutations
of the BAX gene highly occur in CRC with the microsatellite Beclin 1: Beclin 1 was identified as a BCL-2 interaction factor in
mutator phenotype.493 BAX mutations are found in ~21% of the yeast two-hybrid screen in 1988.512,513 Human Beclin 1, the
human hematopoietic malignancies such as ALL.494 Reduced BAX mammalian orthologue of yeast Atg6, consists of a BCL-2-
expression is a major factor in cisplatin resistance of ovarian homology 3 structural domain,514 a flexible helical domain,515 a
cancer cells,495 5-FU resistance of CRC cells,496 and zoledronate coiled coil domain,516 and an evolutionarily conserved domain.514
resistance of lung cancer cells.497 The decreased BAX/BCL-2 ratio Moreover, Beclin 1 contains a leucine-rich nuclear export signal
can be induced by BAX abnormalities, which affects the that is essential for its autophagic and tumor suppressor
temozolomide-induced resistance in U87MG cells and paclitaxel- functions.517 Beclin 1 is phosphorylated by ULK1 and acts as an
resistant breast cancer cells. Thus, the activation of BAX could be integral component of the PI3K complex to localize autophagy
used to promote apoptotic cell death and overcome resistance.498 proteins to the phagosome. Furthermore, Beclin 1 interacts with
Furthermore, the high BAK expression is correlated with and is inhibited by BCL-2/BCL-XL in the BH3 structural domain,
improved OS and PFS in patients with advanced gastric cancer. which blocks the formation of the Beclin 1-VPS34 complex and
BAK is a predictive and prognostic biomarker for the therapeutic inhibits Beclin 1 interacting with UVRAG, thereby inhibiting
effect of docetaxel in patients with advanced gastric cancer.499 autophagy. The bind of AMBRA1 to Beclin 1 stabilizes the Beclin
BAX-BAK heterodimer is also used as a pharmacodynamic 1-VPS34 complex, thus promoting autophagosome
biomarker of on-target drug action of MCL1 inhibitors.500 formation.518,519
Studies have found that Beclin 1 is a prognosis biomarker for
Autophagy: In 1955, Christian de Duve discovered the lyso- various cancers. Reduced expression of Beclin 1 has been
some,501 a key organelle for intracellular degradation, and observed in brain tumors and cervical cell carcinomas.520 The
subsequently introduced the term “autophagy” at the CIBA absence of BECN1 has been found in 40 to 75% of sporadic breast
Foundation Symposium on Lysosomes in 1963.502 In 2016, cancer and ovarian cancer,521 and 40% of prostate cancer.522 Low
Yoshinori Ohsumi was awarded the 2016 Nobel Prize for Medicine expression of Beclin 1 is associated with the malignant phenotype
or Physiology for elucidating the mechanism of autophagy, which and poor prognosis of gastric cancer.523 Beclin 1 inhibits the
led to increasing attention to autophagy in health and dis- proliferation of human breast cancer cells MCF7 in vitro and
ease.460,503 To date, there are three main types of autophagy: in vivo through regulating autophagy.524 On the contrary, the
macroautophagy, microautophagy, and chaperone-mediated elevated Beclin 1 expression is related to distant metastasis and
autophagy. The general term “autophagy” usually means poor prognosis in CRC patients, and reduced survival in CRC
macroautophagy.504 patients with 5-FU treatment.525 Taken together, Beclin 1 may
Autophagy is a multistep, highly conserved degradation serve as a valid prognostic indicator and therapeutic target for
process: the initiation and nucleation of the autophagosome, cancers although further research is needed to determine its
the expansion, and elongation of the autophagosome membrane, specific mechanism in different cancers.526,527
the closure and fusion with the lysosome, and the degradation of
products.460,505 Briefly, autophagy is triggered by a variety of LC3B: The microtubule-associated protein 1 light chain 3B (LC3B
factors, including nutrient or growth factor deprivation, energy or MAP1LC3B) is a classical autophagy marker, is cleaved by
status, hypoxia, ROS, and other stress inducers.506 Subsequently, a protease at the C-terminus to form free LC3B-I, and LC3B-I binds to
flat membrane named the phagophore or isolation membrane PE to form membrane LC3B-II in autophagy occurrence. The
sequesters cytoplasmic constituents. The elongating phagophore process in which LC3B-I converts to LC3B-II is essential for
results in complete sequestration and the formation of a double- phagophore expansion and the formation of autophago-
membraned organelle autophagosome. Then, the autophagosomes.528,529 Thus, LC3B is a marker for the detection of multiple
some fuses with the lysosome, and the inner membrane of the autophagic fluxes.530 Accordingly, LC3B-II is one of the most
autophagosome and the cytoplasm-derived materials it contains commonly used biomarkers to detect the number of autophago-
are subsequently degraded by the lysosome, resulting in the somes and autophagosome-related structures.518
production of amino acids and lipids which are exported to the The high LC3B expression is closely associated with the
cytoplasm for recycling.504,507,508 aggressive progression, and poor prognosis of multiple tumors,
Mechanistically, autophagy-associated (ATG) proteins, a group including gastric cancer,531 CRC,532 TNBC,533 melanoma,534 astro-
of evolutionarily conserved proteins, are responsible for this cytoma,535 esophageal cancer,536 and OSCC.537 Studies have
process.509 Autophagy begins with the activation of unc-51 like found that LC3B has the highest expression in TNBC cells in
autophagy activating kinase 1 (ULK1) (also known as ATG1) different molecular subtypes of breast cancer,538 and its high
complex which includes ULK1, ULK2, ATG13, FIP200, and ATG101. expression is related to the progression and poor prognosis of
The ULK1 complex subsequently activates class III PI3K complex TNBC patients.533 Moreover, LC3B is closely connected with the

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vascular invasion and lymph node metastasis of HCC, and is a molecular, T-cell receptors, interferon receptors, Toll-like receptors,
potential therapeutic target for HCC.539 Collectively, LC3B is a cellular metabolism, genotoxic stress, and various anticancer
meaningful prognostic biomarker in cancer management.527,534 compounds.565,567 Common morphological features of necrotic
cells include moderate chromatin condensation, cytoplasmic
ULK-1/2: ULK1, a conserved Ser/Thr kinase, plays a pivotal role in organelle swelling, and the rupture of plasma membrane.568 The
autophagy induction.540 High expression of ULK-1 is associated biochemical characters of necrotic cells include a drop in ATP
with poor prognosis in various tumors, including esophageal level, the activation of RIP1, RIP3, and MLKL, the release of
SCC,541 HCC,542 NPC,543 prostate cancer,544 and CRC.545 Studies damage-associated molecular pattern molecules (e.g., HMGB1),
have found that HCC patients with ULK1 and LC3B overexpression the hyperactivation of poly(ADP-ribose) polymerase 1 (PARP1).568
have larger tumors and a higher frequency of lymph node The basic feature that distinguishes necrosis from apoptosis is the
metastasis. The combination of ULK1 and LC3B is an independent rapid loss of cell membrane potential. Cellular energy depletion,
predictor of OS and PFS in HCC patients.546 After androgen membrane lipid damage, and the impairment of steady-state ion
deprivation therapy, prostate cancer patients with high levels of pump function lead to loss of membrane potential which in turn
ULK1 have shorter PFS and OS.544 In addition, elevated expression leads to cytoplasmic swelling, plasma membrane rupture, and cell
of ULK1 has been connected to lymph node metastasis547 and lysis, thus promoting necrotic cell death.461
functions as a prognostic biomarker in patients with CRC.545 Studies have identified that necrosis is an essential predictor for
Interestingly, low expression of ULK1 is associated with operable prognosis and treatment response in various tumors, including
breast cancer progression and is a poor prognostic biomarker for pancreatic cancer,569 RCC,570 breast cancer, lung cancer, CRC,571
patient survival.548 In human NPC, ULK1 is also a promising and soft tissue sarcoma.571,572 Tumor necrosis is closely associated
biomarker for the prediction of poor prognosis and treatment with cancer-specific survival, OS, RFS, and PFS in patients with
response.543 Furthermore, ULK2 has been found to be expressed RCC, and it can be a prognostic biomarker of patients in clinical
at higher levels in prostate cancer tissues compared with that in practice.570 Therefore, the discovery of biomarkers that identify
normal tissues.549 To better determine the prognostic value of necrosis and molecular mechanisms of necrosis enables the
ULK1 and ULK2 in different cancer types, comprehensive studies development of necrosis-based antitumor therapies.569
in prospective cohorts are necessary.530
RIPK3: The serine/threonine kinase RIPK1 is a key regulator of
p62: p62 (also known as sequestosome-1, SQSTM 1) was necrosis, and RIPK3 is a downstream regulator of RIPK1.573,574 The
originally identified as an atypical protein kinase C (aPKC) RIPK1-RIPK3-MLKL complex, also known as the “necrosome”,
interacting protein.550 p62 consists of several structural domains, mediates upstream cell death receptors and downstream signal-
including the N-terminal PB1 domain, the ZZ-type zinc finger (ZZ) ing.565 Necrosome is a multiprotein complex that contributes to
domain, the tumor necrosis factor receptor-associated factor 6 TNF-induced cell death.575,576 Necrotic cells trigger caspase 8
(TRAF6) binding (TB) domain, the LIR domain, the Kelchlike ECH- inactivation and activate RIPK1 and RIPK3, followed by autopho-
associated protein 1 (Keap1)-interacting region (KIR), and the sphorylation and cross-phosphorylation between RIPK1 and RIPK3
C-terminal UBA domain.551,552 Each structural domain of p62 has a to form necrosome. Then, MLKL is phosphorylated, followed by
different function. The PB1 domain is essential for the formation of oligomerizing and translocating to the plasma membrane and
homodimeric aggregates that regulate autophagic degradation. stimulating the necroptosis.577,578
Moreover, p62 can interact with other proteins containing the PB1 The RIPK3 expression is significantly reduced in AML
domain, such as MAPK.551 ZZ structural domain is involved in the patients,579,580 which is consistent with the high methylation level
activation of NF-kB signaling pathway,553 and the TB structural near the transcriptional start site of RIPK3.581 RIPK3 deficiency
domain can interact with TRAF6 which induces protein poly- promotes leukemogenesis by enhancing the accumulation of
ubiquitination.554 The LIR structural domain affects autophago- leukemia-initiating cells, and hinders myeloid differentiation
some formation and autophagic degradation by mediating LC3- through reducing cell death and IL-1β production.579,580 In
p62 interactions,551,555 and the KIR structural domain activates addition, RIPK3 expression plays an important role in solid tumors.
Nrf2 by binding with Keap1.556,557 UBA structural domain is RIPK3 has been discovered to be downregulated in various cancer
involved in autophagic lysosomal degradation558 and apoptosis cells, including breast cancer,581 melanoma,582 lung cancer,583 and
signaling pathways.551 CRC.584 RIPK3 is downregulated in human CRC tissues compared
As a marker for autophagic flow detection, p62 accumulation with normal tissues,585,586 and the deletion of RIPK3 accelerates
usually represents the inhibition of autophagy.460,559 Upregulation colorectal tumorigenesis in mice through sustained inflamma-
or reduced degradation of p62 is associated with tumor tion.577,585 Consistent with the above observations, low RIPK3
progression and anticancer drug resistance.552 p62 expression is levels are strongly correlated with poor prognosis in patients with
increased in 60% of lung adenocarcinomas and 90% of lung CRC586 and breast cancer.581 On the contrary, the expression of
SCCs.550 Numerous studies have shown that high p62 expression RIPK3 is elevated in several other tumors, such as serous ovarian
is correlated with the aggressiveness and poor prognosis of cancer,587 pancreatic cancer,588 and colitis-associated cancer and
cancers, including endometrial cancer,560 OSCC,537 epithelial colon cancer.589 RIPK3 promotes colitis-associated CRC through
ovarian cancer,561 and NSCLC.562 In addition, elevated p62 tumor cell proliferation and CXCL1-induced immunosuppression,
expression is also correlated with the high-grade, distant and RIPK3 deficiency significantly reduces colitis-associated CRC
metastasis and reduced 5-year survival of breast cancer development in mice.577,589 In conclusion, RIPK3 is a potential
patients,563 especially in patients with TNBC cancer.564 In short, prognostic biomarker for tumors, although its role needs to be
p62 is a meaningful prognostic biomarker and a potential target analyzed on a case-by-case basis.
for cancer therapy.551,552
MLKL: MLKL is a key factor in necroptosis execution,574,576,590
Necrosis: Necrosis is derived from the Greek “nekros” for and a vital determinant of treatment response and poor prognosis
corpse.461 Necroptosis is a programmed necrotic cell death type in cancer patients.579,591 The low expression level of MLKL is
in a caspase-independent f manner, induced by TNFR superfamily significantly associated with lower OS in gastric cancer,592 ovarian
and mediated by receptor-interacting protein kinase 1 (RIPK1, also cancer,593 cervical SCC,594 colon cancer,577,595 and pancreatic
known as RIP1), RIPK3 (also known as RIP3), and mixed lineage cancer.591 Moreover, in resected PADC patients receiving adjuvant
kinase domain-like (MLKL).565,566 Necrosis is caused by numerous chemotherapy, the low expression level of MLKL is related to
stimuli such as cytokines, viral infection, pathogen-associated decreased RFS. Thus, MLKL has become a prognostic biomarker

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Fig. 5 The cancer invasion and metastasis and its targeted therapy. The tumor metastasis process consists of multiple steps. Initially, tumor
cells invade the surrounding stroma and extracellular matrix from the primary tumor site, and then intravasate into the bloodstream or the
lymphatics. Subsequently, tumor cells arrest in the circulation and arrive at distant organ sites, followed by extravasating and invading the
parenchyma of distant tissues. Finally, tumor cells adapt to the new microenvironment and grow to form metastatic colonization. EMT is the
basic embryonic developmental process that transforms polarized non-motile epithelial cells into motile and invasive mesenchymal cells.
Multiple cellular stress conditions including hypoxia, inflammation, metabolic stress, and signaling cascades, can induce the expression of EMT
transcription factors and prompt tumor metastasis. Meanwhile, MET amplification and mutation, the transcriptional dysregulation of c-MET,
degradation deficiency, and abnormal HGF production result in the abnormal expression of HGF/c-MET and tumor progression. Various
inhibitors including MMP inhibitors and HGF/c-MET inhibitors have been developed and emerging as promising tools in the suppression of
tumor metastasis. c-MET mesenchymal-epithelial transition factor, EMT epithelial-mesenchymal transition, HGF hepatocyte growth factor,
MMPs matrix metalloproteinases
for patients with early-stage resected PDAC.591 However, high lymphatics; (3) tumor cells survive in the circulation; (4) tumor
levels of MLKL are tied to poor prognosis in patients with colon cells arrest in the circulation and arrive at distant organ sites;
and esophageal cancers.596 The mRNA expression level of MLKL in (5)tumor cells extravasate and invade into the parenchyma of
gastric cancer tissues is significantly higher than that in normal distant tissues; (6) survival in the microenvironment and grow
tissues.592 The possible reason for this difference is that some to form metastatic colonization599–601 (Fig. 5). In 1889, Stephen
cancer cells activate necrosis to modulate the immune system, Paget vividly compared tumor metastasis to fertile “seeds”
and the exact mechanism needs to be further investigated.577 In (tumor cells) falling on “congenial soil” (the metastatic
short, MLKL is a potential prognostic biomarker for cancer microenvironment). 602,603 Many changes occur in “seeds”
patients. during the metastasis process, including proteolytic degrada-
tion of basement membranes and ECM, changes in tumor cell
Activating invasion and metastasis. Tumor metastasis is a adherence to cells and the ECM, and physical motility of tumor
process of transferring tumor cells from the primary lesion cells. 599,604 Meanwhile, homeostasis of “soil” is also altered
tumor to distant tissues and organ cascades.597,598 Tumor before tumor cells arrival by modulating the cellular composi-
metastasis is divided into multiple steps: (1) tumor cells invade tion, immune status, blood supply, and ECM of the metastatic
the extracellular matrix (ECM) and the surrounding stroma; (2) site to create a microenvironment conducive to tumor cell
tumor cells enter into the bloodstream directly or the colonization.605

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Activated invasion and metastasis have been recognized as one cells, EMT promotes tumor cell invasion and metastasis, induces
of tumor hallmarks299,600 and a major cause of death in patients cancer stem cell (CSC) stemness, chemoresistance, immune
with solid tumors.606 Predicting tumor metastasis facilitates the evasion, and cellular metabolic reprogramming,636,637 and inhibits
implementation of personalized therapy in the clinical treatment senescence.638
of tumors, leading to better outcomes for cancer patients. Thus, EMT is regulated by EMT transcription factors which are
identifying metastatic biomarkers helps to detect initial tumor classified according to their direct or indirect repression of
metastasis or recurrence in clinical practice, thus improving the E-cadherin.634 The direct repressors include zinc finger proteins
potential treatment and management strategy for cancer patients. of the Sail superfamily Snail1 (also known as Snail), Snai2 (Slug),
and Snai3 (Smuc), zinc finger E-box binding protein (ZEB) family
E-cadherin: Cadherins are a superfamily of at least 80 specific members ZEB1 and ZEB2. The indirect repressors including the
types of adhesion molecules characterized by the ability to form basic helix-loop-helix proteins Twist1 and Twist2.634,639 Tumor cell
calcium-dependent intercellular homophilic bonds,607 which are stress conditions, such as hypoxia, inflammation, or metabolic
involved in the regulation of tumor cell recognition, tumor stress, stimulate signaling cascades, such as Wnt, Notch, TGF-β,
suppression, and tissue morphogenesis.608 Common family and RAS, and induce the expression of EMT transcription factors
members include Epithelial (E)-cadherin, Neuronal (N)-cadherin, Snail, Slug, Twist, and ZEB.634,640 Then, EMT transcription factors
and Placental (P)-cadherin.609 E-cadherin, a homophilic cell-cell induce downstream effects of EMT by a series of processes
adhesion molecule,610 is a type I cadherin expressed in epithelial including regulating epithelial marker-related or mesenchymal
cells. E-cadherin is the first member of the cadherin superfamily to marker genes, activating matrix metalloproteinases (MMPs)
be identified.607 The human E-cadherin gene (CDH1) is located on expression or interacting with epigenetic regulators to promote
chromosome 16q22.1. The structure of mature E-cadherin consists oncogenic transformation, modulating CSCs, generating chemore-
of three parts: a highly conserved carboxyterminal cytodomain sistance, and increasing tumor angiogenesis, and ultimately
that is identical in all cadherin family members, a single-pass promoting tumor cell motility and metastasis.634,638,641 In addition,
transmembrane domain, and an extracellular domain that consists EMT transcription factors also regulate tumor prosurvival pheno-
of five cadherin-motif subdomains with putative calcium-binding types, such as participating in tumor cell DNA repair, the evasion
sites.610,611 E-cadherin mediates cell adhesion through calcium- of senescence and apoptosis, and immune evasion, providing
dependent trans-homodimeric interactions of its EC1 structure survival advantages for tumor cells under various stress
with the EC1 domain of adjacent cells, while the cytoplasmic part conditions.642
interacts with adherens junctions-related molecules such as A significant enrichment analysis of 244 differentially expressed
β-catenin.612 E-cadherin plays a significant role in normal EMT-related genes in CRC has revealed that EMT-related signaling
embryonic development, organ morphogenesis, and tissue pathway genes are highly related to the prognosis prediction of
formation by regulating proliferation, migration, or maintaining CRC patients, where higher risk scores indicate poor prognosis.643
epithelial cell polarity.613 Thus, E-cadherin is a biomarker of the In conclusion, EMT transcription factors have been considered as
epithelial cell layer.613 prognostic biomarkers for tumor aggressiveness and metastasis in
The embryonic program EMT is of great importance in the clinical practice.644–646
progress of epithelial-derived tumors from benign lesions to
invasive carcinomas and metastases.613,614 This process is Twist: Twsit1 and Twsit2 are highly conserved basic helix-loop-
accompanied by changes in cadherin expression599,615: from helix transcription factors,638,647 which are pivotal regulators of
E-cadherin which promotes tumor adhesion and blocks invasion, embryonic morphogenesis.648 Twist is expressed in mesodermal
to N-cadherin which is expressed in mesenchymal cells to and ectodermal-derived tissues, and it has been found that Twsit1
promote tumor cell invasion,599 and E-cadherin dysfunction is an and Twsit2 which are structurally homologous are overexpressed
EMT landmark in this process.613 The causes of abnormal in multiple human cancers.647,648 Twsit1 overexpression has been
E-cadherin in tumor cells include reduced or absent E-cadherin confirmed to be strongly associated with aggressiveness and
expression, mutations or reduced transcription of E-cadherin metastasis in cancer patients, including sarcoma, glioma, mela-
genes, abnormal redistribution of E-cadherin within cells, the noma, ESCC,649 neuroblastoma,650 cervical cancer,651 RCC,652 and
shedding of E-cadherin from the cell surface, and competition hematological malignancies including AML, chronic myeloid
with other proteins for binding.610 In addition, E-cadherin is an leukemia (CML), ALL, CLL, lymphomas.653 In CML patients, the
important tumor growth suppressor,616 and inhibits tumor cell increased expression of Twist is related to tumor progression,
growth by upregulating p27-induced cell cycle arrest. Inhibition of tumor staging, and drug resistance, and Twist can be applied as a
E-cadherin leads to a decrease in cell adhesion, which promotes biomarker to assess MRD.653 Inhibition of Twist expression has
tumor metastasis.610 been found to impair the high metastasis of breast cancer cells
Several investigations have demonstrated the critical role that from the mammary gland to the lung.648 Collectively, Twist is a
E-cadherin plays in tumor progression. E-cadherin is closely meaningful biomarker for tumor prognosis and metastasis.647
connected to pathological and clinical characteristics of tumor
patients, such as the degree of differentiation, aggressiveness, Snail: Snail, the first member of the snail superfamily, was first
venous permeation, peritoneal seeding, infiltrative growth, liver described in Drosophila melanogaster,654,655 and is essential for
and bone metastasis, lymph node metastasis, tumor staging, and cellular mesoderm formation.654 The three members (Snail, Snai2,
poor prognosis.609,617–619 The deletion or downregulation of and Snai3) of the Snail family share a similar structure: a highly
E-cadherin promotes tumor invasion, infiltrative growth, and conserved C-terminal domain containing four to six C2H2-type
dedifferentiation.610,616 Thus, E-cadherin can be utilized as a zinc finger.656 In cancer cells, Snail functions as a transcriptional
prognostic biomarker of tumor metastasis for multiple tumors,609 repressor by binding to the E-box motif (CAGGTG) of Snail-related
including CRC,620 gastric cancer,621 pancreatic cancer,622 esopha- genes with its C-terminal structural domain, thus inhibiting the
geal cancer,623 liver cancer,624 lung cancer,625 bladder cancer,626 transcription of target genes.654,656 For example, Snail down-
prostate cancer,627,628 breast cancer,629 endometrial cancer,630 regulates E-cadherin expression and thereby induces EMT and
ovarian cancer,631 thyroid cancer,632 and HNSCC.633 basal-like phenotype conversion.636 The overexpression of Snail is
associated with poor prognosis in patients with breast cancer,657
EMT transcription factors: EMT is the basic embryonic develop- CRC,658 and liver cancer.659 Snail expression is significantly higher
mental process that transforms polarized non-motile epithelial in the high-stage, high-grade, and significant lymphovascular
cells into motile and invasive mesenchymal cells.634,635 In tumor invasion patients with upper urinary tract urothelial carcinoma.660

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Slug, a member of the Snail family, also has a striking impact on such as gastric cancer, metastatic CRC, gastroesophageal cancer,
EMT. Slug expression is an independent prognostic biomarker for and esophageal adenocarcinoma.677,679 c-MET mRNA and protein
poor survival in CRC661 and esophageal SCC patients.662 levels are significantly higher in liver metastasis of CRC than in
primary CRC, and its expression is positively correlated with tumor
ZEB1/2: ZEB1 (also known as Zfhx1a and Zfhep) and ZEB2 (also stage in CRC liver metastasis.684 Besides gastrointestinal cancers,
known as SIP1 and Zfhx1b),663 members of the ZEB transcription c-MET mutations are found in papillary renal cancer,685 ovarian
factor family,638 which are encoded by the ZFHX1a and ZFHX1b cancer,685 SCLC,686 HNSCC,687 and childhood HCC.679,685 Elevated
genes.664 Both ZEB1 and ZEB2 possess two separated clusters of HGF levels are found in various cancers including head and neck
C2H2-type zinc fingers which bind to paired E-box promoter cancer,688 cervical cancer,689 HCC,690 and lung cancer,691 and are
elements.664 ZEB1 is a key regulator of tumor cell plasticity and associated with poor prognosis. HGF promotes HCC migration and
metastasis.665 Mechanically, ZEB1 binds directly to the E-box in the invasion, and is positively correlated with HCC metastasis.692 HGF
promoter of the CDH1 gene which encodes E-cadherin, blocking has been observed to be an independent blood-based predictive
CDH1 transcription and inducing EMT.666 ZEB1 overexpression is biomarker and primary diagnostic marker in ovarian cancer
strongly associated with highly aggressive precursor lesions and patients.693 Moreover, HGF/c-MET can be used as a prognostic
poor prognosis of pancreatic cancers.665,667,668 ZEB1 deficiency biomarker in various hematologic tumors, such as B-cell lym-
reduces stemness, tumorigenic, and colonization capacities in phoma, T and natural killer (NK) cell lymphoma, and Hodgkin
CSCs of pancreatic cancer, thereby inhibiting the formation of lymphoma.694
undifferentiated high-grade cancers, invasion, and metastasis.665 Furthermore, c-MET activation mediates resistance to TKIs,
ZEB1 overexpression serves as a significantly independent adverse chemotherapy, cetuximab, and radiotherapy in CRC patients.677
prognostic factor for RFS and OS in metaplastic breast cancer.669 c-MET mediates radio-resistance by increasing cell motility and
The knockdown of ZEB1 in human breast cancer cells results in inhibiting apoptosis through autocrine and paracrine signaling.695
approximately 230 gene changes, most of which are related to HGF co-amplification leads to clinical resistance in MET-amplified
epithelial differentiation and intercellular adhesion.666 Moreover, esophagogastric cancer.696 In conclusion, c-MET/HGF overexpres-
aberrant expression of ZEB1 is associated with multiple tumor sion is an independent biomarker of poor prognosis and drug
progression and metastasis, including uterine cancer, osteosar- resistance in patients with various hematologic and solid tumors.
coma, lung cancer, liver cancer, and gastric cancer, which reveals
the importance of ZEB1 in EMT induction and tumor N-cadherin: N-cadherin, also known as cadherin 2 or CDH2,697
development.666 was identified in the 1980s.698 N-cadherin is a single-pass
ZEB2 is a DNA-binding transcriptional repressor consisting of transmembrane calcium-binding glycoprotein that mediates
multiple functional domains which interact with various transcrip- intercellular adhesion,699,700 and consists of five extracellular
tional effectors.670 ZEB2 is proven to be highly expressed in substructural domains (EC1-EC5).701 In addition to expression in
human cancer cell lines lacking E-cadherin protein. Overexpres- normal cells such as neuronal cells, osteoblasts, stromal cells, and
sion of ZEB2 blocks E-cadherin protein-mediated intercellular endothelial cells.702 Studies have found that N-cadherin is highly
adhesion and promotes tumor cell metastasis.671 ZEB2 promotes expressed in various tumors including melanoma,703 neuroblas-
the migration and invasion of breast cancer,672 bladder cancer, toma,704 breast cancer,705 urothelial cancer,702 ovarian cancer,706
ovarian cancer, stomach cancer, CRC,673 OSCC,674 and pancreatic and multiple myeloma.701 Abnormal expression of N-cadherin
cancer.667 promotes tumor cell survival, proliferation, invasion, and metas-
tasis by regulating signaling pathways, such as fibroblast growth
HGF/c-MET: c-MET, also known as RTK Met, was first identified as factor receptor signaling, canonical Wnt signaling,701,702 and
a proto-oncogene in the 1980s.675,676 c-MET is a disulfide-linked signalings involved in neovascularization and vascular stability
heterodimer composed of an extracellular α-subunit and a single- regulation.701,707 In addition, N-cadherin exhibits great importance
pass transmembrane β-subunit, which is translated and cleaved in hematological malignancies, such as leukemia and multiple
form pro-c-MET, a 170 kDa single-stranded precursor pro- myeloma,702 and is closely associated with poor prognosis in
tein.677,678 The β-subunit of c-MET is involved in the regulation multiple myeloma.708 The N-cadherin antagonist ADH-1 induces
of kinase activity and effector signaling by forming extracellular cell apoptosis in various tumors including neuroblastoma,704
and partially intracellular structural domains.677 Hepatocyte multiple myeloma,709 and pancreatic cancer,710 and improves the
growth factor (HGF, also known as scatter factor) is the only efficacy of tumor-infiltrating lymphocyte therapies.711 Blocking
known c-MET ligand,677 which is a 90 kDa heterodimer composed N-cadherin effectively inhibits prostate cancer invasion, metasta-
of an α chain and a β chain.677 HGF consists of six structural sis, and castration resistance, which has become an important
domain groups: amino-terminal domain (N), four kringle domains therapeutic target and biomarker for prostate cancer.712
(K1–K4), and a serine proteinase homology (SPH) domain,675 of
which the N-terminal and the first kringle region are c-MET high- MMPs: MMPs, also called matrixins, are highly conserved zinc-
affinity binding sites. HGF induces c-MET dimerization and dependent endopeptidases belonging to the metzincin super-
phosphorylates c-MET residues Y1349/1356, subsequently activat- family.713,714 MMP1, the first matrix metalloproteinase, was
ing various downstream signaling pathways including the ERK1/2, discovered in 1962 in the tadpole tail, which exerted the ability
p38/MAPK, and PI3K-AKT, ultimately promoting cell proliferation to degrade collagen.715 The members of the MMP family can be
and survival.675,677 divided into six major groups: the astacins, the adamalysins (a
Under normal physiological conditions, HGF/c-MET is involved proteinase with a disintegrin and metalloproteinases, ADAMs, the
in cellular processes such as embryogenesis, angiogenesis, wound ADAMs with thrombospondin motif, the pappalysins, the serraly-
healing, and organ regeneration. While abnormal expression of sins, and the MMPs.714–716 Most catalytic domains of MMPs are
HGF/c-MET in tumor cells including MET amplification and highly homologous and basically consist of four structural
mutation, the transcriptional dysregulation of c-MET, degradation domains. However, differences between each MMP still exit
deficiency, and abnormal HGF production are closely related to including substrate specificity, cellular and tissue localization,
tumor progression.677,679 c-MET activation enhances tumorigeni- membrane binding, and regulation.713,717 MMPs consisting of 23
city, invasion, and metastasis.680,681 High expression of HGF/c-MET members with different structural domains in humans are widely
is revealed in various cancers and is closely associated with the expressed in various organs and tissues.713
poor prognosis of cancer patients.675,682,683 For example, c-MET The ECM is a fundamental component of body tissues and
locus amplification occurs in patients with gastrointestinal cancers organs, which maintains tissue integrity by homeostatic balance

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between ECM production and its degradation.715 MMPs are nascent DNA and promoting the HR repair of broken replication
proteolytic enzymes capable of degrading the basement mem- forks to regulate DNA replication.733,736,737 The loss of BRCA1/2
brane and the most of ECM components, thus remodeling the function leads to the accumulation of DNA damage and genomic
ECM.604 In addition, MMPs can also act as extracellular processing alterations including insertions, deletions, and chromosomal
enzymes to regulate protein functions, as well as participate in rearrangements, ultimately damaging genomic integrity and
various homeostatic regulations in tumor cells, such as immunity, promoting tumorigenesis.733 The overexpression of BRCA1/2 is
angiogenesis, cell adhesion, cell proliferation, apoptosis, and significantly associated with worse OS and clinicopathological
EMT.713,718 characteristics in breast cancer.738 High expression of cytoplasmic
The upregulation of MMPs has been observed in different BRCA1 and BRCA2 is significantly associated with favorable OS in
tumors, such as breast cancer,719 CRC,720 gastric cancer,721 digestive cancers, whereas BRCA1 nuclear expression usually
esophageal cancer,722 urinary bladder cancer,718 and lung predicts poor outcomes. Thus, BRCA1/2 could be used as
cancer,723 which increases tumor metastasis and promotes cell clinicopathological biomarkers to evaluate the prognosis of
invasion.604,724 In particular, MMP-9 is critical in cancer cell digestive system cancers.739 Moreover, BRCA1/2 mutations are
invasion and metastasis, and has been demonstrated to be a closely related to the progression of multiple cancers, including
key biomarker in different cancers including NSCLC,725 cervical breast cancer, ovarian cancer, prostate cancer, and pancreatic
cancer,726 gastric cancer,727 ovarian cancer,728 breast cancer,729 cancer.733,740 BRCA1/2-deficient cells are highly sensitive to PARP
osteosarcoma,730 and pancreatic cancer.731 The expression of inhibition,741 which is due to inhibition of PARP-dependent SSB
MMP1, MMP2, and MMP16 are positively correlated with OS and repair resulting in the accumulation of DNA lesions (SSBs and
DFS in patients with uveal melanoma.732 Collectively, MMPs can DSBs) during replication.733 In conclusion, BRCA1/2 serves as a
be potential biomarkers in various cancers. biomarker for prognosis and treatment response in cancer.
Genome instability and mutation. DNA is a relatively stable ATR-CHK1/ ATM-CHK2: Ataxia telangiectasia mutated (ATM) is a
organic molecule and genomic maintenance systems monitor and kinase responsible for orchestrating cellular responses to DSB and
resolve damaged DNA, thus ensuring low mutation frequency replication stress, including DNA repair, checkpoint activation,
within cells. During tumor development, cancer cells induce the apoptosis, senescence, chromatin structural change, and tran-
accelerated accumulation of mutations by compromising genomic scription.742 Ataxia telangiectasia and Rad3-related protein (ATR),
integrity or forcing genetically damaged cells to senescence or an essential regulator of the cellular replication stress response, is
undergo apoptosis.299 DNA damage response (DDR) coordinates involved in cell-cycle arrest, inhibiting the beginning of replication
DNA repair by regulating cell cycle checkpoints and other global origins, regulating global fork speed, and promoting fork
cellular responses. Genome instability and mutation caused by stabilization.743 ATM and ATR respond to DNA damage by
DDR defects are important hallmarks of cancer.733 phosphorylating hundreds of substrates.744 The checkpoint kinase
1 (CHK1) and checkpoint kinase 2 (CHK2) are the major substrates
PARP: DNA single-strand break (SSB) or single-strand nick are downstream of ATR and ATM, respectively, and are responsible for
primarily recognized by PARP1 or PARP2, which catalyze the downregulating the activity of CDKs, thereby preventing cell cycle
formation of poly (ADP-ribose) (PAR) chains on themselves and progression under stress. ATM is recruited to DSB sites and
neighboring target proteins.733 PARP1 and its activity in poly(ADP- promotes histone H2AX phosphorylation. Phosphorylated H2AX in
ribosyl)ation (PARylation) at SSBs recruit the scaffold protein turn recruits the mediator of DNA damage protein MDC1, and
XRCC1 which drives DNA ligase 3 (LIG3) and accessory repair subsequent MDC1 phosphorylation by ATM leads to recruitment
factors to rejoin disruptions. The poly(ADP-ribose) polymerase of DNA damage mediator proteins 53BP1 and BRCA1, thereby
(PARP) plays an important role in many cancer types, including promoting DSB repair.733
ovarian cancer, breast cancer, pancreatic cancer, and prostate ATM is frequently mutated or inactivated in a variety of tumors,
cancer.733 Furthermore, PAR chains are rapidly degraded by PAR including lung cancer, breast cancer, brain cancer,745 and
glycohydrolase which restores PARP and PARylated proteins to a pancreatic cancer.746 Endometrial cancer patients with ATM
de-(ADP-ribosylated) state to promote SSB repair. As PARylation is mutations exhibit a higher tumor mutational burden, a higher
a highly dynamic and transient process, the inhibition of both PAR neoantigen load, and increased expression levels of immune
glycohydrolase and PARP could reduce the repair efficiency of checkpoints. Thus, ATM mutations can act as an independent
SSBs, exhibiting their anticancer efficiency.733 Especially, PARP prognostic factor and a potential biomarker for immune
inhibitors have been demonstrated to block the SSB repair checkpoint therapy in endometrial cancer.747 Moreover, ATM
pathway and trigger synthetic lethality in cancers with homo- mutations are independently associated with longer OS in
logous recombination (HR) deficiency which results in impaired patients with metastatic CRC.748 ATM deficiency also renders
DNA double-strand breaks (DSB) repair.734,735 cancer cells sensitive to topoisomerase I inhibitors or PARP
inhibitors. PARP and topoisomerase I inhibitors lead to single-
BRCA1/2: In addition to SSB, DSB exerts a vital role in genome ended DSB, while ATM inactivation delays DNA damage repair,
integrity. There are two major DSB repair pathways in human cells: leading to toxic chromosome fusions.733
the nonhomologous end joining pathway and the HR pathway.733
The HR pathway uses the homologous DNA molecule (usually the Tumor-promoting inflammation. As one of the tumor hall-
sister chromatid) as the repair template. HR is initiated when marks,299 persistent inflammation plays an essential role in a
nuclease digests double-stranded DNA ends at DSB sites to variety of human cancers by manipulating cancer development,
produce ssDNA overhangs. Immediately afterward, BRCA1 facil- angiogenesis, malignant transformation, invasion and migration,
itates the recruitment of BRCA2 to DSB sites through interaction immune surveillance, and response to therapy.749,750
with PALB2, which loads RAD51 directly onto ssDNA ends. Inflammation-related regulators, including tumor necrosis factor-
Nucleoprotein filaments are formed on ssDNA by RAD51, which α (TNF-α), nuclear factor-κB (NF-κB), and Nod-like receptor protein
subsequently promotes strand invasion and displacement loop (D- 3 (NLRP3), are potential tumor prognostic biomarkers.
loop) ss formation. Finally, the invasion strand is replaced and
strand annealing contributes to the HR completion.733 TNF-α: TNF-α, a vital member of the multifunctional TNF
BRCA1/2 maintain genomic integrity after DNA damage by superfamily, is a 17 kDa type II transmembrane protein that was
promoting accurate DNA repair via the HR pathway.733 BRCA1/2 first isolated from the serum of mice infected with Bacillus
regulate DNA replication by preventing nuclease degradation of Calmette-Guérin and endotoxin by E. A. Carswell in 1975.751,752 As

