International Journal of

Molecular Sciences

Review
An Overview of EGFR Mechanisms and Their Implications in
Targeted Therapies for Glioblastoma
Silvia Mara Baez Rodriguez 1 , Amira Kamel 1 , Gheorghe Vasile Ciubotaru 1 , Gelu Onose 2 ,
Ani-Simona Sevastre 3 , Veronica Sfredel 4 , Suzana Danoiu 4 , Anica Dricu 5, * and Ligia Gabriela Tataranu 1,6

1 Neurosurgical Department, Clinical Emergency Hospital “Bagdasar-Arseni”, Soseaua Berceni 12,

041915 Bucharest, Romania; [email protected] (S.M.B.R.); [email protected] (A.K.);
[email protected] (G.V.C.); [email protected] (L.G.T.)
2 Neuromuscular Rehabilitation Department, Clinical Emergency Hospital “Bagdasar-Arseni”, Soseaua Berceni
12, 041915 Bucharest, Romania; [email protected]
3 Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Medicine and Pharmacy of
Craiova, Str. Petru Rares nr. 2–4, 710204 Craiova, Romania; [email protected]
4 Department of Physiology, Faculty of Medicine, University of Medicine and Pharmacy of Craiova, Str. Petru
Rares nr. 2–4, 710204 Craiova, Romania; [email protected] (V.S.); [email protected] (S.D.)
5 Department of Biochemistry, Faculty of Medicine, University of Medicine and Pharmacy of Craiova, Str. Petru
Rares nr. 2–4, 710204 Craiova, Romania
6 Department of Neurosurgery, Faculty of Medicine, University of Medicine and Pharmacy “Carol Davila”,
020022 Bucharest, Romania
* Correspondence: [email protected]

Abstract: Despite all of the progress in understanding its molecular biology and pathogenesis,
glioblastoma (GBM) is one of the most aggressive types of cancers, and without an efficient treatment
modality at the moment, it remains largely incurable. Nowadays, one of the most frequently studied
molecules with important implications in the pathogenesis of the classical subtype of GBM is the
epidermal growth factor receptor (EGFR). Although many clinical trials aiming to study EGFR
targeted therapies have been performed, none of them have reported promising clinical results when
used in glioma patients. The resistance of GBM to these therapies was proven to be both acquired
Citation: Rodriguez, S.M.B.; Kamel,
and innate, and it seems to be influenced by a cumulus of factors such as ineffective blood–brain
A.; Ciubotaru, G.V.; Onose, G.;
barrier penetration, mutations, heterogeneity and compensatory signaling pathways. Recently, it was
Sevastre, A.-S.; Sfredel, V.; Danoiu, S.;
shown that EGFR possesses kinase-independent (KID) pro-survival functions in cancer cells. It seems
Dricu, A.; Tataranu, L.G. An
Overview of EGFR Mechanisms and
imperative to understand how the EGFR signaling pathways function and how they interconnect
Their Implications in Targeted with other pathways. Furthermore, it is important to identify the mechanisms of drug resistance and
Therapies for Glioblastoma. Int. J. to develop better tailored therapeutic agents.
Mol. Sci. 2023, 24, 11110. https://
doi.org/10.3390/ijms241311110 Keywords: glioblastoma; RTK; growth factor; EGFR; signaling pathways; EGFR targeted therapy

Academic Editor: Doriano Fabbro

Received: 4 June 2023

Revised: 29 June 2023 1. Introduction
Accepted: 3 July 2023
Glioblastoma multiforme (GBM, World Health Organization [WHO] grade 4 glioma)
Published: 5 July 2023
represents the most common, aggressive, and malignant central nervous system (CNS)
tumor. Despite advances made in the molecular characterization of GBM and newly avail-
able targeted therapeutic approaches, this form of brain cancer has a very poor prognosis
Copyright: © 2023 by the authors.
with a median survival time estimated at 15 months after maximal treatment [1]. The
Licensee MDPI, Basel, Switzerland. standard care for glioblastoma includes radical surgery followed by radiotherapy with
This article is an open access article concomitant chemotherapy with temozolomide (TMZ), an alkylating agent that causes
distributed under the terms and apoptosis generating single and double strand breaks in DNA [2]. Gliomas appear when
conditions of the Creative Commons glial cells—supportive cells of the CNS—become malignant [3–8]. Malignant gliomas are
Attribution (CC BY) license (https:// highly heterogeneous tumors comprised of multiple populations of cells in different phases
creativecommons.org/licenses/by/ of differentiation.
4.0/).

Int. J. Mol. Sci. 2023, 24, 11110. https://doi.org/10.3390/ijms241311110 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2023, 24, 11110 2 of 24

Grade 4 gliomas are characterized by necrosis, vascular proliferation, mitoses, nuclear