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a key molecule mediating the tumor-promoting inflammatory cytoplasmic compartments, and the NLRP3 inflammasome has
process, TNF-α drives inflammation directly by promoting been intensively studied for its involvement in broad ranges of
inflammatory gene expression, or indirectly by triggering the human diseases. Especially, dysregulation of the NLRP3 inflamma-
inflammatory immune response and regulating cell death.753 some is closely associated with the development of different
Mechanistically, TNF-α binds as a homotrimer to two distinct cancers, including gastric cancer, CRC, HCC, head and neck cancer,
homotrimeric receptors on the cell surface: TNFRI (p55 receptor) lung cancer, breast cancer, prostate cancer, skin cancer, cervical
and TNFRII (p75 receptor),754 thus inducing downstream inflam- cancer, and central nervous system tumors.749 A high NLRP3 level
matory mediators and growth factors, which further activates is correlated with the advanced tumor stage, distant metastasis,
NF-κB and AP1.754 NF-κB signaling is a major mediator of and the vascular invasion of cancers.775 It has been found that
protumor activity of inflammatory cytokines.755 NLRP3 inflammasome promotes cancer cell differentiation by
TNF-α levels are abnormally elevated in various precancerous regulating cell cycle proteins and inducing the production of IL-1β
lesions, such as gastric lesions754,756 and inflammatory bowel which activates NF-κB by binding to its receptor, which ultimately
disease, compared with normal tissues.754,757 In addition, TNF-α is leads to proliferation and invasion of gastric cancer.749 NLRP3
overexpressed in the tumor and stroma of multiple malignancies, inflammasome activation in glioblastoma cells leads to IL-1β in
including breast cancer, ovarian cancer, CRC, prostate cancer, aberrant expression.776 In addition, the NLRP3 inflammasome has
bladder cancer, esophageal cancer, renal cell cancer, melanoma, been demonstrated to be elevated in HNSCC tissues, and its level
lymphoma, and leukemia.752 For example, ovarian cancer cells is correlated with tumor prognosis.777 Activation of the NLRP3
express 1000-fold more TNF-α mRNA than normal ovarian surface inflammasome promotes the progression of prostate cancer,778
epithelial cells.758 The combination of upregulated TNF-α and while reduced expression of NLRP3 inflammasome and IL-1β
C-reactive protein in the patient’s plasma is significantly related to inhibits melanoma development.779 Furthermore, a high NLRP3
shorter survival in HNSCC patients.759 In conclusion, TNF-α is a key level is associated with a low 5-year and 10-year survival rate in
regulator linking inflammation and tumorigenesis, and it may CRC patients.780 Targeting NLRP3 inflammasome effectively
serve as a promising prognostic and therapeutic biomarker for inhibits HCC proliferation and metastasis.781 In conclusion, NLRP3
tumor inflammation.755 inflammasome activation leads to an inflammatory response that
promotes cancer development and progression, and NLRP3 may
NF-κB: NF-κB, first identified as a nuclear factor essential for serve as a prognostic and therapeutic biomarker for tumors.
immunoglobulin kappa light chain transcription in B cells in
1986,760 is a dimeric transcription factor. The mammalian NF-κB Deregulating cellular metabolism. Otto Warburg first discovered
family consists of RELA (p65), NF-κB1 (p50; p105), NF-κB2 (p52; the tendency of tumors to convert glucose to lactate in the
p100), c-REL, and RELB,761,762 all of which share a conserved presence of oxygen in 1924, known as “aerobic glycolysis“,782
amino-terminal region containing dimerization, nuclear localiza- which subsequently came to be termed the “Warburg effect“.783
tion, and DNA-binding domains. External stimuli, including Tumor cells reprogram glucose metabolism even in the presence
infection factors, proteins, stress signals, and proinflammatory of oxygen by restricting energy metabolism mainly to glycolysis,
cytokines released by necrotic cells can activate NF-κB.762 The thus reprogramming energy production. Extensive alterations in
main activated form of NF-κB is a heterodimer of the p50 or energy metabolism in cancer cells are considered to be important
p52 subunit associated with the p65 subunit.762 NF-κB proteins are hallmarks of cancer299 (Fig. 6).
present in the cytoplasm and are associated with inhibitory
proteins of IκB. Activated IκB proteins are phosphorylated and GLUT1: Tumor cells require the high uptake of glucose and
ubiquitinated and then degraded by the proteasome, which glutamine to meet sustained proliferation.784 The polarity and
induces NF-κB proteins to translocate to the nucleus.762 The hydrophilicity of glucose result in its inability to penetrate
nucleus NF-κB binds to cognate DNA-binding sites, promoting the hydrophobic cell membranes. The transmembrane glucose
transcription of various genes involved in cell cycle, proliferation, transporter protein 1 (GLUT1, also known as SLC2A1) is the major
apoptosis resistance, and metastasis-promoting, ultimately enhan- glucose transporter protein, and GLUT1 expression is significantly
cing cell growth, angiogenesis, stem cell formation, and cell upregulated in tumor cells.784 The crystal structure of human
metabolism.762–764 GLUT1 was first reported in 2014,785 and the expression of GLUT1
As a key regulator of inflammation,765 NF-κB is activated in is regulated by various signaling pathways. The PI3K-AKT signaling
various hematological and solid tumors and is closely associated pathway increases GLUT1 mRNA expression and drives GLUT1
with tumor development.766 A meta-analysis of 44 studies with a protein transport from the inner membrane to the cell surface,
total of 4418 patients has revealed that NF-κB expression is thereby promoting glucose uptake.786 RAS upregulates GLUT1
connected to poor 3-year and 10-year OS in solid tumors.767 NF-κB mRNA expression and increases cellular glucose consumption.787
level is significantly associated with large tumor size and high Tumor suppressor gene mutations, such as P53, block glycolysis
tumor grade in breast cancer patients.768 NF-κB also plays an by inhibiting GLUT1 expression.788 Additionally, the TME upregu-
important role in the TME. Activated NF-κB in cancer cells initiates lates GLUT1 expression through HIF-1α.787
and maintains the TME by upregulating chemokines that recruit Overexpression of GLUT1 is an important biomarker for poor
immune response cells, inflammatory cells, and progenitors of prognosis in multiple cancers, including breast cancer, ovarian
cancer-associated fibroblasts.769 In addition, NF-κB regulates the cancer, prostate cancer, thyroid cancer, gastric cancer, HNSCC,
EMT transition through the induction of EMT transcription glioblastomas, retinoblastomas, CRC, NSCLC, OSCC, esophageal
factors.770,771 In conclusion, NF-κB is a prognostic biomarker of cancer, urothelial papilloma, meningioma, brain cancer, diffuse
tumor inflammation in cancer. large B-cell lymphoma, RCC, HCC, and cervical cancer.789–792
Studies have demonstrated that inhibitors targeting GLUT are an
NLRP3: NLRP3, belonging to the NLR protein family, is one of the effective strategy for cancer treatment.787 In conclusion, GLUT1 is
most characterized inflammasomes.749 The NLR protein family has an essential target for tumor glucose metabolism, and it can be
22 members in humans.772 After the first inflammasome was used as a diagnostic biomarker for tumors.
discovered by Fabio Martinon in 2002,773 multiple PRRs have been
identified and shown to be involved in inflammatory vesicle IDH1/2: Isocitrate dehydrogenase 1 (IDH1) is localized to
formation, such as NLRP1, NLRP2, NLRP3, and NLRC4.774 peroxisomes and cytoplasmic lysosomes, whereas isocitrate
The inflammasome is a type of intracellular multiprotein dehydrogenase 2 (IDH2) is localized to mitochondria. Wild-type
hexamers or heptamers signaling complex that forms in IDH1 and IDH2 metabolic enzymes catalyze the oxidative

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Fig. 6 The potential inhibitors that target cancer metabolic process. Glucose is taken up into the cell by glucose transporters GLUT1/4 and
phosphorylated by hexokinases HK1 and HK2. Glucose 6-phosphate (P) and its downstream intermediates can either be converted to
pyruvate or fuel biosynthesis through different pathways, such as the pentose phosphate pathway which provides ribose 5-P for nucleotide
synthesis. Fructose-6-P is involved in the hexosamine biosynthesis pathway. Glycerol 3-P production contributes to the serine and glycine
biosynthesis pathways which are regulated by the key enzymes PHGDH and SHMT1/2. Moreover, serine biosynthesis plays an essential role in
amino acid metabolism and nucleotide metabolism by regulating one-carbon metabolism which is mediated by the methylenetetrahy-
drofolate dehydrogenase MTHFD1. Pyruvate can be converted to lactate by LDH and exported through the monocarboxylate transporter
MCT-1. Besides, pyruvate can enter the TCA cycle as acetyl-CoA through the mitochondrial pyruvate carrier and pyruvate dehydrogenase.
Various pathways influence the production of the mitochondrial acetyl-CoA, including fatty acid β-oxidation, glucose metabolism, and other
sources that can condense with oxaloacetate to form citrate, which can then be exported from the mitochondrion. Citrate via ACLY is a vital
source of cytoplasmic acetyl-CoA which forms malonyl-CoA by acetyl-CoA carboxylase ACC1 and ACC2. Subsequently, malonyl-CoA is
cyclically extended by the addition of carbons from acetyl-CoA by FASN to make saturated fatty acids. Fatty acid catabolism is initiated with
the formation of fatty acyl-CoA which is then converted by CPT1 to an acylcarnitine. Pyrimidine synthesis, a multistep process regulated by
key enzymes such as CAD and DHODH, can produce pyrimidine nucleotides from glutamine, carbonate, and aspartate. Meanwhile, glutamine
is taken up by transporters SLC1A5. Glutamate produced from glutamine by glutaminase enzymes can be used in glutathione synthesis. In
addition, the complex V (ATP synthase) and the electron transport chain consisting of four complexes including complex I/II/III/IV (CI–IV), are
promising targets for drug development. Inhibitors (red), key enzymes or transporters (blue), and key metabolites (purple) are shown. ACC
acetyl-CoA carboxylase, ACLY ATP-citrate lyase, BP bisphosphate, CAD carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and
dihydroorotase, CoA coenzyme A, CI–IV, complex I/II/III/IV, CV complex V, CPT1 carnitine palmitoyltransferase 1, DHODH dihydroorotate
dehydrogenase, FASN fatty acid synthase, GLUT1/GLUT4 glucose transporter 1/4, HK hexokinase, IDH1 isocitrate dehydrogenase 1, LDHA/B
lactate dehydrogenase A/B, MCT-1 monocarboxylate transporter 1, MTHFD1 methylenetetrahydrofolate dehydrogenase 1, P phosphate,
PHGDH phosphoglycerate dehydrogenase, PKM2 pyruvate kinase M2, SHMT serine hydroxymethyl transferase, SLC1A5 solute carrier family 1
member 5, TCA tricarboxylic acid
decarboxylation of isocitrate to generate α-ketoglutarate (α-KG). levels competitively inhibit α-KG-dependent lysine demethylases,
Cancer-associated IDH1 and IDH2 mutations occur almost leading to D2HG-induced dysregulation of histone and DNA
exclusively at different arginine residues in the active site of the methylation in cells, ultimately promoting tumor progression.793
enzyme.793 IDH1 and IDH2 mutations occur in a wide variety of IDH1/2 mutations have many advantages as easily detectable,
hematologic and solid tumors, including glioma, AML, intrahepatic reliable, and specific biomarkers. First, IDH1 and IDH2 mutations
cholangiocarcinoma, chondrosarcoma, thyroid cancer, and occur in highly restricted tumor types. Second, almost all tumor-
angioimmunoblastic T cell lymphoma.793–795 Mutant IDH1/2 derived mutant loci can be identified by simple PCR amplification
catalyzes the conversion of α-KG to D-2-hydroxyglutarate and sequencing with a low volume of tumor samples. Third, IDH1
(D2HG). D2HG is maintained at normal levels under physiological mutations can be identified by routine IHC.796 Fourth, techniques
conditions, whereas mutant IDH leads to a large intracellular for the noninvasive detection of 2-hydroxyglutarate (2-HG)
accumulation of D2HG in IDH mutant cancer. Elevated D2HG accumulation in glioma patients have been developed.797

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Moreover, given that D2HG is upregulated in tumors with IDH urothelial carcinoma, and other cancers, has proven that patients
mutations, elevated D2HG level in tumor tissues is used as a with high expression levels (>11.0 pg/μL) of soluble PD-L1 are
noninvasive detection biomarker for clinical IDH mutated more likely to exhibit progressive than those with low expression
tumors.793 In conclusion, IDH1/2 mutations are meaningful levels of PD-L1 (41.8 versus 20.7%). Moreover, a high expression
diagnostic biomarkers of tumor metabolism. level of soluble PD-L1 is also associated with a worse prognosis,
the median PFS is 2.9 months versus 6.3 months, and the median
HK2: Hexokinases regulate the first step of glycolysis which OS is 7.4 months versus 13.3 months. Thus, high soluble PD-L1 is a
produces and captures negatively charged glucose 6-phosphate predictive and prognostic biomarker for both decreased PFS and
ions within the cells. The hexokinases family has five isoforms in OS in advanced cancer patients who receive immune checkpoint
mammals including hexokinase 1–4 (HK1, HK2, HK3, HK4), and blockade treatment.811 Moreover, a study of 293 HNSCC patients
HKDC1.798 HK2 is the most active isozyme of the hexokinase has concluded that strong PD-L1 expression is correlated with
family.799 In addition to being expressed in the muscle and distant metastases, and dominates as the strongest prognostic
heart,798 HK2 has been evaluated in various cancers, and is factor of patient outcome.812 The PD-L1 expression level is a
induced solely or synergistically by HIF-1 and MYC.800 An analysis negative prognostic factor for patients with RCC813 and gastric
of 21 studies with 2532 patients has revealed that HK2 over- cancer.814 Immune checkpoint inhibitors that block PD-1/PD-L1
expression is significantly associated with worse OS and PFS in interactions effectively prolong the survival of patients with
solid tumors. For example, the negative effect of HK2 on OS is various cancers and are promising cancer therapy.803 In conclu-
observed in HCC, gastric cancer, and CRC patients.801 HK2 sion, PD-/PD-L1 expression can be used as a predictive and
expression is correlated with advanced-stage and high-grade prognostic biomarker for cancers.
ovarian cancer.798 HK2 downregulation inhibits tumor occur-
rence.802 Thus, HK2 is a meaningful prognostic tumor biomarker CATL-4: Cytotoxic T lymphocyte antigen 4 (CTLA-4, also known
and a potential tumor treatment target. as cluster of differentiation 152, CD152) is a receptor present on
the surface of activated T cells, was discovered in 1987 by
Evading immune destruction. The immune system is responsible screening a cDNA library of mouse cytolytic T cell origin.815 CTLA-4
for monitoring and eliminating most early cancer cells, thereby is normally expressed upon T cell activation,816 and activated
inhibiting tumor formation. However, a significant increase in CTLA-4 inhibits T cell proliferation and induces cell cycle arrest by
cancers due to low immune function is the evidence of defects in cross-talk with PI3K and MAPK pathways that regulate cell
tumor immune surveillance. It has been found that cancer cells proliferation.816 CTLA-4 is an inhibitory checkpoint commonly
generate immune escape by disrupting the immune system, found in activated T cells and has been discovered to be the most
which ultimately promotes tumor progression, dissemination, and reliable target for the treatment of cancer.817 CTLA-4 facilitates the
metastasis. Tumor cells suppress the action of cytotoxic lympho- tumor evasion of host immune surveillance, and participates in
cytes by recruiting inflammatory cells with active immunosup- immune dysregulation in multifarious cancers, including lung
pressive effects, such as regulatory T cells and myeloid-derived cancer, cervical cancer, breast cancer, skin cancer, gastric cancer,
suppressor cells. Thus, immune evasion is another valuable CRC, B-cell CLL, and non-Hodgkin’s lymphoma.818 Moreover,
hallmark of cancer.299 targeting CTLA-4 significantly improves outcomes in multiple
advanced cancers, including melanoma, lung cancer, breast
PD-1/PD-L1: PD-1 and PD-L1 participate in the evasion of the cancer, head and neck cancer, bladder cancer, cervical cancer,
immune system by cancer cells.803 PD-1 (also called CD279), liver cancer, gastric cancer, squamous cell skin cancer, classical
encoded by the PDCD1 gene,804 was cloned and identified from Hodgkin’s lymphoma, and B-cell lymphoma.816
an apoptotic immune cell line in 1992.805 PD-1 is a type I However, the correlation between CTLA-4 expression and
transmembrane protein receptor consisting of 288 amino acids, patient prognosis in different cancers is controversial. Studies
whose structure consists of an IgV-like extracellular domain, a have found a significant correlation between the high expression
transmembrane domain, and a cytoplasmic (intracellular) of CTLA-4 and OS in single nucleotide polymorphisms subgroup
domain.803 As a negative regulator of the immune response,806 cancers, including NPC, esophageal cancer, glioblastoma, and
PD-1 is mainly expressed in memory T cells in peripheral tissues, hematologic malignancy, in which CTLA-4 is a good prognostic
and less in B cells, activated monocytes, dendritic cells, and NK biomarker.818 CTLA-4 overexpressed NSCLC is associated with a
cells.803,804 Two ligands of PD-1, PD-L1 (also known as B7-H1 or reduced death rate. Conversely, malignant pleural mesothelioma
CD274) and PD-L2 (also known as B7-DC or CD273),807 are type I with high CTLA-4 exhibits poor prognosis.818 Higher CTLA-4 mRNA
transmembrane protein receptors. PD-L1 is a 290 amino acid levels in breast cancer indicate higher clinical stage and axillary
protein receptor encoded by the Cd274 gene and includes two lymph node metastasis.819 A combined analysis of 844 ESCC
extracellular structural domains (IgV- and IgC-like domains), a patients has found that patients with both a low CTLA-4 and
transmembrane domain, and a cytoplasmic domain. Activated PD- platelet lymphocyte ratio (PLR) level have longer OS.820 In
1/PD-L1 signaling negatively regulates T cell-mediated immune conclusion, CTLA-4 is a prognostic biomarker in cancers and its
responses in peripheral tissues, thereby limiting effector T cell positive or negative effects depend on specific cancer conditions.
responses and protecting tissues from damage.803,804
PD-1 signaling in the TME promotes tumor progression and Unlocking phenotypic plasticity. Cell development and organo-
survival by evading tumor immune surveillance. PD-1 is highly genesis are accompanied by terminal differentiation that in most
expressed in tumor-infiltrating lymphocytes in many types of cases results in antiproliferative outcomes and suppresses tumor
cancers. PD-L1 is expressed on different types of tumor cells, formation. It has been found that unlocking phenotypic plasticity
including melanoma, ovarian cancer, lung cancer, and kidney to evade the state of terminal differentiation is a pivotal
cancer.808 The innate and adaptive immune resistance mechan- component of cancer development.300
isms contribute to the upregulated expression of PD-L1 in Tumor cell differentiation is regulated by multiple factors. Liver
multifarious human cancers.809 A meta-analysis that analyzed enriched transcription factors are crucial regulators of hepatocyte
1251 patients from eight different microarray gene expression differentiation and are essential for the maintenance of hepato-
datasets has revealed that the expression levels of PD-1 and PD-L1 cyte phenotype and function. Noncoding single-stranded RNA
individually or jointly are potential prognostic factors for predict- microRNAs are involved in the post-transcriptional regulation of
ing the outcomes of patients with lung cancer.810 A study with 128 gene expression, which is closely correlated with tumor dediffer-
patients who are diagnosed with NSCLC, SCLC, melanoma, entiation. The expression of miRNAs is negatively correlated with

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the degree of differentiation in HCC. Moreover, differentiation- A study has searched the PubMed database for literature
related genes such as HMGCS2, BDH1, ALDH2, PIPOX, HAO1, related to DNA methylation-based cancer biomarkers and
AQP9, and PAH, have been identified to predict survival and poor retrieved a total of 14,743 research papers, which ultimately
prognosis in multifarious cancers.821 yields ~1800 tumor biomarkers through calculation and screening.
Differentiation and dedifferentiation are also essential for the However, only 13 DNA methylation-based biomarkers are
developmental processes of many tumors. Melanocytes undergo currently commercially available and detectable, including GSTP1,
dedifferentiation during tumorigenesis, and the malignant pro- APC, RASSF1, NDRG4, BMP3, SEPT9, SHOX2, TWIST1, OTX1,
gression of pancreatic islet cell cancers to metastasis-prone ONECUT2, MGMT, BCAT1, and IKZF1. Only nine of them (GSTP1,
carcinomas is associated with dedifferentiation. HDAC inhibitors APC, RASSF1, NDRG4, BMP3, two SEPT9 biomarkers, SHOX2 and
induce the myeloid leukemia cell differentiation into mature MGMT) have been included in the clinical guideline application.826
myeloid morphology cells, thereby hindering the progression of In addition, only two tests have been approved by the FDA:
leukemia.300 Furthermore, cellular plasticity in HCC is presented by Cologuard (NDRG4 and BMP3), which analyzes stool DNA samples
the dynamic interconversion of cancer cell subpopulations in collected as part of a CRC screening protocol, and Epi proColon
multiple developmental lineages and differentiation stages. (SEPT9), which analyzes blood samples collected for the same
Regardless, differentiation therapy unlocks phenotypic plasticity purpose.826
in HCC and induces terminal differentiation of CSCs, promoting As a promising biomarker for tumor diagnosis, prognosis, and
their transformation into precursor cells that have lost self-renewal prediction,833 DNA methylation has many advantages: frequent
capacity, or converting them into non-CSCs that are sensitive to DNA methylation at the early stages of cancer, mature detection
anticancer drugs.821 In conclusion, differentiation-related factors technology, good stability of DNA methylation in fixed samples,
can be used as diagnostic and prognostic biomarkers for cancers. and presence in various body fluids and cell type specificity.
Methylation at specific genomic sites can be a beneficial
Nonmutational epigenetic reprogramming. Since first described biomarker under the following conditions: clinically significant
by Conrad Waddington in 1942,822 the epigenetic program of differences in methylation expression between the two groups,
gene expression has become a hallmark of cancer that initiates including diagnostic biomarkers in tumor versus nontumor tissues;
and promotes tumorigenesis. The process of gene expression prognostic biomarkers between tumor samples from patients with
changes through pure epigenetic regulation is called “nonmuta- high-risk disease versus those with low-risk disease. In conclusion,
tional epigenetic reprogramming”, which is different from DNA methylation in cancer is a clinically valuable biomarker for
genomic DNA instability and mutational mechanisms. Epigenetic tumor management.826
alterations such as DNA methylation, histone modifications,
chromatin remodeling, and noncoding RNA contribute to the Histone modification: Modification of histone proteins at amino-
signature ability during tumor progression.300 terminal tails such as acetylation, methylation, phosphorylation,
and ubiquitination could alter the chromatin condensation, and
DNA methylation: DNA methylation is a chemical modification DNA accessibility, subsequently interfering with gene expression.
that plays a crucial role in chromatin-based transcriptional regula- Histone modification is a dynamic process that is controlled by
tion, epigenetic gene expression, genomic stability, DNA repair, and writers, such as histone acetyltransferases (HATs), histone methyl-
replication. DNA methylation is mainly catalyzed by three DNA transferases (HMTs), readers, such as proteins containing bromo-
methyltransferases (DNMTs), DNMT1, DNMT3A, and DNMT3B.823 domains, and erasers, such as histone deacetylases (HDACs) and
DNMTs are overexpressed in multiple cancers, including AML, CML, lysine demethylases. Histone modifications coregulate processes,
glioma, breast cancer, gastric cancer, CRC, HCC, pancreatic cancer, such as DNA transcription, DNA replication, and DNA repair.834
prostate cancer, and lung cancer.824 DNA methylation may lead to Altered post-translational modifications of histones have been
tumor suppressor gene silence, cell cycle dysregulation, DNA repair, found in cancer cells, and changes in the overall level of histone
and the misregulation of chromosomal stability genes, resulting in modifications are found to predict clinical outcomes in various
genomic instability in tumor cells.825 DNA methylation-based cancers.835
biomarkers have been hailed as an important event in cancer
biomarker research.826 DNA methylation occurring mainly in HATs: The Nε-acetylation of lysine residues is a major histone
centromeres, telomeres, inactive X-chromosomes, and repeat modification involved in the regulation of gene transcription,
methylation is altered in 70% of mammalian promoter CpG islands, chromatin structure, and DNA repair. Acetylation neutralizes the
which are essential for gene transcriptional regulation and tumor positive charge of lysine, thereby weakening the electrostatic
malignant transformation.827,828 It has been found that 5–10% of interaction between histones and negatively charged DNA. Thus,
CpG promoter islands are aberrantly methylated in various cancer histone acetylation is associated with an open chromatin
genomes. DNMT1 is a CpG dinucleotides methyltransferase that conformation. The HATs and HDACs family regulate the acetyla-
recognizes hemimethylated DNA produced during DNA replication tion of histones.828 HATs are involved in a number of solid tumors
and methylates newly synthesized.828 and hematologic malignancies, and their expression levels are
The downregulation of tumor suppressor genes by hyper- altered during tumor progression.836
methylated CpG-rich regions of promoters is a typical example in
tumor cells.826 Methylation of CpG dinucleotides (e.g., gene HMTs: Histones are methylated on the side chains of arginine,
promoters) may serve as a clinically valuable biomarker. Moreover, lysine, and histidine residues and their methylation does not
CpG methylation is related to poor prognosis in patients with change the total charge of the molecule. The most characteristic
ccRCC.829 Methylation of GSTP1 has been discovered to be a sites of histone methylation are mono-, dimethyl- or trimethyla-
promising diagnostic biomarker for HCC.826 The promoter tion of lysine residues, including H3K4, H3K9, H3K27, H3K36,
methylations of NMDAR2B and PGP9.5 are linked to poor H3K79, and H4K20. Among them, H3K4, H3K36, and H3K79 are
prognosis in patients with ESCC, and are meaningful clinical correlated with active genes in euchromatin, while H3K9, H3K27,
diagnostic and prognostic biomarkers for ESCC.830 The methyla- and H4K2 are associated with heterochromatic regions of the
tion status of single CpG dinucleotides affects the regulation of genome.828,837 In addition, different methylation states on the
gene expression, and it can be utilized as a prognostic biomarker same residue have different functions. For example, H3K4me2/3
for CLL.831 O6-methylguanine-DNA methyltransferase (MGMT) usually spans the transcriptional start site of the active gene,828
methylation can also be used as a prognostic biomarker in while H3K4me1 is linked to active enhancers.838 Trimethylation of
glioblastoma patients.832 H3K9 is involved in the repression of gene expression.828

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KMTs are specific enzymes that target certain lysine residues, F. nucleatum, and P. gingivalis, are involved in several digestive
including the members of the EZH2 family. EZH2 is the catalytic cancers. P. gingivalis is enriched in ESCC at higher levels than
subunit of polycomb repressive complex 2 and is primarily normal tissues, and it utilizes the miR-194/GRHL3/PTEN/AKT
responsible for the methylation of H3K27. Studies have shown signaling pathway to promote ESCC proliferation and migration.
that EZH2 overexpression is strongly associated with poor The concentration of F. nucleatum nucleic acid is significantly
prognosis in prostate and breast cancers.828 Loss-of-function higher in esophageal cancer tissues than in normal esophageal
mutations in the EZH2 gene in myeloid malignancies and T cell tissues. P. gingivalis and F. nucleatum are associated with a high
acute lymphoblastic leukemia (T-ALL) also lead to poor risk of pancreatic cancer, and P. gingivalis can promote the
prognosis.839 proliferation of pancreatic cancer cells.853 Collectively, certain
microbiota can be carcinogenic by stimulating chronic inflamma-
HDACs: HDACs are enzymes that reverse lysine acetylation and tory response. In addition, microbiota can alter key intracellular
restore the positive charge on the side chain. HDACs can be signaling pathways and attack gastric mucosa by utilizing various
classified into four major groups based on sequence homology: virulence factors.857 In conclusion, the microbiota is essential for
class I (HDAC 1–3 and HDAC8), class II (HDAC 4–7 and HDAC 9-10), cancer development, and the microbiota can be used as a
class III HDAC (sirtuin 1–7), and class IV only (HDAC11).828 HDACs potential biomarker for tumors.
promote leukemia development by mediating abnormal gene
silencing in malignant tumors. Inhibition of HDACs induces Senescent cells. Cellular senescence is a classic form of irrever-
growth arrest, differentiation, and apoptosis in tumor cells.840 In sible proliferative arrest characterized by the shutdown of the cell
addition, studies have demonstrated that HDACs are usually division cycle, changes in cell morphology and metabolism, and
connected to poor tumor prognosis.834 the activation of the senescence-associated secretory phenotype
(SASP), which is capable of transmitting signaling molecules in a
HDMTs: LSD1 (KDM1A) is a class of demethylases that demethy- paracrine manner to neighboring living cancer cells as well as to
late lysine through an oxidation reaction with flavin adenine other cells in the TME. SASP involves the release of a large number
dinucleotide, which is restricted to demethylating mono- and of bioactive protein chemokines, cytokines, and proteases. A
dimethyl lysine. Jumonji is a class of demethylases with a variety of conditions, such as microenvironmental stress, and
conserved JMJC structural domain that demethylates all three telomere erosion, induce cell senescence.862–864 The cellular
methyl lysine states through an oxidative mechanism and radical senescence is a significant biomarker of tumor cells.
attack.828 The most studied LSD1 is increased in a variety of
cancers, and it is related to the differentiation of neuroblastoma SASP: SASP is mediated through the proinflammatory transcrip-
cells. In addition, HDMTs are involved in the development of tion factor NF-kB or through transcriptional processes that depend
breast cancer, PDAC, and other tumorigenic processes.824 on epigenetic changes.863 The NF-κB, p38, mTOR and C/EBPβ
signaling pathways induce the formation of SASP865 which include
Polymorphic microbiomes. The microbiota, an increasingly hot proinflammatory cytokines (e.g., IL-1α, IL-1β, IL-6, and IL-8),
topic in recent years, has been demonstrated to influence the chemokines (e.g., CCL2, CCL5, and CXCL1), growth factors (e.g.,
microenvironment, tumorigenesis, and metastasis of various HGF, EGF, and TGFα), MMPs, and various oxylipins SASP factors.
malignancies.841 An increasing number of studies have uncovered These factors are the main paracrine messengers between
that the microbiota is critical for the development of various senescent cells and their surrounding cells (including stromal
cancers,842 including gastric cancer,843 ovarian cancer,844 CRC,845 bystander cells, immune cells, precancerous cells, and cancer
pancreatic cancer,846 prostate cancer,847 HCC,848 lung cancer,849 cells).865
breast cancer,850 and cholangiocarcinoma.851 The SASP exerts a double-edged sword effect in tumorigenesis,
There are three major categories of regulatory mechanisms by which might be beneficial or detrimental to tumorigenesis.866 On
which microbiota promote carcinogenesis: altering the balance of the one hand, SASP exhibits tumor suppressive effects by
host cell proliferation and death such as DNA damage and DNA maintaining the senescence program, permanently blocking
repair; regulating the tumorigenic inflammatory environment tumor transformation of normal cells, and recruiting immune
within the tissue and immune system function; and affecting cells to remove damaged or oncogene-expressing cells from the
host metabolism.852,853 Therefore, small molecule drugs targeting organism.867 The SASP factor IL-6 inhibits osteosarcoma formation
microbiota have become a hot research topic in antitumor by inducing and enhancing senescence.868 Precancerous lesions
therapy.854 Known oncogenic gut microbiota include Salmonella in RAS-driven pancreatic cancer are accompanied by extensive
typhi855 and Helicobacter spp856 in biliary tract cancer, Helicobacter senescence and SASP.869 On the other hand, some SASP factors
pylori857 in gastric cancer, etc. Helicobacter pylori has been are tumorigenic. Studies have revealed that SASP factors promote
identified as a class I carcinogen by the World Health Organiza- tumorigenesis due to paracrine mitogenic or metastatic effects on
tion, and it is associated with gastric cancer and mucosa- other premalignant cells, as well as interactions with surrounding
associated lymphoid tissue.858 CRC is a classic case of dysregula- endothelial cells, stromal cells, and tissues.867 Senescent cells and
tion of the gut microbiota that promotes cancer development.859 SASP factors favor the promotion of cell transformation, metas-
Certain microbiota species can ultimately exert a pro-carcinogenic tasis, and tumor growth. Specific PTEN deficiency in mouse
effect by stimulating inflammatory states, including the induction prostate cancer tissues leads to precancerous lesion development
of proinflammatory toxins, the increase of ROS production,860 the with extensive senescence and SASP.867,870 In general, SASP
aberration of signaling pathways,861 and the blockage of factors regulated by NF-kB have tumor-suppressive, immunosur-
antitumor immune function.858 It has been confirmed that F. veillance effects, while SASP factors regulated by signal transducer
nucleatum is crucial in the progression of CRC,861 which was and activator of transcription 3 (STAT3) have tumor-promoting
detected in lymph nodes and distant metastasis samples from and immunosuppressive effects.867
patients.858 Peptostreptococcus anaerobius is more enriched in
stool samples from CRC patients, and its ability to induce Lamin B1: Lamins are intermediate filament proteins that line
phosphorylation of adherent spot kinase in CRC cells activates the inner surface of the nuclear envelop which contribute to the
NF-κB signaling, ultimately promoting the cause of chronic size, shape, and stability of the nucleus.871 Nuclear lamins are type
inflammation and tumor progression. In addition, microbiota in V intermediate filaments ranging in size from 60 to 80 kDa. Lamins
other sites still play carcinogenic roles. Oral micro-common are divided into A type (lamin A, C) or B type (lamin B1, B2)
pathogenic bacteria including Streptococcus anginosus, Veillonella, according to isoelectric points. Lamin regulates nuclear and