and cellular atypia [9], and pseudopalisading features [10]. The management of these
tumors presents many challenges: their location often limits the surgical accessibility, the
blood–brain barrier is almost impossible to penetrate by conventional medical therapies,
the infiltrative tumor cells migrate into normal brain parenchyma, the mass effect and also
the treatment of these tumors can cause important functional disability, high resistance to
radio- and chemotherapy, faulty well-developed neovasculature, the complex and dynamic
genome of the tumors, and their molecular and morphological heterogeneity [11,12].
The extent of tumor aggressiveness is determined by several pathological attributes,
such as nuclear atypia, necrosis, microvascular proliferation, and intratumoral hemor-
rhages [13,14]. The microenvironment poses many challenges for the glioma cells, including
hypoxia, acidity, and the lack of nutrients; therefore, they modulate their metabolic activity
in order to maintain the rapid growth [15,16]. At a microscopic level, GBMs invade the
brain beyond the gross radiographic margins of the tumor [17], making it impossible to
perform a complete surgical resection.
Until recently, the classification of gliomas was made based only on histological and
immunohistochemical criteria. Alongside classical diagnostic methods like functional
magnetic resonance (MRI), positron emission tomography, and computed tomography,
for GBMs, it is also useful to perform a liquid biopsy—a non-invasive technique which
confirms the diagnosis and helps to establish the correct treatment [18,19]—and an addi-
tional molecular diagnosis, which helps to provide a more personalized prognosis and
enhances the efficacy of therapy [20]. The fifth edition of the WHO Classification of Tumors
of the CNS, published in 2021 [21], included the molecular classification of the tumors.
This classification describes three genetic criteria for the diagnosis of GBM, IDH-wildtype:
telomerase reverse transcriptase (TERT) promoter mutation, epidermal growth factor re-
ceptor (EGFR) amplification, and the combined gain of the entire chromosome 7 and loss
of entire chromosome 10 [21].
GBMs have been demonstrated to possess intratumoral [22] and intertumoral [23]
heterogeneity together with a temporal evolution of their genome [24]. They also present an
abundance of cell signaling networks supporting tumor growth and, further, the tumor’s
resistance to therapy. The GBM genomic changes were first analyzed in the Cancer Genome
Atlas. GBM was classified into four subtypes based on the analysis of mutational changes
in 601 genes:
• Proneural—most common in younger patients. It presents an oligodendrocytic lineage
associated with secondary GBM, and enhancing mutations in tumor protein 53 (TP53)
and IDH1 genes;
• Neural—appears in older patients. Derived from astrocytes and oligodendrocytes, it
expresses neuron-related genes and no specific mutations;
• Classical—with no TP53 mutations and enhancing expression of EGFR;
• Mesenchymal—presents an astroglial lineage, with mutations in neurofibromin 1,
phosphatase, and tensin homolog (PTEN) and TP53 genes.
Evidence shows that almost every GBM tumor presents attributes of various subtypes,
indicating high levels of intra- and intertumoral heterogeneity [23,25,26] with individual
cell variations in their gene expression patterns [27].
A major part of modulating the invasion in GBMs is played by the members of the
protein tyrosine kinase family (PTK) [28,29]. The human protein kinase genome encloses
518 protein kinase genes, some of them being responsible for encoding transmembrane
receptor tyrosine kinases (RTKs) [30]. RTKs consist of an extracellular ligand-binding
domain, a single transmembrane helix domain, and an intracellular region comprised of
a juxtamembrane regulatory region, a tyrosine kinase domain (TKD), and a carboxyl (C-)
terminal tail [31]. More than 58 mammalian RTKs have been identified [30,32]. Tyrosine
phosphorylation plays an important role in signal transduction, modulating a large series
of cellular events under both physiological as well as pathological conditions, resulting in
cell differentiation, proliferation, migration, and survival [33,34]. The activation of receptor
Int. J. Mol. Sci. 2023, 24, 11110 3 of 24

tyrosine kinases (RTKs) results in their oligomerization, a process that occurs when specific
ligands bind to their extracellular domain, leading to the activation of the intracellular
domain. Then, with the help of recruitment proteins, it initiates a signaling cascade, with
numerous specific signaling pathways determining particular cellular responses depending
on the cell type and the activated signal transduction pathway [35].
The RTK dimerization can take place in four ways:
1. completely ligand mediated, with no direct contact between the extracellular regions
of the two receptors [36];
2. completely receptor mediated, with no physical interaction between two activating
ligands—as in the case of EGFR [37];
3. ligand homodimers attach themselves to two receptor molecules and then interact
across the dimer interface [38];
4. a combination of direct receptor–receptor contacts and bivalent ligand binding, with
the cooperation of accessory molecules in the receptor dimerization [39,40].
A portion of the RTKs can form dimers or high-order oligomers without the participa-
tion of activating ligands, with monomers and dimers in a dynamic equilibrium. In the
case of EGFR, the monomers are predominant before the ligand-binding phase [41]. The
predetermined dimers can be active or inactive, both forms being in dynamic equilibrium
with each other. Ligand-induced dimerization occurs after ligand binding, a process that
will help to stabilize the active dimer or to activate the inactive one [41–43].
The physiological information will be transmitted from the exterior of the cells to the
interior by a cascade of processes. First, intra-molecular interactions, that are different for
all the receptors, participate in maintaining the TKD in a cis-autoinhibition state [44,45].
After the ligand induces dimerization, this cis-autoinhibition is cleared and the transphos-
phorylation of key tyrosine residues is induced, leading to damage of the autoinhibitory
interactions, with an active conformation of the kinase the end of this process.
As the activation and the autophosphorylation of RTKs occurs, many downstream
signaling proteins are recruited. The majority of autophosphorylation sites work as binding
sites for Src homology-2 (SH2) and phosphotyrosine-binding (PTB) domain. Both domains
contain signaling proteins. The SH2 domain can be recruited either to the receptor directly
or indirectly with the participation of docking proteins that use their PTB domains to
bind to the RTKs. Additional signaling molecules that contain SH2 or other domains
are recruited by the docking proteins [33,46]. The activated RTKs may regulate various
signaling pathways with the participation of several docking proteins. Some of these
pathways are represented by RAS/MAPK, PI-3 K/AKT, and JAK2/STAT [47].
During glioma initiation and progression, aberrant RTK activation may occur. The
abnormal activation of RTK in cancers could be the result of four mechanisms: gain-of-
function mutation, genomic amplification, chromosomal rearrangements, and/or autocrine
activation. Evidence shows that these tumor-initiating cascades, through signaling cross-
talks, may be responsible for the malignant transformation of cells, resistance to treatment,
and relapse.
The most amplified RTK in GBM is the epidermal growth factor receptor (EGFR) [48].
EGFR gene amplification is detected in 57.4% patients presenting primary GBM [23] specific
to the classical subtype of glioma [25]. It results in high levels of EGFR protein, promoting
tumorigenesis and progression [23].
In recent years, clinical trials testing new drugs and therapies have been conducted,
but targeted therapies against EGFR have not yet shown any clinical benefit. Ineffective
blood–brain barrier penetration, heterogeneity, the presence of mutations, and the compen-
satory signaling pathways may play a significative role in the drug resistance. A deeper
understanding of the EGFR signaling networks and their interrelations with other path-
ways is essential to determine the mechanisms of resistance and to develop more optimized
and effective therapeutic strategies.
Int. J. Mol. Sci. 2023, 24, 11110 4 of 24

Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 4 of 24

In this review, we present the process of the activation of EGFRs under physiological
conditions and several mechanisms of aberrant activation in glioblastomas that present
important implications for theis essential
pathways development of anti-cancer
to determine therapies.
the mechanisms of resistance and to develop more op-
timized and effective therapeutic strategies.
2. EGFR Biology In this review, we present the process of the activation of EGFRs under physiological
conditions and several mechanisms of aberrant activation in glioblastomas that present
Epidermal growth
important belongs
factor tofor
implications thethelarge family of
development ofanti-cancer
RTKs. The EGFR family contains
therapies.
four members: designated EGFR (known as ErbB1/HER1) located on chromosome 7p12,
ErbB2 (HER2/Neu), ErbB32. EGFR(HER3),
Biology and ErbB4 (HER4) [49]. EGFR has the same structure
Epidermal
as all RTKs: an extracellular domain growth factor belongs
(ECD), a singleto the large family of RTKs.
transmembrane The EGFR(TMD),
domain family con-
an
tains four members: designated EGFR (known as ErbB1/HER1) located on chromosome
intracellular juxtamembrane domain (JMD), a tyrosine kinase, and a C-terminal
7p12, ErbB2 (HER2/Neu), ErbB3 (HER3), and ErbB4 (HER4) [49]. EGFR has the same struc- end.
EGFR is activated byastwo
ture large
all RTKs: groups of domain
an extracellular ligands: thea activators,
(ECD), also called
single transmembrane domainthe EGF
(TMD),
an intracellular
agonists, and the neuregulins juxtamembrane
[50,51]. More than domain (JMD), aare
40 ligands tyrosine kinase,in
involved and a C-terminal
signaling end.
control:
EGFR is activated by two large groups of ligands: the activators, also called the EGF
high-affinity ligands like EGF, transforming growth factor alpha (TGF-α), heparin-binding
agonists, and the neuregulins [50,51]. More than 40 ligands are involved in signaling con-
EGF-like growth factor (HB-EGF),
trol: high-affinitybetacellulin,
ligands like EGF, and low-affinity
transforming growthligands like(TGF-α),
factor alpha amphiregulin
heparin-
and epiregulin [52,53].binding
The EGF-like
ECD ofgrowthEGFR factor (HB-EGF),by
is formed betacellulin, and low-affinity
two homologous ligands like
domains, am-
I and
phiregulin and epiregulin [52,53]. The ECD of EGFR is formed by two homologous do-
III, where ligands bind, and two domains, II and IV, that are rich in cysteine [54]. After the
mains, I and III, where ligands bind, and two domains, II and IV, that are rich in cysteine
binding of extracellular
[54].ligands
After the like reactive
binding astrocytes
of extracellular ligandsandlikemicroglias secreted
reactive astrocytes by glioma
and microglias se-
cells and tumor microenvironmental
creted by glioma cells andcellstumor
[55],microenvironmental
EGFR suffers cells dimerization followed
[55], EGFR suffers by
dimeriza-
tion followed by trans-autophosphorylation of its intracellular domain. This may result
trans-autophosphorylation of its intracellular domain. This may result an active homodimer
an active homodimer when EGFR pairs with another EGFR or a heterodimer when it
when EGFR pairs withbindsanother EGFR
to another or aofheterodimer
member the EGFR family when it binds
[56] (Figure 1). to another member of
the EGFR family [56] (Figure 1).

Figure 1. The EGFR structure and mode of activation. (A) Overall view of the EGFR structure: the
Figure 1. The EGFR structure and mode of activation. (A) Overall view of the EGFR structure: the
extracellular region incorporates
extracellular four
regionsubdomains:
incorporates fourtwo homologous
subdomains: domains
two homologous D I and
domains D DIII,
D I and III,where
where
ligands bind (L1, L2), and D II and D IV, being cysteine rich domains (CR1, CR2); the transmembrane
ligands bind (L1, L2), and D II and D IV, being cysteine rich domains (CR1, CR2); the transmembrane
region, the juxtamembrane domain, the tyrosine kinase composed of the C-lobe and N-lobe, and the
region, the juxtamembrane domain,
C-terminal thedomains.
regulatory tyrosine (B) kinase
Different composed of the C-lobe
steps of EGFR activation: and N-lobe,
autoinhibitory and
tethered mon-
omer, untethered monomer, ligand-stabilized extended and ligand-induced activated dimer
the C-terminal regulatory domains. (B) Different steps of EGFR activation: autoinhibitory tethered
monomer, untethered monomer, ligand-stabilized extended and ligand-induced activated dimer
conformations. EGFR ligands binding to the receptor expose a dimerization motif which determines
structural rearrangements that are expressed in the cytoplasmic domain, allowing for the formation
of asymmetric dimers between the two juxtaposed catalytic domains. The ligand binding process
requires a 130◦ rotation of Domains I and II about the axis of the Domain II/III junction. In the active
state, the residues from Domain IV from the receptor pair form a V-shape. There is an interaction
between the carboxy-terminal portion of the activator kinase domain with the amino-terminal portion
of the receiver kinase domain. The transmembrane helices of the active receptor interact near their
amino-terminal by the extracellular compartment; the inactive receptor transmembrane helices
interact near their carboxy-terminal by the intracellular compartment.
Int. J. Mol. Sci. 2023, 24, 11110 5 of 24

Studies have shown that EGFR has pro-survival kinase-independent functions in

cancerous cells, offering a new perspective on EGFR implications in malignancies and
targeted cancer therapies [57–59].
Furthermore, EGFR was proven to influence the glutamine metabolism in glioma
cells [60]. Glutamate, obtained from glutamine by glutaminase conversion, is indispens-
able for tumor cells because of its role as a forerunner to produce α-KG, which further
generates carbon and nitrogen in the tumor cells. Indeed, the study performed by Yang
et al. demonstrated that the EGFR signaling pathway activated phosphorylated ELK1,
which further induced the GDH1 transcription, increasing the glutamine metabolism [61].
Therefore, EGFR plays a key role in the development and progression of glioma due to its
involvement in regulating the proto-oncogene MYC family and in promoting the glutamine
metabolism via ELK1 activation.

3. EGFR Pathway Activation

The EGFR pathway is activated by more than one mechanism, such as increased ligand
production or overexpression, receptor mutation, or a deficient inactivation.
Under normal physiologic conditions, the expression of EGFR is between 4 and 10 x104
receptors per cell [62]. Stimulating the EGFR-specific transcription factor (ETF) will result in
higher EGFR RNA expression. The epidermal growth factor, alongside other proteins like
E1A, Sp1, and AP2, is capable of regulating the expression of EGFR [58].
Multiple pathways are involved in receptor signaling. EGFR can activate a variety
of signal transduction pathways at the same time. One of the most frequently studied
pathways is the recruitment and activation of the phosphatidylinositol—3- kinase (PI3K)
signaling network, which is responsible for tumor growth caused by the downstream
activation of AKT and mTOR proteins [63]. PTEN plays a part in the negative regulation of
the pathway. Other important pathways are the RAS/MAPK pathway and the JAK2/STAT
pathway [64]. The genomic alterations of EGFR contribute to receptor activation and
signaling increase.