Zhou et al.
27
cytoskeletal organization, mechanical stability, chromatin organi- resistance. ctDNA analysis identifies CRC resistance to HER2-
zation, gene regulation, genomic stability, differentiation, and targeted therapy. The NCCN Guidelines for Gastric Cancer (version
tissue-specific functions by binding to a variety of nuclear protein 2.2020) and the Esophageal and Esophagogastric Junction Cancer
complexes.871 Lamin B1 is essential for the regulation of normal Guidelines (version 2.2020) recommend the use of plasma ctDNA
organogenesis and organism survival.872 Lamin B1 knockdown analysis to detect drug sensitivity in patient treatment.884
triggers the formation of H3K27me3-enriched mesas and DNA Osimertinib resistance in NSCLC patients with EGFR mutation
hypomethylation regions overlapping with lamin B1-associated can be detected by plasma ctDNA analysis.889 Prostate cancer
domains in cancer and accelerates the replicative and oncogene- plasma ctDNA is used to detect BRCA reversion which mediates
induced senescence. Reduced lamin B1 expression has been PARPi treatment resistance.890 In summary, ctDNA is widely used
discovered in multiple senescent cells, and its overexpression in patients with advanced solid tumors for the detection of MRD,
delays senescence.873,874 Silencing lamin B1 expression slows cell the monitor of early recurrence, the prediction of treatment
proliferation and induces premature senescence in WI-38 cells.873 response, and drug resistance monitoring.58,888
Oncogenic Ras-induced premature senescence also reduces lamin
B1 expression through a pRb-dependent mechanism. In addition, CTCs. In 1869, Thomas Ashworth, an Australian physician, first
senescence is induced by DNA damage, replication failure, or identified CTCs, a type of cells shed into the bloodstream from
oncogene expression when lamin B1 is lost in human and mouse primary or metastatic tumor sites.891 CTCs are cancer cells isolated
primary cells. Lamin B1 loss is not dependent on p38-MAPK, NF- from the primary tumor site and transported via the circulation to
κB, or ROS signaling pathways which are positive regulators of distant organs.892 CTC characteristics are defined as a nucleated
senescence phenotypes.872 circulating cell larger than 4 μm expressing the epithelial cell
On the other hand, Lamin B1 upregulation is widely observed in protein EpCAM and cytokeratin 8, 18, and 19, while not expressing
tumor tissues of most cancer types. A high level of lamin B1 the leukocyte-specific antigen CD45.893 This is the main basis and
expression predicts poor OS and DFS for cancer patients.875 Lamin foundation of CTC testing.891
B1 is overexpressed and facilitates cell proliferation and metastasis CTCs are clinically significant as biomarkers for the clinical
in HCC, and increased lamin B1 expression indicates a dismal management of patients with metastatic cancer and the
prognosis and immunotherapy response in HCC.876 Besides, lamin implementation of precision medicine.894 More than 400 clinical
B1 has been proposed as a prognostic senescence biomarker in trial studies have been conducted using CTCs as biomarkers. The
ccRCC877 and lung adenocarcinoma.878 To summarize, lamin B1 key research contents and objectives are assessing prognostic
loss is a senescence-associated biomarker. information, the risk of recurrence and metastasis, stratifying and
monitoring treatment in real-time, and identifying therapeutic
Liquid biopsy tumor biomarkers targets and resistance mechanisms of cancer patients.895 CTC
Liquid biopsies have become a pivotal strategy for cancer assays and molecular characterization have been applied to
diagnosis, real-time monitoring, and prognosis through minimally stratify patients with breast cancer, CRC, prostate cancer, lung
invasive detection of biofluids, such as blood, saliva, urine, pleural cancer, pancreatic cancer, glioblastoma multiforme, and mela-
fluid, and ascites.879 Liquid biopsy tumor diagnostic biomarkers, noma, and to monitor disease progression.892 Studies have
including circulating tumor DNA, circulating tumor cells, and discovered that CTC count is a prognostic indicator for multiple
exosomes, are all effective monitoring tools for tumor diagnosis myeloma.896 The application of CTCs in the clinical assessment of
and treatment. gastric cancer has shown that CTCs are correlated with metastasis,
poor prognosis, recurrence, and treatment response in gastric
ctDNA. Cancer cells release naked DNA molecules into the cancer patients.891 The comprehensive analyses of CTCs in
circulation known as ctDNA which has become an essential metastatic breast cancer confirmed heterogeneous mechanisms
biomarker for liquid biopsies to predict response to targeted of patient resistance to targeted therapies.897 CTCs can monitor
therapies and immunotherapies to guide clinical anticancer therapeutic response and be utilized as a screening tool for brain
treatment.880 micro-metastasis detection in breast cancer.898 In addition, CTCs
ctDNA consists of small nucleic acid fragments that are not have important clinical utility in the selection of therapeutic-
associated with cells or cellular fragments.881 Plasma ctDNA refers specific biomarkers for the treatment of patients with prostate
to tumor-derived DNA fragments and was detected in human cancer. The response of prostate cancer to anticancer drugs is
plasma in 1948, and includes plasma cfDNA, circulating DNA strongly correlated with the expression of AR-V7, a treatment-
derived from the death of hematopoietic cells.882,883 A study has specific biomarker, in the CTC nuclei.899 The CTCs in the NSCLC
revealed that ctDNA is detected in >75% of patients with pulmonary vein are independent predictors of NSCL recurrence
advanced pancreatic cancer, ovarian cancer, CRC, bladder cancer, after surgery.900 In addition, CTC analysis captures the tumor
gastroesophageal cancer, breast cancer, melanoma, HCC, and heterogeneity noninvasively and real-timely in cancer patients,
head and neck cancer, and in 50% of patients with primary brain which effectively explains the existence of heterogeneous drug
cancer, kidney cancer, prostate cancer, and thyroid cancer.881 As resistance mechanisms in patients with refractory tumors901 and
the plasma ctDNA content is less than 0.01%884 and its half-life is promotes the development of precise targeting strategies.901 In
very short, plasma ctDNA levels allow for real-time dynamic conclusion, CTC analysis is a clinically relevant noninvasive tool to
assessment of tumor evolution through serial sampling, and monitor the progression and prognosis of cancers, and to deeply
represent intrapatient and interpatient variability to guide clinical explore the biology of metastatic cancers, and has the potential to
drug use. In addition, plasma ctDNA captures tumor heterogeneity facilitate personalized precision treatment of cancers. Of course,
and effectively reflects DNA shed from multiple metastatic sites.884 the difficulties undoubtedly need to be solved in the future,
The NCCN Guidelines for Breast Cancer (version 4.2020) recom- including that the efficiency of CTC targeting approaches varies by
mend the use of ctDNA analysis for the evaluation of PIK3CA cancer types, and the large patient cohorts are needed to flesh out
mutations in breast cancer.884,885 the arguments in CTC application.894
Moreover, ctDNA analysis is used for clinical real-time monitor-
ing of treatment response in a variety of tumors, including breast Exosomes. Exosomes, first discovered in 1983 by Pan and
cancer,886 NSCLC,887 prostate cancer, gastroesophageal cancer,884 Johnstone,902 are extracellular lipid bilayer vesicles of endosomal
HCC,50 and CRC.888 Plasma ctDNA analysis has been applied to origin with an average size range of approximately 100 nm in
monitor clinical cancer therapy resistance. Metastatic CRC is the diameter.903 The process of exosome biogenesis is well defined,
first disease that utilizes liquid biopsy to evaluate treatment starting with double invagination of the plasma membrane and

Zhou et al.
28
the formation of intracellular multivesicular bodies containing ascites, tears, and breast milk.905 Therefore, it is easy and
intraluminal vesicles, followed by exocytosis of multivesicular convenient to collect samples. Second, exosomes can reveal
bodies fused to the plasma membrane, and the intraluminal specific proteins from parental cells and target cells, which isolates
vesicles are finally secreted as exosomes with a diameter of the origin-specific exosomes and predict organ-specific metasta-
~40–150 nm.904 Exosomes released from the cell surface can fuse sis.117 Third, the concentration of exosomes is higher than that of
with the plasma membrane of recipient cells, thereby transporting other liquid biopsy biomarkers, such as CTCs, thereby reducing the
their contents into the cytoplasm. In addition, proteins on the amount of sample collection. Fourth, exosomes are highly stable
surface of exosomes can bind to the surface receptors of recipient compared with other liquid biopsy biomarkers, such as ctDNA,
cells to promote intracellular signaling.905 The exosomes are which is rapidly degraded in the blood.926 In conclusion,
highly heterogeneous given their size, content, functional impact exosomes are valuable biomarkers for the early diagnosis,
on recipient cells, and cellular complexity of origin.903 Among prognosis prediction, and the therapeutic efficacy assessment of
them, the content of exosomes is a key factor in the execution of cancers. The possibility of combining protein, lipid, RNA, and
functions. Recent advances have revealed that the contents of miRNA exosome cargos in cancer may enhance the diagnostic and
exosomes include proteins, DNA, mRNA, microRNA, long noncod- prognostic potential of exosomes, which is being considered.
ing RNA, circular RNA, and other components.906
Exosomes have been demonstrated to be involved in cancer
development, angiogenesis, metastasis, and therapeutic resis- TUMOR BIOMARKER-BASED CANCER THERAPY
tance, and can be used as diagnostic and prognostic biomarkers Optimizing the precise medical care of patients according to
for tumor patients.906 For example, exosomes are useful in liquid genetic and molecular characteristics can maximize the benefits of
biopsy to diagnose various cancers, including lung cancer,907 precision medicine. Therefore, discovering and developing tumor
pancreatic cancer,908 gastric cancer,909 prostate cancer, breast biomarkers and related cancer therapies contribute greatly to
cancer, ovarian cancer, glioblastoma, and melanoma.905 The effective precision medicine. Compared with traditional che-
exosomal DNA in serum exosomes has been proven to be of motherapy drugs, targeted cancer therapy including small-
significant value in detecting cancer-related mutations, such as molecule targeted drugs, monoclonal antibodies (mAbs),
KRAS and TP53.910,911 The specific miRNAs that are differentially antibody-drug conjugates, and the proportion of biological drugs
expressed in exosomes between cancer cells and normal cells specifically targeting proteins or genes in cancer cells, results in
have important diagnostic or prognostic value in the early high potency and low toxicity. During the past 30 years,
detection of cancers.912 Studies have revealed that elevated remarkable progress has been achieved in this field. Targeted
serum-derived exosomal miR-21 is associated with multiple therapy has been the mainstream strategy for cancer therapy, and
cancers, including pancreatic cancer, ovarian cancer, and lung targeted drugs approved by the FDA, European Medicines
cancer.913 The tumor suppressor miRNAs, such as miR-146a and Agency, Ministry of Health Labour and Welfare and National
miR-34a, are found in exosomes, and their low levels are Medical Products Administration for cancer treatment have
correlated with poor prognosis of liver cancer, breast cancer, increased accordingly.927,928 Here, we summarize the pivotal
CRC, pancreatic cancer, and hematologic malignancies.903,912 The tumor biomarker-based cancer therapies in preclinical and clinical
upregulation of other exosomal oncogenic microRNAs, such as studies in recent years and hope to provide comprehensive
miR-155, miR-17-92, and miR-1246, have also been shown to be insights into cancer therapy.
connected to the progression of multiple cancers.903,912,914
Moreover, as the exosomal cargo exchange between cancer Targeting tumor cell proliferative signaling
cells and stromal cells in the TME, exosomes from multiple cancer There is an increasing number of inhibitors related to tumor
cells can effectively regulate TME angiogenesis and extracellular growth signaling pathways, including inhibitors of RAS, PI3K-AKT-
matrix remodeling at metastatic sites, thereby enhancing tumor mTOR, and RAF-MEK-ERK signaling pathways. In particular, multi-
growth and metastasis.903,904 Breast cancer-derived exosomes ple protein kinases have engaged in cell proliferation, and
have been demonstrated to impair vascular integrity and enhance targeted drugs have rapidly developed and been applied for
vascular permeability, thereby promoting breast cancer metasta- clinical use since the approval of the first small-molecule TKI,
sis.915 Exosomes from glioblastoma cells promote tumor cell imatinib, by the FDA in 2001.929 Although their targets and
proliferation and induce angiogenesis.916 Ovarian cancer cell mechanisms of action are different, all of them can effectively
exosomes are involved in promoting their peritoneal dissemina- inhibit tumor cell proliferation.
tion.917 Moreover, cancer cell-derived exosomes also facilitate
metastasis in melanoma,918 pancreatic cancer,919 gastric cancer, RAS inhibitors. Since the discovery of RAS in the rat sarcoma
etc.920,921 Exosomes from different cellular sources, such as virus, targeting RAS has attracted attention because of its vital role
immune cells, cancer cells, epithelial cells, and mesenchymal cells, in tumorigenesis and progression. However, the development of
can influence the activity of recipient cells of both the innate and RAS targeting therapy is extremely challenging.930 The RAS
adaptive immune system. Exosomes stimulate or inhibit the protein surface is very smooth, and the lack of drug-binding
function of CCD4+ and CD8+ T cells.903 pockets makes targeting RAS problematic.931 The existence of
In addition, exosomes secreted by cancer cells can benefit cell high intracellular GTP concentrations, the activation of compensa-
survival by interacting directly with drugs and reducing their tion pathway, and drug-resistant mutations in RAS-RAF-ERK
antitumor efficacy, or by regulating cancer cell gene expression pathway genes after RAS inhibitors administration, lead to poor
through TEM cell-derived exosomes. CAF-derived exosomes inhibitory effects of RAS inhibitors.319,932 In recent years, due to
stimulate chemotherapy resistance in CRC922 and breast cancer,923 the consecutive failure in the discovery of RAS inhibitors, RAS was
and promote resistance of ovarian cancer cells to paclitaxel.924 once considered an undruggable target. Significantly, Shokat and
Moreover, macrophage-derived exosomes induce resistance to colleagues opened a new chapter in RAS-targeted therapy by
gemcitabine in pancreatic cancer cells.925 Thus, exosomes discovering a binding pocket containing the mutant cysteine
secreted by cancer cells can induce resistance to chemother- residue in KRASG12C in 2013, which prompted the fast develop-
apeutic drugs.903 ment of the first small-molecule KRASG12C inhibitor sotorasib.319
Exosomes have numerous advantages as liquid biopsy diag- Subsequently, due to the importance of RAS in tumorigenesis and
nostic biomarkers for cancer. First, exosomes can be secreted by progression, alternative strategies such as targeting RAS muta-
all types of cells and are present in various biological fluids, such tions, and upstream and downstream effectors have been
as blood, urine, semen, saliva, amniotic fluid, cerebrospinal fluid, attempted. For example, drug exploitation in targeting RAS

Zhou et al.
29
mutated malignancies can be accomplished using various mainly influenced by the KRAS status in the cells. The cancer cells
strategies (Table 1), including interfering with RAS maturation carrying KRAS mutations can be divided into two categories,
and transport, promoting its localization to the plasma membrane, KRAS-dependent and KRAS-independent,944 the latter of which
and inhibiting its downstream signaling.933 To date, inhibitors may be less sensitive to KRAS inhibitors. Similarly, KRAS knock-
against KRASG12C have been approved for clinical use.308 Mean- down PDAC cells are able to maintain cell proliferation through
while, RAS inhibitor combination options are increasingly devel- PI3K alternative bypass pathway.931,945 The mechanisms of
oped, i.e., combination with inhibitors that inhibit the RAS acquired resistance can be broadly classified into three categories:
signaling pathway, maintain the GDP-binding status of KRAS (1) KRAS alterations: mutations in the KRAS Y96, R68, and H95
proteins, and modulate the immune system.319 residues. KRASY96D mutation can affect the binding pocket of
inhibitors to KRAS protein switch-II.931,946 KRAS G12D/V/R, G13D,
KRASG12C mutation inhibitors. Extensive studies on the structure and Q61H mutations similarly cause resistance to KRAS G12C.
and biofunction of KRAS mutations have shed light on the Certain tumor cells produce new KRASG12C mutation after inhibitor
development of drugs targeting KRAS mutations. In particular, the treatment which maintains KRAS active and results in drug
development of KRAS mutation inhibitors has been encouraged resistance.931 (2) Altered vertical signaling cascades: changes in
by the approval of the first small-molecule KRASG12C inhibitor upstream signaling pathways affecting KRAS-GTP binding affinity,
sotorasib (AMG510) for the treatment of KRASG12C mutant NSCLC downstream signaling pathways, such as MEK-ERK947 and EGFR,947
patients by the FAD in 2021.934,935 Sotorasib is an FDA-approved and other upregulation can all induce drug resistance.931 (3)
KRASG12C-specific covalent inhibitor that irreversibly binds to the Changes in the TME such as immune escape pathways produce
GDP-binding inactive conformation of KRASG12C, thus blocking its resistance to KRASG12C inhibition931,948 Therefore, the combination
activity.931 Clinical trials have shown that sotorasib exerts antic- of KRASG12C inhibitors with other drugs is considered an
ancer activity in advanced solid tumor patients with KRASG12C efficacious way to improve their antitumor effect. The results of
mutations, including NSCLC,936 CRC, pancreatic cancer, endome- clinical trials have proven that KRASG12C inhibitor combination
trial carcinoma, appendiceal cancer, and melanoma.937 The first strategies are well tolerated with no serious adverse effects in
randomized phase III trial of targeting the KRASG12C inhibitor has most patients. Of note, many clinical trials of the KRASG12C
revealed that sotorasib significantly increases PFS in NSCLC inhibitor combination are currently underway for the treatment of
patients with KRASG12C mutation.938 Meanwhile, phase II clinical NSCLC and other solid tumors.931
trials of sotorasib used for the treatment of previously treated In summary, KRASG12C inhibitors have promising therapeutic
locally advanced or metastatic NSCLC subjects with KRASG12C prospects for both monotherapy and combination use in cancer
mutation or comorbidities are ongoing (NCT05631249 and treatment. Given that KRAS inhibitor resistance is still a serious
NCT05311709). However, the objective response rate to sotorasib challenge in clinical practice, it remains to be elucidated in deeper
monotherapy is still far from satisfactory. A phase II clinical trial has detail to achieve precise therapies for cancer patients.
shown that the objective response rate to sotorasib monotherapy
in patients with advanced KRASG12C-mutated CRC is only 9.7%, Non-KRASG12C mutation inhibitors. The promising advances in
indicating that its combination treatment strategy for KRASG12C- KRASG12C inhibitors have brought light to target other KRAS
mutated CRC needs to be further evaluated.939 Following the mutations. The KRASG12C mutation only represents a small part of
successful development of sotorasib, adagrasib (MRTX849), an the mutations, and other mutations include G12D, G12V, G12S,
irreversible KRASG12C inhibitor received its first approval by the and G12R.931 MRTX1133 is a selective noncovalent KRASG12D
FDA for the treatment of advanced or metastatic NSCLC patients inhibitor949 with a high affinity for KRASG12D and effectively
with KRASG12C mutation in December 2022. Adagrasib has a inhibits the tumor growth of PDAC xenograft mouse models in
favorable pharmacokinetic profile, such as a long half-life (~24 h), vivo.950 RMC-9805 is a selective, orally bioavailable covalent
broad tissue distribution, and dose-dependent pharmacoki- KRASG12D inhibitor that effectively blocks the growth of KRASG12D
netics.940 Notably, adagrasib penetrates the cerebrospinal fluid mutant cancer cells in vitro and in vivo.931 In addition, RMC-6236 is
and causes the regression of lesions in patients with KRASG12C a pan-RAS inhibitor that inhibits all RAS mutant subtypes,931 and is
mutant NSCLC brain metastases.941 Clinical trials have demon- currently in phase I clinical trial (NCT5379985) in advanced solid
strated that adagrasib monotherapy is well tolerated with a tumors with KRAS mutations. However, due to the lack of active
disease control rate of 87% in 46 patients, and adagrasib in residues and intrinsic hydrolytic activity in these mutated proteins,
combination with cetuximab has shown clinical activity in patients the development of highly potent inhibitors against the above
with KRASG12C-mutated CRC. Moreover, adagrasib in combination KRAS mutations remains a big challenge.931
with cetuximab is currently in a phase III clinical trial The fast development of the above inhibitors targeting KRAS
(NCT04793958) in patients with KRASG12C mutant CRC.942 The mutations may take advantage of the “addiction” of tumors to
approval of sotorasib and adagrasib has opened the door to the mutant RAS and create more effective regimens for more patients
possibility of developing more effective RASG12C inhibitors. ARS- such as pancreatic cancer patients. The in-depth study of KRAS-
853 is a highly cell-active, KRASG12C mutant-specific covalent specific mechanisms by which they enhance or hinder cancer
inhibitor that targets the GDP-bound inactive state of KRAS and proliferation will provide new insights for subsequent inhibitor
prevents its activation, resulting in the abrogation of KRAS development.
mutation-induced signaling.930 ARS-853 is the first direct targeting
KRAS inhibitor by covalently reacting with the RAS-GDP complex Other RAS targeting strategies. Existing KRASG12C inhibitors have
to trap it in its inactive state.930 Other KRASG12C mutation narrow therapeutic windows and are only effective in a small
inhibitors such as JNJ-74699157 (ARS-3248), divarasib (GDC- proportion of cancer patients with KRAS mutation. Many studies
6036), garsorasib (D-1553), JDQ443, and RMC-6291 are under- are currently dedicated to discovering alternative strategies for
going different phases of clinical trials in cancer patients (Table targeting RAS. Pan-RAS inhibitors broaden the therapeutic
1).Although extensive development of KRASG12C inhibitors is windows by directly targeting multiple RAS-mutated cancers.951
ongoing, they only work in a small fraction of patients with The pan-RAS inhibitor RAS-IN-3144 can block the growth of
KRASG12C mutation, and the median PFS is fairly short, less than 1 KRASG13D mutant MDA-MB-231 cell-derived mouse xenograft
year. Thus, an in-depth study of the resistance mechanism of tumors in vivo.952 A synthetic sos protein mimic has been found
KRASG12C mutation inhibitors is urgently needed. To date, KRAS to suppress RAS activation as a pan-RAS inhibitor.953 Moreover,
inhibitor resistance mechanisms are distinguished into intrinsic rigosertib is a RAS analog that inhibits RAS-mediated pancreatic
resistance and acquired resistance.931,943 Intrinsic resistance is cancer growth by interacting with the RAS-binding domain of RAF

30
Table 1. The FDA-approved and clinically developed RAS inhibitors in cancer therapies
Target Drug Highest Phase Indications Company/Identifier Status
Farnesyltr-ansferase Tipifarnib Approved Head and neck squamous cell carcinoma harboring HRAS mutations who have Kura Oncology /
progressed following platinum-containing chemotherapy
Lonafarnib (SCH66336) Approved Hutchinson-Gilford progeria syndrome and progeroid laminopathies Merck & Co /
III Carcinoma, non-small cell lung, metastases, neoplasm NCT00050336 Terminated
III Myelodysplastic syndromes, leukemia, myelomonocytic, chronic, myelodysplasia, NCT00109538 Terminated
myelomonocytic
Salirasib II Non-small cell lung cancer NCT00531401 Completed
SOS BI-1701963 II Advanced solid tumors, KRASG12C mutation NCT04185883 Recruiting
MRTX0902 I/II Solid tumor, advanced solid tumor, non-small cell lung cancer, colorectal cancer NCT05578092 Recruiting
SHP2 RMC-4630 II Non-small cell lung cancer NCT05054725 Active, not
recruiting
I/II Solid tumor NCT03989115 Completed
I/II Advanced solid tumors, KRASG12C mutation NCT04185883 Recruiting
I/II Metastatic neoplasm NCT04418661 Active, not
recruiting
TNO155 I/II KRASG12C mutant solid tumors, non-small cell lung, carcinoma, colorectal, lung NCT04699188 Recruiting
cancer, pulmonary cancer
I/II Advanced cancer, metastatic cancer, malignant neoplastic disease NCT04330664 Active, not
Zhou et al.
recruiting
I/II Advanced solid tumors, KRASG12C mutation NCT04185883 Recruiting
RLY-1971 I Advanced solid tumors, metastatic solid tumors NCT05487235 Recruiting
I Solid tumor, unspecified, adult NCT04252339 Completed
I Colorectal cancer, non-small cell lung cancer NCT05954871 Recruiting
KRASG12C Adagrasib (MRTX849) Approved Solid tumors harboring KRASG12C oncogenic driver mutation, including non-small Mirati Therapeutics /
cell lung cancer and colorectal cancer
Sotorasib (AMG510) Approved KRASG12C-mutated locally advanced or metastatic non-small cell lung cancer Amgen /
JNJ-74699157 (ARS- I Neoplasms, advanced solid tumors, non-small cell lung cancer, colorectal cancer NCT04006301 Completed
3248)
Divarasib (GDC-6036) III Non-small cell lung cancer NCT03178552 Recruiting
D-1553 II Non-small cell lung cancer NCT05383898 Recruiting
II Non-small cell lung cancer NCT05492045 Recruiting
II Solid tumor, non-small cell lung cancer, colorectal cancer NCT04585035 Recruiting
II Solid tumor NCT05379946 Not yet recruiting
JDQ443 III Non-small cell lung cancer NCT05132075 Recruiting
RMC-6291 I Non-small cell lung cancer, colorectal cancer, pancreatic ductal adenocarcinoma, NCT05462717 Recruiting
advanced solid tumor
Non-KRASG12C MRTX1133 I/II Solid tumor, advanced solid tumor, non-small cell lung cancer, colorectal cancer, NCT05737706 Recruiting

pancreatic adenocarcinoma
RMC-6236 I Non-small cell lung cancer, colorectal cancer, pancreatic ductal adenocarcinoma, NCT05379985 Recruiting
advanced solid tumors
siRNA strategies KRASG12D iExosomes I KRASG12D metastatic pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma NCT03608631 Recruiting
Antisense oligonucleotide AZD4785 I Non-small cell lung cancer, advanced solid tumors NCT03101839 Completed
Zhou et al.
31
kinase, resulting in the inability of RAF.954 Additionally, oligonu-
cleotides can inhibit protein synthesis by boosting mRNA
degradation or interfering with translation.306 AZD4785 is an
antisense oligonucleotide that targets KRAS mRNA with high
Completed
Completed
Completed
Completed
Recruiting
affinity. It inhibits downstream signaling pathways by depleting
Company/Identifier Status
cellular KRAS mRNA and proteins, thus exerting antitumor

effects.955 A phase I clinical trial of AZD4785 (NCT03101839) in
patients with KRAS mutated solid tumors has been completed, but
no results have been posted. In addition, KRAS dimerization is
essential for downstream signaling when KRAS is localized at the
NCT01360853
NCT01928537
NCT01241500
NCT03948763
NCT05735717
plasma membrane.951 NS1, a synthetic binding protein antibody,
interferes with RAS dimerization and blocks CRAF-BRAF hetero-
Source: All the information is derived from ClinicalTrials.gov (https://www.clinicaltrials.gov) and the United States Food and Drug Administration.gov (https://www.fda.gov/)
dimerization and activation.951,956 Recently, proteolysis-targeting
chimeras (PROTACs) technology can directly degrade targeted
proteins,308 which have been used to specifically degrade KRAS in
cancer cells.957 In addition, CRISPR/Cas9 screening is also applied
in the identification of RAS synthetic lethal gene.958 In addition,
fibroblasts-derived exosomes loaded with G12D siRNA (iExo-
somes) can effectively restrain PDAC tumor growth.959,960
Mesenchymal stromal cells-derived exosomes with KRAS are
currently in a phase I clinical trial (NCT03608631) evaluating
efficacy in patients with pancreatic cancer. A phase I clinical trial
Myelodysplastic syndromes, chronic myelomonocytic leukemia
Myelodysplastic syndromes, chronic myelomonocytic leukemia
(NCT03948763) of the KRAS mRNA vaccine V941 (mRNA-5671) in

KRAS-mutated NSCLC, pancreatic cancer, and CRC has been
completed, but no results have been posted.
Targeting upstream of RAS: Targeting RAS plasma membrane

localization. Only plasma membrane localization of RAS proteins
can stimulate downstream effectors and signaling pathways. If
post-translational modification and membrane localization are
blocked, RAS proteins will be inactive, suggesting that the
Metastatic pancreatic adenocarcinoma
inhibition of RAS membrane localization is an effective therapeutic

strategy.951
Farnesyltransferase plays a vital role in RAS localization. The
farnesyltransferase inhibitors (FTIs) can prevent RAS localization in
Non-small cell lung cancer
the plasma membrane by suppressing farnesyltransferase, leading

Hematologic malignancy,
acute myeloid leukemia,
to the blockade of downstream signaling.931 Tipifarnib, an orally

bioavailable, nonpeptidomimetic quinolinone FTI,961 was granted
acute lymphoblastic
pancreatic cancer,
colorectal cancer
a fast track designation by the FDA in December 2019 for the

acute leukemia,
treatment of patients with HNSCC harboring HRAS mutations who

have progressed following platinum-containing chemotherapy.
Highest Phase Indications
The objective response rate of tipifarnib in a phase II clinical study

of HRAS-mutated recurrent and/or metastatic HNSCC was 55%,
with common adverse effects of anemia and lymphocyte
reduction.962 Subsequently, tipifarnib was given fast track status
again by the FDA for the treatment of adult patients with different
subtypes of peripheral T-cell lymphoma. Moreover, tipifarnib has
been demonstrated to be effective in advanced refractory
uroepithelial carcinoma with HRAS mutation,963 in recurrent and
III
III
III
metastatic salivary gland carcinoma with HRAS mutation,964 and in

II
I
HRAS-driven dedifferentiated thyroid cancers.965 Lonafarnib,

originally discovered by Merck & Co as an investigational oncology
drug966 and known as the world’s first drug approved by the FDA
in November 2020 for the treatment of progeria and progeroid
mRNA-5671
laminopathies, is also an orally active FTI that blockades RAS

Rigosertib
localization in the plasma membrane. Salirasib is a farnesylcysteine

mimetic that blocks the function of all RAS isoforms (H-RAS, K-RAS,
Drug
NS1
and N-RAS) by interfering with RAS binding to the plasma

membrane.967 Numerous preclinical studies have shown the
ability of salirasib to inhibit the proliferation of various human
cancer cells,967 including breast cancer,968 glioblastoma,969,970
Table 1. continued
CRC,971 melanoma,972 ovarian cancer,973 HCC,974 and pancreatic

Cancer vaccines
cancer.975 However, a phase II clinical study revealed poor

therapeutic activity of salirasib in KRAS-mutant lung cancer,976
RAS analog
and its development in subsequent clinical trials has been

Antibody
discontinued.931
Target
Phosphodiesterase-δ phosphodiesterase-δ (PDEδ) is a prenyl-

binding protein involved in regulating the membrane localization

Zhou et al.
32
and signaling of farnesylated RAS. PDEδ binds and solubilizes protein tyrosine phosphatase structural domain. SHP099 effec-
farnesylated RAS proteins to enhance their diffusion in the tively suppressed tumor cell proliferation in vivo and in vitro by
cytoplasm and transfers RAS from the Golgi apparatus and blocking RAS-ERK signaling.990 However, SHP2 inhibitor-based
endomembranes to the plasma membrane, thereby facilitating development has come a long way in the past few years. Earlier
RAS enrichment at the plasma membrane and signaling developed SHP2 inhibitors targeting the catalytic site failed in
transduction.977–979 Deltarasin inhibits KRAS-dependent prolifera- clinical practice due to poor selectivity and low bioavailability.
tion of human PDAC cells by competitively binding the farnesyl- Therefore, the development of SHP2 metathesis inhibitors has
binding pocket of PDEδ and reducing RAS enrichment at the become an important research direction. Moreover, SHP2
plasma membrane.978 Deltazinone has a similar mode of action to inhibitors have a wide scope in drug combinations. In addition,
deltarasin, but possesses a higher selectivity and lower cytotoxi- due to the extensive expression of SHP2, a strategy that induces
city.979 However, the fast release of KRAS-PDEδ inhibitors from toxic effects and increases the safety of SHP2 inhibitors on normal
PDEδ hindered drug-binding affinity, which resulted in poor cells is a primary problem to be solved.
antiproliferative activity of PDEδ inhibitors.980 Novel strategies,
such as PDEδ degraders are underway.980 Targeting RAS downstream effectors: The most classical down-
SOS1 inhibitors. Son of sevenless homolog 1 (SOS1), a guanine stream effector pathways of RAS are the RAF-MEK-ERK cascade
nucleotide exchange factor (GEF), catalytically promotes the pathway and the PI3K-AKT-mTOR pathway. A large number of
activation of RAS which in turn consecutively enhances the GEF inhibitors targeting the RAF-MEK-ERK and PI3K-AKT-mTOR path-
function of SOS1.306 Targeting SOS1 to disturb its interaction with ways have been developed and are under clinical evaluation.958
KRAS in tumors is referred to as an effective way to inhibit a wide PI3K-AKT-mTOR pathway inhibitors. Dysregulation of the PI3K-
panel of KRAS-driven cancers.930,981,982 Multiple SOS1 inhibitors AKT-mTOR pathway is critical to the oncogenesis and progression
have been developed, mainly including quinazoline-based SOS1 of many human tumors, and their inhibitor development is of
inhibitors such as BAY-293 and BI-3406. BAY-293, first reported by great significance991 (Table 2). However, the PI3K-AKT-mTOR
Bayer and identified by combining high throughput screening and pathway inhibitors have various resistance mechanisms. For
fragment screening in 2019, is a selective SOS1 inhibitor that example, the treatment of PI3K pathway inhibitors can result in
suppresses the KRAS-SOS1 interaction at an IC50 value of 21 the feedback activation of upstream signaling pathways, thus
nM.983,984 BI-3406, an orally selective SOS1 inhibitor with quinazo- limiting their efficacy992
line structure developed by Boehringer Ingelheim, can bind to the PI3K inhibitors. Numerous PI3K inhibitors have been developed
SOS1 catalytic domain, reduce GTP-RAS formation, and impair and can be divided into three major classes: pan-PI3K inhibitors,
MEK inhibitor-induced feedback activation, eventually inhibiting isoform-specific PI3K inhibitors, and dual PI3K/mTOR inhibitors
KRAS-driven cancer cell proliferation.985 The most advanced SOS1 (Table 2). Most PI3K inhibitors are currently in clinical trials, and
inhibitor is BI-1701963, an analog of BI-3406, which has some of them (copanlisib, alpeilisib, idelalisib, duvelisib, and
demonstrated safety in clinical phase I trials and is under phase umbralisib) are approved by the FDA. However, the approvals/
I clinical trial as monotherapy and in combination with trametinib accelerated applications of some PI3K inhibitors, such as idelalisi,
in patients with KRAS mutated advanced or metastatic solid duvelisab, and umbrailisib, have been withdrawn due to frequent
tumors (NCT04111458).931 MRTX0902 is a selective, orally bioavail- and severe adverse effects.993
able SOS1 inhibitor with antitumor effects in combination with Pan-PI3K inhibitors simultaneously inhibit the four catalytic
MRTX849 in KRASG12C mutant NSCLC,931 and a phase I/II study of subunits of class I PI3K p110α (PIK3CA), p110β (PIK3CB), p110γ
MRTX0902 in solid tumors with mutations in the KRAS MAPK (PIK3CG), and p110δ (PIK3CD). LY294002 and wortmannin are the
pathway is ongoing (NCT05578092). In addition to the above first generation of pan-PI3K inhibitors, which build the foundation
representative small-molecule agonists, ZZ151, a potent, coopera- for the exploitation of novel high-efficiency and low-toxicity PI3K
tive, and selective SOS1 PROTAC, has shown superior anticancer inhibitors. Unfortunately, the clinical applications of LY294002 and
activities in KRASG12D and KRASG12V mutant xenografts in mice, wortmannin are seriously limited by obvious adverse effects.
which is worth further optimization.986 However, there is no LY294002 has poor aqueous solubility and adverse effects
marketable SOS1 compound, and only two cases are in clinical including severe respiratory depression and lethargy. Wortmannin
studies, with a lack of publicly disclosed clinical data. Exploiting has similar poor pharmacological properties, including a short
highly selective and low-toxicity SOS1 inhibitors is a major half-life, chemical instability, and side effects, such as liver
research focus in the future. dysfunction and lymphocytopenia.993 Copanlisib (BAY80-6946) is
SHP2 inhibitors. SHP2, a nonreceptor protein tyrosine phos- an intravenous Pan-PI3K inhibitor developed by Bayer that inhibits
phatase encoded by the PTPN11 gene, promotes GEF-mediated α, β, γ, and δ isoforms with varying degrees of affinity,994 and
RAS-GTP interactions and activates the downstream RAS-RAF-ERK received accelerated FDA approval in 2017 for the treatment of
pathway.987,988 RMC-4630 is a selective orally bioavailable recurrent follicular lymphoma.993 Buparlisib (BKM120) is an oral
allosteric SHP2 inhibitor,988 which is undergoing a clinical phase ATP-competitive pan-PI3K inhibitor, being used for the treatment
I trial (NCT03634982) of monotherapy in participants with of stage II ESCC.995 The good brain penetration of buparlisib
advanced relapsed or refractory solid tumors. A clinical phase I makes it a promising drug for the treatment of intracranial
trial (NCT04916236) combining RMC-4630 and the ERK inhibitor tumors.996 Buparlisib is currently being evaluated in a clinical
LY3214996 for the treatment of KRAS-mutated cancer is ongoing. phase III trial (NCT04338399) in combination with paclitaxel for the
In addition, RMC-4630 and sotorasib are being used in combina- treatment of HNSCC. However, the lack of selectivity of pan-
tion in a clinical phase II trial (NCT05054725) to explore antitumor inhibitors results in nonselective inhibition of the PI3K pathway,
effects in patients with KRASG12C-mutated NSCLC. Similarly, leading to serious adverse side effects.
TNO155, a selectively orally bioavailable SHP2 inhibitor,989 is Researchers developed selective ATP-competitive inhibitors for
being used alone or in combination with EGF816 (nazartinib) in each isoform of PI3K to limit the emergence of toxic effects.
phase I clinical trial (NCT03114319) for advanced solid tumor Isoform-specific PI3K inhibitors selectively inhibit p110α, p110β,
treatment. Other SHP2 inhibitors are also being tested in clinical p110δ, or p110γ subunits with greatly improved off-target effects.
trials. A clinical phase I trial of RLY-1971 in patients with advanced Alpelisib (BYL719), an oral inhibitor targeting PI3Kα developed by
or metastatic solid tumors (NCT04252339) has been completed, Novartis, was approved by the FDA for ER+/HER2-advanced
but results have not yet been disclosed. SHP099 is a potent and metastatic breast cancer treatment in 2019.993 Inavolisib (GDC-
selective small molecule SHP2 inhibitor that inhibits SHP2 activity 0077) is also an oral inhibitor targeting PI3Kα with multiple
by binding to the N-terminal and C-terminal ends of SH2 and the ongoing clinical trials,997 including a phase III clinical trial

Table 2. The FDA-approved and clinically developed PI3K-AKT-mTOR inhibitors
Type Target Drug Highest Phase Indications Company/Identifier Status
PI3K Pan-PI3K Copanlisib (BAY80-6946) Approved Relapsed follicular lymphoma Bayer /

Buparlisib (BKM120) III Head and neck cancer NCT04338399 Recruiting
III Metastatic breast cancer NCT01633060 Terminated
III Breast cancer NCT01610284 Completed
SF1126 (prodrug of I Neuroblastoma NCT02337309 Terminated
LY294002)
p110α Alpelisib (BYL719) Approved ER + /HER2-advanced metastatic breast cancer Novartis Pharmaceuticals /
Inavolisib III Breast cancer NCT05646862 Recruiting
III Breast cancer NCT04191499 Recruiting
III Metastatic breast cancer NCT05894239 Recruiting
p110β AZD8186 I Advanced castrate-resistant prostate cancer, squamous non-small cell NCT01884285 Completed

lung cancer, triple-negative breast cancer
GSK2636771 II Melanoma and other malignant neoplasms of skin, metastatic melanoma NCT03131908 Active, not
recruiting
II Advanced lymphoma, NCT04439188 Active, not
advanced malignant solid neoplasm, hematopoietic and lymphoid cell recruiting
neoplasm, refractory lymphoma, refractory malignant solid neoplasm,
refractory multiple myeloma
II Advanced lymphoma, advanced malignant solid neoplasm, NCT04439149 Active, not
hematopoietic and lymphoid cell neoplasm, refractory lymphoma, recruiting
refractory malignant solid neoplasm, refractory multiple myeloma
I/II Advanced gastric adenocarcinoma NCT02615730 Completed
Zhou et al.
p110γ IPI-549 II Head and neck squamous cell carcinoma, head and neck cancer, head and NCT03795610 Recruiting
neck carcinoma, head and neck cancer stage IV, head and neck cancer
stage III, HPV-Related carcinoma, HPV-Related malignancy, HPV-Related
squamous cell carcinoma
II Bladder cancer, urothelial carcinoma, solid tumor, advanced cancer NCT03980041 Completed
II Breast cancer, renal cell carcinoma NCT03961698 Active, not
recruiting
p110δ Idelalisib Approved Relapsed chronic lymphocytic leukemia Gilead Sciences /
Duvelisib Approved Relapsed or refractory chronic lymphocytic leukemia, small lymphocytic Verastem /
lymphoma
Umbralisib Approved Relapsed or refractory marginal zone lymphoma TG Therapeutics /
Dual PI3K/ Omipalisib I Solid tumors NCT00972686 Completed
mTOR (GSK2126458)
AKT Capivasertib (AZD5363) Approved HR + , HER2- locally advanced or metastatic breast cancer with one or AstraZeneca /
more PIK3CA/AKT1/PTEN-alterations
Ipatasertib (GDC0068) III Breast cancer NCT04060862 Active, not
recruiting
GSK2141795 II Cervical cancer NCT01958112 Terminated
II Melanoma NCT01941927 Completed

II Recurrent adult acute myeloid leukemia, untreated adult acute myeloid NCT01907815 Terminated
leukemia
33
34
Table 2. continued
Type Target Drug Highest Phase Indications Company/Identifier Status
II Estrogen receptor negative, HER2/Neu negative, invasive breast NCT01964924 Completed

carcinoma, progesterone receptor negative, recurrent breast carcinoma,
stage IV breast cancer, triple-negative breast carcinoma
MK-2206 II Colorectal neoplasms NCT01333475 Completed
II Ovarian sarcoma, recurrent fallopian tube carcinoma, recurrent ovarian NCT01283035 Completed
carcinoma, recurrent primary peritoneal carcinoma
II Recurrent nasopharyngeal carcinoma NCT01370070 Completed
mTOR mTORC1 Everolimus Approved Advanced renal cell carcinoma following one prior antiangiogenic Novartis /
therapy
Approved HR+ and HER2- breast cancer Novartis /
Temsirolimus Approved Advanced renal cell carcinoma Wyeth Pharmaceuticals /
mTORC1/ AZD2014 II Meningioma NCT03071874 Active, not
mTORC2 recruiting
II Neurofibromatosis 2 NCT02831257 Completed
meningioma
II Diffuse large B-cell lymphoma NCT02752204 Completed
I/II Endometrial carcinoma, NCT02730923 Active, not
metastatic carcinoma, recruiting
HR+ tumor
Zhou et al.
II Metastatic breast cancer NCT02299999 Active, not

recruiting
II Non-small cell lung cancer metastatic NCT02117167 Active, not
recruiting
II Hepatocellular carcinoma NCT03591965 Terminated
INK128 II Metastatic castration-resistant prostate cancer NCT02091531 Completed
II merkel cell carcinoma NCT02514824 Completed
II anaplastic thyroid cancer, NCT02244463 Active, not
thyroid cancer recruiting
OSI-027 I Any solid tumor or lymphoma NCT00698243 Completed
Source: All the information is derived from ClinicalTrials.gov (https://www.clinicaltrials.gov) and the United States Food and Drug Administration.gov (https://www.fda.gov/)

Zhou et al.
35
(NCT05646862) for the treatment of HR+, HER2−, PIK3CA mutated and metformin in combination with other drugs are currently in
breast cancer, and a phase II clinical trial (NCT05306041) for HR+, clinical phase I trials to evaluate their safety and dosage
HER2+, PIK3CA mutated breast cancer. There are a few appropriateness in tumor patients. Researches on rapalogs have
PI3Kβ-specific inhibitors, including GSK2636771,998,999 and been relatively mature, but are further challenged by defects of
AZD8186, developed by AstraZeneca.999 In addition, PI3Kδ large molecular weight, complex structure, difficulty in synthesis,
inhibitors have mostly been approved by the FDA for clinical and limited modification sites.1007 Therefore, the second-
treatment. Idelalisib, developed by Gilead Sciences, was approved generation of small molecule-based mTOR inhibitors with
by the FDA in 2014 for the treatment of patients with relapsed simplified structures is a promising direction in the field of mTOR
CLL. Duvelisib, developed by Verastem, was approved by the FDA inhibitor development.1007 The second-generation mTOR inhibi-
in 2018 for the treatment of relapsed or refractory CLL and small tors are structurally quite different from the first-generation mTOR
lymphocytic lymphoma, and subsequently received accelerated inhibitors. They selectively target the active kinase site of mTOR
approval for adult patients with relapsed or refractory FL.1000 and thus act as ATP-competitive inhibitors. A large number of ATP-
Umbralisib was developed by TG Therapeutics and received competitive mTOR inhibitors are currently in clinical trials,
accelerated approval by the FDA in 2021 for the treatment of adult including MLN01289 (INK128) in a phase II clinical trial
patients with relapsed or refractory marginal zone lymphoma.1001 (NCT02244463) for the treatment of anaplastic thyroid cancer.
Finally, no PI3Kγ inhibitors are currently available for clinical AZD2014 is in a phase I clinical trial (NCT02398747) for the
treatment. IPI-549, a potent and highly selective PI3Kγ inhibitor treatment of advanced solid malignancies and a phase II clinical
developed by Infinity Pharmaceuticals,1000 is being used in clinical trial (NCT03071874) for the treatment of meningioma. OSI-027, an
phase II trials as a single agent for the treatment of HNSCC oral selective mTOR inhibitor, is being tested in a phase I clinical
(NCT03795610) and in combination therapy for TNBC trial for advanced solid tumors or lymphoma (NCT02398747). A
(NCT03961698). Furthermore, dual-targeted PI3K and mTOR phase I clinical trial for AZD8055 in adults with recurrent gliomas
inhibitors also exert potential roles in cancer therapy. Omipalisib (NCT01316809) has been completed, but no results have been
(GSK2126458), a dual-targeted PI3K and mTOR inhibitor devel- disclosed. The third-generation mTOR inhibitor RapaLinks are
oped by GlaxoSmithKline,993 has completed a phase I clinical trial important research breakthroughs that link rapamycin to ATP-
in solid tumors (NCT00972686), but no results have been disclosed competitive mTOR inhibitors via linkers to improve drug
yet. efficacy.1007 In addition, some natural products such as resveratrol
In summary, although the side effects of PI3K inhibitors that can can directly or indirectly regulate mTOR and mTOR signaling
lead to serious and fatal immune-related adverse reactions remain pathways.1007 Moreover, differences in mTOR inhibitor adminis-
an urgent issue,1002 PI3K inhibitors have great potential in clinical tration formula, doses, and oral bioavailability are found to result
treatment. Exploring PI3K inhibitors in combination with other in differences in drug exposure and efficacy.1011 Improving the
targeted therapies may be an effective strategy to reduce toxicity administration formula and combination with targeted drugs may
and improve clinical activity.346 be effective strategies to improve mTOR inhibitors.
AKT inhibitors. AKT belongs to the serine/threonine kinases
family,343 which contains three isoforms with highly similar RAF-MEK-ERK inhibitors: The RAF-MEK-ERK pathway regulates
structures: AKT1, AKT2, and AKT3, making the development of intracellular growth signaling1012 and is activated in more than
their isoform-specific inhibitors challenging.346 Meaningfully, 30% of human cancers.1013
tumors with AKT1 mutations and AKT2 and AKT3 amplifications BRAF is a commonly mutated protein kinase in human cancers,
are highly sensitive to AKT inhibitors, while many PIK3CA mutant particularly frequent BRAF V600 point mutation in melanoma, and
cancer cells were considerably less dependent on AKT,1003 which it has been thought to be an ideal target for cancer therapy.354
guides the rational use of AKT inhibitors. Currently, various The first-generation selective RAF inhibitors, such as vemurafenib
selective ATP-competitive pan-AKT inhibitors (Table 2), including and dabrafenib, have been well-demonstrated to possess
capivasertib by AstraZeneca Pharmaceuticals,1004 ipatasertib therapeutic effects in patients with BRAFV600E or BRAFV600K
(GDC0068),1005 and GSK2141795,1006 have been developed which mutations although their efficacy was abrogated by quick-rising
can inhibit all three AKT isoforms.1003 Significantly, on November drug resistance.1014 To improve the efficiency and overcome drug
16, 2023, the FDA approved capivasertib (truqap) with fulvestrant resistance of first-generation RAF inhibitors, second-generation
for adult patients with HR+, HER2− locally advanced or metastatic novel RAF inhibitors such as pan-RAF inhibitors and RAF dimer
breast cancer with one or more PIK3CA/AKT1/PTEN-alterations. In breakers have been developed.1015 Until now, DAY101 is the
addition, AKT allosteric inhibitors such as the potent allosteric pan- fastest progressing pan-RAF inhibitor in a phase II study to
AKT inhibitor MK-2206 possess better AKT specificity.1006 However, evaluate its safety and efficacy in patients with recurrent or
concerns such as the poor selectivity and toxicity of AKT inhibitors progressive low-grade glioma or advanced solid tumors harboring
remain a burning challenge. Presently, PROTAC and AKT drug a known BRAF alteration (NCT NCT04775485, NCT05760586). KIN-
combination strategies may change the landscape of AKT drug 2787 is another pan-RAF inhibitor that is under a phase I clinical
development. trial in adults with BRAF/NRAS-mutated advanced or metastatic
mTOR inhibitors. The mTOR pathway participates in multiple solid tumors (NCT04913285). The phase I clinical trials of the pan-
tumor cell processes, and hyperactivated mTOR signaling is RAF inhibitor LY3009120 and LXH254 in advanced or metastatic
observed in different types of cancers. Suppression of mTOR cancers have been terminated because of their unfavorable
was approved by the FDA and the EMA as an effective strategy efficiency.1016–1018 However, a phase I clinical trial of LXH254 in
capable of inhibiting the PI3K-AKT-mTOR signaling.343 Currently, combination with LTT462 or trametinib or ribociclib for the
drug development against mTOR is a hot track, and mTOR treatment of KRAS or BRAF mutant NSCLC or NRAS mutant
inhibitors can be categorized into three generations: antibiotic melanoma (NCT02974725) is ongoing.951 In addition, PLX8349 is
allosteric inhibitors (first generation), ATP-competitive inhibitors an orally available, second-generation BRAF inhibitor with IC50
(second generation), and novel mTOR inhibitors (third generation) values of 3.8 nM, 14 nM, and 23 nM for BRAFV600E, BRAF, and CRAF,
(Table 2).1007 Rapamycin and its analogs (termed rapalogs) are respectively. Importantly, PLX8349 effectively suppresses mutant
members of the first generation of allosteric inhibitors of mTOR. BRAF cells without activating the MAPK pathway, thereby over-
Significantly, the rapamycin analogs everolimus (RAD001) has coming the resistance of first-generation RAF inhibitors.1019
been approved for the treatment of RCC,1008 HR+ and HER2− Moreover, the inhibitors of MEK and ERK which are typical
breast cancer.1009 On May 30, 2007, temsirolimus (CCI-779) was effectors of the RAS-RAF signaling pathway have been developed.
approved for the treatment of advanced RCC.1010 Temsirolimus Most MEK inhibitors are allosteric inhibitors rather than ATP-

Zhou et al.
36
competitive inhibitors that block ERK phosphorylation via MEK.951 approved palbociclib in combination with fulvestrant for the
The oral ERK inhibitor MK-8353 is well tolerated and has good treatment of HR+, HER2− advanced or metastatic breast cancer in
antitumor activity in BRAFV600 mutant melanoma patients.1020 The women with disease progression following endocrine therapy,
ATP-competitive ERK inhibitor SCH772984 effectively inhibited followed by the approval of palbociclib in combination with an
ERK1 and ERK2 activity and significantly suppressed the prolifera- aromatase inhibitor for women with HR+, HER2− advanced or
tion of BRAF, NRAS, or KRAS mutated tumor cells.1021 Clinical metastatic breast cancer and men in April 2019.1035 In preclinical
phase I trials have indicated that the ATP-competitive ERK1/2 studies, palbociclib exhibits significant antiproliferative effects on
kinase inhibitor ulixertinib (BVD-523) has antitumor effects in Rb-positive cells and leads to the selective arrest of the G1 phase
patients with advanced solid tumors.1013 In addition to mono- in a series of tumor cells.1036 In a phase II clinical trial of
therapy by the above inhibitors, the combination of MEK palbociclib, the efficacy and safety of letrozole with or without
inhibitors or ERK inhibitors with RAF inhibitors effectively blocks palbociclib in the treatment of ER+, HER2− postmenopausal
the feedback activation pathway induced by RAF inhibitors or ERK breast cancer patients were compared. The PFS of palbociclib plus
inhibitors in BRAF-mutated or KRAS-mutated cancer cells.1022 In letrozole versus letrozole alone was 20.2 months versus
addition, the combination of the RAF-MEK-ERK pathway and PI3K- 10.2 months. The adverse effects especially hematologic aspects
AKT-mTOR pathway for tumor-targeted therapy is a promising have been proven to be higher in the combination group
therapeutic strategy,951 which potently curbs the growth of compared with the letrozole monotherapy group.1037
various cancers such as KRAS-mutant lung cancer,1023 NRAS- Ribociclib is the second selective inhibitor of CDK4/6 developed
mutated melanoma,1024 pancreatic cancer,1025 and many other by Novartis and approved by the FDA in 2017 for the treatment of
cancers.1026 patients with HR+, HER2− advanced or metastatic breast
Taken together, inhibitors of the RAF-MEK-ERK pathway exhibit cancer.1030,1038 The IC50 values of ribociclib for CDK4 and CDK6
promising anticancer efficiency, although drug resistance impedes are much lower than those of other kinases, at 10 and 39 nM,
their further use. The in-depth investigation of the interaction of respectively.1029 Ribociclib is used as a single agent or in
these inhibitors and growth regulatory mechanisms in specific combination with other drugs in preclinical studies. A phase III
tumor cell contexts and environments will improve the therapeu- clinical trial of ribociclib has evaluated the efficacy and safety of
tic effects and benefit future drug development. The combination ribociclib plus letrozole in patients with HR+, HER2− relapsed, or
of drug strategies based on RAS-targeted therapies and new metastatic breast cancer. Results have shown PFS after 18 months
technological approaches such as RNAi and CRISPR technologies in the ribociclib group versus placebo group 63.0 versus 42.2%.
will shed light on tumor therapy.992 Moreover, adverse effects in the ribociclib group included nausea,
infection (mainly upper respiratory tract infections and urinary
Targeting evading growth suppressors tract infections), fatigue, diarrhea, neutropenia, leukopenia,
Cancer cells can maintain tumor progression by circumventing hypertension, elevated alanine aminotransferases, lymphopenia,
processes that negatively regulate cell proliferation which are and QTc interval prolongation.1034 In the clinical trial MONALEESA-
supported by numerous tumor suppressor genes, such as RB and 7, the efficacy and safety of ribociclib in combination with
TP53. Therefore, targeting evading growth suppressors is a tamoxifen or nonsteroidal aromatase inhibitors have been
promising antitumor strategy.299 Indeed, CDK4/6 and MDM2 observed in the treatment of advanced breast cancer with HR+
inhibitors are currently being used in clinical treatment or under and HER2-. Moreover, ribociclib plus nonsteroidal aromatase
development.1027,1028 inhibitors have been found to benefit more in advanced breast
cancer without new adverse effects.1039
CDK4/6 inhibitors. Relaxation of cell cycle mechanisms is Abemaciclib is an oral CDK4/6 inhibitor developed by Eli Lilly
associated with the dysregulation of CDKs, which ultimately with IC50 values of 2 and 10 nM for CDK4 and CDK6, respectively,
promotes abnormal tumor proliferation and disease progres- and is approved by the FDA for the treatment of patients with HR
sion.1027 Over the past few decades, three generations of CDK +, HER2− advanced or metastatic breast cancer.1040 Abemaciclib
inhibitors (CDKIs) have been developed. First- and second- is more active and less neutropenic than palbociclib and
generation CDKIs receive few clinical attention in the treatment ribociclib.1041,1042 In addition, numerous studies have shown that
of cancer patients due to their limited specificity and high abemaciclib can cross the blood-brain barrier into the central
toxicity. The development of third-generation CDKIs has made system, suggesting the possibility of treating primary or metastatic
significant progress in clinical practice. Preclinical and clinical brain cancer.1043 In preclinical studies of abemaciclib, both
results suggest that these selective CDK4/6 inhibitors significantly monotherapy and in combination with endocrine therapy with
reduce the progression of multiple malignancies.1029 Early in the abemaciclib have been found to have significant inhibitory effects
cell cycle, mitotic signaling increases cyclin D expression, which on cancers.1043,1044 In the clinical study MONARCH-1, the safety
binds to and activates CDK4/6. CDK4/6 phosphorylates the Rb and efficacy of abemaciclib monotherapy in patients with
protein which is further phosphorylated by CDK2, followed by advanced breast cancer with refractory HR+, HER2− were
releasing the E2F transcription factor which allows the cell to evaluated. Results have shown that patients using abemaciclib
enter S phase.1030 Selective CDK4/6 inhibitors bind to ATP have a significantly longer duration of response and PFS with
pockets, prevent CDK4/6 from binding to cyclin D, and prevent milder adverse effects.1045 In a follow-up, a clinical study of
Rb phosphorylation, leading to G1 phase arrest and tumor cell abemaciclib in combination with fulvestrant or letrozole showed
death.1031 Currently, the typical CDK4/6 inhibitors palbociclib, significant increases in PFS in the abemaciclib combination group
ribociclib, and abemaciclib have been approved by the FDA for compared with abemaciclib monotherapy group, and most
the treatment of different cancers alone or in combination with patients were not severely neutropenic.1044,1046
established therapies (Table 3).1032–1034 Trilaciclib, a short-acting CDK4/6 inhibitor developed by G1
Palbociclib, an orally reversible small molecule inhibitor Therapeutics, has received approval from the FDA to decrease the
developed by Pfizer, is the first selective CDK4/6 inhibitor. The incidence of chemotherapy-induced myelosuppression in adult
chemical structure of palbociclib was identified in 2004,1030 and patients when administered before a platinum/etoposide-contain-
subsequently followed by 11 years of development. In February ing regimen or topotecan-containing regimen for extensive-stage
2015, Palbociclib in combination with letrozole was accepted for SCLC in 2021.1047 Other clinically CDK4/6 inhibitors have been
accelerated approval by the FDA for the treatment of ER+, HER2− developed including TQB3616, SPH4336, dalpiciclib (SHR6390),
advanced breast cancer as initial endocrine-based therapy in and flavopiridol (L86-8275). TQB3616, a novel CDK4/6 inhibitor,
postmenopausal women. Thereafter, in February 2016, the FDA exhibits high selectivity and effectiveness in preclinical cancer

Zhou et al.
37
Table 3. The FDA-approved and clinically developed CDK4/6 inhibitors
Target Drug Highest Indications Company/ Status

Phase identifier
CDK4/6 Palbociclib Approved HR+, HER2− advanced breast cancer in combination with Pfizer /
(PD0332991) hormonal therapy
Ribociclib (LEE-011) Approved HR+, HER2− advanced breast cancer in combination with Novartis /
hormonal therapy
Abemaciclib Approved HR+, HER2− advanced breast cancer in combination with Eli Lilly /
(LY2835219) hormonal therapy, monotherapy for advanced HR+, HER2−
breast cancer, adjuvant therapy for high-risk, early-stage HR+,
HER2− breast cancer in combination with hormonal therapy
Trilaciclib Approved Approved to reduce chemotherapy-induced bone marrow G1 Therapeutics /
suppression in patients with extensive-stage small cell lung
cancer
TQB3616 III HR+, HER2− in advanced breast cancer NCT05375461 Recruiting
III Breast cancer NCT05780567 Recruiting
III HR+, HER2− breast neoplasms NCT05365178 Not yet recruiting
SPH4336 III Locally advanced or metastatic breast cancer NCT05860465 Not yet recruiting
III Locally advanced or metastatic breast cancer NCT05744687 Recruiting
Dalpiciclib III Advanced breast cancer NCT05861830 NCT05861830
(SHR6390) III Female breast cancer NCT04842617 NCT04842617
III Advanced breast cancer NCT03966898 NCT03966898
Flavopiridol (L86- I/II Lymphoma NCT00445341 NCT00445341
8275) I/II B-cell chronic lymphocytic leukemia, recurrent small NCT00058240 NCT00058240
lymphocytic lymphoma, refractory chronic lymphocytic
leukemia, waldenström macroglobulinemia
II Leukemia, lymphocytic, chronic NCT00464633 Recruiting
II B-cell chronic lymphocytic leukemia, refractory chronic NCT00003620 Completed
lymphocytic leukemia, stage I–IV chronic lymphocytic
leukemia,
II Refractory multiple myeloma, stage I–III multiple myeloma NCT00047203 Completed
II Adenocarcinoma of the pancreas, recurrent pancreatic cancer, NCT00331682 Completed
stage IV pancreatic cancer
II Sarcoma NCT00005974 Completed
II Prostate cancer NCT00003256 Completed
II Adenocarcinoma of the gastroesophageal junction, diffuse NCT00991952 Completed
adenocarcinoma of the stomach, intestinal adenocarcinoma
of the stomach, mixed adenocarcinoma of the stomach,
recurrent gastric cancer, stage IIIA gastric cancer, stage IIIB
gastric cancer, stage IIIC gastric cancer, stage IV gastric cancer
II Lymphoma NCT00003039 Completed
II Kidney cancer NCT00016939 Completed
II Melanoma (skin) NCT00005971 Completed
II Endometrial cancer NCT00023894 Completed
II Liver cancer NCT00087282 Completed
I/II Recurrent adult acute lymphoblastic leukemia, recurrent adult NCT00016016 Completed
acute myeloid leukemia, secondary acute myeloid leukemia,
untreated adult acute lymphoblastic leukemia, untreated
adult acute myeloid leukemia
II B-cell chronic lymphocytic leukemia, prolymphocytic NCT00098371 Terminated
leukemia, refractory chronic lymphocytic leukemia
II Head and neck cancer NCT00020189 Completed
II Esophageal cancer NCT00006245 Completed
II Breast cancer NCT00020332 Completed
Source: All the information is derived from ClinicalTrials.gov (https://www.clinicaltrials.gov) and the United States Food and Drug Administration.gov (https://
www.fda.gov/)

Zhou et al.
38
models.1048 SPH3643, a highly selective CDK4/6 inhibitor, can been completed with no results posted (NCT01760525). Sirema-
efficiently and stably cross the blood‐brain barrier.1049 Dalpiciclib dlin (HDM201) is an orally bioavailable and selective inhibitor of
(SHR6390), a novel, highly selective CDK4/6 inhibitor, reveals high the p53-MDM2 interaction designed by Novartis.1062 Currently,
activity with IC50 of 12.4 nM and 9.9 nM against CDK4 and CDK6, three clinical trials of siremadlin monotherapy or in combination
respectively.1050 Flavopiridol, a pan-CDK inhibitor originally with other drugs for the treatment of patients with advanced soft-
purified from Dysoxylum binectariferum,1051 is the first CDK tissue sarcoma (NCT05180695), AML (NCT05447663,
inhibitor entering clinical trials and can target CDK1, CDK2, CDK4, NCT05155709) are ongoing. MK-8242 (SCH900242) is a potent,
CDK6, and CDK7.1052 orally bioavailable, small-molecule inhibitor of the MDM2-p53
interaction. Its common adverse events include anemia, leukope-
MDM2 inhibitors. Since the first discovery of the co-crystal nia, pancytopenia, nausea, hyperbilirubinemia, hypophosphate-
structure of MDM2-p53 complex in 1996,1053 an increasing mia, and anorexia.1063 A phase I trial of MK-8242 in patients with
number of researches have been dedicated to uncovering its refractory/recurrent AML has been completed.1064 Furthermore,
functions in tumors.1054 Gene amplification, increased transcrip- Milademetan (DS3032b) is an orally active MDM2 inhibitor by
tion, and accelerated translation cause the aberrant elevation of disrupting the MDM2-p53 interaction.1065 A phase I study of
MDM2, which promotes p53 ubiquitination and increases p53 milademetan in combination with low-dose cytarabine with or
degradation. Thus, targeting MDM2-p53 interaction is a particu- without venetoclax for the treatment of AML has been completed,
larly attractive therapeutic strategy for p53 reactivation in and has revealed modest therapeutic responses with recognizable
tumors.1054,1055 Numerous small-molecule MDM2 inhibitors have gastrointestinal toxicity.1065
been discovered so far, and nine of them, including RG7112, Several MDM2 inhibitors are currently being evaluated clinically
idasanutlin, AMG-232, SAR40583, APG-115, NVP-CGM097, sirema- for cancer therapy. However, there is no MDM2 inhibitor approved
dlin, and MK-8242, and milademetan, have undergone or been for clinical application.387 Challenges such as acquired resistance
undergoing clinical trials for the treatment of cancers.1028 and toxicity remain. In addition, MDM2 PROTAC degraders have
Nutlins, including Nutlin-1, Nutlin-2, and Nutlin-3, are the first received heightened attention in recent years with higher cancer
selective and potent MDM2 inhibitors synthesized in 2004,1055 therapeutic efficacy, and their safety needs further
which lay the foundation for the following development of MDM2 determination1066.
inhibitors. RG7112, an MDM2 inhibitor developed by Roche based
on the structure of Nutlins, can bind to the p53 pocket on MDM2 Targeting enabling replicative immortality
and suppress the p53-MDM2 interaction, and is the first MDM2 Studies have illustrated that the protection of telomeres at the
inhibitor to be assessed in clinical trials.387,1054 RG7112 has ends of chromosomes is essential for the replicative immortality
demonstrated clinical activity in a phase I trial for the treatment of ability of tumor cells. Tumor cells avoid senescence or apoptosis
patients with leukemia.1056 However, the poor tolerability and the by upregulating telomerase expression to maintain telomeric DNA
adverse events, such as gastrointestinal toxicity, myelosuppres- length. Therefore, targeting telomerase is of great importance.
sion, sepsis, and hemorrhage, hinder its further develop- Various telomerase targeting strategies have been developed,
men.387,1054,1056 Subsequently, idasanutlin (RG7388), another such as vaccines, antisense oligonucleotides, and small molecule
highly potent and selective MDM2 antagonist, exhibits superior inhibitors.390,1067
potency, selectivity, and bioavailability compared with RG7112,
although sharing the same action mechanism.1057 Idasanutlin is Telomerase inhibitors. Given the critical role of telomere length in
the second MDM2 inhibitor to be evaluated in clinical trials.387 The tumor proliferation, targeting telomerase is a promising antitumor
most common adverse events of idasanutlin include gastrointest- treatment approach.392 It has been observed that the initial
inal toxicity (diarrhea and nausea) and hypokalemia.1054 A phase telomere length affects the therapeutic effect of telomerase
Ib study of idasanutlin in combination with XPO1 inhibitor inhibitors.1068 Therefore, it is valuable to detect telomerase length
selinexor for the treatment of children with progressive or during clinical telomerase-targeted therapy.1069
recurrent atypical teratoid/rhabdoid tumors and malignant The key goals of antitelomerase therapy are to induce tumor
rhabdoid tumors is ongoing (NCT05952687). Meanwhile, a phase cell death and to reduce normal cytotoxicity. Numerous telomer-
I/II study of idasanutlin monotherapy or in combination with ase therapeutic options inhibit hTERT or hTR activity.390,1067
chemotherapy or venetoclax for the treatment of acute leukemias Imetelstat (GRN163L), developed by Geron, is a competitive
or solid tumors is recruiting (NCT04029688). In addition, AMG-232 inhibitor of telomerase that binds to the nucleotide region of the
(navtemadlin, KRT-232) is an investigational oral, selective MDM2 telomerase holoenzyme at a specific active site with high affinity,
inhibitor. Its most common adverse events include nausea, resulting in complete inhibition of telomerase activity.390 It has
diarrhea, vomiting, decreased appetite, anemia, thrombocytope- been identified that imetelstat has inhibitory effects on a series of
nia, and leukopenia.1058 Currently, five clinical trials of AMG-232 cancer cells, including breast cancer,1070 lung cancer,1071 prostate
monotherapy or in combination with other drugs for the cancer,1072 pancreatic cancer,1073 osteosarcoma,1074 glioblas-
treatment of patients with cancers are recruiting (NCT03031730, toma,1075 HCC, and bladder cancer.390 In August 2023, Geron
NCT03041688, NCT03107780, NCT03217266, and NCT04190550). corporation announced the FDA acceptance of a new drug
Furthermore, SAR405838(MI-77301) and APG-115 are all application for imetelstat for the treatment of transfusion-
spirooxindole-based MDM2 inhibitors developed by Wang Shao- dependent anemia in patients with lower-risk myelodysplastic
meng’s research group.387,1059 APG-115 is designed to overcome syndromes (MDS). Imetelstat is currently in a phase II clinical trial
stability-related issues observed in SAR405838.1054,1060 APG-115 to evaluate its efficacy and safety in participants with high-risk
can strongly bind with MDM2 protein, and has good chemical MDS or AML that is relapsed/refractory to hypomethylating agent
stability and excellent oral pharmacokinetic parameters.387 A treatment (NCT05583552).
phase I study of SAR405838 in patients with advanced cancer has In addition, an increasing number of studies have identified
been completed (NCT01636479), but no result has been posted. telomerase as a promising anticancer immunotherapy target, and
Significantly, seven clinical trials of APG-115 monotherapy or in telomerase-based immunotherapy is a potential antitumor ther-
combination with other drugs for the treatment of patients with apeutic strategy.1076 Antitelomerase immunotherapy exerts its
tumors are recruiting. NVP-CGM097(CGM097), a highly potent and antitumor effects mainly by enhancing the sensitivity of the
selective MDM2 inhibitor, has good cell activity, metabolic immune system to tumor cells expressing telomerase-specific
stability, and PK parameters.1061 A phase I study of CGM097 in antigenic epitopes, thereby activating hTERT-specific CD8+ cells.
patients with advanced solid tumors with p53 wild-type status has The protein fragments or peptides formed by telomerase

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degradation in tumor cells are expressed on the surface of tumor adenocarcinoma, uterine cancer, CRC, and ovarian can-
cells as tumor-associated antigens via the human leukocyte cer.425,1083,1084 Aflibercept, also known as VEGF-Trap, is a soluble
antigen (HLA) class I pathway, which in turn triggers antitumor recombinant fusion protein consisting of the extracellular binding
cytotoxic T lymphocyte responses.390 CD4+ or CD8+ cytotoxic T domains of VEGFR-1 and VEGFR-2, which was approved by the
lymphocytes can target telomerase-specific antigenic epitopes to FDA in 2012 for the treatment of metastatic CRC.1084 Aflibercept
kill tumor cells.1076 Current telomerase-based cancer immunother- interacts with circulating VEGF, thus preventing its binding to
apy mainly includes the hTERT vaccine and dendritic cell strategy. receptors on endothelial cells.1085 There are clinical studies using
Immunotherapy-based hTERT peptide (GV1001), cryptic peptides aflibercept in combination with the chemotherapeutic agent for
(Vx001), and dendritic cells (GRVAC1), three promising telomerase- the treatment of various malignancies, such as patients with
targeted vaccines with low toxicity to normal cells and no advanced CRC.401 In addition, ziv-aflibercept was approved for the
autoimmunity,390 have been used for antitelomerase immune treatment of patients with metastatic CRC in combination with
response therapy in cancer patients.1077 GV1001 is a 16 amino- FOLFIRI (folinic acid, fluorouracil, and irinotecan) in 2012.425,1086
acid hTER peptide vaccine containing the hTERT active site which Since angiogenesis contains multiple signaling pathways, such
significantly activates CD4+ or CD8+ cytotoxic T lymphocyte as the VEGFR family and FGFR family, selective antiangiogenic
responses.1078 Vx001 is a vaccine containing the hTERT amino acid agents especially TKIs can minimize the induction of toxicities.1087
sequence with a high affinity for HLA class I. GRNVAC1, a dendritic Compared with macromolecules, small molecule TKIs possess
cell-based vaccine with good activity and tolerance, exerts the multitarget inhibitory efficiency, and penetrate into cells easily
antitumor effect by stimulating lysosomal degradation of hTERT due to hydrophobic properties, thus blocking the activation of
into small peptide.390 Currently, GV1001, GRNVAC1, and Vx001 various signaling pathways, which ultimately increases treatment
vaccines are in clinical trials. A phase II clinical trial (NCT00510133) efficiency.1087 In addition, small molecule TKIs can be adminis-
of active immunotherapy with GRNVAC1 in patients with acute tered orally as inhibitor salt form.1087
myelogenous leukemia has been completed, but no results have Sorafenib (BAY43-9006) is a selective inhibitor targeting VEGFR-
been posted. 2 and VEGFR-3, PDGFR, FMS-like tyrosine kinase 3 (FLT-3), and
Although targeting telomerase is an attractive antitumor c-Kit401 and is the first TKI approved by the FDA as a first-line
strategy, some limitations still exist. First, as telomere shortening treatment for advanced HCC.1088 A phase III clinical trial uncovered
induced by cell division is a slow and long-term process, therapies significant improvements in OS in patients with advanced HCC
that inhibit telomerase activity are hysteretic and are not suitable treated with sorafenib.1089 Common clinical adverse effects of
for first-line cancer therapy. Second, since telomerase is expressed sorafenib include diarrhea, fatigue, skin reactions on the hands
in highly proliferative cells such as hematopoietic precursor cells and feet, rash or desquamation, and anorexia.416 Lenvatinib, an
and epidermal stem cells, telomerase targeting therapies are oral TKI that targets VEGFR, FGFR, PDGFR, KIT, and RET, exerts
potentially toxic and cause side effects. Third, preclinical antiproliferative and immunomodulatory activity in preclinical
telomerase targeting therapy lacks a suitable model for evalua- cancer models, and has been approved as a first-line treatment for
tion.1068 It has been observed that mouse telomeres are 5–10 HCC. Compared with sorafenib, levatinib has improved OS and
times longer than human,1068,1079 and their telomerases are PFS in patients with HCC.1090 In addition, lenvatinib suppresses the
widely expressed at low levels in adult tissue.1080 Thus, the development of various cancers by inhibiting VEGFR signaling,
dependence of mice on replicative immortality induced by such as kidney cancer,1091 pancreatic cancer,1092 SCLC,1093 breast
telomerase activation is much lower than that of humans. Fourth, cancer,1094 differentiated thyroid cancer,1095 and anaplastic
the structure characteristics of human telomerase holoenzymes thyroid cancer.1090 The most common adverse effects of
need to be further resolved, which is crucial for telomerase lenvatinib treatment include hypertension, diarrhea, loss of
inhibitor development.1068 appetite, weight loss, and fatigue.416 Sunitinib, an oral TKI
targeting VEGFR1-3, PDGFR, FLT-3, and c-Kit,401,1085 is approved
Antiangiogenesis therapy by the FDA for the treatment of refractory GIST and metastatic
The concept of “antiangiogenesis” therapy was proposed by Dr. RCC.401 There are clinical studies using sunitinib for the treatment
Judah Folkman in 1971, who found that tumor growth requires of advanced or metastatic breast cancer and NSCLC.401 Pazopanib
neovascularization for maintenance.1081 Antiangiogenic therapy (GW786034) has been approved for treating advanced RCC. Its
has become an invaluable strategy to hinder tumor proliferation common adverse effects are diarrhea, hypertension, hair color
and metastasis. The current antiangiogenic inhibitors mainly change, nausea, anorexia, and a 2% occurrence of myocardial
include VEGF inhibitors, FGF inhibitors, and PDGF inhibitors. infarction or ischemia. Axitinib, a second-generation inhibitor of
VEGF-1, 2, and 3, is more selective for VEGF without blocking
VEGF inhibitors. Antiangiogenic therapies target angiogenesis via PDGF, B-RAF, FLT-3, and KIT targets. In January 2012, axitinib was
two major mechanisms: blocking intracellular receptor tyrosine approved by the FDA for the treatment of RCC after failure of a
kinases or neutralizing angiogenic factors such as VEGF or its prior systemic therapy.401
receptor. The current drugs targeting VEGF include mAbs, VEGF Despite the emerging number of antiangiogenic therapeutic
decoy receptors, and small molecule TKIs. These drugs can be agents, challenges remain to be solved in antiangiogenic drug
used as monotherapy or in combination with other chemother- development and application.398 First, the tumor vascular system
apeutic agents for clinical treatment.401 in preclinical mouse models is more sensitive to antiangiogenic
In 2004, the anti-VEGF-A humanized mAb bevacizumab therapy than the human tumor vascular system, which enhances
(avastin), the first antiangiogenic agent, was first approved by the inconsistency between preclinical and clinical practice.398
the FDA for the treatment of advanced CRC. In May 2009, Second, the effect of antiangiogenic monotherapy is limited, and
bevacizumab was approved as a second-line cancer therapy for the appropriate strategy for the combination of antiangiogenic
the treatment of glioblastoma. Subsequently, in July 2009, the FDA drugs with other drugs needs to be discovered. For example,
approved bevacizumab in combination with interferon-alpha for immune checkpoint inhibitors and antiangiogenic agents have
the treatment of RCC.401 Currently, combination chemotherapy is been proven to be a promising therapeutic strategy. Antiangio-
used for treating patients with advanced CRC, NSCLC, and breast genic agents can reshape the immune TME, while immune
cancer.398,1082 Ramucirumab (cyramza), a mAb that binds VEGFR2 checkpoint inhibitors can downregulate vascular endothelial
to block the VEGF signaling pathway, has been approved for the growth factor expression and alleviate hypoxic conditions to
treatment of many types of solid tumors, such as advanced or combat angiogenesis.1096 Moreover, withdrawal of antiangiogenic
metastatic gastric cancer, gastroesophageal junction drugs may result in tumor growth and metastasis, and the strategy