3.1. Activation of the Extracellular Domain

At the level of EGFR, the dimerization is mediated by receptors. There is no physical
contact between the activating ligands. In the inactive state, the receptors’ extracellular
domain has a tethered configuration, with the dimerization arm being blocked by intra-
molecular links and the tyrosine kinase domain being in an inert state. After ligand binding,
the dimerization arm is exposed, followed by the dimerization of the extracellular domain.
At the intracellular domain level, the kinase activation is facilitated by the configuration
changes [65].

3.2. Activation of the Intracellular Domains

After the ligand-induced dimerization, the cis-autoinhibition is discharged—the kinase
activity of EGFR being activated—by creating a physical contact between the C-terminal
tail of the activator and the N-terminal tail of the receiver. This process results in the
trans-phosphorylation of the C-terminal domain of the activator and structural changes to
the N-lobe of the receiver [66].

3.3. Downstream Signaling

The activation and autophosphorylation of EGFR are followed by the recruitment
of downstream signaling proteins such as Src Homology 2 (SH2) and phosphotyrosine
binding (PTB) signaling proteins. The SH2 proteins are capable of binding either directly to
the receptor or indirectly with the help of docking proteins using PTB domains [46]. Many
signaling pathways are then recruited and regulated by EGFR [34,48].
The PI-3K/AKT signaling pathway regulates apoptosis and cell survival. Following
the activation and the phosphorylation of EGFR, PI3K is recruited and transported to
the cell membrane where it phosphorylates phosphatidylinositol 4,5-biphosphate (PIP2),
Int. J. Mol. Sci. 2023, 24, 11110 6 of 24

resulting phosphatidylinositol (3,4,5)-triphosphate (PIP3). Then, PIP3 reacts with AKT and
can be either phosphorylated by phosphoinosite-dependent protein kinase-1 (PDK1) at
Threonin308, or by the mammalian target of rapamycin complex 2 (mTORC2) at Serine 473.
The PI3K/AKT pathway is negatively regulated by the phosphatase and tensin homolog
(PTEN). PTEN has the ability to dephosphorylate PIP3 and to remove it from the cellular
membrane. This process results in the relocation of AKT in the cytoplasm, with the loss of
the ability of being reactivated [67,68].
After the dimerization with human HER3, or through the docking protein GRB2-
associated binder 1 (GAB1), activated EGFR is associated with regulatory p85, absolving p85
of its inhibitory effect [69]. GAB1 is a scaffolding protein that is implicated in the attraction
of other signaling proteins like PI3K, SHP2, and p120RasGap. It plays an important part in
the association of EGFR with the PI3K/Akt signaling pathway, being implicated in others
EGFR signaling outputs as well [70,71]. In the future, GAB1 may be an important potential
therapeutic target due to its significant role in different types of cancers [72].
The RAS/MAPK signaling pathway requires growth-factor-receptor bound-2 (GRB2)
to form a complex with Son of Sevenless (SOS), which is a guanine-nucleotide exchange
factor (GEF), and the activation of the RAS G-protein, resulting in the conversion of guano-
sine diphosphate (GDP) into guanosine triphosphate (GTP) [73]. After these processes, a
downstream signaling cascade is activated by RAS and MAPK, resulting in the phospho-
rylation of the nuclear protein Jun, which, by binding to several nuclear proteins, leads
to the formation of the key transcription factor activator protein (AP-1). This protein is
responsible for the translation and transcription of the proteins that are responsible for
cellular growth and division. GTPase activating proteins (GAPs), such as tumor suppressor
neurofibromin 1 (NF1), are responsible for the negative regulation of activated RAS [74].
Signal transduction and activator of transcription 3 (STAT3) is activated by the EGFR
regulation of interleukin-6 (IL-6) expression, resulting in pSTAT3. This mechanism is
involved in the IL-6/Janus kinase (JAK)/STAT3 loop [34,75–77].

4. Role of EGFR in the Molecular Pathogenesis of Glioblastoma

4.1. Oncogenic Activation of RTKs
Normally, the activity of RTKs is managed by various already-described mechanisms
and by various molecules, one of them being tyrosine phosphatase [78]. The result of the
transforming abilities of RTKs provoked by several mechanisms, is the broken equilib-
rium between cell growth and proliferation and cell destruction [33,79]. Normal cells can
develop oncogenic properties as a result of constitutive activation causing RTK-induced
oncogenesis [80]. This type of activation can be the cause of four mechanisms in human
cancers [66]:
1. Gain of function mutations: these types of mutations lead to atypical downstream
signal transduction. One of these types of mutations are the “driver mutations” that are
able to give a selective growth advantage to the cells [81]. It is possible that the further
study of these “driver mutations” could help us to understand how the cancer initiates
and progresses and to bring new perspectives for targeted treatments. EGFR TKD is
encoded by exons 18–24, while the EGFR mutations appear mostly in exons 18–21,
close to the ATP binding pocket [82]. The kinase and the downstream signaling are
hyperactivated by these mutations, giving them oncogenic properties [82–84]. It was
demonstrated that patients with tumors that present somatic EGFR TKD mutations
are more sensitive to EGFR TKIs [85–91]. Other types of mutations can appear in
the extracellular domain (ECD), transmembrane domain (TMD), or juxtamembrane
domain (JMD) of RTKs. In glioblastoma, missense mutations at the level of EGFR
ECD were discovered and were associated with higher expression of EGFR protein,
which undergoes phosphorylation when not stimulated by ligand [92–94]. Patients
with EGFR ECD mutations had poor clinical results when under treatment with EGFR
TKIs erlotinib and gefitinib [95,96]. The ECD mutations seem to adopt an inactive
form, suggesting that they may be more responsive to EGFR targeted therapies that
Int. J. Mol. Sci. 2023, 24, 11110 7 of 24