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to prevent this rebound needs to be developed.1097 In conclusion, amplification copy numbers are more sensitive to FGFR inhibitors,
tumor angiogenesis is essential for tumor growth. The regulatory whereas the fraction of patients with high FGFR amplification is
factors of tumor angiogenesis and targeted therapies are diverse not frequent. Thus, monitoring the status of FGFR and choosing a
and need to be further elucidated in the future. subgroup of patients could be beneficial for FGRF-targeted
therapy.438,1098 The precise treatment for patients with tumor
FGF/FGFR inhibitors. Studies have found that abnormal FGF heterogeneity and specific FGFR abnormalities remains an urgent
signaling contributes to tumor angiogenesis and subsequently task.1098 Moreover, the side effects and toxicity of nonselective
promotes tumor growth.422 Researches on FGF-targeted therapies FGFR inhibitors remain an unresolved challenge.1109
have flourished in recent years, but no FGF-targeted therapy has
been approved for cancer treatment. Current FGF-targeted PDGF/PDGFR inhibitors. PDGF is an essential angiogenic factor
therapies include multitargeted small molecule TKIs, selective and its expression is upregulated in a diverse range of tumor cells.
FGFR-targeted TKIs, mAbs against FGFR, and FGF ligand Therefore, blocking PDGF/PDGFR signaling is a promising strategy
traps.1098,1099 for targeted therapy.
Dovitinib (TKI258) is an orally active and nonselective TKI that
targets VEGFR1-3, FGFR1-3, and PDGFR.1100 It has good antitumor Targeting PDGFR: PDGFR-targeted therapies include specific
activity against RCC1101 and breast cancer.1100 Clinical trials inhibitors, nonspecific inhibitors, mAbs, and RNA aptamers,
evaluating the efficacy and safety of dovitinib alone or in among which inhibitors have been extensively studied. PDGFR
combination with other anticancer drugs in patients with solid inhibitors are classified into two categories including specific and
tumors and hematologic malignancies are ongoing. Other nonspecific inhibitors, based on their binding properties to the
nonselective multitargeted anti-FGFR TKIs include nintedanib receptor, and many PDGFR inhibitors are currently used in the
(BIBF1120),1102 lucitanib (E3810),1103 and ponatinib clinical treatment of different cancers.443 CP-673451, a specific
(AP24534),1104 which reveal antitumor activity in advanced solid ATP-competitive PDGFR inhibitor, effectively inhibits PDGFRβ
tumors. Given the toxicity of multitargeted TKIs, several FGFR- activity.1110 CHMFL-PDGFR-159 is a highly selective inhibitor of
selective inhibitors have been developed that hinder FGFR1-3 PDGFRα, which significantly inhibits the proliferation of chronic
kinase activity to varying degrees and are currently in clinical trials. eosinophilic leukemia cells.1111 In addition, there are a large
Infigratinib (NVP-BGJ398), an oral ATP-competitive FGFR inhibitor number of nonspecific PDGFR inhibitors. As tyrosine kinases have
developed by QED Therapeutics, was granted accelerated a conserved ATP-binding pocket, most small molecule TKIs are in a
approval by the FDA on May 28, 2021, for the treatment of adults multitarget binding mode and inhibit both PDGFRα and PDGFRβ
with previously treated, unresectable locally advanced or meta- activity, including imatinib, ponatinib, sorafenib, nilotinib, ninte-
static cholangiocarcinoma with an FGFR2 fusion or other danib, dasatinib, midostaurin, ripretinib, sitravatinib, masitinib,
rearrangement.1105 In addition, infigratinib is currently in a phase sunitinib, axitinib, pazopanib, crenolanib.1112 Imatinib, the mile-
I clinical trial for renal pelvis and ureter urothelial carcinoma stone in the history of TKI development, is a multitarget TKI that
(NCT04228042), and phase II clinical trials for gastric cancer targets PDGFR signaling1112 and is used for the first-line treatment
(NCT05019794), advanced solid tumors (NCT04233567), cholan- of patients with BCR-ABL-positive leukemia.443 Sunitinib (SU11248)
giocarcinoma (NCT04233567), and metastatic and refractory is a multitarget inhibitor that targets PDGFR, VEGFR, c-Kit, FLT-3,
malignant solid neoplasm (NCT04233567) treatment. Erdafitinib and Ret kinase,1113 which was approved by the FDA in 2006 for
(JNJ-42756493), an FGFR inhibitor developed by Janssen Pharma- the first-line treatment of metastatic RCC.1114 Sorafenib is an
ceutical, was granted accelerated approval by the FDA on April 12, inhibitor targeting PDGFR, c-Kit, FLT-3, and VEGFR for the
2019, for the treatment of patients with locally advanced or treatment of patients with advanced HCC.443,1115 Ponatinib
metastatic urothelial carcinoma with susceptible FGFR3 or FGFR2 (AP24534) is a multitarget TKI targeting BCR-ABL, PDGFR, VEGFR,
genetic alterations.1106 Erdafitinib is currently in a phase II clinical FGFR, and Src, which has been applied as a third-line agent for
trial for urinary bladder neoplasms (NCT04172675) and advanced CML treatment.443 Regorafenib is a multitargeted TKI that
solid tumor (NCT04083976) treatment. Moreover, futibatinib suppresses PDGFR, VEGFR, c-Kit, BRAF, as well as EGFR, and
(TAS120), an oral, covalently binding, irreversible inhibitor of ERK1116 for the treatment of advanced HCC, CRC, and
FGFR1-4, is being developed by Taiho Oncology and Taiho GIST.443,1116,1117 Nilotinib is a TKI that blocks PDGFR α/β, c-Kit,
Pharmaceutical for the treatment of various cancers.1107 On and is primarily used in the treatment of patients with CML and
September 30, 2022, the FDA granted accelerated approval to ALL.443,1118 Nintedanib is an inhibitor that blocks PDGFRα/β,
futibatinib for the treatment of adult patients with previously VEGFR1-3, and FGFR1-3.443 Dasatinib is a multitarget inhibitor that
treated, unresectable, locally advanced or metastatic intrahepatic targets PDGFR α/β, BCR-ABL, YES, and c-Kit and is approved by the
cholangiocarcinoma harboring FGFR2 gene fusions or other FDA for the treatment of chronic granulocytic leukemia.443,1119
rearrangements.1108 Futibatinib is currently in a phase II clinical Midostaurin (PKC412) blocks PDGFR, Kit, VEGFR2, and PKCα, and is
trial for the treatment of patients with metastatic breast cancer primarily used in the treatment of AML with FLT-3 muta-
(NCT04024436), advanced and metastatic HCC (NCT04828486), tions.443,1120,1121 Ripretinib (DCC-2618) inhibits PDGFRα/β, VEGFR2,
and advanced and metastatic urothelial cancer (NCT04601857), and Kit activity and is approved for the treatment of adult patients
phase I and II clinical trials for NSCLC (NCT04965818), and a phase with advanced GIST.443,1122 Sitravatinib (MGCD516) and masitinib
III clinical trial for soft tissue sarcoma (NCT03784014). In addition (AB1010) broadly target the PDGFR family.443 Axitinib is also a
to small molecules, there are few clinical anti-FGFR mAbs. multitarget inhibitor targeting PDGFR, VEGFR1-3, and c-Kit, and is
MGFR1877S is an anti-FGFR3 mAb that is being studied in a being used to treat patients with advanced RCC.443,1123 Pazopanib
phase I clinical trial in patients with solid tumors (NCT01363024). is an inhibitor that targets PDGFR, VEGFR, and c-Kit, and is useful in
FP-1039 (GSK3052230) is a soluble fusion protein that belongs to the treatment of RCC and soft tissue sarcoma.443,1124 Crenolanib
an FGF ligand trap that isolates the FGF ligand and prevents its (CP-868596) inhibits PDGFRα/β, FLT-3, and c-Kit and is a highly
binding to the receptor.1099 FP-1039 was studied in a phase I trial selective inhibitor of PDGFRβ. Moreover, lenvatinib and avapritinib
in patients with advanced tumors (NCT00687505) and a phase I are both oral inhibitors that target PDGFRα and c-Kit.443 Although
trial was withdrawn in endometrial cancers with FGFR2 mutations a large number of TKIs targeting PDGFR have been approved for
(NCT01244438) due to substandard recruitment of patients. clinical practice, their low specificity and activity against PDGFR
Although there are numerous inhibitors and antibodies target- make accuracy evaluation of anti-PDGFR effect difficult. Moreover,
ing FGF/FGFR under investigation, the development of anti-FGF/ the side effects and increased toxicity of PDGFR inhibitors induced
FGFR agents remains a tough challenge. Tumors with higher FGFR by multitargets or in combination with other anticancer drugs are

Zhou et al.
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big issues and cannot be ignored. It is worthwhile to explore the as a metamorphic inhibitor of mTORC1.1136,1139 Rapamycin, Torin-
structure-function relationship of PDGF inhibitors in order to 1, and AZD8055 can selectively inhibit mTORC1 kinase activ-
discover PDGF/PDGFR inhibitors with high specificity and ity.1136,1140 In addition, the sodium voltage-gated channel blocker
selectivity in the future. The nanotechnology may provide new carbamazepine and the L-type calcium channel blocker felodipine
perspectives for PDGF/PDGFR-targeted therapy by enhancing are mTORC1 nondependent autophagy inducers and have been
target specificity and drug delivery accuracy.443 approved by the FDA.1136 The covalent acrylamide-based autop-
Compared with extensive progress that has been made in TKIs hagy inducer EN6 indirectly induces mTORC1 inactivation and
targeting PDGFR, the progress on mAbs targeting PDGFR is increases lysosomal acidification, leading to enhanced autophagic
limited. Olaratumab (IMC-3G3), a high-affinity human anti-PDGFRα flow.1136 Additionally, disaccharide trehaloses SMER-28 and
mAb,1125 is currently in a phase I clinical trial for soft tissue BRD5631 can induce autophagy in an mTORC1-independent
sarcoma (NCT03126591). Tovetumab (MEDI-575) is also a human manner.1141,1142 The natural product OSW-1 induces autophagy by
anti-PDGFRα mAb that is well tolerated and has a favorable blocking oxysterol-binding protein to inhibit cholesterol transport
pharmacokinetic profile in patients with advanced solid tumors.443 to lysosomes, thereby inhibiting mTORC1.1143,1144 Mucolipin 1
Gint4.T, a high-affinity RNA aptamer that specifically binds to (TRPML1), an activator of the transient receptor potential cation
PDGFRβ,1126 inhibits TNBC lung metastases1127 and glioma.443 The channel, induces autophagosome biogenesis.1136,1145
high specificity of mAbs offers a significant opportunity to The second category is the inhibitors of autophagy initiation.
selectively target PEGFR resulting in specific therapeutic effects, Autophagy inhibitors targeting PI3K and ULK1 can inhibit
which warrant further investigation. autophagy by preventing autophagosome formation. The pan-
PI3K inhibitors 3-methyladenine and wortmannin are widely used
Targeting PDGF: Compared with the extensive studies targeting in autophagy studies.1136 VPS34 is an important component of the
PDGFR, only a few studies are reporting PDGF targeting therapy. class III PI3K complex. Autophinib and cinchona alkaloid-derived
MOR8457 is a high-affinity PDGF mAb1128 that selectively binds azaquindole-1 are both potent inhibitors of VPS34,1146,1147 and
PDGF-BB and masks its receptor PDGFRβ-binding epitope, thereby SAR405 and VPS34-IN1 are better selective VPS34 inhibitors.1136
blocking receptor dimerization and tyrosine kinase activation, ULK inhibitors include MRT68921 and SBI-0206965,1148,1149 which
ultimately effectively preventing PDGF-BB-induced cell prolifera- selectively inhibit ULK1 and ULK2. In addition, the inhibitor ULK-
tion.443 6B3, a highly selective mAb, blocks PDGF-CC-induced 101 is a more selective and active ULK1 inhibitor, and is currently
PDGFRα phosphorylation and activation.1129 AX102 is a high- considered to be the most promising ULK1 inhibitory tool
affinity single-stranded DNA aptamer that blocks PDGF-B and compound.1136 The cysteine protease autophagin-1 (ATG4B), a
prevents activation of downstream proliferative signals.443,1130 key protein for autophagosome formation and maturation, cleaves
E10030 is a PEG-modified aptamer that specifically binds to and the C-terminus of LC3B and then binds to PE via ATG3. The
restrains the function of PDGF-B.425 development of ATG4B inhibitors is an effective way to inhibit
In summary, antiangiogenic drugs offer hope for antitumor autophagy. The styrrylquinoline-derived ATG4B inhibitor LV-320
therapy, but their resistance remains a pressing clinical issue. has cysteine protease selectivity.1136,1150 The fluoromethylketone-
There are various reasons for antiangiogenic drug resistance. It has based peptidomimetic FMK-9a, an irreversible covalent inhibitor
been revealed that lack of VEGF or VEGFR in certain metastatic of ATG4B, effectively inhibits ATG4B protein hydrolysis activity. In
tumors leads to poor efficacy in VEGF-targeted therapy, e.g., addition, altering the lipid composition of the nascent phago-
blocking the VEGFR2 pathway inhibits the growth of human RCC phore is also a new strategy for the development of autophagy
RBM1-IT4 cells implanted in the kidney but not in the bone of inhibitors. The cholesterol transport protein GRAMD1A can
nude mice.1131 Tumor cells maintain sustained growth through effectively inhibit autophagosome biogenesis.1136
existing blood vessels in organs with rich vascular systems (e.g., The third category is inhibitors of autophagosome maturation
lungs), thus developing drug resistance.1132 Moreover, The and lysosomal activity. The calcium channel TRPML1 is essential for
abnormal induction of various proangiogenic factors such as autophagosome formation, and its small molecule inhibitor ML-SI3
bFGF,406 circulating PlGF, VEGF,1133 and FGF,1134 can cause can inhibit autophagic flow.1145,1151 In addition, inhibition of
acquired resistance to antiangiogenic therapy.406 In addition, the autophagosome-lysosome fusion by enhancing enhancement of
vascular dependence of tumor cells is heterogeneous and 20S proteasome activity is an effective strategy to reduce
variable. Tumors with p53 mutation are less dependent on autophagic flux.1152 The small molecule TCH-165 activates the
vascular supply and are more resistant to antiangiogenic drug 20S proteasome and specifically degrades important
treatment.1135 Therefore, an in-depth investigation into the autophagosome-lysosome fusion regulators. Targeting autophagic
mechanisms of antiangiogenic drug resistance and tumor terminal lysosomal activity is an effective method to inhibit
heterogeneity is a promising strategy for tumor treatment. autophagic flow.1136 These inhibitors inhibit lysosomal acidification
by inhibiting v-ATPase or by directly increasing lysosomal pH and
Targeting resisting cell death promoting lysosomal hydrolase inactivation. v-ATPase is a multi-
Autophagy inhibitors/inducers. The multiple steps of the autop- subunit proton pump responsible for maintaining low lysosomal
hagic pathway present many opportunities for the development pH. Natural products have been a rich source of highly potent
of targeted autophagy inhibitors, which currently include small v-ATPase inhibitors, including the macrolide antibiotics bafilomycin
molecule autophagy inducers and autophagy inhibitors, the latter A1, concanamycin, benzolactone enamides salicylihalamide A, and
consisting of inhibitors of autophagy initiation, autophagosome lobatamide.1136,1153,1154 Lys01 is a tenfold more active autophagy
maturation, and lysosomal activity, hijacking the autophagosome inhibitor than hydroxychloroquine (HCQ), and its water-soluble salt
and lysosome for targeted protein degradation1136,1137 (Table 4). Lys05 effectively promotes lysosomal deacidification and inhibits
The first category of autophagy inducers is small molecule the proliferation of multiple tumor cell lines in vitro and the growth
compounds which act primarily by directly inhibiting mTORC1 or of tumor xenograft models in vivo.1155 Other lysosomal inhibitors,
activating AMPK. The cellular energy state sensor AMP can inhibit such as quinacrine, VATG-027, and VATG-032, also showed
biosynthesis in response to energy stress by suppressing mTORC1. antitumor activity. VATG-027 is a potent inhibitor of autophagy
Thus, inhibition of mTORC1 blocks phosphorylation of ATG13, with high cytotoxicity.1156 To date, the lysosomotropic agents
ULK1, and ULK2 in the ULK1 complex, and promotes AMPK chloroquine (CQ) and HCQ have been the main clinically applied
activation or RAPTOR phosphorylation, thereby increasing autop- autophagy inhibitors and are commonly used to alleviate acute and
hagic flux.1136,1138 Moreover, rapalogs induce autophagy by chronic inflammatory diseases, although a variety of autophagy
forming a complex with FK506-binding protein (FKBP12), acting inhibitors have been developed.1136,1157 They block the fusion of

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Table 4. The FDA-approved and clinically developed autophagy inhibitors/ inducers in cancer therapies
Drug Highest Indications Identifier Status

phase
Rapamycin IV Non-hodgkin’s lymphoma NCT01180049 Completed

IV Angiomyolipoma NCT01217125 Completed
IV Refractory solid Tumors NCT02688881 Completed
IV Hemangioendothelioma of liver NCT04406870 Not yet
recruiting
Everolimus (RAD001) Approved Progressive, well-differentiated non-functional, neuroendocrine Novartis /
tumors of gastrointestinal or lung origin with unresectable, locally
advanced or metastatic disease
Approved Advanced renal cell carcinoma following one prior antiangiogenic Novartis /
therapy
Approved Advanced hormone receptor-positive, HER2-negative breast cancer in Novartis
postmenopausal women
AZD8055 I Glioblastoma multiforme, anaplastic astrocytoma, anaplastic NCT01316809 Completed
oligodendroglioma, malignant glioma, brainstem glioma
I Cancer, solid tumors, advanced solid malignancies NCT00973076 Completed
I Solid tumors NCT00731263 Completed
I Advanced hepatocellular carcinoma, cancer NCT00999882 Completed
Chloroquine (CQ) Approved Prophylactic treatment of malaria Bayer /
II Astrocytoma, grade IV, glioblastoma NCT02432417 Not yet
recruiting
Hydroxychloroquine (HCQ) Approved Chronic, discoid or systemic lupus erythematosus and acute or chronic / /
rheumatoid arthritis
II Advanced cancer, pancreatic cancer NCT04386057 Recruiting
II Melanoma NCT04464759 Recruiting
II Breast cancer NCT04841148 Recruiting
II Hepatocellular cancer NCT03037437 Recruiting
II Breast cancer NCT04523857 Recruiting
II Metastatic colorectal cancer NCT05843188 Recruiting
www.fda.gov/)
autophagosomes with lysosomal fusion to block organelle and Antiapoptotic therapy. Controlling cancer growth by promoting
protein degradation processes, thereby inhibiting nutrient recy- apoptosis is an effective antitumor strategy, and various
cling.460 CQ can enter the lysosome as a freely diffusing apoptosis-based targeted therapies for tumors have been devel-
lysosomotropic agent and is deprotonated and trapped inside as oped. Currently, the main focus on inhibitor development is
a diacidic base in the lysosome.1156,1158 By sequestering the free targeting antiapoptotic family members. The inhibitors against
hydrogen ions required to maintain an acidic pH, CQ increases the BCL-2 are the most extensively studied, which include oligonu-
basicity of the lysosome, which renders pH-dependent lysosomal cleotides that target BCL-2 expression, and proapoptotic BH3
hydrolases and proteases, blocks lysosomal turnover, and inhibits mimetics that bind to antiapoptotic BCL-2 members (Table 5).463
the final stage of autophagy.1156 CQ and HCQ have been PNT-2258, a 24-base, single-stranded, chemically unmodified
extensively studied as safety autophagy distributors for the phosphodiester DNA oligonucleotide encapsulated in a specia-
treatment of various cancers, including breast cancer, melanoma, lized liposome, can target the regulatory region upstream of the
lung cancer, multiple myeloma, glioma, kidney cancer, prostate BCL-2 gene.1163 Navitoclax (ABT-263) is the second-generation,
cancer, CRC, and other advanced solid tumors.1156,1159–1161 potent, and orally bioavailable Bad-like BH3 mimetic with an oral
The fourth category is the degradation of the autophagosome bioavailability of 20 to 50% in preclinical models. ABT-263 disrupts
and lysosome for targeted proteins. PROTACs have been applied the interaction of BCL-2/BC-XL with pro-death proteins and
to selectively target the degradation of autophagosomes or induces BAX translocation, cytochrome c release, and ultimately
lysosomes and have promising applications.1136 apoptosis.463,1164 Navitoclax inhibits growth in multiple preclinical
In conclusion, there are various action mechanisms of tumor models1165 and is currently being evaluated in combination
autophagy inhibitors, which increase tumor chemotherapy drug with the PARP inhibitor olaparib for the treatment of TNBC and
sensitivity and inhibit tumor cell proliferation and metastasis.1159 ovarian cancer in a phase I clinical trial (NCT05358639). Palcitoclax
Small molecule autophagy inhibitors remain excellent tools for (APG-1252) is a dual BCL-2 and BCL-XL inhibitor with safe and well-
autophagy-targeted therapy with the advantages of easy admin- tolerated properties for treating patients with metastatic solid
istration, rapid onset of action, and mostly reversible.1136 Given tumors.1166 ABT-737 is the first small molecule designed to
the nonautophagic targeted effects of most current autophagic selectively bind the hydrophobic pocket within BCL-2, BC-XL, and
targets1162 and the existence of mitochondrial autophagy path- BCL-W, which is not bioavailable for oral administration. ABT-737
ways, the development of highly selective autophagy inhibitors is displaces BIM from BCL-2’s BH3-binding pocket, subsequently
crucial.1136 activates BAX and induces mitochondrial permeability, ultimately

Zhou et al.
43
Table 5. The FDA-approved and clinically developed antiapoptotic inhibitors in cancer therapies
Targets Drug Highest Indications Company/Identifier Status

Phase
BCL-2 PNT-2258 II Non-hodgkin’s lymphoma NCT01733238 Completed

II Diffuse large B-cell lymphoma NCT02226965 Completed
Dual BCL-2 and Navitoclax (ABT- II Small cell lung cancer, NCT00445198 Completed
BCL-XL inhibitors 263) Small cell lung carcinoma
II Chronic lymphocytic leukemia NCT01557777 Completed
II Metastatic malignant solid neoplasm, recurrent NCT03366103 Terminated
lung small cell carcinoma
II Refractory acute lymphoblastic leukemia, relapsed NCT05192889 Recruiting
acute lymphoblastic leukemia
II Metastatic malignant solid neoplasm, refractory NCT02079740 Active, not
malignant solid neoplasm, unresectable malignant recruiting
solid neoplasm
II Malignant solid neoplasm, melanoma NCT01989585 Active, not
recruiting
II Chronic lymphocytic leukemia NCT01087151 Completed
II Platinum-resistant or Refractory ovarian cancer NCT02591095 Completed
I/II Chronic lymphocytic leukemia NCT00481091 Completed
I/II Chronic lymphoid leukemia, follicular lymphoma, NCT00406809 Completed
lymphoid malignancies, mantle cell lymphoma,
non-hodgkin’s lymphoma, peripheral T-cell
lymphoma
II Prostate cancer NCT01828476 Terminated
Palcitoclax (APG- II Small cell lung cancer NCT04210037 Terminated
1252)
ABT-737 / Ovarian cancer NCT01440504 Completed
AZD0466 II Hematological malignancies NCT04865419 Recruiting
Selective BCL-2 Venetoclax (ABT- Approved Chronic lymphocytic leukemia and acute myeloid AbbVie Inc. and /
inhibitors 199) leukemia Genentech Inc
S55746 (S-055746, I B-cell non-hodgkin lymphoma, chronic lymphocytic NCT02920697 Completed
BCL201) leukemia, multiple myeloma
I Follicular lymphoma, mantle cell lymphoma NCT02603445 Completed
I Acute myeloid leukemia, myelodysplastic syndrome NCT02920541 Completed
Lisaftoclax (APG- II Chronic lymphocytic leukemia, small lymphocytic NCT05147467 Recruiting
2575) lymphoma
II Multiple myeloma, amyloidosis NCT04942067 Recruiting
II Breast cancer solid tumor, adult NCT04946864 Recruiting
II Multiple myeloma NCT04674514 Recruiting
II Chronic lymphocytic leukemia, small lymphocytic NCT04494503 Recruiting
lymphoma
II Relapsed/refractory acute myeloid leukemia, NCT04501120 Recruiting
myeloid malignancy
I/II Acute myeloid leukemia NCT04964518 Recruiting
BCL-XL inhibitors ABBV-155 I Advanced solid tumors NCT03595059 Active, not
recruiting
www.fda.gov/)
leading to apoptosis.463,475,1167 ABT-737 has antitumor activity FLT-3 TKIs gilteritinib or sorafenib synergistically inhibited FLT-3/
against hematologic and solid tumors, including CLL, lymphoma, ITD mutant AML proliferation and promoted apoptosis.1172 The
and SCLC.475,1168,1169 In addition, ABT-737 exerts synergistic toxic reactions of venetoclax include mild diarrhea (52%), upper
cytotoxicity with chemotherapy and radiotherapy.475 AZD0466, a respiratory tract infection (48%), nausea (47%), and grade 3 or 4
novel BH3-mimetic inhibitor targeting BCL-XL and BCL-2, has neutropenia (41%).1173 S55746 (S-055746, BCL201) is an orally
potent antitumor activity in preclinical models of malignant selective BCL-2 inhibitor and can effectively impair hematological
pleural mesothelioma.1170 tumor growth.1174 Lisaftoclax (APG-2575), a selective oral BCL-2
Venetoclax (ABT-199), a selective inhibitor of BCL-2, has been inhibitor, demonstrates potent antitumor activity in preclinical
approved by the FDA for the treatment of CLL and AML.1171 models of hematologic malignancy.1175
Venetoclax inhibits the proliferation of BCL-2 overexpressing small ABBV-155 is a first-in-class selective BCL-XL inhibitor. ABBV-155
lymphocytic lymphoma.463 Venetoclax in combination with the monotherapy or in combination with paclitaxel or docetaxel is

Zhou et al.
44
currently in a phase I clinical trial in advanced solid tumors to protein disulfide isomerase PDIA6,1197 death receptor ligand
assess its safety and preliminary activity (NCT03595059). In TRAIL,1198 and mitochondrial activator of caspases mimetics
addition, as the BAX serine 184 regulatory site is responsible for (Smac).1184 Moreover, necrocytes can initiate adaptive immunity
subcellular localization and insertion into mitochondrial mem- and recruit macrophages through activation of NF-κB, thereby
branes, the agonists targeting BAX have been developed for activating immune cells and enhancing immunotherapy efficacy.
cancer treatment. The small molecule BAX agonists SMBA1, The combined use of checkpoint inhibitors and necrocyte
SMBA2, and SMBA3 selectively bind to BAX and inhibit S184 vaccines significantly improves the clinical outcomes of cancer
phosphorylation, thereby promoting BAX insertion into mitochon- patients.566,1184
drial membranes and the formation of BAX oligomers, and
inducing conformational changes in BAX, ultimately leading to Targeting invasion and metastasis
cytochrome c release and apoptosis.1176 Other BAX-activating There are still limitations and tough challenges in the targeted
compounds, such as BAM-7 and BTSA1, also have antitumor treatment of tumor metastasis. Tumor cells have already spread to
activity in glioblastoma and AML cells.463,1177 Meanwhile, some the blood, bone marrow, and distant organs by the time some
other preclinical inhibitors are also under investigation.463 cancer patients are first diagnosed with tumors.165,1199 Therefore,
Overall, apoptosis resistance is a hallmark of human cancers. antimetastatic therapies need to take into account not only cells
The abnormal expression of antiapoptotic proteins and the that metastasize from the primary tumor site, but also the
downregulation or mutation of proapoptotic proteins promote inhibition of cancer cells that have already spread. Currently,
the acquisition of apoptosis resistance in tumors.491 Tumor- strategies for preventing metastasis have been demonstrated
targeted therapy inducing apoptosis is an effective approach to preclinically (Fig. 5). However, drug development has been
overcome apoptosis resistance and open up new directions for hindered due to poor trial design and therapeutic strategies.600
cancer treatment strategies. Encouragingly, potent and specific MMP inhibitors are being
developed that may further improve efficacy and attenuate
Necroptosis-inducing anticancer agents. Various compounds and toxicity.1200 In addition, targeted HGF/c-MET inhibitors are bring-
anticancer drugs with various mechanisms of action are capable of ing light to tumor treatment.677 In conclusion, more than 90% of
inducing necrosis in cancer cells, which include chemotherapeutic cancer mortality is now attributed to metastasis, and the prospect
agents, natural compounds, and classical necrosis inducers.578,1178 of targeted tumor metastasis therapy is unlimited.165
Chemotherapeutic agents for necrosis have been widely
developed in recent years. Necrostatin-1 is a small molecular MMP inhibitors. Since cancer cells require MMPs to degrade
alkaloid that was first considered an inhibitor of necrotic cell death collagen to promote cell metastasis, the first generation of MMP
in 20051179 and was subsequently identified as a specific inhibitor inhibitors is structural analogs of collagen, among which the first
of RIPK1. Necrostatin-1 blocks RIPK1 kinase activity by interacting compound belongs to the hydroxamic acid zinc-binding
with an essential structure for the death domain receptor group.715,1201 Developed by British Biotech, batimastat (BB-94) is a
engagement T loop and blocking RIPK1 kinase activity.1180 In potent pan-MMP inhibitor that acts by chelating zinc ions at the
addition, necrostatin-1 analogs were also effective in inducing active site of each MMP enzyme. As the first anti-MMP drug to be
necrotic cell death.1181,1182 BI2536, a small molecule inhibitor of tested in clinical trials,604 batimastat effectively inhibits breast cancer,
mitotic kinase polo-like kinase 1, induces necroptotic cell death in ovarian cancer, and CRC tumor growth and metastasis in vivo,
prostate cancer cells.1183 although its poor solubility in water hampers its further develop-
Moreover, natural compounds also hold an essential place in ment.604,1202,1203 The subsequent development of marimastat (BB-
necrosis-based cancer therapy. Shikonin, a naturally occurring 2516) is an orally available second-generation synthetic MMP
naphthoquinone, triggers necrotizing cell death, circumvents drug inhibitor.604 Marimasta has been tested in phase III clinical trials to
transporter proteins, and antiapoptotic BCL-2 protein-mediated evaluate its effectiveness in patients with SCLC (NCT00003011), stage
apoptosis resistance.1184,1185 Shikonin inhibits glioma cells,1186 III NSCLC (NCT00002911), and metastatic breast cancer
primary osteosarcoma, and pulmonary metastatic osteosar- (NCT00003010), but no results have been disclosed. In addition,
coma1187 by inducing necrosis. Shikonin analogs such as deoxy- the bryostatins are naturally occurring macrocyclic actone products
shikonin, acetylshikonin, isobutyrylshikonin, beta- that inhibit MMPs by modulating upstream regulators of MMPs.604
dimethylacrylshikonin, isovalerylshikonin, and alpha-methyl-n Tanomastat (BAY12-9566), a bryostatin compound developed by
butylshikonin are able to induce necrosis, thus overcoming tumor Bayer, is a nonpeptide biphenyl MMP inhibitor that is effective
resistances which are mediated by the resistance factors such as against a wide range of tumors.1204,1205 However, a phase III
P-gp, BCL-2 and BCL-XL, MRP1, and BCRP1.1188 Obatoclax (GX15- randomized trial of tanomastat as maintenance therapy in patients
070) is an indole bipyrrole compound antagonizing BCL-2, BCL-XL, with advanced ovarian cancer responsive to primary surgery and
BCL-W, and MCL1, which triggers necrotizing cell death by paclitaxel/platinum-containing chemotherapy has shown that tano-
promoting necrosomes on autophagosomal membranes. Obato- mastat is generally well tolerated but had no impact on PFS or
clax induces nonapoptotic forms of cell death in rhabdomyosar- OS.1206 Other MMPs inhibitors such as COL-3 (NSC-683551),1207
coma cells.1189 In addition, polyphenon E(R), a natural product of neovastat (AE-941),1208 prinomastat (AG3340),1209 BMS-275291,1210
green tea extract, induces endoplasmic reticulum stress and and metastat(COL-3),1211 have been evaluated in clinical trials (Table
causes necrotic death of prostate cancer cells.1190 Staurosporines, 6).
isolated from the bacterium Streptomyces staurosporeus in 1977, In conclusion, MMPs are a large family with different functions
are protein kinase inducers of the intrinsic apoptotic pathway and in tumor cells.1212 Depending on cell and tissue localization,
trigger necrosis in the leukemia cell line U937 when cystatin disease type, etc., MMPs can be used as both drug targets and
proteases are impaired.1191,1192 FTY720, a sphingolipid analog that anti-targets.715 Therefore, given the cell or tumor specificity of
mimics ceramide, induces necroptotic cell death by modulating MMPs, it is important to explore in-depth the contribution of
lipid signaling in glioblastoma cells.1193,1194 5′-Benzylglycinyl- MMPs in tumor progression and metastasis in various tumor types,
amiloride (UCD38B) induces mitochondrial swelling, endoplasmic which will facilitate the rational development of specific MMP
reticulum expansion, and nuclear condensation, and induces a inhibitors.604
nonapoptotic form of cell necrosis in glioma cells.1195
In addition to the above observations, other cell necrosis HGF/c-Met inhibitors. There is an increasing number of studies on
inducers are found to induce necrotic cell death via various HGF/c-MET-targeted therapies, which mainly include anti-HGF/c-
mechanisms, such as selenosemicarbazone metal complexes,1196 MET mAbs and small molecule inhibitors targeting the structural