bind to the inactive form of the receptor [97]. It seems that both intra- and extracellular
GBM mutations have, as a result, ligand-independent oncogenic activation.
2. Genomic amplification: EGFR is the most commonly overexpressed RTK in GBM [98].
The prevalence of EGFR gene amplification and overexpression is higher in primary
GBM than secondary GBM [99]. The consequence of overexpression is a higher local
concentration of the receptor, resulting in elevated RTK signaling and overpowered
antagonizing regulatory effects [100]. This overexpression is caused by multiple
mechanisms, the most important being gene amplification, followed by transcrip-
tional/translational enhancement [101,102], oncogenic viruses [103], degradation of
normal regulatory mechanisms such as the loss of phosphatases [104], and other
negative regulators. Gene amplification leads to an increase in the copy number of a
specific region of the genome [105] in the form of extrachromosomal elements (dou-
ble minutes) that are usually high-level amplifications with more than 25 copies, or
repeated units at a single locus or throughout the genome (distributed insertions) char-
acterized by low-level amplification with 5 to 25 copies [98,106]. These amplifications
can be determined by flaws in the DNA replication, fragile sites at the chromosomal
level, or telomere dysfunction [105]. The amplification pattern is quite different in the
same tumor type [98].
3. Chromosomal rearrangements: studies have shown that the formation of new tyrosine kinase
fusion oncoproteins is caused by numerous chromosomal rearrangements [23,107,108]. It may
be of significant importance to identify these fusion proteins, as they can be therapeutically
targetable with small molecule inhibitors. Some risk factors are thought to participate in the
gene fusion events—topoisomerase poisons [109], exposure to ionizing radiation [110,111],
and oxidative stress [112]—but the exact way in which these mechanisms function is not
yet known. Although a great number of tyrosine kinase fusions have been described, the
structure of the fusion oncoproteins is quite similar. The fusion can arise in either the N-
terminal or the C-terminal of the RTK, the tyrosine kinase domain being preserved either
way. The genomic breakpoint can appear either downstream of the exons that encode
the full kinase domain, in which case the ECD, TMD, and JMD are conserved and the
resultant fusion protein will behave like a membrane-bound receptor, or upstream thereof, in
which case loss of the ECD, TMD, and JMD occurs and the protein that appears as a result
will not be membrane bound. The chimeric fusion proteins that appear as a result of the
chromosomal rearrangements lead to oncogene addiction [113,114]. The use of target-specific
TKIs against RTK fusions may be a good weapon in the battle against numerous types of
RTK fusion-driven cancers.
4. Autocrine activation: communication between cells is carried out with the help of messengers,
like growth factors and cytokines, released by secretory cells. When the target cells are also
the secretory cells, the signaling is called “autocrine” [115]. This type of autocrine activation
can lead to tumor formation and clonal expansion [116]. In association with other autocrine
growth pathways, the autocrine activation loop of RTKs can lead to tumor formation. The
autocrine pathways can be used as a target for cancer therapy [117]. The wild-type EGFR
ligands, like TGF-alpha and HB-EGF, are generally increased in glioblastoma, leading to an
autocrine loop that results in the growth of glioma cells [118]. GBM expresses EGFRvIII,
which does not bind ligands and is thought to be more tumorigenic than wild-type EGFR.
TGF-α and HB-EGF induce the expression of EGFRvIII, implying that EGFRvIII may create
an autocrine loop with wild-type EGFR, which plays an important role in signal transduction
in glioblastoma cells [119].
Another type of RTK activating mechanism is special intragenic partial duplication
-kinase domain duplication (KDD) [120]. It is represented by a type of chromosomal
rearrangement that gives cancer cells the capacity to achieve new protein isoforms [121].
As an example, EGFR-KDD is present in human cancers, including glioma, with good
response to the targeted therapies against EGFR [122]. Amplification of the EGFR-KDD in
the post-treatment biopsy leads to the conclusion that KDD may contribute to the achieved
resistance of EGFR TKI, afatinib [122].
Int. J. Mol. Sci. 2023, 24, 11110 8 of 24

4.2. EGFR Mutations

Glioblastoma is thought to be a copy number disease, almost 20% of them with-
holding EGFR point mutations, which are sometimes associated with copy alterations or
rearrangements [23]. These point mutations appear more commonly in the extracellular
domain [92,97,123] and may be enhanced in EGFR-amplified tumors, but they can also
be detected without amplification [23,124]. Clinical detection of the mutations plays an
important part in treatment with EGFR inhibitors.
EGFR was one of the first oncogenes to be identified in glioblastoma. In cancer cells,
more than 106 EGFR receptors/cell were observed [125]. There are two types of patho-
logical alterations of EGFR in cancers: one is represented by a kinase-activating mutation
in EGFR, which can lead to increased tyrosine kinase activity of EGFR and can be pri-
mary or secondary to anti-EGFR therapies [126,127], and the second is represented by the
overexpression of the EGFR protein and may or may not be associated with gene amplifica-
tions [128–131]. The molecular alterations of EGFR include overexpression, deletion, and
amplification. In glioblastoma, the amplification of the EGFR gene is the most frequent RTK
mutation (approximately 40%) [132] and is mostly observed in primary GBM. The EGFR
amplification is a promoter for invasion, proliferation, and drug resistance [133]. EGFR has
also been associated with structural alterations, the most common being EGFR variant III
(EGFRvIII). This variant has an in-frame deletion of exons 2–7 from the extracellular domain
and is able to transmit growth signaling in a ligand-independent way [134,135]. The expres-
sion of EGFRvIII in glioma cells leads to the expression of TGF-α and HB-EGF, resulting
in EGFRvIII being involved in generating an autocrine loop with wild-type EGFR expres-
sion [119]. Various downstream pathways are activated when EGFR signaling is influenced
by genetic alterations. Some of these pathways are represented by phosphatidylinositol 3-
kinase (PI3K)/Akt and Ras/Raf/MEK (MAPK kinase)/MAPK (mitogen-activated protein
kinase), and they are responsible for cell proliferation, survival, and mobility [136,137]. The
tumor suppressor phosphatase and tensin homolog deleted from chromosome 10 (PTEN)
negatively regulate the PI3K/Akt pathway. PTEN is emblematic for GBM, being mutated
in 20% to 40% of cases [138].
The secondary mutations that contribute to acquired resistance to anti-EGFR therapies
are represented by the T790M mutation, accounting for 50% of resistance in non-small cell
lung cancer patients that were treated with first- and second-generation TKIs [139,140].
Studies have shown that EGFR overexpression or mutation causes shorter overall
survival in patients with GBM [141–143].