Zhou et al.
45
Table 6. The typical and clinically developed MMPs inhibitors in cancer therapy
Drug Highest Indications Identifier Status

phase
COL-3 (NSC-683551) I Lymphoma, melanoma, neoplasm metastasis, renal cell carcinoma NCT00001683 Completed
I Unspecified adult solid tumor, protocol specific NCT00003721 Completed
Neovastat (AE-941) III Kidney cancer NCT00005995 Completed
III Adenocarcinoma of the lung, adenosquamous cell lung cancer, large-cell lung NCT00005838 Completed
cancer, squamous cell lung cancer, stage IIIA non-small cell lung cancer, stage IIIB
non-small cell lung cancer
Prinomastat (AG3340) III Lung cancer NCT00004199 Completed
III Prostate cancer NCT00003343 Completed
II Brain and central nervous system tumors NCT00004200 Completed
Marimastat (BB-2516) III Lung cancer NCT00003011 Completed
III Lung cancer NCT00002911 Completed
BMS-275291 II/III Lung cancer NCT00006229 Completed
Metastat(COL-3) I/II Brain and central nervous system tumors NCT00004147 Completed
Source: All the information is derived from ClinicalTrials.gov (https://www.clinicaltrials.gov)
domain of c-MET kinase (Fig. 5 and Table 7). The mechanism of demonstrated that the serious adverse events of foretinib include
action of HGF/c-MET inhibitors is mainly neutralization or abdominal pain (6.25%), dehydration (6.25%), malignant neoplasm
competition with HGF to inhibit receptor dimerization or induce progression (4.17%)(NCT00725712). Tivantinib (ARQ197) is a
c-MET degradation.677 highly selective, non-ATP competitive inhibitor of c-MET.1223
Tivantinib inhibits the autophosphorylation of c-MET in many
c-MET inhibitors: RTK is a cell surface receptor that binds to cancer cells and is highly selective for inactive or non-
growth factor ligands such as HGF, VEGF, EGF, etc., and activates phosphorylated forms of c-MET, thus effectively blocking the
downstream signaling pathways. A number of small molecule TKI activation of c-MET downstream effectors such as RAS, MAPK, and
have been developed that selectively and nonselectively inhibit STAT3, ultimately inhibiting tumor proliferation, invasion and
the catalytic activity of c-MET.1213 Significantly, two c-MET metastasis.1222,1224 Other nonselective c-MET inhibitors include
inhibitors, tepotinib developed by EMD Serono, and capmatinib glesatinib (MGCD-265), golvatinib (E-7050) and merestinib (LY-
developed by Novartis, have been approved by the FDA. Tepotinib 2801653), while selective c-MET inhibitors include tepatinib (EMD-
inhibits MET kinase activity with an IC50 value of 1.7 nM and 1214063), AMG-337 and capatinib (INC-280), which are approved
showed high selectivity for MET in screening against >400 for clinical use or undergoing clinical study.
kinases.1214 On February 3, 2021, the FDA granted accelerated
approval to tepotinib for adult patients with metastatic NSCLC Anti-c-MET antibodies: Onartuzumab, an Escherichia coli-derived,
harboring MET exon 14 skipping alterations.1215 Capmatinib, an humanized mAb against c-MET, can block the high-affinity
ATP-competitive, highly potent (IC50 value of 0.13 nM) and binding of HGFα chain but not HGFβ chain to c-MET.1225
selective MET inhibitor, was granted regular approval by the Preclinical studies found that onartuzumab effectively inhibits
FDA for the treatment of adult patients with metastatic NSCLC human glioblastoma and pancreatic cancer cell growth although
whose tumors have MET exon 14 skipping mutations in May further development of onartuzumab has been halted.1226
2020.1216 In addition, crizotinib (PF-02341066) is an orally Emibetuzumab (LY2875358) is a humanized anti-c-MET bivalent
bioavailable TKI that competitively inhibits the ATP-binding site antibody that effectively promotes internalization and degrada-
of tyrosine kinases and inhibits c-MET, ALK, AXL and TIE2 tion of c-MET, thereby blocking HGF-c-MET binding and HGF-
activity.679 Crizotinib effectively inhibits c-MET phosphorylation induced c-MET phosphorylation.1227 Emibetuzumab combined
and c-MET-dependent proliferation, migration, or invasion of with erlotinib is in a phase II clinical trial to evaluate their efficiency
tumor cells.679,1217 Notably, crizotinib has been approved for as a first-line treatment for patients with metastatic NSCLC with
patients with ALK-positive and ROS1-positive metastatic NSCLC.677 activated EGFR mutations (NCT01897480). LY3164530 is a
Cabozantinib (XL184) is an orally available TKI that targets a bispecific anti-EGFR/c-MET antibody produced by fusing an anti-
variety of kinases including MET, VEGFR2, RET, FLT-3, and KIT.1218 It EGFR single-chain variable fragment to the N-terminal end of the
has shown efficacy in patients with prostate cancer1219 and emibetuzumab heavy chain.1226 LY3164530 disrupts signaling by
advanced RCC.1220,1221 Foretinib (GSK1363089) is an oral and binding and internalizing c-MET and EGFR.1226 Its phase I clinical
potent TKI targeting c-MET and VEGFR2. It binds to the ATP- trial in patients with advanced or metastatic cancer has been
binding pocket of the above kinases and makes kinase completed. However, significant toxicities associated with EGFR
conformational changes. Foretinib has been tested in clinical inhibition and the lack of a potential predictive biomarker limit its
trials in a variety of tumors including papillary renal cancer, gastric future development.1228 Amivantamab (JNJ-61186372) is a bispe-
cancer, and head and neck cancer.1222 A phase II study of foretinib cific EGFR/c-MET antibody that binds the extracellular structural
in adults with HNSCC revealed that 50.0% of participants have domain of each receptor, thereby avoiding resistance at the TKI
stable disease, and 21.4% have progressive disease binding site.1229 Amivantamab effectively inhibits tumors in
(NCT00725764). Other phase II clinical trials of foretinib in solid various contexts, including tumors with T790M second-site
tumors (NCT00742131) and breast cancer (NCT01138384) have resistance mutation in EGFR, c-MET pathway activation,1230 and
been completed, but no results have been posted yet. Moreover, a EGFR exon 20 insertion driver mutations.1229 Amivantamab is
phase II study of foretinib in adults with gastric cancer currently being administered in monotherapy or combination

46
Table 7. The clinically developed HGF/c-MET inhibitors
Type Drug Targets Highest Indications Identifier Status

phase
Anti-c-MET mAbs Emibetuzumab(LY2875358) c-MET II Carcinoma, non-small-cell lung NCT01900652 Completed

II Advanced cancer, gastric adenocarcinoma, gastroesophageal NCT02082210 Completed
junction adenocarcinoma, hepatocellular cancer, renal cell
carcinoma, non-small cell lung cancer
ABT-700 c-MET I Advanced solid tumors NCT01472016 Completed
Onartuzumab c-MET III Solid tumor NCT02488330 Completed
III Non-squamous non-small cell lung cancer NCT01456325 Completed
III Gastric cancer NCT01662869 Completed
III Non-squamous non-small cell lung cancer NCT01887886 Completed
III Non-small cell lung cancer NCT02031744 Completed
LY2875358 c-MET II Gastric cancer NCT01874938 Completed
II Non-small cell lung cancer NCT01897480 Active, not
recruiting
II Non-small cell lung cancer NCT01900652 Completed
I/II Advanced cancer, gastric adenocarcinoma, gastroesophageal NCT02082210 Completed
junction adenocarcinoma, hepatocellular cancer, renal cell
carcinoma, non-small cell lung cancer
Zhou et al.
Anti-HGF mAbs Rilotumumab HGF III Gastric cancer NCT02137343 Terminated

III Gastric cancer NCT01697072 Terminated
III Recurrent squamous cell lung carcinoma, stage IV squamous NCT02926638 Terminated
cell lung carcinoma
II Recurrent fallopian tube carcinoma, recurrent ovarian NCT01039207 Completed
carcinoma, recurrent primary peritoneal carcinoma
Ficlatuzumab HGF II Resistant, recurrent or metastatic head/neck squamous cell NCT03422536 Completed
carcinoma
II Non-small cell lung cancer NCT02318368 Terminated
YYB-101 HGF Ib/IIa Colorectal cancer NCT04368507 Completed
Nonselective TKI (ATP- Capmatinib c-MET IV Non-small cell lung carcinoma NCT05110196 Recruiting
competitive) Tepotinib c-MET II Non-small cell lung cancer NCT03940703 Active, not
recruiting
II Solid tumor, MET exon 14 skipping mutation, MET NCT04647838 Recruiting
amplification
II Advanced (Stage IIIB/IV) non-small cell lung cancer with MET NCT02864992 Active, not
exon 14 skipping alterations or MET amplification lung recruiting
adenocarcinoma stage IIIB/IV
II Colorectal neoplasms NCT04515394 Terminated
II Recurrent lung non-small cell carcinoma, stage IV lung cancer NCT06031688 Not yet

recruiting
II Gastric cancer, gastroesophageal-junction cancer NCT05439993 Recruiting
I/II Advanced non-small cell lung cancer with MET mutations NCT04739358 Recruiting
I/II Non-small cell lung cancer NCT01982955 Completed
I/II Hepatocellular carcinoma NCT02115373 Completed
Table 7. continued
Type Drug Targets Highest Indications Identifier Status
phase
I/II Hepatocellular carcinoma NCT01988493 Completed

II Breast cancer, NCT04591431 Active, not
gastrointestinal cancer, recruiting
non-small cell lung cancer, other cancer
AMG-337 c-MET II Stomach neoplasms NCT02016534 Terminated
II Solid tumor NCT03147976 Withdrawn
II Clear cell sarcoma NCT03132155 Terminated
I/II Stomach neoplasms NCT02096666 Completed
Nonselective TKI Tivantinib c-MET III Hepatocellular carcinoma NCT01755767 Completed
(allosteric) III Non-squamous, non-small cell lung cancer NCT01244191 Terminated
III Non-small cell lung cancer NCT01377376 Terminated
III Liver cancer NCT02029157 Completed

Nonselective TKI (ATP- Crizotinib c-MET, ALK, RON, AXL, TIE2, IV Non-small cell lung cancer, anaplastic large-cell lymphoma, NCT05160922 Recruiting
competitive) ROS1 inflammatory myofibroblastic tumor
IV Anaplastic lymphoma kinase or ROS1-positive non-small cell NCT03672643 Active, not
lung cancer recruiting
IV Systemic anaplastic large-cell lymphoma NCT02487316 Withdrawn
Cabozantinib c-MET, c-RET, VEGFR1-3, c-Kit, IV Hepatocellular carcinoma NCT03963206 Completed
FLT-3, TIE2, TRKB, AXL IV Medullary thyroid cancer NCT01896479 Active, not
recruiting
Foretinib c-MET, VEGFR2, RON, ERK, AKT, II Recurrent breast cancer NCT01147484 Completed
PDGFRβ, c-Kit, TIE2 II Neoplasms, head and neck NCT00725764 Completed
Zhou et al.
II Carcinoma, renal cell NCT00726323 Completed

II Neoplasms, gastrointestinal tract NCT00725712 Completed
I/II Breast cancer NCT01138384 Completed
I/II Lung cancer NCT01068587 Completed
Glesatinib c-MET, AXL II Non-small cell lung cancer NCT02954991 Completed
Golvatinib c-MET, VEGFR2, RON, Eph, c-Kit I/II Advanced solid tumors NCT01433991 Terminated
I/II Platinum-resistant squamous cell carcinoma of the head and NCT01332266 Completed
neck
Merestinib c-MET, MST1R, FLT-3, AXL, II Carcinoma, non-small-cell lung, solid tumor NCT02920996 Active, not
MERTK, TEK, ROS1, NTRK1/2/3, recruiting
DDR1/2, MKNK1/2 II Biliary tract cancer, metastatic cancer, advanced cancer NCT02711553 Active, not
recruiting
47
Zhou et al.
48
with various drugs for cancer treatment in 15 clinical trials HGF for binding to MET. NK4 effectively inhibits neovasculariza-
(NCT04077463, NCT02609776, NCT05845671, and NCT05653427). tion, growth, invasion, and metastasis of many tumor cells.1243,1244
Encouragingly, amivantamab has been approved for marketing for The HGF/c-MET pathway serves a critical role in cancer and is an
the treatment of patients with metastatic NSCLC with EGFR exon attractive therapeutic target for cancer therapy. Over the past
20 insertion mutations and platinum-based chemotherapy resis- decade, great efforts have been devoted to the development of
tance (NCT04599712). selectively c-MET inhibitors. Although small molecule c-MET
SAIT301 is a monoclonal humanized antibody developed by inhibitors and antibody-based drugs have shown meaningful
Samsung that promotes c-MET degradation.1226,1231 SAIT301 clinical efficacy, the challenges of resistance and side effects
inhibits nasopharyngeal cell invasion and migration by down- remain to be addressed. The c-MET amplification and over-
regulating EGR-1 via the degradation of c-MET.1232 Its clinical expression, c-MET mutations, the activation of parallel signaling
phase I trial in c-MET-overexpressed metastatic CRC has been pathways, and the induction of HGF secretion are associated with
completed, and the most common adverse effects were acquired resistance after initial response to HGF/c-MET inhibitors.
decreased appetite (50.0%), hypophosphatemia, fatigue, and Therefore, how to overcome the acquired resistance as well as
dizziness (25.0%), diarrhea, and dyspnea (18.8%).1233 ABT-700 improve the safety of c-MET inhibitors needs to be solved
(h224G11) is a humanized bivalent mAb that inhibits c-MET urgently.1245 Moreover, stratifying patients appropriately based on
dimerization and activation. A phase I clinical trial of ABT-700 in the discovery of biomarkers may help identify the subgroups of
subjects with advanced solid tumors containing MET amplification patients who can benefit from anti- HGF/c-MET therapy.
or c-MET overexpression (NCT01472016) has been completed.
ARGX-111 is an afucosylated IgG1 antibody that competitively Targeting DDR pathways
binds c-MET, inhibits c-MET activity, and downregulates c-MET Multiple DDRs related small molecule inhibitors have been
expression on the cell surface.1234 A clinical phase I trial of ARGX- approved for clinical use or are under clinical investigation,
111 in patients with advanced cancer overexpressing c-MET has including PARP inhibitors, ATM inhibitors, ATR inhibitors, and
been completed (NCT02055066). In addition. DN30 is a mono- CHK1 inhibitors (Table 8). Herein we mainly focus on the inhibitors
valent chimeric Fab that induces the cleavage of the extracellular of PARP, ATM, ATR, and CHK1.
portion of c-MET, leading to the shedding of its ectodo-
main.1235,1236 DN30 inhibits tumor growth in human gastric PARP inhibitors. PARP inhibitors are selective for targeting tumors
cancer, lung cancer, and glioblastoma.1226 deficient in the HR DNA repair factor BRCA1 or BRCA2 (BRCA1/
2)741 or compromised HR.733 Six PARP inhibitors are currently
Anti- HGF antibodies: Rilotumumab (AMG-102), an anti-HGF approved for the clinical treatment of cancer patients including
mAb binding to HGFβ chain structural domain, specifically blocks the specific subgroups with BRCA1/2 mutation: olaparib, ruca-
the activation of c-MET.1237 In particular, rilotumumab selectively parib, niraparib, talazoparib, fuzuloparib, and pamiparib733 (Table 9).
alters the mature HGF, but shows no effect on the proteolytic Olaparib is the first PARP inhibitor introduced into clinical
activation process of pro-HGF.1238 To date, rilotumumab alone or practice.1246 In 2014, olaparib was approved for the treatment of
in combination with other anticancer drugs, such as antiangio- patients with BRCA1/2-mutated metastatic ovarian cancer who
genic agents, EGFR inhibitors, and chemotherapeutic agents, has had received three or more prior lines of chemotherapy.
been studied in clinical trials in patients with various solid tumors Subsequently, in 2016, rucaparib, a second PARP inhibitor, was
such as prostate cancer, kidney cancer, and advanced authorized for the treatment of patients with advanced-stage
NSCLC.677,1239 Ficlatuzumab (AV-299), a humanized anti-HGF ovarian cancers harboring deleterious BRCA1/2 mutations who
antibody, has been studied in clinical trials as a monotherapy or had received two or more prior lines of chemotherapy. In 2019,
in combination with chemotherapeutic agents in patients with niraparib was approved for the treatment of patients with HR-
advanced pancreatic cancer (NCT03316599) and HNSCC deficient advanced-stage ovarian cancers who had received three
(NCT02277197). YYB-101 is a humanized HGF antibody which or more prior chemotherapy regimens.1246 Recently, in March
inhibits c-MET activation by binding to the HGFα chain.1226 A 2022, olaparib was approved by the FDA for the adjuvant
clinical phase I trial of YYB-101 in patients with refractory treatment of patients with hereditary BRCA1/2 mutations and
advanced solid tumors (NCT02499224) has shown that YYB-101 HER2- high-risk early breast cancer1247 as well as for the
exhibited favorable safety and efficacy in patients with refractory maintenance treatment of patients with BRCA1/2-mutated ovarian
solid tumors. A clinical phase Ib/ IIa trial of YYB10 in combination cancers who are in complete or partial remission after platinum-
with irinotecan in patients with metastatic or recurrent CRC based chemotherapy.1248 Moreover, PARP inhibitors are also used
(NCT04368507) has been completed, but no results are currently in patients with germline or somatic BRCA1/2-mutated ovarian
available. cancer as maintenance therapy (olaparib)1249 or post-
chemotherapy therapy (olaparib and rucaparib).1250 The FDA has
Antibody mimetic engineered protein against HGF: MP0250, an approved olaparib and talazoparib for the treatment of advanced
ankyrin repeat protein capable of neutralizing VEGF and HGF, or metastatic HER2- breast cancer patients carrying deleterious
effectively inhibits multiple myeloma-mediated osteolysis and germline BRCA1/2 mutations.1251,1252 Olaparib is also used for
myeloma cell invasion.1226 Meanwhile, MP0250 can effectively maintenance therapy in patients with germline BRCA1/2-mutated
improve bortezomib efficacy without increasing toxicity, suggest- metastatic pancreatic cancer.1253 Meanwhile, rucaparib has been
ing that MP0250 combined with cytotoxic therapy may be a applied for second-line treatment of patients with metastatic
promising therapeutic approach.1240 A phase II clinical evaluation castration-resistant prostate cancer with germline or somatic
of MP0250 in combination with bortezomib and dexamethasone BRCA1/2 mutations.1254 Significantly, PARP inhibitors have been
in patients with multiple myeloma (NCT03136653) has been approved as first-line systemic therapies for patients with ovarian
completed, although the result has not yet been disclosed. cancer.1246 However, acquired resistance to PARP inhibition is still
an urgent question, which usually results from three types of
Competitive analogs of HGF: NK4 is a synthetic intramolecular mechanisms: drug target-related effects including the upregula-
fragment of HGF, originally purified as a fragment from elastase- tion of drug efflux pumps or mutations of PARP or functionally
digested HGF samples.1241 NK4 contains the HGF α-chain related proteins; restoration of BRCA1/2 function leading to
N-terminal hairpin domain and 4 kringle domains (K1–K4),1242 restoration of HR; or loss of DNA end-protection and/or restoration
and lacks the 16 amino acids of the HGF C-terminus.1226 NK4 of replication fork stability.1246 Therefore, targeted strategies to
inhibits c-MET phosphorylation and activation by competing with overcome resistance to PARP inhibitors remain to be explored,

Table 8. The clinically developed DDR inhibitors in cancer therapies
Type Drug Highest phase Indications Identifier Status
ATM inhibitor AZD0156 I Advanced solid tumors NCT02588105 Completed

AZD1390 I Recurrent glioblastoma multiforme, primary glioblastoma multiforme, brain neoplasms, NCT03423628 Recruiting
malignant, leptomeningeal disease
I Glioblastoma, glioma, glioblastoma multiforme, glioma, malignant NCT05182905 Recruiting
I Solid tumor, metastatic solid tumor, solid carcinoma, solid tumor, adult, metastatic NCT05678010 Recruiting
tumor, metastatic cancer
I soft tissue sarcoma adult NCT05116254 Recruiting
I non-small cell lung cancer NCT04550104 Recruiting
M4076 I Advanced solid tumors NCT04882917 Completed
I Metastatic or locally advanced unresectable solid tumors NCT05396833 Recruiting
ATM and DNA-PKcs XRD-0394 I Metastasis, locally advanced solid tumor, recurrent cancer NCT05002140 Active, not recruiting
inhibitor

ATR inhibitor ART0380 II Advanced solid tumor, recurrent endometrial cancer, metastatic cancer NCT05798611 Recruiting
II Advanced cancer, metastatic cancer, ovarian cancer, primary peritoneal cancer, fallopian NCT04657068 Recruiting
tube cancer
ATRN-119 II Advanced solid tumor NCT04905914 Recruiting
BAY1895344 I Advanced solid tumor NCT04095273 Completed
I Advanced solid tumor, non-hodgkin’s lymphoma, mantle cell lymphoma NCT03188965 Recruiting
I Advanced solid tumors (excluding prostate cancer), ovarian cancer NCT04267939 Recruiting
Berzosertib (VX-970, M6620, II Small cell lung cancer NCT04768296 Completed
VE-822) II Small cell lung cancer, small cell cancer, advanced solid tumor, high grade NCT04802174 Recruiting
neuroendocrine cancers
II small cell lung cancer, advanced solid tumors NCT04826341 Recruiting
Zhou et al.
II Lung non-small cell squamous carcinoma, stage IV lung cancer NCT04216316 Recruiting
II Leiomyosarcoma, adult NCT04807816 Recruiting
I Advanced solid tumor NCT05246111 Completed
II Castration-resistant prostate carcinoma, metastatic prostate carcinoma, stage IV NCT03517969 Active, not recruiting
prostate cancer
II Ovarian serous tumor, recurrent fallopian tube carcinoma, recurrent ovarian carcinoma, NCT02595892 Active, not recruiting
recurrent primary peritoneal carcinoma
II Bladder small cell neuroendocrine carcinoma, extensive stage lung small cell carcinoma, NCT03896503 Active, not recruiting
extrapulmonary small cell neuroendocrine carcinoma, limited stage lung small cell
carcinoma, platinum-resistant lung small cell carcinoma, platinum-sensitive lung small
cell carcinoma, prostate small cell neuroendocrine carcinoma, recurrent lung small cell
carcinoma
II Metastatic malignant solid neoplasm, refractory malignant solid neoplasm, NCT04266912 Active, not recruiting
unresectable malignant solid neoplasm
II Metastatic bladder urothelial carcinoma, metastatic renal pelvis and ureter urothelial NCT02567409 Active, not recruiting
carcinoma, metastatic ureter urothelial carcinoma, stage IV bladder urothelial carcinoma
Ceralasertib (AZD6738) III Advanced or metastatic non-small cell lung cancer NCT05450692 Recruiting
IMP9064 I Solid tumor, advanced solid tumor NCT05269316 Recruiting

M4344 II Ovarian cancer recurrent NCT04149145 Withdrawn (never
opened)
49
50
Table 8. continued
Type Drug Highest phase Indications Identifier Status
I Solid tumor, advanced solid tumor NCT02278250 Completed

RP-3500 II Advanced solid tumor, adult NCT04972110 Recruiting
II Solid tumor, metastatic cancer NCT05566574 Recruiting
I Advanced solid tumor NCT04855656 Recruiting
II Advanced solid tumor NCT04497116 Recruiting
CHK1 inhibitor Prexasertib (LY2606368) II Small cell lung cancer NCT02735980 Completed
II Ovarian cancer NCT03414047 Completed
II Triple-negative breast cancer NCT04032080 Completed
II Advanced cancers NCT02873975 Completed
II Desmoplastic small round cell tumor, rhabdomyosarcoma NCT04095221 Active, not recruiting
II Platinum-resistant ovarian cancer, endometrial adenocarcinoma, urothelial carcinoma NCT05548296 Recruiting
SRA737 II Advanced solid tumors or non-hodgkin’s lymphoma NCT02797964 Completed
II Advanced solid tumors NCT02797977 Completed
LY2880070 II Ewing sarcoma, Ewing-like sarcoma NCT05275426 Recruiting
II Solid tumors, colorectal cancer, breast cancer, ovarian cancer, colon cancer, rectal NCT02632448 Recruiting
cancer, neoplasms, endometrial cancer, soft tissue sarcoma, triple-negative breast
cancer, pancreatic cancer
II Castration-resistant prostate carcinoma, metastatic malignant neoplasm in the lymph NCT04071236 Recruiting
Zhou et al.
nodes, metastatic prostate carcinoma, stage IV prostate cancer

II Locally advanced rectal cancer NCT03770689 Completed
II Cholangiocarcinoma, gallbladder carcinoma, malignant solid neoplasm NCT04068194 Suspended

Zhou et al.
51
Table 9. The FDA-approved PARP inhibitors in cancer therapies
Drug Company Indications FDA approvals
Olaparib (Lynparza) AstraZeneca Ovarian (2014) Olaparib capsules in patients with BRCA1/2 mutant advanced-stage ovarian cancers who
have received ≥3 types of chemotherapies
Ovarian (2017) Maintenance therapy for advanced -ovarian cancer patients with PR or CR to platinum-
based chemotherapy
Ovarian (2018) First-line maintenance therapy for patients with BRCA1/2 mutant advanced-stage ovarian
cancers
Breast (2018) Patients with BRCA1/2 mutant HER2-negative metastatic breast cancer who have been
treated with chemotherapy
Breast (2022) Patients with BRCA1/2 mutant HER2-negative high-risk early breast cancer who have been
treated with adjuvant chemotherapy
Pancreatic (2019) Adult patients with germline BRCA-mutated metastatic pancreatic adenocarcinoma
Prostate (2020) Adult patients with HRR gene mutated metastatic castration-resistant prostate cancer
Rucaparib (Rubraca) Clovis Oncology Ovarian (2016) Patients with BRCA1/2-mutant ovarian cancer refractory to ≥ prior lines of treatment
Ovarian (2018) Maintenance treatment of patients with recurrent ovarian cancer
Prostate (2020) BRCA-mutated metastatic castration-resistant prostate cancer
Niraparib Tesaro Ovarian (2019) Patients with HR deficiency -positive, advanced ovarian cancer
Ovarian (2020) First-line maintenance treatment of patients with advanced ovarian cancer
Talazoparib Pfizer Breast (2018) Patients with germline BRCA-mutated, HER2-negative locally advanced or metastatic
breast cancer
Source: All the information is derived from the United States Food and Drug Administration.gov (https://www.fda.gov/)
and the identification of vulnerabilities of PARP inhibitor-resistant pharmacokinetic properties.1263 Phase II clinical trials of cerala-
tumors is still challenging. Illustrating the properties of HR- sertib in patients with osteosarcoma (NCT04417062) and
deficient cancers will rationalize the treatment strategies to advanced solid tumors (NCT04564027) are undergoing. Berzoser-
overcome resistance and improve the survival of patients.1246 tib (VX-970) is a highly potent, selective, and intravenous ATR
inhibitor with an IC50 value of 19 nM.1264 A phase II clinical trial of
ATM inhibitors. ATM is the apical DDR kinase that coordinates berzosertib in patients with NSCLC (NCT04216316) is ongoing.
DSB repair, and a variety of compounds have been developed for BAY1895344, another novel potent and selective ATR inhibitor,
selective inhibition of ATM.733 AZD0156 is a potent, selective, and exhibits strong monotherapy efficacy in cancers, and synergistic
orally active inhibitor of ATM,1255 and a phase I clinical trial of activity in combination with DNA damage therapies.1265 A phase II
AZD0156 (NCT02588105) for safety and preliminary efficacy in clinical trial of BAY1895344 in patients with recurrent solid tumors
advanced solid tumors has been completed, but no results have (NCT05071209) is in progress. Other ATR inhibitors, such as
been posted. AZD1390, belonging to the same potent series as ART0380, ATRN-119, IMP9064, M4344, and RP-3500, are also in
AZD0156, is an exquisitely potent, highly selective, and orally clinical trials. Studies also have found synergistic antitumor effects
bioavailable ATM inhibitor.1256 AZD1390 effectively sensitizes the of ATR inhibitors with immunotherapy and other anticancer
brain metastasis of breast cancers with DDR mutation to radiation drugs.733 ATR inhibitors in combination with PARP inhibitors are
therapy.1257 Multiple clinical trials of AZD1390 for cancer used in the treatment of tumors with BRCA1/2 mutations.733,1266
treatment are ongoing. M4076, an ATP-competitive ATM inhibitor Exploiting combination therapy based on ATR inhibitors may be a
with an IC50 value <1 nM, inhibits tumor cell growth by blocking promising strategy for cancer therapy.
DSB repair and enhances the sensitivity of tumor cells to radiation
therapy both in vitro and in vivo.1258,1259 A phase I clinical trial of CHK1 inhibitors. CHK1, a downstream effector of ATR, is activated
M4076 in advanced solid tumors is active (NCT04882917). The by DDR, and its inhibitors effectively suppress the proliferation of
dual ATM and DNA-PK inhibitor XRD-0394 is a novel, potent, and cancer cells with high levels of replication stress.1267 Some CHK1
orally active dual inhibitor.733 A phase I clinical trial of XRD-0394 inhibitors have been evaluated or are currently under evaluation
for the treatment of metastatic locally advanced solid tumors and in clinical trials, especially in combination with DNA damaging
recurrent cancer is recruiting, but no data are yet publicly available agents such as gemcitabine, cisplatin, and camptothecin.733,1268
(NCT05002140). LY2603618 is the first selective and potent CHK1 inhibitor.1269
Given the important role of ATM in DSB signaling and repair, However, the phase II evaluations of LY2603618 in combination
ATM inhibition combination therapy is currently an attractive with pemetrexed in patients with advanced NSCLC revealed no
strategy for cancer therapy in various clinical trials. ATM inhibitors significant clinical activity and increased risk of thromboembolic
enhance the anticancer activity of DNA damage agents such as events, which hindered its further development (NCT00988858,
topoisomerase inhibitors1260 and PARP inhibitor.1261 Collectively, NCT01139775).1270,1271 MK-8776 (SCH900776) is a selective CHK1
ATM-targeted therapy has a promising potential in cancer therapy. inhibitor that induces cell death when it combines with
antimetabolite drugs, such as hydroxyurea, gemcitabine, or
ATR inhibitors. ATR kinase maintains accurate DNA replication by pemetrexed in xenograft models.1269 However, clinical trials of
regulating the DNA replication initiation and the process of MK-8776 combined with gemcitabine or cytarabine in patients
replication forks, supporting that ATR is an important target for with solid tumors or hematological malignancies have been
cancer therapy. To date, several ATR inhibitors have been completed and the results have shown no positive efficacy but
developed.733,1262 Ceralasertib (AZD6738) is a selective and potent some adverse events such as mucositis, nausea, and prolonged QT
ATR inhibitor with good solubility, bioavailability, and interval (NCT01870596, NCT00779584). LY2880070 is an oral,

Zhou et al.
52
selective competitive CHK1 inhibitor,1272 and phase II studies in IL-6 antibody may exert therapeutic efficacy and benefit to
patients with solid tumors (NCT02632448) and Ewing sarcoma cancer patients. Tocilizumab, a recombinant humanized mAb
(NCT05275426) are in progress. In addition, LY2606368 (prexa- against the human IL-6 receptor, specifically binds to soluble and
sertib) is a CHK1/2 dual inhibitor,1269,1273 and a phase I/II study to membrane-bound IL-6 receptor and inhibits signal transduction.
evaluate the efficacy and safety of LY2606368 in combination with Multiple clinical trials of tocilizumab for the treatment of patients
irinotecan and temozolomide in participants with desmoplastic with refractory AML (NCT04000698), NHL (NCT05171647), diffuse
small round cell tumors and rhabdomyosarcoma (NCT04095221) is large B-cell lymphoma (NCT04408638), and relapsed or refrac-
ongoing. SRA737, an orally bioavailable and selective CHK1 tory follicular lymphoma (NCT04712097) are ongoing. In addi-
inhibitor, exhibits preclinical activity in MYC-amplified models of tion, siltuximab, a mAb against the human IL-6 receptor, was
neuroblastoma and lymphoma.1274 Its phase II clinical trials in approved by the FDA for treating patients with multicentric
patients with advanced solid tumors or NHL (NCT02797964) and in Castleman disease on April 22, 2014. Its most common adverse
patients with advanced solid tumors (NCT02797977) have been events include pruritus, increased weight, rash, hyperuricemia,
completed. The results have shown that SRA737 is well tolerated. and upper respiratory tract infection.1281 To date, clinical trials of
However, further clinical development of SRA737 needs to be siltuximab are being conducted to evaluate the efficacy for the
performed in combination therapy due to its poor monotherapy prevention of CAR-T cell related cytokine release syndrome in
activity.1274 AZD7762, another ATP-competitive CHK1/2 dual patients with NHL (NCT05665725), for the treatment of patients
inhibitor, suppresses the CHK1-mediated phosphorylation of with metastatic pancreatic cancer (NCT04191421), large granular
CDC25C with an IC50 value of 5 nM. However, phase I clinical lymphocytic leukemia (NCT05316116), and multiple myeloma
trials of AZD7762 have been terminated due to its cardiac toxicity (NCT03315026).
and adverse effects.1269,1275 The significant role of the Janus kinase/signal transducers and
In summary, although CHK1 inhibitors are beneficial in activators of transcription (JAK/STAT) signaling pathway in
preclinical studies, their clinical benefit remains to be confirmed. cancer suggests targeting this pathway as a potential anticancer
Studies have found that p53 mutation status may be a key factor strategy.1276 Inhibition of the JAK/STAT signaling pathway has
in affecting the cell sensitivity to CHK1 inhibitors, suggesting that been demonstrated to downregulate cellular proliferation and
biomarkers affecting the efficacy of CHK1 inhibitors need to be survival, decrease stem cell properties and inflammatory
identified.1269 Moreover, the development of novel CHK1 inhibi- response, suppress invasion and metastasis, ameliorate immu-
tors with reduced toxicity is meaningful in the near future. nosuppress in malignant tumors.1282 Ruxolitinib is a first-in-class,
potent, ATP-competitive, and small molecule JAK1/2 inhibitor
Targeting tumor inflammation pathways (IC50 = 3.3 nM for JAK1, 2.8 nM for JAK2) developed by Incyte
Inflammation is considered to be one of the key characteristics Corp.1283 Following its approval by the FDA for the treatment of
of tumor initiation, progression, invasion, metastasis, and diseases, such as multiple sclerosis and vitiligo, the potential of
treatment resistance.299 The drugs modulating tumor inflamma- ruxolitinib in cancer therapy has garnered widespread interest.
tion pathways mainly include nonspecific agents, such as Several clinical trials of ruxolitinib in patients with pancreatic
nonsteroidal anti-inflammatory drugs (NSAIDs), statins, and cancer, breast cancer, relapsed or refractory or post myelopro-
corticosteroids, and targeted drugs, such as neutralizing liferative AML, HNSCC, or NSCLC were either terminated or
antibodies, small molecule inhibitors, and recombinant cyto- completed with results suggesting insufficient efficacy to justify
kines1276 (Table 10). NSAIDs, including aspirin, celecoxib, and further investigation. There are still multiple clinical trials
ibuprofen, mainly exhibit anticancer efficiency by inhibiting COX undergoing to evaluate ruxolitinib monotherapy or in combina-
activity and prostaglandin synthesis. NSAIDs have been dis- tion with decitabine for the treatment of accelerated/blast phase
covered to reduce cancer mortality, and have shown good myeloproliferative neoplasms (NCT04282187), with trametinib
therapeutic and preventive effects in cancer patients with CRC, for CRC and pancreatic adenocarcinoma (NCT04303403), with
breast cancer, prostate cancer, and head and neck cancer.1277 paclitaxel and carboplatin for stage III-IV epithelial ovarian and
Aspirin, one of the most widely used and typical anti- primary peritoneal cancer (NCT02713386), and with preoperative
inflammatory drugs, has been applied as a broad-spectrum chemotherapy for triple-negative inflammatory breast cancer
cancer-preventive agent. Phase III clinical trials of aspirin for the (NCT02876302), etc. Pacritinib, a potent inhibitor of JAK2 and
treatment of patients with gastric cancer (NCT04214990) and FLT-3 with the IC50 values of 23 and 22 nM, respectively, was
CRC (NCT02467582) are underway. Celecoxib, a COX-2 inhibitor, granted accelerated approval by the FDA on February for the
reveals anticancer activity in CRC, breast cancer, prostate cancer, treatment of adult patients with intermediate or high-risk
and head and neck cancer.1278 A phase IV trial of celecoxib as primary or secondary myelofibrosis.1284 Although some clinical
adjuvant therapy to chemotherapy in subjects with metastasis trials of pacritinib, such as those for the treatment of
CRC (NCT03645187) is ongoing. However, long-term treatment myeloproliferative neoplasmsrefractory CRC or AML, have been
with NSAIDs can lead to side effects including mucosal lesions, affected by increased side effects. Several clinical trials are still
bleeding, and peptic ulcers. Therefore, balancing the benefits of undergoing, including pacritinib in combination decitabine or
taking NSAIDs for the prevention and treatment of cancers is the treatment of accelerated/blast phase myeloproliferative
essential.1276 Statins, consisting of a series of compounds like neoplasms (NCT04282187), prostate cancer (NCT04635059),
rosuvastatin, can reduce blood cholesterol concentration by relapsed/refractory T-cell lymphoproliferative neoplasms
inhibiting the 3-hydroxy-3-methylglutaryl coenzyme A (HMG- (NCT04858256). Moreover, other JAK inhibitors such as itacitinib,
CoA) reductase. Statins exert a significant role in antiangiogenic STAT3 inhibitors like OPB-31121, and TTI-101, and STAT3
and anti-inflammatory therapy in preclinical studies. However, antisense oligonucleotide danvatirsen (AZD9150) have been
their clinical benefits remain to be further confirmed.1279 evaluated or are under investigation for their anticancer
Corticosteroids, such as dexamethasone which is usually used potential. It is important to note that the clinical studies
as anti-inflammatory drugs for various chronic inflammatory targeting the JAK/STAT pathway have revealed the complexity
diseases, are found to improve the efficacy of chemotherapy for of this approach and have underscored the necessity for in-
glioma, breast cancer, lung cancer, and CRC in preclinical depth investigation to combat cancer more effectively.
studies.1280 The strategies that specifically target inflammation Inhibition of NF-κB is also an effective way to slow down tumor
pathways mainly include neutralizing antibodies, small molecule development and induce apoptosis in cancer cells. However, NF-
inhibitors, and recombinant cytokines.1276 As chronic inflamma- κB deficiency can lead to severe immunodeficiency and long-term
tion cytokine IL-6 plays a pivotal role in cancer progression, the inhibition of NF-κB causes serious side effects, suggesting that an