4.3. EGFR Wild-Type

Overexpression of wild-type EGFR is tumorigenic in a variety of types of cells [144–146],
concluding that EGFR wild-type is oncogenic. The protein levels of EGFR, independent of its
phosphorylation status, is strongly associated with the progression of the disease and a bad
prognosis of a variety of types of cancers not expressing mutated EGFR [147,148]. Most of the
GBMs and low-grade astrocytomas as well as many other types of solid cancers overexpress
wild-type EGFR protein [149] throughout the entire tumor. The presence of the overexpression
of this protein is mostly associated with negative prognosis [150–153], being a more common
phenomenon than EGFR mutations and promoting disease progression [57]. It was observed
by immunohistochemistry (IHC) that almost all cells within the tumor express high levels of
EGFR protein.
The reason why cancers expressing/overexpressing wild-type EGFR mutation don’t
have a good response to EGFR TKIs is connected to the fact that these types of cancers are
not addicted to the EGFR functions for growth and/or survival. However, it seems that
TKIs are highly capable to inhibit the growth of wild-type EGFR expressing cells [154,155].
Studies are also showing that cells expressing wild-type EGFR cannot survive after EGFR
knockdown by siRNA [156–160]. All this information helps to conclude that although
EGFR wild-type cancer cells survival may not be dependent on EGFR’s kinase activity, it
may be sustained by EGFR without involving its kinase activity.
Int. J. Mol. Sci. 2023, 24, 11110 9 of 24

4.4. EGFR Copy Number Alterations

Focal gene amplification causes an increased number of copies of the gene and is the
most frequent EGFR anomaly in adult GBM. This amplification is the result of specific mech-
anisms, which include the formation of extrachromosomal double minutes (dmins) and the
heterogeneously staining regions (HSR) that affect a specific gene locus or chromosomal region.
The consequence of this amplification is the increase of the copy number. There is a need to
differentiate the terms “gain” that indicates a lower-level increase of gene number caused by
different mechanism like tandem gene duplication or polysomy, and the term “amplification”
that is used to indicate any level of increase without mechanism implications.
The most common EGFR copy number alterations in glioma is the polysomy of the
entire chromosome 7. A substantial percentage of gliomas present other alterations that
can lead to a gain of 7q that contains the EGFR gene. Although these chromosomal gains
appear frequently in gliomas, they are not GBM specific. More than 40% of the GBM’s
present focal EGFR gene amplification having more than 50–100 copies of the gene [23,161].
These amplifications may also appear in association with the co-amplification of other RTK
genes [162].
The EGFR copy number alterations can be used clinically for the diagnosis and classi-
fication of GBM’s, helping to distinguish them from other tumors with similar histological
features, these amplifications being almost specific exclusively to glioblastomas. Although
initial studies demonstrated the poor prognosis of the presence of EGFR amplification, later
investigations have not confirmed these results [138]. The role of EGFR amplifications as a
predictive marker was highly studied in a variety of EGFR inhibitors clinical trials, failing
to show good results as an independent biomarker [163,164].

4.5. EGFR Rearrangements

4.5.1. EGFRvIII
Almost 30% of the GBMs present the variant EGFRvIII, the most frequent intragenic
deletion of EGFR. It appears only in tumors that present with EGFR amplification and
presents with intragenic in-frame deletion of exons 2–7, resulting in the overexpression of
a truncated receptor that has some missing parts of the ECD. It is not capable of binding
ligands, and it is constitutively active due to its ligand-independent dimerization [165]. It is
a strong oncogenic promoter, being able to transform cells and generate a more aggressive
phenotype in GBMs. The presence of EGFRvIII is specific to GBM, and it can be used
as a glioblastoma-specific marker. Although the identification of EGFRvIII is not usually
conducted for all patients, it can be performed to identify patients that could be included
in specific clinical trials directed against this antigen. The use of IHC helped to identify the
heterogeneous extensive expression of EGFRvIII within the tumor, information that was
also proved by the patterns observed in genomic assays [166], representing an important
hypothesis of the resistance in nonexpressing cells.

4.5.2. Other Deletion Variants

In 71% of the EGFR-amplified tumors, one can identify intragenic deletions. EGFRvII
is one of the variants that may be clinically important to the pathogenesis of GBM [161].
Other deletions that were capable of performing cell transformation without ligand were
those involving the carboxy-terminal intracellular domain [135,167]. These presented high
sensitivity to cetuximab in a xenograft model [168].

4.6. EGFR Fusions

When a large RNA-sequencing dataset of glioblastoma was investigated, several in-frame
gene fusion and elaborated rearrangements were exposed [169,170]. EGFR was the most
common gene fusion in GBM. The most common EGFR fusion was to intron 9 of SEPT14 or
to the gene PSPH, leaving the tyrosine kinase domain unharmed [169]. Due to the lacking
expression of EGFRvIII in the before-mentioned fusions, the C-terminal deletion of EGFR
through gene fusion could be an additional mechanism involved in EGFR activation.
Int. J. Mol. Sci. 2023, 24, 11110 10 of 24

4.7. MicroRNAs
MicroRNAs (miRNAs) are endogenous noncoding single-stranded RNAs consisting
of approximately 20–22 nucleotides. They regulate gene expression by binding to target
protein-encoding mRNA at the posttranscriptional level. MiRNAs have the ability to
regulate a large variety of carcinogenic pathways, including the EGFR signaling pathway.
MiRNAs have important roles in the initiation, progression, and prognosis of various
human illnesses, including cancers. The expression of RTKs can be directly modulated
by the activity of microRNAs. These are able to function as both tumor suppressors and
as oncogenes [171,172], and they have been proved to play a part in RTK signaling and
the regulation of tumor formation. Chromosomal regions that encode oncogenic miRNAs
related to the negative regulation of tumor suppressor genes are amplified. Usually,
miRNAs that target oncogenes are located at fragile sites where deletions or mutations may
appear, resulting in the reduction or loss of expression of miRNAs and the overexpression
of targeted oncogenes, thereby initiating cancer development [173].
Recent studies showed that miRNAs participate in the occurrence and development of
GBM, regulating numerous biological activities [174,175] such as tumor growth, migration,
invasion, and chemotherapy resistance. They may also be used as biomarkers for diagnosis,
treatment, and prognosis [176]. Due to their function of regulating the expression of
relevant proteins that are involved in the EGFR signaling pathway, they cause tumor cell
apoptosis, proliferation, migration, and invasion.
MicroRNA-219-5p can suppress GBM development by silencing EGFR expression, as
it is able to bind directly to its 30 -UTR [177]. Alongside others RTKs, EGFR can regulate
microRNA-134 in GBM, which acts as a tumor-suppressive center [178]. Further advanced
study of microRNAs and RTKs signaling will bring improvements in the cancer therapies
domain. It is already known that the association of an inhibitor of microRNA-21 and a
monoclonal antibody against EGFR is able to improve the treatment outcome in GBM [179].
MicroRNAs could be an important prognostic marker and help with patient stratification,
and further understanding of their role in RTK signaling may help in cancer detection,
therapy, and prognosis.

4.8. Crosstalk
Many RTKs lead to activation of PI3K/Akt and Rac1 signaling pathways in gliomas,
suggesting that they play an important part in downstream signaling for invasion. In his
study, Stommel revealed the co-expression of multiple activated RTKs in individual disso-
ciated cells in a primary GBM [180]. Individual cells have the capacity to express a diverse
number of RTKs, which in turn have the ability to interact with each other. The phosphory-
lation of the c-Met receptor as a function of EGFRvIII receptor levels suggests a crosstalk
between c-Met and EGFRvIIII signaling [181]. Axl RTK has a similar response [181]. EGFR
and EphA2 co-localize on the cell surface and are both expressed in GBM. EphA2 phospho-
rylation is modulated by EGFR activity. EGFR phosphorylation, downstream signaling,
and EGF- induced cell viability are inhibited by EphA2 downregulation [119].
Due to the multiple correlations between RTKs, one single RTK inhibitor is not capable
of successfully suppressing the invasion signaling.

5. Targeted Therapy—EGFR as Therapeutic Agent

In recent years, many agents, TKIs, and monoclonal antibodies have been developed
and used in the clinical treatment of different cancer types with the goal of inhibiting the
signaling pathways of EGFR [182,183]. The activity of EGFR can be regulated by binding
it to either the extracellular domain or the tyrosine kinase domain. Three generations of
TKIs have been developed and approved for use in a variety of cancer types. These TKIs
work by targeting signal transduction, binding to the EGFR tyrosine kinase domain, and
inhibiting its activity. The first generation of TKIs works by competitive binding with ATP
and inhibiting the receptor. As the emergence of acquired resistance to targeted therapies is
inevitable [184,185], new agents of second-, third-, and fourth-generation inhibitors and
Int. J. Mol. Sci. 2023, 24, 11110 11 of 24

combinations of both TKIs and monoclonal antibodies are now being studied [91,186–189].
Second-generation TKIs have the ability to irreversibly inhibit all four ERBB receptors, third-
generation TKIs were designed to target the T790M resistance mutation that is responsible
for about 50% of acquired resistance to the earlier generation of TKIs [190–193], and fourth-
generation TKIs preferentially inhibit the T790M/C797S EGFR mutant that leads to some
resistance to the third-generation TKIs [193]. The monoclonal antibodies are receptor
blockers and work by preventing EGFR from binding to the ligand by binding to its ECD.
The therapeutic effect of anti-EGFR antibodies cannot be solely attributed to the inhibition
of the tyrosine kinase activity of EGFR.
The existing data suggest that the tyrosine activity of EGFR is primarily involved in
promoting cell proliferation rather than cell survival [194]. Under physiologically relevant
conditions, EGFR TKIs and mAbs constantly present anti-proliferative effects [195]. Concern-
ing the impact that TKIs have on cell survival, studies have shown that they are able to induce
cytoprotective autophagy, which has the capacity to promote cell survival [196,197].

The Kinase-Independent Pro-Survival Function of EGFR in Cancer Cells

Studies made in recent years have shown that EGFR has pro-survival functions that are
independent of its tyrosine kinase activity in cancer cells. This type of kinase-independent
(KID) pro-survival function has been encountered in different types of cancer cells, includ-
ing several cellular functional domains, such as the plasma membrane, the autophagic
machinery, and the mitochondrion. The discovery of this pro-survival function has pro-
found implications for overcoming the challenges of EGFR targeted therapies as it offers an
alternative point of view on the clinical failures of already-existing EGFR kinase inhibitors.
It may also yield new perspectives on cancers that are expressing/overexpressing EGFR
wild-type, as these cancers are innately resistant to EGFR inhibitors. It may be that these
cancers cells are more dependent on EGFR’s KID function for survival rather than on its
kinase activity for growth.
An explanation of how the acquired TKI resistance develops is that the EGFR’s kinase
dependent function is shifted toward its KID pro-survival function, giving cancer cells
that are dependent on EGFR kinase activity for growth a way to establish alternative
proliferative mechanisms, bypassing the EGFR kinase-dominated pathway that is under
constant exposure to TKIs. It has been proven that EGFR can be found in two states in
cancer cells: with the capacity to be activated or without this capacity [198]. EGFR with the
capacity to be activated behaves according to the canonical mechanisms, while EGFR that
cannot be activated is characterized by the inability to autophosphorylate while physically
interacting with other proteins at its C-terminal kinase domain [198].
On the basis of existing evidence, it was concluded that the pro-survival function
of EGFR is regulated by mechanisms that are not dependent on EGFR kinase function.
Targeting the KID pro-survival function of EGFR by lowering its protein level may be
helpful in finding more effective ways of targeting EGFR for cancer therapy.

6. The tyrosine Kinase Inhibitors

6.1. First-Generation EGFR Inhibitors
Gefitinib is the first selective inhibitor of EGFR tyrosine kinase to be made with
low molecular weight. Early studies may have shown signs of activity in patients with
malignant gliomas [96,163], but in a multicenter phase II study, it showed limited efficacy
both as part of the initial treatment regimen and for recurrent disease [96,199]. In a phase
I/II trial, when it was combined with radiation for the treatment of newly diagnosed GBM,
gefitinib showed no improved overall survival [200].
Erlotinib is an orally active, reversible tyrosine kinase inhibitor (TKI). Studies have not
yet demonstrated its efficacy in the treatment of newly diagnosed GBM either used alone
or in combination with temsirolimus or bevacizumab [163,164,201,202].
Int. J. Mol. Sci. 2023, 24, 11110 12 of 24

Lapatinib, a first-generation EGFR inhibitor, is a dual TKI that acts as an interrupter on

the HER2/neu growth receptor factor. It is being used successfully in breast cancer therapy,
but it is not demonstrating results in patients with recurrent GBM [203].

6.2. Second-Generation EGFR Inhibitors

Afatinib showed limited efficacy when used as a single agent in a clinical trial in
recurrent glioblastoma [204].
Tesevatinib is currently being evaluated for glioblastoma therapy. Tesevatinib had
good efficacy results in EGFR-amplified PDX GBM models in vitro but achieved relatively
modest performance in vivo despite a high brain-to-plasma ratio [205].
Dacomitinib had poor results in a phase II clinical trial for recurrent glioblastoma [206].
Vandetanib was only studied in association with other therapies, either radiotherapy
or therapeutic agents, and yielded very poor results.