Zhou et al.
53
Table 10. The clinically anti-inflammatory inhibitors in cancer therapies
Drug Target Highest phase Indications Identifier Status
Aspirin COX-1/2 III Gastric cancer NCT04214990 Recruiting

III Colon cancer NCT02467582 Active, not
recruiting
Celecoxib COX-2 IV Colon cancer stage NCT03645187 Recruiting
IV Hepatocellular carcinoma NCT02961998 Completed
IV Bile duct cancer, pancreatic cancer NCT01111591 Unknown
IV Colorectal cancer NCT00473980 Completed
Rosuvastatin HMG-CoA IV Prostate cancer metastatic NCT04776889 Completed
Dexamethasone Undefined IV Metastatic prostate cancer NCT03432949 Recruiting
IV Cancer NCT02815319 Completed
IV Early-stage breast cancer NCT03348696 Completed
IV Pancreatic cancer NCT04025840 Recruiting
IV Lung cancer NCT02275702 Completed
IV Multiple myeloma NCT01731886 Completed
IV Ovarian cancer NCT00817479 Completed
IV Nasal and nasal-type NK/T-cell lymphoma NCT01501149 Unknown
IV Relapsed refractory multiple myeloma NCT03934684 Active, not
recruiting
IV Peripheral T cell lymphoma NCT03071822 Unknown
IV Hemophagocytic syndrome T/NK-cell lymphoma NCT04999878 Recruiting
Not Applicable Mammary cancer NCT05408676 Completed
IV Primary CNS lymphoma NCT01960192 Unknown
IV PH+ acute lymphoblastic Leukemia NCT02690922 Unknown
Tocilizumab IL-6R-specific III Refractory acute myeloid leukemia Refractory acute NCT04000698 Unknown
antibody lymphoblastic leukemia
III Non-hodgkin lymphoma NCT05171647 Recruiting
III Diffuse large B-cell lymphoma NCT04408638 Recruiting
III Relapsed or refractory follicular lymphoma NCT04712097 Recruiting
Siltuximab anti-IL-6 antibody II Lymphoma, non-Hodgkin, multiple myeloma acute NCT04975555 Recruiting
lymphoblastic leukemia
II Multiple myeloma AL amyloidosis NCT03315026 Active, not
recruiting
II High-risk smoldering multiple myeloma NCT01484275 Completed
II Multiple myeloma NCT00402181 Completed
II Prostate cancer NCT00433446 Completed
II Myeloma NCT01531998 Completed
II Carcinoma, renal cell NCT00265135 Completed
II Ovarian neoplasms, pancreatic neoplasms, colorectal NCT00841191 Completed
neoplasms, head and neck neoplasms, lung neoplasms
II Metastatic pancreatic adenocarcinoma, stage IV pancreatic NCT04191421 Completed
cancer AJCC v8
Itacitinib CXCR4 IV Lymphoma NCT05510544 Recruiting
IV Non-hodgkin’s lymphoma NCT01164475 Completed
Ruxolitinib JAK1/2 IV Hemophagocytic syndrome, T/NK-cell lymphoma NCT04999878 Recruiting
Pacritinib JAK2 II T-Cell neoplasm lymphoproliferative disorders NCT04858256 Recruiting
II Prostate cancer NCT04635059 Recruiting
II Breast cancer NCT04520269 Unknown
Bortezomib NF-κB IV Multiple myeloma NCT02268890 Completed
Source: All the information is derived from the United States Food and Drug Administration.gov (https://www.clinicaltrials.gov)
appropriate dosage regimen and administration time will facilitate Targeting tumor cell metabolism
NF-κB targeted therapy in the clinic.764 Moreover, multiple other The precedent of targeting the metabolism of cancer cells was
inhibitors such as antibodies, cytokines and chemokines inhibitors, created by Sydney Farber and colleagues in the 1940s, who
and inhibitors of inflammatory transcription factors, are in clinical successfully used antifolate agents such as aminopterin to induce
trials and the results are eagerly anticipated. remission in childhood ALL.1285 This discovery led to the

Zhou et al.
54
development of chemotherapy drugs including methotrexate, 5- capecitabine for PIK3CA mutant CRC (NCT02861300), with temo-
FU, gemcitabine, and pemetrexed which are widely used to treat zolomide for IDH-mediated diffuse astrocytoma (NCT03528642),
various types of cancers by targeting one-carbon metabolic with carfilzomib and dexamethasone for recurrent or refractory
pathways and their downstream effectors, such as nucleotide multiple myeloma (NCT03798678), and with chemoradiation for
metabolism. However, these drugs exhibit many deleterious side advanced cervical cancer (NCT05521997). Sirpiglenastat (DRP-104),
effects due to their nonspecific effect and the importance of one- a glutamine analog that broadly targets glutamine metabolism, is
carbon pathways in healthy cells.1286 under early-phase clinical trials for examining its efficacy as a single
Sixty years after Sidney Farber introduced antifolates for the agent or in combination with immune checkpoint inhibitors for
treatment of childhood ALL, aberrations in cancer metabolism advanced cancer (NCT04471415 and NCT06027086).1292 The results
attracted much attention and have been extensively studied, from the above trials are still pending.
although nearly one century has passed since Otto Warburg
discovered aerobic glycolysis in cancer cells in the 1920s. Targeting fatty acid metabolism. Cancer cells rely on de novo
However, therapeutic progress in targeting cancer metabolism fatty acid synthesis for proliferation; thus, cancer cells are
remains limited and only a few metabolism-based drugs have expected to be vulnerable to the inhibition of fatty acid synthetic
been developed, and entered clinical trials for cancer therapy.1286 enzymes. Inhibitors targeting fatty acid synthase, a key enzyme for
de novo fatty acid synthesis, have been developed1293 (Fig. 6).
Targeting glycolysis. As glucose supplies the major source of Candidates such as TVB316 and TVB2640 have been demon-
energy, carbon intermediates and NADH for biosynthesis, targeted strated to be effective and less toxic than their predecessors, and
inhibition of glucose uptake and utilization in cancer cells is a more than 10 clinical trials of TVB2640 for the treatment of NSCLC,
promising therapeutic strategy1287 (Fig. 6). Multiple inhibitors prostate cancer, and HER2-positive metastatic breast cancer are
against glycolytic enzymes and glycolytic product transporter underway (NCT03808558, NCT03179904, and NCT05743621). The
proteins have been studied, such as GLUT1 inhibitors (STF-31, field is anxiously awaiting the results of these studies, decades
glutor, and BAY-876), HK2 inhibitors (2-deoxyglucose, benitroben- after fatty acid synthase was identified as a potential cancer
razide), PKM2 inhibitors (TEPP-46 and mitapivat), LDHA inhibitors therapeutic target. ATP-citrate lyase (ACLY), a key enzyme for fatty
(GNE-140, NCI-006, and GSK28387808A), and MCT-1 inhibitors acid chain elongation, converts citrate acetyl-CoA into the cytosol.
(AZD3965).1286–1288 Although these inhibitors have exhibited The ACLY inhibitor bempedoic acid was approved by the FDA in
potent anticancer efficiency in various cancers both in vitro and 2020 as a lipid-lowering drug.1294 Furthermore, a series of
in vivo preclinically, only a few of them have entered clinical trials, allosteric ACLY inhibitors with low (nanomolar) competitive
and no clinical success has been achieved thus far due to limited inhibitory activity were discovered, such as the allosteric inhibitor
efficacy and toxicity. For example, the MCT-1 inhibitor AZD3965 NDI-091143, which binds to homotetramer ACLY, shows potent
blocks lactate-mediated tumor progression and has significant inhibition and is competitive with citrate and noncompetitive with
anticancer effects alone or combined with metformin. However, a ATP.1295 PF-05221304, an orally administered inhibitor of acetyl-
phase I clinical trial in patients with advanced cancers including CoA carboxylases (ACC1 and ACC2), is currently undergoing
diffuse large B-cell lymphoma (DLBCL) and Burkitt’s lymphoma, clinical studies (NCT03248882) in nonalcoholic fatty liver disease
has shown that a number of patients experienced dose-limiting with fibrosis. Its potential in cancer therapy needs to be further
AZD3965 related toxicities such as hematological, cardiac, and evaluated. ND-646, another allosteric inhibitor of ACC1 and ACC2,
ophthalmic toxicities (NCT01791595). reduces tumor growth in NSCLC subcutaneous xenografts,
suggesting potential avenues for therapeutic application.1291
Targeting amino acid metabolism. Similarly, amino acids, espe-
cially glutamine, participate in various cellular processes in cancer Targeting mitochondria metabolism. The pivotal role of mito-
progression, which provide a major source of energy, cell chondria as metabolic and biosynthetic organelles makes them
component building blocks, and redox homeostasis, thereby attractive anticancer targets (Fig. 6). Although this approach thus
providing a scientific rationale for targeting their metabolism for far has been limited by toxicity due to difficulties in identifying
cancer treatment.1289 Anticancer drug candidates against gluta- specific compounds targeting metabolic enzymes, several com-
mine metabolism and closely linking metabolic networks, such as pounds or candidates targeting the tricarboxylic acid (TCA) cycle
glutamine transporter SLC1A5, glutaminase (GLS), and amino- and oxidative phosphorylation are currently in clinical trials for the
transferase, have shown promising effects in cancer treatment treatment of both solid and hematological tumors.1296 The first
(Fig. 6). The amino acid analog L-g-glutamyl-p-nitroanilide, the successful antimetabolite drug came from targeting TCA. IDH
originally discovered compound V-9302, and specific synthetic catalyzes the oxidative carboxylation of isocitrate to produce α-KG,
mAbs (i.e., KM4008 and KM4012) were developed for the whereas its mutations result in the gain of function, converting α-
inhibition of SLC1A5. Although they suppressed glutamine- KG to the oncometabolite 2-HG.1297 In 2017, the FDA approved
dependent growth of cancer cells to some extent, none of them enasidenib, a first-in-class IDH2 mutation inhibitor developed by
entered clinical trials, for their specificity, efficiency, and safety Celegene, for the treatment of recurrent or refractory AML with
profile need to be further evaluated and optimized. In addition IDH2 mutation. Subsequently, ivosidenib, developed by Agios
to targeting glutamine transporters, targeting GLS, which trans- Pharmaceuticals against IDH1 mutations, was approved by the
forms glutamine into glutamate in the mitochondria, is a notable FDA for the treatment of AML and cholangiocarcinoma with IDH1
drug development strategy. Among GLS1 inhibitors, telaglena- mutation.1298 Moreover, AG-881 is undergoing clinical trials for the
stat (CB-839), a derivative of the allosteric inhibitor BPTES, has treatment of AML-carrying IDH2 or IDH1/2 mutations
attracted much attention.1290,1291 CB-839 has been assessed in (NCT02492737). Similarly, CPI-613, targeting both the α-KG
more than 10 completed clinical trials alone for the treatment of dehydrogenase complex and pyruvate dehydrogenase, is in phase
hematological and solid tumors, or in combination with I/II clinical trials for leukemias, lymphomas, and SCLC
everolimus for RCC (NCT03163667), talazoparib or palbociclib (NCT03699319 and NCT03793140). In addition to the TCA cycle,
for solid tumors (NCT03875313, NCT03965845), paclitaxel for the electron transport chain, also known as the respiratory chain
TNBC (NCT03057600), and azacitidine for myelodysplastic which consists of four complexes (CI–IV), is the main target for
syndrome (NCT03047993). Disappointingly, CB-839 did not prove drug development. Metformin, the most well-known inhibitor of
efficacious in the above clinical trials or some results were not complex I, is approved for type-2 diabetes and has been found to
disclosed. Moreover, some clinical trials of CB-839 alone or in exhibit anticancer effects against various cancers in preclinical
combination with anticancer drugs are ongoing, such as with studies and clinical trials. In contrast to metformin which is a

Zhou et al.
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nonspecific complex I inhibitor, IACS-010759 is a specific inhibitor and intermittent fasting sensitize anticancer medicines. For
of complex I that has undergone clinical trials for AML and example, the combination of metformin and intermittent fasting
advanced cancers. However, its toxicity has hindered its further is effective at targeting the metabolic plasticity of cancer.
development.1299 Rotenone and deguelin also inhibit complex I, Understanding the interactions between cancer and diet is crucial
while their neurotoxic effects are prominent. Antimycin A is an for establishing diet as a line of treatment. Elucidating altered
inhibitor of complex III commonly used in experimental research, drug efficacy under a differential metabolic context will be
while resveratrol has enrolled in clinical trials for different types of important for future enhancing the dietary interventions in
cancer. Complex IV can be inhibited by doxorubicin, a DNA specific cancer therapies due to the heterogeneous nature of
intercalating chemotherapeutic drug, and the porphyrin photo- cancers and host metabolisms.1309,1310
sensitizer photofrin, which is approved for esophageal cancer and Although extensive efforts have been made to develop targeted
NSCLC. No promising inhibitors have been reported to date for therapy against cancer metabolism, few have achieved clinical
Complex V, except for oligomycin, which is only suitable for success, and metabolism-targeted therapy is still challenging for
experimental use. Employing mitochondrial uncouplers is an the following reasons. First, metabolism plays a crucial role in all
alternative approach to impair the function of the electron kinds of cells including tumor cells, cells in the TME, such as
transport chain. Niclosamide is in phase I/II clinical trials for immune cells, macrophages, cancer-related fibroblasts and other
prostate and colon cancer, while nitazoxanide is in phase II for stromal cells, and normal cells. Moreover, extensive interactions of
different forms of advanced cancers.1296 metabolites in these cells exist. These factors make strong
antitumor activity and low toxicity by regulating metabolism
Targeting one-carbon and nucleotide metabolism. In addition to extremely difficult. Second, both metabolite enzymes and
the metabolism mentioned above, other metabolic processes also metabolites possess unclassical functions such as acting as kinases
perform significant roles in tumors, such as one-carbon metabo- or second messengers in addition to acting as enzymes and
lism and nucleotide metabolism which have close connections. metabolites. Thus, inhibiting enzyme function may exhibit only
One-carbon metabolism provides one carbon unit in the form of moderate anticancer efficiency. Third, cancer cells reprogram their
methyl groups to several metabolic pathways and is responsible metabolism very quickly after various stimuli by increasing
for the synthesis of methionine, serine/glycine, purine, and metabolic flexibility, uptake of extracellular metabolites via
pyrimidine.1300 After a landmark study by Farber and colleagues compensatory transporters and macropinocytosis, and upregula-
revealed that the folate antagonist aminopterin induced remission tion of nutrient stress-response proteins. Blockage of one pathway
in children with ALL, a series of classical inhibitors in this field, by targeting a key enzyme could result in the activation of another
including methotrexate, pemetrexed, gemcitabine, and 5-FU, were metabolic hub. In this regard, targeting cancer metabolism must
used as frontline chemotherapy for a diverse range of cancers.1301 be based on a thorough understanding of how metabolic
After that, multiple attempts were made to develop targeted pathways affect the whole metabolic status of cancer hubs, which
molecules. MTHFD2 inhibitors LY345899 and DS18561882, have could promote the successful development of anticancer drugs
shown anticancer activity both in vitro and in vivo.1302 PHGDH targeting metabolism.
catalyzes the transformation of the glycolytic intermediate 3-PG
into 3-phosphohydroxy pyruvate, and its allosteric inhibitors, such ICIs-based immunotherapy
as CBR-5884, NCT-503, α-ketothioamide derivatives, and com- Since Tasuku Honjo’s group at Kyoto University discovered PD-1 in
pound b36, and orthosteric inhibitors which are indole derivatives 1992,806 and Allison’s team at MD Anderson Cancer Center
such as BI-4916, inhibit PHGDH activity and moderately suppress reported that blocking CTLA-4 by its antibody could increase the
cancer cell proliferation preclinically.1303 SHMT catalyzes the antitumor activity of T cells and inhibit tumor growth in 1996,1311
conversion of serine and tetrahydrofolate into glycine and 5,10- immunotherapy has been considered as a breakthrough in clinical
methylenetetrahydrofolate, thus providing one carbon unit for cancer treatment due to the promising efficacy.1312 Immune
nucleotide synthesis. Optimization has generated several experi- checkpoint inhibitor (ICI) therapies, including anti-CTLA-4,
mental dual SHMT1/2 inhibitors, including SHIN1 and SHIN2, anti-PD-1, and anti-PD-L1 therapies, have revolutionized the
which have revealed some extent of anticancer effects preclini- systemic treatments for advanced hematological and solid tumors
cally.1304,1305 Human dihydroorotate dehydrogenase (hDHODH) is in the area of antitumor immunotherapy. Compared with
the fourth and rate-limiting enzyme of de novo pyrimidine chemotherapy and targeted therapies, ICIs induce unprecedented
synthesis, and inhibition of hDHODH is an effective strategy for improvements in response rate and better survival rate in partial
the treatment of cancers. To date, classical DHODH inhibitors, such patients, even after cessation of treatment.1313,1314
as leflunomide and teriflunomide, and several novel hDHODH Ipilimumab, the antibody against CTLA-4, was the first ICI
inhibitors, such as brequinar, ASLAN003, BAY2402234, AG-636, approved by the FDA in 2011, which is a milestone in cancer
PTC299, and JNJ-74856665 (NCT04609826), have been evaluated immunotherapy. Ipilimumab successfully hindered cancer pro-
in clinical trials to investigate their safety and antitumor gression in patients with refractory metastatic melanoma.1315
efficacy.1306 Tremelimumab is another human IgG2 CTLA-4 antibody against
HCC and was approved by the FDA in 2022.1316
Targeting dietary interventions. Dietary interventions alone or in
combination with various anticancer strategies have become PD-1/PD-L inhibitors. To date, PD-1/L1 inhibitors are the most
promising tools for cancer therapy, including preventing tumor- widely applied ICIs, which undoubtedly changed the paradigm of
igenesis, delaying tumor growth, and improving the effectiveness cancer therapy. They have shown clinical efficacy against many
of existing cancer treatments.1307 Dietary interventions potentially different solid and hematologic malignancies. The binding of PD-
improve tumor therapy in several ways, such as eliminating L1 (initially identified as B7-H1) to its receptor PD-1 inhibits T-cell
specific nutrients that tumors use as fuel and building blocks, migration, proliferation, and secretion of cytotoxic mediators,
potentiating other forms of therapy including chemotherapy, thereby limiting tumor cell killing. Inhibitors of PD-1 and PD-L1
radiotherapy, and targeted therapy by depriving tumors of reverse T cell suppression by disrupting the PD-1 axis, thereby
nutrients, enhancing the antitumor immune response by mod- enhancing the endogenous antitumor immune response.1312 Until
ulating the growth factors or altering the systemic immune now, multiple PD-1/L1 inhibitors have been approved for
system.1308 Dietary interventions come in various forms, such as commercialization in the US and China, which include pembro-
the restriction of energy or macronutrients, defined by timing lizumab, nivolumab, dostarlimab, cemiplimab, sintilimab targeting
such as intermittent fasting regimens.1308 Fasting mimicking diet PD-1, and atezolizumab, avelumab, and durvalumab targeting PD-

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Table 11. The FDA-approved immune checkpoint inhibitors
Target Drugs Indications Company
CTLA-4 Ipilimumab Colorectal cancer, hepatocellular carcinoma, melanoma mesothelioma, non-small cell lung Bristol Myers Squibb
cancer, renal cell carcinoma
PD-1 Cemiplimab Basal cell carcinoma, cervical squamous cell cancer, non-small cell lung cancer Regeneron Pharmaceuticals
Nivolumab Colorectal cancer, esophageal squamous cell carcinoma, hepatocellular carcinoma, gastric Bristol Myers Squibb
cancer, hodgkin lymphoma, head and neck squamous cell carcinoma, melanoma,
mesothelioma, non-small cell lung cancer, renal cell carcinoma, urothelial carcinoma
Pembrolizumab Breast cancer, cervical cancer, colorectal cancer, esophageal squamous cell cancer, Merck & Co
endometrial carcinoma, esophageal carcinoma, gastric carcinoma, hepatocellular
carcinoma, hodgkin lymphoma, non-small-cell lung cancer, melanoma, mesothelioma,
Merkel cell carcinoma, non-small cell lung cancer, primary mediastinal large B-cell
lymphoma, renal cell carcinoma, small-cell lung cancer, urothelial carcinoma, biliary tract
cancer
Dostarlimab -gxly Advanced or recurrent mismatch repair-deficient/microsatellite instability-high GlaxoSmithKline
endometrial cancer, recurrent or advanced mismatch repair-deficient solid tumors
PD-L1 Atezolizumab Breast cancer, hepatocellular carcinoma, melanoma, non-small cell lung cancer, small-cell Genentech
lung cancer, urothelial, alveolar soft part sarcoma, urothelial carcinoma
Avelumab Merkel cell carcinoma, renal cell carcinoma, urothelial carcinoma EMD Serono
Durvalumab Non-small cell lung cancer, small-cell lung cancer, urothelial carcinoma, biliary tract cancer, AstraZeneca UK Limited
hepatocellular carcinoma
Source: All the information is derived from the United States Food and Drug Administration.gov (https://www.fda.gov/)
L1 (Table 11). These inhibitors are now widely used for the stage III NSCLC in 2018.1319 Avelumab is a fully human IgG1 mAb
treatment of various cancers, including NSCLC, melanoma, with a wild-type IgG1 crystallizable fragment (Fc) region, which
uroepithelial carcinoma, HNSCC, CRC, HCC, and Hodgkin’s enables avelumab to utilize both adaptive and innate immune
lymphoma.1316 Nivolumab (Opdivo) and pembrolizumab (Key- mechanisms to suppress cancer cells.1320 Similarly, avelumab
truda) are particularly extensively used in clinical therapy. obtained accelerated approval for the treatment of patients with
Nivolumab, developed by Bristol Myers Squibb, is the first clinical locally advanced or metastatic urothelial carcinoma in 2017, and
anti-PD-1 antibody approved in 2015 for the treatment of subsequently approval for first-line treatment of patients with
advanced SCLC and metastatic squamous NSCLC. After that, advanced RCC in combination with axitinib in 2019.1320 Compared
pembrolizumab, developed by Merck & Co, was approved by the with many oncology regimens, PD-1/PD-L1 blockade is associated
FDA for the first-line treatment of patients with metastatic NSCLC with fewer adverse events including fatigue, diarrhea, and
in 2016.1312 Different from nivolumab, the prescription of decreased appetite which are well tolerated. Moreover, there are
pembrolizumab requires confirmed PD-L1 overexpression on still a large number of clinical trials undergoing to evaluate the
tumors. At the same time, atezolizumab (Tecentriq) by Roche therapeutic potential of the above inhibitors.
was approved by the FDA in 2016 to treat patients with advanced In addition to antibodies, novel strategies targeting PD-1/PD-L1
and metastatic urothelial carcinoma. Another two new PD-L1 were developed. For example, AC-1, an antibody-based PROTAC
antibodies, durvalumab (Imfinzi) and avelumab (Bavencio), were termed AbTAC, simultaneously bound PD-L1 and E3 ligase RNF43
approved in 2017. Furthermore, Innovent Biologics in China to degrade cell-surface PD-L1 via lysosomal degradation in
developed a PD-1 antibody named sintilimab which achieved different cell lines with high PD-L1 expression levels.1321,1322
good efficiency after neoadjuvant administration.1317 Dostarlimab Considering only a small fraction of cancer patients (lower than
(Jemperli) developed by GSK was approved in 2021 and used in 50%) respond to PD-1/L1 inhibitors which are far from satisfactory,
patients with mismatch repair-deficient advanced solid immune combination therapy which may improve the efficacy
tumors.1318 and expand the beneficiary population attracted much attention.
There are currently three PD-L1 mAbs, atezolizumab, durvalu- The combinations of immune checkpoint blockade and costimu-
mab, and avelumab, approved by the FDA for the treatment of latory receptor activation, such as PD-L1 × 4-1BB (MCLA-145) and
NSCLC and merkel cell carcinoma. Atezolizumab, a humanized PD-1 × ICOS (XmAb23104), are under clinical investigation
IgG1 mAb, abrogates antibody-dependent cytotoxicity and pre- (NCT03922204, NCT03752398). Monovalent trispecific antibody
vents depletion of PD-L1-expressing T cells.1316 Based on its NM21-1480 (αPD-L1, α4-1BB and αHSA) and GNC-038, a tetra-
favorable safety and efficacy profile, the FDA accelerated the specific IgG-scFv conjugated antibody (αCD19/CD3/4-1BB/PD-L1)
approval of atezolizumab in May 2016 for the treatment of locally are in phase I clinical trials (NCT04442126 and NCT05192486).
advanced or metastatic urothelial carcinoma treatment after the Checkpoint blockades incorporation with BsAbs achieved tumor-
failure of cisplatin-containing chemotherapy, and subsequently localized and TAA-dependent checkpoint blockage. For example,
approved for the treatment of advanced metastatic NSCLC during IBI315, an anti-PD-1 × HER2 developed by Innovent Bridge, is
or following platinum-containing chemotherapy in October under phase I clinical study for patients with HER2-expressing
2016.803 In addition, atezolizumab is the first ICI approved in advanced solid tumors (NCT04162327). Anti-PD-1 × CTLA-4 BsAbs,
combination with carboplatin and etoposide to treat advanced including AK104, MEDI5752, and MGD019, are expected to
SCLC. Durvalumab is a fully human IgG1 mAb that binds PD-L1 synergistically inhibit PD-1 and CTLA-4 double-positive lympho-
with high affinity and specificity.803 Durvalumab obtained cytes, which are under clinical investigations (NCT06035224,
accelerated approval of the FDA in 2017 for the treatment of NCT04522323, and NCT05293496).
patients with advanced or metastatic urothelial carcinoma who
have disease progression following platinum-containing che- Other ICIs. In addition to PD-1/PD-L1 and CTLA-4, novel immune
motherapy, and for the treatment of patients with unresectable checkpoints including lymphocyte activation gene-3 (LAG-3), T cell

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immunoglobulin and mucin-domain-containing 3 (TIM-3), and T immunotherapy as a mainstream cancer treatment alongside
cell immunoglobulin and ITIM domain (TIGIT) that mediate chemotherapy, radiotherapy, and surgery.
inhibitory signals through different mechanisms have been
identified, and their inhibitors have been emerging for cancer Differentiation therapy
immunotherapy.1312 The mAb drugs targeting these immune The concept of differentiation therapy originates from the fact
checkpoints transmissed inhibitory signals following ligand that hormones or cytokines can promote differentiation in vitro
engagement, and their synergistic antitumor effect with PD-1/ and thus irreversibly alter the phenotype of cancer cells. Certain
PD-L1 inhibitors were evaluated in preclinically and in multiple signaling molecules and drugs, such as retinoic acid, cAMP,
clinical trials (NCT03219268, NCT03708328, and NCT03440437). sodium butyrate, and cytokines, can induce terminal ex vivo
Induced on CD4+ and CD8 + T cells under antigen stimulation, differentiation in AML, embryonic carcinomas, or
LAG-3 has become one of the most promising new targets of neuroblastoma.1327
immune checkpoint blockage after PD-1 with great application Differentiation therapy is a meaningful tumor-targeting strat-
prospects. Relatlimab is the most advanced mAb targeting LAG-3, egy, and many inhibitors have been developed to induce
which is under phase II/III clinical trial in unresectable or untreated differentiation. The combination of small molecule inhibitors all-
metastatic melanoma in combination with nivolumab. The study trans retinoic acid and arsenic trioxide for the treatment of acute
resulted in a median PFS of 10.12 months in the combination promyelocytic leukemia is a watershed for differentiation therapy.
group compared with 4.63 months in the monotherapy group, In addition, retinoic acid is used to treat solid tumors. For example,
supporting its approval by the FDA for the treatment of metastatic retinoic acid induces the differentiation of tumor-initiating cells in
melanoma combined with nivolumab. Relatlimab represents the HCC, suppresses the expression of stem cell markers, and induces
third type of ICI to enter the market.1316 TM-3 is a T-cell surface the expression of liver-specific genes, ultimately increasing the
inhibitor that is mainly expressed on CD4 + T helper cell 1 (Th1) sensitivity of cisplatin therapy. The small molecule drug arsenic
and CD8 + CTL cells, and some innate immune cells including trioxide, approved by the FDA for leukemia treatment, has shown
dendritic cells, NK cells, and macrophages. LY3321367, an anti- effectiveness in various hematological malignancies and solid
Tim-3 antibody, demonstrated good tolerability as monotherapy tumors. The natural product oroxylin A, a bioactive flavonoid in
or in combination with an anti-PD-L1 antibody in phase I studies, Scutellaria baicalensis with strong anticancer effects and safety,
and further clinical studies are needed to verify its efficacy and can induce tumor cell differentiation. Oncostatin M, a glycoprotein
safety in larger cohorts of patients.1323,1324 TIGIT, a type I belonging to the IL-6 family of cytokines, is involved in cell growth
transmembrane protein which belongs to the immunoglobulin and development and can induce differentiation and inhibit the
superfamily (IgSF), is expressed on T cells, regulatory T cells, proliferation of HCC cells.821
memory T cells, and NK cells. Tiragolumab is the mAb targeting Since most solid tumor oncogenic signaling pathways are far
TIGIT which is currently under phase III clinical trial in extensive- more genetically complex than the genetic basis of leukemia, the
stage SCLC in combination with atezolizumab (NCT04256421). In efficacy of solid tumor differentiation inducers has not yet reached
addition, mAbs targeting fibrinogen-like protein 1 (FGL1), a ligand that of hematologic malignancies. Conventional cancer therapy
for Lag-3 for NSCLC, nuclear receptor subfamily 2 group F member aims to kill rapidly proliferating tumor cells, which can damage
6 (NR2F6), an intracellular IC molecule, and V-set immunoregula- normal cells and lead to serious side effects. In contrast,
tory receptor (VISTA), an immunomodulatory protein expressed in differentiation therapies have low cytotoxicity and are effective
lymphoid organs and bone marrow cells, are now being evaluated in combination with classical tumor-killing cytotoxic compounds.
in phase I clinical studies for the treatment of solid tumors.1312 Differentiation therapy reduces malignancy and inhibits the
At present, approximately5000 registered clinical studies listed aggressiveness of tumors. Tumor differentiation therapy has many
on the US trial registry site ClinicalTrials.gov are ongoing to benefits, including reversing the malignant phenotype of tumors,
evaluate the effectiveness of ICIs involving PD-1, PD-L1, or CTLA-4, restoring normal cellular functions, enhancing the immunogeni-
both individually and in combinations against various hematolo- city of tumor cells, and enhancing the therapeutic sensitivity of
gical and solid tumors. Although ICIs have achieved great success tumor cells to conventional tumor therapy and ICIs.821 Therefore,
in clinical treatment, some challenges still remain to be solved in the induction of cancer cell differentiation is a valuable tumor
this field. First, only parts of patients significantly benefit from ICI treatment strategy, which warrants further study.
treatment. Thus, accurate prediction biomarkers by integrating
multiple approaches to determine which patients are likely to Epigenetic reprogramming inhibitors
benefit from ICIs is urgently needed. The combination and Epigenetic dysregulation in cancer has led to the exploration of
development of multiple functional approaches, including large- epigenetic machinery as a promising target for drug development.
scale genomic sequencing, single-cell transcriptomic techniques, Consequently, the field of developing epigenetic drugs, which
multi-omics, and computational immunogenomics, which inte- target enzymes involved in regulating genome function through
grate intratumour heterogeneity, tumor mutational burden, epigenetic mechanisms, has gained significant attention.1328
neoantigen expression, and immunogenicity, could improve the Currently, the focus of epigenetic drug development revolves
prediction of response to ICIs.1325 Second, although ICIs initially around enzymes responsible for introducing (writers), recognizing
exhibited strong efficiency against tumor growth, patients still (readers), and removing (erasers) epigenetic marks on DNA or core
have relapse and/or develop acquired resistance. Combinational histones.1329 Inhibitors have been designed to target these
therapeutic strategies based on a deep understanding of the enzymes, including DNMTs, and HMTs EZH2 and DOT1L as
tumor and TME, and coordination of systemic and local intratumor writers, HDM LSD1 and HDACs as erasers, and BET proteins as
immune responses enable to improve and maximize the potential readers (Table 12).
benefit to more tumor patients.1326 Third, the development of
novel cancer immunotherapy targets based on the mechanistic DNMT inhibitors. DNMT inhibitors, also known as hypomethylat-
study can lead to the discovery of effective approaches, which in ing agents, have become effective epigenetic therapies for cancer
turn improve the efficacy of tumor immunotherapy. In summary, due to the crucial role of DNMTs in DNA methylation. There are
ICIs opened a new era of immunotherapy and changed the two main classes of DNMT inhibitors: nucleoside analog inhibitors
landscape of cancer treatment. They are promising treatment that are incorporated into newly synthesized DNA and recognized
options although the response rate is far from satisfactory. by DNMTs, and nonnucleoside inhibitors that interfere with DNMT
Combination therapy and mechanism studies may improve binding.1330 Nucleoside inhibitors, such as 5-azacitidine (Vidaza),
efficacy, expand the beneficiary population, and further support 5-aza-2′-deoxycytidine (decitabine, Dacogen), and SGI-110

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(guadecitabine), belong to the first class. These cytidine analogs HMT inhibitors. In addition to broad epigenetic reprogrammers
irreversibly sequester DNMT proteins into DNA, resulting in global such as DNMT inhibitors and HDAC inhibitors, targeted therapy
DNA hypomethylation and reactivation of silenced genes in has been developed for specific mutations in epigenome-
cancers. Currently, 5-azacitidine and decitabine have been modifying enzymes.1343 For instance, tazemetostat (EPZ-6438,
approved for treating AML, chronic myelomonocytic leukemia, E7438) is used for patients with EZH2 mutations. Tazemetostat is
and MDS.1331 However, these drugs have notable side effects, the only approved EZH2 inhibitor granted FDA approval in 2020
including cellular and clinical toxicity, as well as chemical for treating epithelioid sarcoma and follicular lymphoma based on
instability.1331 Next-generation nucleoside analog DNMT inhibi- clinical trials (NCT01897571 and NCT02601950).1344 Ongoing
tors, such as guadecitabine, exhibit improved pharmacokinetic clinical trials are assessing its efficacy against various hematologic
profiles with longer plasma half-life and lower peak plasma malignancies and solid tumors (NCT05567679, NCT04179864,
concentrations, leading to reduced toxicity. Guadecitabine has NCT05023655, NCT04705818, and NCT04846478). Studies have
shown promising outcomes in clinical studies for AML treat- shown that loss of Bap1 in mice can increase EZH2 expression and
ment.1332 Another novel hypomethylating agent called NTX-301 H3K27me3 levels. Moreover, mesothelioma cells with BAP1
has demonstrated superiority over conventional agents such as mutations have shown sensitivity to EZH2 inhibition, leading to
5-azacitidine and decitabine in preclinical studies, which supports clinical trials investigating the use of tazemetostat in treating
ongoing clinical development efforts.1333 Clinical trials with NTX- malignant mesothelioma with BAP1 inactivation
301 are currently underway (NCT04167917, NCT03366116, and (NCT02860286).1345
NCT04851834). GSK126 is a potent and highly selective EZH2 inhibitor that
Nonnucleoside DNMT inhibitors directly target the catalytic site of targets both wild-type EZH2 and Y641 mutant EZH2.1346 However, a
specific DNMT enzymes, unlike nucleoside analogs that are phase I clinical trial assessing its safety, pharmacokinetics,
incorporated into DNA. Researchers have discovered reversible pharmacodynamics, and clinical activity in patients with relapsed
and selective nonnucleoside DNMT inhibitors, including RG108, SGI- or refractory DLBCL, other NHLs, multiple myeloma, and solid
1027, GSK3685032, DC-05, and CM-272.1334 Additionally, MG98 is an tumors was terminated. The results showed insufficient antitumor
antisense oligodeoxynucleotide that targets the 3′UTR of DNMT1 activity and a relatively short half-life, which limited effective
mRNA to achieve DNA demethylation.1335 However, further exposure and did not support further clinical study (NCT02082977).
preclinical and clinical studies are necessary to determine if their On the other hand, CPI-1205 is an orally bioavailable EZH2 inhibitor
outcomes are more favorable than those of nucleoside analogs. with a unique indole-based structure different from pyridine-based
compounds such as GSK126 and tazemetostat.1347 Preclinical
HDAC inhibitors. Many HDAC inhibitors target Zn2+ in the active studies have demonstrated that CPI-1205 significantly inhibits
site of HDACs to inhibit their enzymatic activity, leading to tumor growth in DLBCL xenograft models, with good oral
changes in chromatin structure and gene expression. Five HDAC bioavailability and an acceptable safety profile in rats and dogs.
inhibitors have been globally approved, including vorinostat Currently, CPI-1205 is undergoing evaluation in a phase I clinical trial
(SAHA), romodepsin, belinostat, panobinostat (approved by the in patients with B-cell lymphoma (NCT02395601), advanced solid
FDA), and chidamide (also known as tucidinostat, approved by the tumors (NCT03525795), and metastatic castration-resistant prostate
NMPA) for specific hematologic malignancies.1336 In 2006, the FDA cancer (CRPC) (NCT03480646).
approved the first HDAC inhibitor, vorinostat, for the clinical DOT1L, the sole identified H3K79 methyltransferase, has been
treatment of cutaneous T-cell lymphoma.1337 Additionally, the targeted for cancer treatment, especially in acute leukemias with
FDA has successively approved romidepsin, belinostat, and MLL gene rearrangements.1348 Pinometostat, the first clinical
panobinostat as HDAC inhibitors for the treatment of peripheral inhibitor of DOT1L, exhibits improved potency, longer plasma half-
T-cell lymphoma, T-cell lymphoma, and multiple myeloma, life, enhanced selectivity, and efficacy in reducing leukemic cell
respectively.1336 Since these drugs are all pan-inhibitors of HDAC proliferation. Phase I clinical trials have been conducted for MLL-
and may exhibit potential toxic side effects, the first selective rearranged leukemia using pinometostat. Although well tolerated,
HDAC subtype inhibitor, tucidinostat, has been approved for the pinometostat requires continuous IV infusion due to rapid
treatment of recurrent or refractory PTCL and breast cancer.1338 clearance and shows modest clinical effectiveness (NCT02141828
Currently, available HDAC inhibitors are nonselective or pan- and NCT01684150).1349,1350
HDAC inhibitors, which have drawbacks such as poor efficacy on
solid tumors, limited therapeutic efficacy, drug resistance, and HDM inhibitors. Preclinical studies have shown LSD1 inhibitor-
toxicity.1339 Thus, developing HDAC inhibitors with better activity dependent differentiation and growth inhibition, which has led to
and higher selectivity is an important area of research. Three the initiation of several clinical trials to assess its efficacy. These
schemes for designing HDAC inhibitors include drug design based trials include iadademstat (ORY-1001), INCB059872, IMG-7289,
on zinc-binding groups, selective inhibitors targeting different GSK2879552, seclidemstat, and pulrodemstat (CC-90011).1351
subtypes of HDAC, and dual mechanism or multitarget HDAC Oryzon Genomics developed ORY-1001 in 2012, which uses TCP
inhibitors.1340 Many HDAC inhibitors have been synthesized based as the lead compound and is currently being studied in clinical
on these schemes, with more than ten entering clinical trials.1341 trials for AML and SCLC (NCT05546580 and NCT02913443).1352
For example, abexinostat, a potent oral pan-HDAC inhibitor INCB059872, an irreversible LSD1 inhibitor reported by Lee et al. in
designed based on ZBG, is currently undergoing phase II clinical 2016, has undergone five clinical trials to test its safety and
trials for recurrent/refractory DLBCL and follicular lymphoma as a efficacy in treating solid tumors and hematologic malignancies
single-drug treatment (NCT03936153 and NCT03934567). Addi- (NCT02712905, NCT02959437, NCT03132324, NCT03514407, and
tionally, the combination of abexinostat and pazopanib for locally NCT04061421). While one phase I study was terminated due to
advanced or metastatic RCC has entered the phase III trial sickle cell disease risks, other trials have shown potential in
(NCT03592472). Another instance is entinostat, a synthetic treating various cancers. IMG-7289 (Biomedestat), developed by
benzamide derivative HDAC inhibitor that selectively inhibits class Imago BioSciences in 2018, is currently undergoing clinical trials
I and IV HDAC enzymes.1342 Entinostat has been evaluated in for treating AML and MDS (NCT02842827), bone marrow fibrosis
several phase II trials in patients with breast cancer and has (NCT03136185) and essential thrombocythemia (NCT04081220).
improved patient median OS (NCT00676663). Furthermore, the GSK2879552, another irreversible LSD1 inhibitor, has shown
combination of entinostat and exemestane for advanced breast antitumor activity in AML and SCLC but had to be discontinued
cancer has entered phase III clinical trials (NCT03538171 and in three clinical trials due to unfavorable risk-benefit profiles
NCT02115282). (NCT02034123, NCT02177812, and NCT02929498).

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Table 12. The FDA-approved and clinically developed epigenetic reprogramming inhibitor in cancer therapies
Type Drug Highest Indications Company/Identifier Status

Phase
DNMTi Azacitidine (Vidaza) Approved Acute myeloid leukemia, chronic myelomonocytic Celgene /
leukemia, myelodysplastic syndromes
Decitabine (Dacogen) Approved Acute myeloid leukemia, chronic myelomonocytic MGI Pharma & /
leukemia, myelodysplastic syndromes SuperGen
Guadecitabine III Acute myeloid leukemia NCT02920008 Completed
III Myelodysplastic syndromes, leukemia myelomonocytic NCT02907359 Completed
chronic
III Leukemia myeloid acute NCT02348489 Completed
NTX-301 I Acute myeloid leukemia, myelodysplastic syndromes, NCT04167917 Recruiting
chronic myelomonocytic leukemia
MG98 I Unspecified adult solid tumor NCT00003890 Completed
HDACi Belinostat (Beleodaq) Approved Peripheral T-cell lymphoma Spectrum Pharma /
Panobinostat Approved Multiple myeloma Novartis /
(Farydak)
Romidepsin (Istodax) Approved Cutaneous T cell lymphoma Gloucester Pharma /
Vorinostat (Zolinza) Approved Cutaneous T cell lymphoma Merk Sharp
Abexinostat III Renal cell carcinoma NCT03592472 Recruiting
ACY-241 I Multiple myeloma NCT02400242 Active, not
recruiting
I Malignant melanoma NCT02935790 Completed
I Advanced solid tumors NCT02551185 Completed
I Non-small cell lung cancer NCT02635061 Active, not
recruiting
AR-42 III Neurofibromatosis type 2 NCT05130866 Recruiting
CUDC-907 II Relapsed and/or refractory diffuse large B-cell lymphoma NCT02674750 Completed
including myc alterations
II Thyroid neoplasms, poorly differentiated and NCT03002623 Terminated
undifferentiated thyroid cancer, differentiated thyroid
cancer
II Prostate cancer NCT02913131 Terminated
CXD101 II Colorectal neoplasms malignant NCT03993626 Unknown
II Diffuse large B-cell lymphoma NCT03873025 Withdrawn
II Hepatocellular carcinoma NCT05873244 Recruiting
Entinostat III Advanced breast cancer NCT03538171 Unknown
III Breast adenocarcinoma, HER2/Neu negative locally NCT02115282 Active, not
advanced breast carcinoma, metastatic breast carcinoma, recruiting
recurrent breast carcinoma
Givinostat (ITF2357) II Multiple myeloma NCT00792506 Terminated
II Chronic myeloproliferative neoplasms NCT01761968 Active, not
recruiting
II Hodgkin’s lymphoma NCT00496431 Terminated
II Hodgkin’s lymphoma NCT00792467 Completed
Mocetinostat II Urothelial carcinoma NCT02236195 Completed
(MGCD0103) II Lymphocytic leukemia chronic NCT00431873 Completed
II Hodgkin’s lymphoma NCT00358982 Terminated
II Myelogenous leukemia acute, myelodysplastic syndromes NCT00374296 Terminated
II Lymphoma relapsed and refractory, diffuse large B-cell NCT02282358 Terminated
lymphoma and follicular lymphoma
II Myelodysplastic syndrome, acute myelogenous leukemia NCT00324220 Completed
Resminostat (4SC- II Hepatocellular carcinoma NCT00943449 Completed
201) II Advanced colorectal carcinoma NCT01277406 Completed
II Hepatocellular carcinoma NCT02400788 Completed
II Hodgkin’s lymphoma NCT01037478 Completed
II Lymphoma NCT02953301 Active, not
recruiting

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Table 12. continued
Type Drug Highest Indications Company/Identifier Status
Phase
Ricolinostat (ACY- II Multiple myeloma NCT01997840 Active, not

1215) recruiting
II Lymphoma, Lymphoid malignancies NCT02091063 Completed
II Multiple myeloma NCT01323751 Completed
EZH2i Tazemetostat Approved Epithelioid sarcoma Epizyme /
GSK126 I Cancer, Neoplasms NCT02082977 Terminated
CPI-1205 II Metastatic castration-resistant prostate cancer NCT03480646 Unknown
CPI-1205 I Advanced solid tumors NCT03525795 Terminated
CPI-1205 I B-cell lymphoma NCT02395601 Terminated
DOT1Li Pinometostat (EPZ- II Recurrent/refractory acute myeloid leukemia NCT03701295 Completed
5676)- II Acute myeloid leukemia NCT03724084 Terminated
LSD1i ORY-1001 I Small cell lung cancer NCT02913443 Completed
I Acute myeloid leukemia NCT05546580 Recruiting
INCB059872 II Solid tumors and hematologic malignancy NCT02712905 Terminated
II Solid tumors NCT02959437 Terminated
II Myeloproliferative neoplasms, myelodysplastic syndrome NCT04061421 Recruiting
IMG-7289 II Acute myeloid leukemia NCT02842827 Completed
II Myelofibrosis NCT03136185 Completed
II Thrombocythemia NCT04081220 Recruiting
I Acute myeloid leukemia NCT05597306 Recruiting
II Extensive stage lung small cell carcinoma NCT05191797 Recruiting
GSK2879552 I Carcinoma small cell NCT02034123 Terminated
I Leukemia myelocytic acute NCT02177812 Terminated
CC-90011 I Lymphoma NCT02875223 Active, not
recruiting
I Small cell lung carcinoma NCT03850067 Active, not
recruiting
II Neoplasms NCT04350463 Active, not
recruiting
I Prostatic neoplasms NCT04628988 Completed
I Leukemia NCT04748848 Terminated
SP-2577 II Ewing sarcoma NCT05266196 Enrolling by
invitation
II Recurrent chronic myelomonocytic leukemia NCT04734990 Active, not
recruiting
BETi TEN‐010 I Acute myeloid leukemia, myelodysplastic syndromes NCT02308761 Completed
I Multiple myeloma NCT03068351 Completed
I Solid tumors NCT01987362 Completed
GSK525762 II Neoplasms NCT01943851 Completed
I NUT midline carcinoma NCT01587703 Completed
OTX105 I Acute myeloid leukemia NCT01713582 Completed
I NUT midline carcinoma NCT02259114 Completed
CPI-0610 I Multiple myeloma NCT02157636 Completed
I Lymphoma NCT01949883 Completed
www.fda.gov/)
Two reversible LSD1 inhibitors, CC-90011 and SP-2577 (Seclidem- inhibitor, which showed a manageable safety profile in a phase I
stat), have undergone preclinical studies and clinical trials, showing clinical trial for advanced solid tumors (NCT03895684). Additionally,
promising results for treating different types of tumors. Celgene clinical trials have been initiated to investigate the combination of
developed CC-90011, the first reversible LSD1 inhibitor, for clinical SP-2577 with topotecan, cyclophosphamide (NCT03600649), and
trials targeting relapsed or refractory solid tumors and NHL azacytidine (NCT04734990) for cancer treatment.
(NCT02875223), as well as advanced solid and hematological tumors
(NCT03850067, NCT04350463, NCT04628988, and NCT04748848). BET inhibitors. In contrast to targeting the enzymatic domains
Salarius Pharmaceuticals developed SP-2577, another LSD1 reversible responsible for modifying epigenetic marks, an alternative

Zhou et al.
61
strategy focuses on inhibiting the proteins that recognize these found in gliomas is the Arg132 mutation, resulting in the
modifications by disrupting protein–protein interactions. A prime production of a tumor-specific neoantigen called IDH1(R132H).
example of this innovative approach is BET inhibitors, which Several IDH1(R132H)-specific peptide vaccines are currently
disrupt the interaction between bromodomains and acetylated undergoing testing as monotherapy or in combination with other
lysine residues. This disruption interferes with the recruitment of therapies (NCT02454634, NCT03893903, NCT02193347, and
transcriptional machinery to specific gene loci.1353 NCT02771301).1360
Initial small molecule BET inhibitors such as JQ1 have revealed Despite the theoretical significance and rationale of epigenetic
the oncogenic role of BET proteins and their impact on oncogene therapy, there are still several issues that need to be discussed and
expression, leading to observed antitumorigenic effects in resolved. The first issue relates to selectivity, specifically how to
preclinical models. However, their clinical application has been selectively target widely expressed epigenetic regulators. Epige-
hindered by poor pharmacokinetics, short half-life, and low oral netic events are generally present in both normal and cancer cells.
bioavailability.1354 TEN-010 (RO6870810), a JQ1 derivative with However, certain cancers rely on specific epigenetic changes and
improved pharmacological properties, has undergone clinical are sensitive to their regulation. It is crucial to identify the most
trials for AML, MDS, and solid tumors (NCT02308761 and critical epigenetic changes in different types of cancers. The
NCT01987362). Other BET inhibitors have also advanced to clinical second issue is the differential susceptibility of hematologic
trials. For instance, GSK525762, evaluated in multiple studies for malignancies and solid tumors to epigenetic intervention. While
hematologic malignancies and solid tumors, has shown encoura- significant progress has been made in epigenetic therapy for
ging results, especially in NUT midline carcinoma, AML, and TNBC hematologic malignancies, solid tumors have been less respon-
patients (NCT01943851 and NCT01587703). Further research is sive. The complexity of the oncogenome and inherent cellular
needed to establish its efficacy and safety. OTX015, another BET differences between hematologic malignancies and solid tumors
inhibitor, has demonstrated favorable antitumor activity, particu- may contribute to this variation in efficacy. Understanding these
larly in combination therapies, in clinical evaluations for various biological principles is essential for expanding the application of
hematologic malignancies and solid tumors (NCT01713582 and epigenetic therapy to solid tumors. Moreover, the heterogeneity
NCT02259114). CPI-0610, a selective BET inhibitor, has entered the and plasticity of human cancer highlight the importance of
clinical trial for hematologic malignancies such as myelofibrosis personalized and precise epigenetic therapies. Precision medicine
and lymphoma to evaluate its safety, tolerability, and potential approaches, such as using high-throughput epigenomics sequen-
efficacy as monotherapy and combination therapy cing technology, can help create genome and epigenome maps of
(NCT01949883). Early studies have shown promising activity, but individual patient’s tumor cells. These maps can then be used for
some patients experienced adverse events such as thrombocyto- drug sensitivity testing and screening, enabling optimized
penia and moderate diarrhea (NCT02157636). treatments tailored to each patient.
In addition, efforts have been made to design PROTACs and
molecular glues that can degrade BET proteins by utilizing the Targeting tumor microbiomes
intracellular ubiquitin proteasome system.1343 A clinical trial is Targeted interference with gut and tissue-resident microbiota or
currently underway for the FHD-609 degrader in the treatment of microorganism-derived products-based therapies are effective
synovial sarcoma (NCT04965753), and the CFT8634 degrader has ways to target tumors854 (Table 13). Several strategies are applied
recently entered phase I/II clinical trials for synovial sarcoma and to target gut and tumor microorganisms, including fecal microbial
SMARCB1-null solid tumors (NCT05355753). transplantation (FMT), single strains or designer consortia-based
targeted microbial strategies, diet-based and prebiotic, probiotic,
IDH inhibitors. Mutations in isocitrate dehydrogenases IDH1 and and postbiotic-based interventions, as well as targeted antibiotic
IDH2, commonly found in lower-grade gliomas as well as in AML approaches.854 FMT, a promising way to modulate the gut
and other malignancies, result in neomorphic enzyme activity, microbiome, acts by transplanting the entire gut microbial
leading to increased production of 2-HG from α-KG.1355 2-HG complement from a donor such as a healthy individual into a
serves as a competitive inhibitor of various α-KG-dependent recipient such as a patient with cancer. The early phase I study of
dioxygenases, such as the Jumonji-C domain family of histone FMT revealed that it is beneficial to treat steroid-refractory GI tract
demethylases and the TET family of DNA demethylases, disrupting graft-versus-host disease, which is a complication of hematopoie-
the global methylation landscape and promoting cancer devel- tic stem cell transplantation to treat leukemia.854,1361 While FMT
opment by impairing cellular differentiation.1356 Consequently, methods transplant the entire donor microbiota, the specific
mutant IDH has emerged as an appealing therapeutic target, transplantation of single microbial species or designer microbial
leading to the development of several IDH inhibitors aimed at consortia to improve treatment is needed in certain circum-
counteracting the effects of 2-HG. stances. For example, CBM588, a formulation that includes a strain
Enasidenib, the first FDA-approved IDH2-mutant inhibitor, of Clostridium butyricum, improves the PFS of patients with RCC in
received approval in 2017 after positive results from a single- combination with immune checkpoint blockade therapy.854,1362
arm trial on relapsed or refractory AML patients with IDH2 Moreover, numerous dietary strategies, such as long-term caloric
mutations (NCT01915498). Similarly, based on favorable outcomes restriction, short-term starvation, and ketogenic diets, have shown
observed in a clinical trial (NCT02074839), the FDA approved the potential to enhance the efficacy of immunotherapy.854 The
ivosidenib, an IDH1-mutant inhibitor, for relapsed or refractory dietary interventions have been widely applied in cancer therapy
AML patients with IDH1 mutations.1357 More recently, the for target gut and tumor microorganism. However, lack of rigorous
combination of ivosidenib and azacitidine received FDA approval standard procedures and poor association between diet and
for newly diagnosed, IDH1-mutated AML following a phase III trial clinical effects have hindered their clinical application.854
(NCT03173248).1358 In 2021, ivosidenib was also approved by the In addition, the probiotic and postbiotic-based interventions
FDA for advanced cholangiocarcinoma with IDH1 mutation after a and targeted antibiotic approaches also play significant roles in
phase III clinical trial (NCT02989857).1359 These examples highlight targeting gut and tumor microorganisms.854 A study has found
the success of biomarker-driven approaches in treating cancers that oral probiotic candidate DTA81 is effective in preventing the
with epigenetic alterations, emphasizing the importance of development of early CRC.853,1363 In addition, H. pylori infection is
considering chromatin changes when evaluating drug targets an important oncogenic factor in gastric cancer and gastric
that indirectly modulate chromatin. mucosa-associated lymphoid tissue lymphoma.1364 Combination
Another strategy to target IDH1 genetic alterations in gliomas is therapy of antisecretory proton pump inhibitors with the
the development of vaccines. The most common IDH1 mutation antibiotics amoxicillin, levofloxacin, clarithromycin, and

Zhou et al.
62
Table 13. The typical and clinically developed microbiota inhibitor
Drug Highest Phase Indications Identifier Status
Bismuth colloidal pectin granules quadruple therapy IV Gastric cancer, helicobacter pylori infection NCT04660123 Completed
IV Gastric cancer, helicobacter Pylori infection NCT04209933 Completed
Itraconazole IV Hematologic neoplasms NCT02895529 Terminated
metronidazole is the standard protocol for H. pylori eradica- BCL-XL mediated resistance to apoptosis in ovarian cancer, breast
tion.853,1364 In addition, phytomedicines and probiotics are also cancer, and prostate cancer. Inhibition of DNA replication through
used to treat H. pylori infections.1365 Bismuth collisional tubercu- small molecule inhibition of the kinase CDC7 using XL413 or TAK-
losis has been used in clinical trials to treat the eradication rate of 931 leads to senescence induction in liver cancers.1374 Inhibition
H. pylori infection in cancer patients, as well as to evaluate the of the telomerase complex has been identified as inducing
improvement of symptoms and the incidence of adverse replicative senescence in anticancer therapy. Imetelstat, GX301,
reactions.853 Itraconazole is a broad-spectrum triazole antifungal and BIBR1532, potent telomerase inhibitors, effectively induce
agent with favorable pharmacodynamic and pharmacokinetic senescence and suppress cancer cell proliferation in preclinical or
profiles and is used for the prevention or treatment of systemic clinical trials.1370,1375
fungal infections.1366,1367 Itraconazole inhibits cell proliferation, Another approach to induce senescence is by modulating the
invasion, and migration of oral OSCC cells by suppressing the epigenome of cancer cells. Decitabine, a DNMT inhibitor, and
Hedgehog pathway-induced cell cycle arrest and apoptosis.1368 vorinostat, an HDAC inhibitor, upregulate the expression of
Moreover, itraconazole can inhibit the proliferation and growth of multiple tumor suppressor genes, such as CDKN2A and TP53, thus
CRC cells by promoting autophagy and apoptosis, and it is an inducing cellular senescence via these pathways in various cancer
effective treatment for CRC.1369 In summary, the microbiota is an cells.865,1376
important driver of cancer, and targeting the microbiota in the gut Moreover, numerous other drugs and antibodies can induce
is meaningful in precision cancer care. However, the field requires senescence in cancer cells. Tamoxifen, an estrogen receptor
further elucidation of the specific mechanisms by which micro- antagonist, and bicalutamide, an androgen receptor antagonist,
organisms impact cancer processes. Shortly, targeting microbiota can induce senescence in breast cancer or prostate cancer.1377
in the gut may emerge as a promising tool for cancer care. Trastuzumab and pertuzumab, which are antibodies targeting
HER2, cause senescence in breast cancer. BRAF and MEK inhibitors,
Therapeutic strategies for targeting cellular senescence such as vemurafenib and trametinib, show great senescence-
Induction of tumor cell senescence has been demonstrated as one inducing effects in melanoma.1378
of the underlying mechanisms by which cancer therapies such as
radiation, chemotherapy, and targeted therapy exert their Senolytics. Although TIS contributes to antitumor effects and
antitumor activity. Paradoxically, lingering senescent cells (SnCs) treatment outcomes, increasing evidence has demonstrated that
in tumor tissues fuel tumor progression, relapse, and metastasis the accumulation of SnCs can stimulate the relapse and metastasis
partly through the expression of the SASP.865 Based on the above of cancers. Thus, selective clearance of SnCs with senolytics will
observations, targeted therapeutics inducing tumor cell senes- prevent tumor relapse and metastasis, overcome drug resistance,
cence followed by senolytics to selectively clear newly induced and minimize toxic side effects.
SnCs, which is called “one-two punch” cancer therapy, represent To date, drugs or compounds targeting the apoptosis modulator
an emerging and promising new strategy in cancer treatment. BCL-2/BCL-XL, PI3K-AKT-mTOR, BET, tyrosine kinases, and GLS have
Moreover, the application of senomorphic drugs which reduce the exhibited promising effects on the clearance of senescent
production and secretion of SASP factors has attracted attention cells.865,1370 Apoptosis resistance is a feature shared by both cancer
in cancer therapy.1370,1371 and senescent cells; thus, blocking antiapoptotic proteins could
selectively eliminate senescent cells. ABT-737 was one of the first
Therapy-induced senescence (TIS). The currently available tar- senolytics selectively targeting BCL-2, BCL-XL, and BCL-W, thus
geted therapeutics for inducing senescence include blocking the removing SnCs by reactivating the apoptotic pathway.475 After that,
cell cycle, triggering DNA damage, manipulating epigenetic Navitoclax (ABT-263), a Bcl-2/Bcl-xL inhibitor, and venetoclax (ABT-
modulators, and regulating tyrosine kinases. As cell cycle arrest 199), a BCL-2 inhibitor, were developed and used as adjuvant
is a hallmark of senescent cells, drugs that inhibit CDK or enhance therapies with radiation to selectively eliminate TIS cells, and
levels of CDK inhibitor proteins are currently being used in increase the survival of tumors, including glioblastoma, melanoma,
senescence-inducing cancer therapy.1372 In particular, CDK4/6 and lung cancer cells.1379,1380 In addition, the PI3K/AKT inhibitors
inhibitors such as palbociclib, abemaciclib and ribociclib, which dasatinib and quercetin selectively kill senescent cells and reduce
are approved by the FDA for the treatment of advanced breast the secretion of proinflammatory cytokines.862 An mTOR inhibitor
cancer, are able to induce senescence in various cancer cells.864 significantly reduced the tumor burden and increased survival in
PF-06873600, a triple CDK2/4/6 inhibitor, is a potent senescence xenograft cancer models after treatment with a DNA-replication
inducer in various cancer models, and is ongoing in breast cancer kinase CDC7 inhibitor, which induced senescent liver and lung
in combination with endocrine therapy.1373 AURKs and PLKs, cancer cells.1374 Another drug screen identified the BET family
which are serine/threonine kinases essential for cell mitosis, are protein degrader as a senolytic drug and validated that ARV825, a
potential targets for senescence-inducing therapy.1370 Multiple PROTAC of BET, possesses strong senolytic activity.1381
PLK1 inhibitors, such as BI-6727, and AURK inhibitors, such as Immune-targeted therapy may be an effective way to clear
alisertib, are currently undergoing clinical investigation senescent cells.862 For example, chimeric antigen receptor T cells
(NCT02273388 and NCT06095505). targeting the cell surface protein, urokinase-type plasminogen
Triggering DNA damage is another strategy to induce activator receptor were found to be effective in clearing senescent
senescence. For example, PARP inhibitors, including veliparib cells after mice with lung adenocarcinomas were exposed to MEK
and olaparib, induce a reversible senescent phenotype caused by and CDK4/6 inhibitors to induce senescence.1382

Zhou et al.
63
Senomorphics. Preventing the development of SASP or improv- achieve individualized management, and the predictive biomar-
ing SASP-related functions can reduce inflammation and cancer kers are utilized to demonstrate the treatment response of
risk. Multiple signaling pathways are involved in regulating SASP patients, thereby identifying the best therapy. Thus, prognostic
function, including p38/MAPK, JAK/STAT, mTOR, NF-κB, and C/ biomarkers can identify patients who are at high-risk of cancer.
EBP-β.1383 The inhibitors that modulate the excretion of SASP are The predictive biomarkers suggest the patients that can benefit
called senomorphics which potentially preserves the SASP- from a specific therapy.114 In addition, biomarkers of therapeutic
dependent protumor effects of senescent cells and exert a targets can identify the molecular targets of novel therapies.
synergistic antitumor effect.865 For example, IL-6 mAb siltuximab, Moreover, surrogate endpoint-related biomarkers are used as
multiple signal inhibitor metformin, and the JAK inhibitor substitutes for clinical endpoints or to assess clinical benefits, such
ruxolitinib reduced SASP, thereby reducing the protumor and as posttherapy PSA changes to evaluate drugs in the clinic.145
damage induced by SASP.865,1383 In addition, the protein arginine Herein, we mainly summarize the development history, detection
methyltransferase (PRMT1) induces SASP at the promoter of methods, and classification of tumor biomarkers, thereby illustrat-
proinflammatory genes. The PRMT1 inhibitor TC-E increases the ing the crucial roles of tumor biomarkers in cancer screening,
apoptosis sensitivity of cancer cells by regulating the NF-κB diagnosis, treatment, prognosis, and targeted therapy.
pathway.862 Rapamycin, an inhibitor of mTOR, has been found to Although tumor biomarkers are increasingly critical in cancer
reduce SASP by inhibiting mTOR and to limit the growth- precision medicine, the biomarkers surviving from discovery to
promoting effect of senescent bystander fibroblasts on prostate clinical trials are small in number.1386 Some challenges are still
cancers.1384,1385 urgently being solved in the future. First, the characteristics and
Although targets and several senolytics have been discovered concentration of tumor biomarkers are influenced by different
and tested in preclinical or clinical settings, the development of biologic factors, such as posttherapy, host heterogeneity, age and
senolytics is still a challenge for the following reasons. First, TIS is the presence of other diseases among different individuals, false
heterogeneous and context (e.g., tissue of origin, time after positive biomarkers generated by other physiologic or pathologic
treatment) dependent. Therefore, a deeper understanding of processes, and exogenous interfering substances, i.e., foods, drugs,
tumor contexts is critical for the usage and development of novel and natural alternative therapies. Second, it is necessary and
drugs that induce senescence. Second, the different types of urgent to explore novel tools or technologies that could discover
prosenescence drugs induce cellular senescence via different novel and accurate biomarkers for the detection of preneoplastic
mechanisms, suggesting that different TIS cells may require neoplasia, micrometastatic spread, and states of early or
different senolytics. Moreover, the characteristics of SnCs, and the aggressive cancer recurrence. Third, limitations in analytical
physiological and pathogenic effects of SnCs have not been sensitivity and specificity still exist. Clinical detection and
adequately identified. Thus, the discovery of novel senolytic measurement assays of biomarkers require sufficient sensitivity
targets, senolytics, and the optimization of prosenescence therapy and improved specificity. Standard procedures, clear guidelines,
and senolytic combinations need further investigation. Third, and quality control schemes are essential to ensure accuracy and
senescence-inducing drugs cause senescence not only in tumor reproducibility for biomarker development.145 Fourth, the high
tissues but also in the TME and normal tissues. Induction of risk of false positives arises when identifying a biomarker from
senescence may induce immune escape which decreases the thousands of molecules. Thus, the cost of massive screening
anticancer effect or causes unwanted side effects associated with multiple times and the increased risk of false positives of
lingering senescent cells. The selectivity of senescence-inducing overdiagnosis need to be balanced. Consistent adherence to
should be improved. Last, as surveillance of SnCs is executed by publishing guidelines of tumor biomarkers can improve transpar-
cytokines and chemokines which are released in a time- ency and better judge the quality of putative biomarker
dependent manner, the timing of senolytic intervention is crucial identification.1386 Artificial intelligence offers an intriguing oppor-
for the efficacy of the one-two punch strategy and to avoid side tunity in large-scale screens of available data and to develop novel
effects on normal tissues. Taken together, despite the various tumor biomarkers.1387 Finally, a rational combination of various
challenges, senotherapies is a promising strategy and is likely to biomarkers could improve the efficiency and accuracy of the
be applied in the clinic in the future as our understanding of application of biomarkers. Extensive research along with the use
senescence improves. of new technologies needs to be performed.
In conclusion, we see great enthusiasm in tumor biomarker
discovery and application from the large number of studies in the
CONCLUSION AND PERSPECTIVES late century. Subsequently, the continuous research and devel-
“It is far more important to know what sort of person the disease opment of innovative tumor biomarkers and the continuous
has than what sort of disease the person has”, which is put development of novel detection technologies will make it possible
forward by Hippocrates and precisely interprets the trend of for sensitive and specific tumor biomarkers to be gradually
cancer management. Current cancer management aims to applied in clinical practice, and make early screening, diagnosis,
combine molecular data with traditional clinical information, such treatment, and prognosis assessment of tumors a reality.
as symptoms, personal history, and histology, to tailor medical
care with the most benefit and minimize risk. Meaningfully,
multifarious biomarkers are conducive to monitoring and predict-
ACKNOWLEDGEMENTS
ing the therapeutic response of precision and personalized This study was supported by 135 Project for Disciplines of Excellence of West China
medicine in clinical practice,1386 which highlights the pivotal Hospital Sichuan University (ZYGD23011), National Natural Sciences Foundation of
position of tumor biomarkers in cancer therapy. China (882272716), Natural Sciences Foundation of Sichuan (2022NSFSC0053). We
Tumor biomarkers, biologically indicating pathogenic processes also thank Biorender.com for creating all the figures which have obtained publication
or pharmaceutical responses to therapeutic interventions,145 licensing right in the manuscript.
consist of six different types of biomarkers that are biomarkers
of early detection, diagnosis, prognosis, prediction, therapeutic
target, and surrogate end point.145 The biomarkers of early AUTHOR CONTRIBUTIONS
detection are essential in screening patients with cancers at an Yue Zhou and Yinglan Zhao designed the review and revised the manuscript. Lei Tao,
early stage. The diagnostic biomarkers mainly contribute to the Jiahao Qiu, Jing Xu, Xinyu Yang, Yu Zhang, Xinyu Tian, and Xinqi Guan revised the
identification of the presence and characteristics of cancers. The manuscript and the tables and figures. Yinglan Zhao and Xiaobo Cen revised the
prognostic biomarkers indicate the disease outcome of patients to draft. All authors have read and approved the review.

Zhou et al.
64
ADDITIONAL INFORMATION 32. Wurdinger, T., In ‘t Veld, S. G. J. G. & Best, M. G. Platelet RNA as pan-tumor
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33. Song, P. et al. Limitations and opportunities of technologies for the analysis of
cell-free DNA in cancer diagnostics. Nat. Biomed. Eng. 6, 232–245 (2022).
34. Zou, J. & Wang, E. Cancer biomarker discovery for precision medicine: new
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Signal Transduction and Targeted Therapy www.nature.

REVIEW ARTICLE OPEN

Tumor biomarkers for diagnosis, prognosis and targeted

Signal Transduction and Targeted Therapy (2024)9:132 ; https://doi.org/10.1038/s41392-024-01823-2

Received: 5 June 2023 Revised: 7 March 2024 Accepted: 2 April 2024

© The Author(s) 2024

Fig. 1 Timeline of the history of tumor biomarker

Signal Transduction and Targeted Therapy (2024)9:132

Signal Transduction and Targeted Therapy (2024)9:132

Fig. 2 Technologies for the detection of tumor biomarkers

RIA technology antigens as tracers, the principle of FIA is similar to enzyme-

Signal Transduction and Targeted Therapy (2024)9:132

Signal Transduction and Targeted Therapy (2024)9:132

Signal Transduction and Targeted Therapy (2024)9:132

Fig. 3 Overview of human tumor biomarkers

Tumor biomarkers derived from blood

AFP. AFP, ﬁrst discovered in 1956 by Bergstrand Czar,146 is a 3–5%

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Signal Transduction and Targeted Therapy (2024)9:132

Signal Transduction and Targeted Therapy (2024)9:132

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Signal Transduction and Targeted Therapy (2024)9:132

Signal Transduction and Targeted Therapy (2024)9:132

Signal Transduction and Targeted Therapy (2024)9:132

Target Drug Highest Phase Indications Company/Identiﬁer Status

Signal Transduction and Targeted Therapy (2024)9:132

cellular KRAS mRNA and proteins, thus exerting antitumor

(NCT03948763) of the KRAS mRNA vaccine V941 (mRNA-5671) in

Targeting upstream of RAS: Targeting RAS plasma membrane

inhibition of RAS membrane localization is an effective therapeutic

the plasma membrane by suppressing farnesyltransferase, leading

acute myeloid leukemia,

to the blockade of downstream signaling.931 Tipifarnib, an orally

a fast track designation by the FDA in December 2019 for the

treatment of patients with HNSCC harboring HRAS mutations who

The objective response rate of tipifarnib in a phase II clinical study

metastatic salivary gland carcinoma with HRAS mutation,964 and in

HRAS-driven dedifferentiated thyroid cancers.965 Lonafarnib,

laminopathies, is also an orally active FTI that blockades RAS

localization in the plasma membrane. Salirasib is a farnesylcysteine

and N-RAS) by interfering with RAS binding to the plasma

CRC,971 melanoma,972 ovarian cancer,973 HCC,974 and pancreatic

cancer.975 However, a phase II clinical study revealed poor