6.3. Third-Generation EGFR Inhibitors

Osimertinib demonstrated an ability to inhibit the constitutive activity of EGFRvIII
tyrosine kinase and its downstream signaling in a new study [207].
CM93 is a novel covalent small molecule inhibitor which targets mutant EGFR [208].
In a study performed by Ni et al., this new inhibitor showed promising results expressed
as better efficacy in preclinical studies compared with other EGFR-TKIs. It also extended
the survival rate of mice with orthotopic GBM allografts [209].

6.4. Fourth-Generation EGFR Inhibitors

Fourth-generation tyrosine kinase inhibitors are still under the early phases of preclin-
ical development. The first fourth-generation TKI to be developed is allosteric inhibitor
EAI045, which has proven its efficiency in inhibiting the kinase activity of T790M/C797S in
combination with Cetuximab but not as a single agent [193].
Recently, the orally bioavailable ERAS-801, a small-molecule EGFR inhibitor, has
received an orphan drug designation by the FDA for malignant glioma [210]. In a mouse
model, WSD-0922 preferentially inhibited the phosphorylation of several proteins, includ-
ing EGFR inhibitor resistance proteins [211]. In clinical studies, ERAS-801 demonstrated
a survival benefit in 93% of EGFR mutant and/or amplified models and had a higher
brain penetrance in addition to prolonged survival compared with other approved EGFR
inhibitors such as osimertinib, lapatinib, or erlotinib [210].

7. The Monoclonal Antibodies

Nimotuzumab is an antibody targeting the L2 domain of EGFR, a humanized mon-
oclonal antibody responsible for the inhibition of tyrosine kinase activation. The use of
nimotuzumab showed relatively favorable results in pediatric diffuse intrinsic pontine
glioma [212,213]. It is currently being studied in a phase III trial in concomitant use with
radiotherapy and temozolomide in adult GBM patients.
Cetuximab is an antibody targeting the L2 domain of EGFR, and it prevents dimer-
ization and cross-activation, thereby inhibiting downstream signal transduction. It is a
chimeric monoclonal IgG1 antibody with high affinity and specificity for EGFR. Studies
showed that cetuximab may enhance the cytotoxicity of radiotherapy in patients with
EGFR-amplified GBM [214,215]. In 2009, another study showed that there is no correla-
tion between survival and EGFR amplification and the use of cetuximab in patients with
recurrent glioblastoma [216].
Panitumumab is being investigated in a phase II trial as a GBM diagnostic agent.
bscEGFRvIIIxCD3 is a bispecific T-cell engager antibody (BiTEs) that can bind to the
CD3 T-cell coreceptor and recruit cytotoxic T cells. It redirects the T cells towards tumors
that express EGFRvIII. Results of the studies made in vitro and in vivo on mice showed
that this agent has the ability to destroy GBM cells that express EGFRvIII [217].
Int. J. Mol. Sci. 2023, 24, 11110 13 of 24

mAB806 targets EGFRvIII, its mechanism of action being still unknown [218]. It has
the ability to raise the sensitivity of glioma xenotransplants to radiotherapy [219].

8. Immunotherapy
CAR T-cells targeting EGFRvIII is a new technology that utilizes a chimeric antigen
receptor (CAR) expressed by engineered T cells to recognize their target, in the case of
glioblastoma, the target being represented by the EGFRvIII [220]. This technology is still
being studied. The results of preclinical studies showed that the use of CAR T-cells targeting
EGFRvIII had impressive results in reducing tumor growth [221]. One study showed a
lower expression of EGFRvIII in resected tumors from patients treated with CAR T-cell
infusion [222]. It has been shown that CAR T cells specific to EGFRvIII have limited efficacy
in GBM patients [223].
Regarding the immunologic target, rindopepimut it is a vaccine that uses an EGFRvIII-
specific peptide conjugated to a keyhole limpet hemocyanin. The results of a phase III
clinical trial showed no improvement in the survival rate of newly diagnosed glioblastoma
patients after the use of the vaccine [224–226].
T-cell bispecific antibodies (TCB) are engineered molecules that bind both the T-cell
receptor and tumor-specific antigens. In a study performed by Iurlaro et al. in patient-
derived models expressing EGFRvIII, the EGFRvIII-TCB demonstrated specificity for the
mentioned receptor and also promoted tumor cell death together with T-cell activation
and cytokine secretion. Furthermore, EGFRvIII-TCB promoted the recruitment of T-cells
in the intracranial tumors. It induced tumor regression in humanized orthotopic GBM
patient-derived xenograft models [227].

9. Targeted Isotopes
125 I-mAb 425, antibody toxin or radioactive isotope conjugated, is an 125 I labeled
anti-EGFR 425 murine monoclonal antibody developed from mice immunized with A-431
epidermoid carcinoma cells. It binds to the tumor and induces direct cell growth inhibition,
complement-dependent cytotoxicity, and activation of the humoral response. A phase II
study conducted in 2010 [228] demonstrated that its use was safe and well tolerated, and
the reported survival was 15.7 months.

10. Nanoparticles
Nanoparticles are vesicular carriers that can protect their composition, resulting in
higher bioavailability with a better capacity to penetrate the blood–brain barrier. A high
number of agents have been entrapped in a variety of nanoparticles targeting EGFR [229].
Studies are still in progress for cetuximab-conjugated liposomes [230].

11. Targeting the Regulation of EGFR Gene Expression

This method is represented by the use of ribozymes, antisense oligonucleotides, siRNA,
and miRNA. The post-transcriptional gene expression of RTKs’ signaling pathways is under
the control of microRNAs. It was demonstrated that EGFR can be carried by extracellular
vesicles (EVs) [231] and that the EVs participating in the cell communication can enhance
the intratumoral heterogeneity of glioblastomas [232]. To date, none of the strategies that
target the regulation EGFR gene expression have been further studied.

12. Challenges to Current Anti-EGFR Therapies

The efficacy of EGFR targeted cancer therapies is obstructed by two major challenges:
acquired and innate resistance towards the available drugs.
Drug resistance may be able to explain why the response to EGFR targeted therapies is not
efficient for glioblastoma [233]. Two mechanisms were proposed to explain this resistance:
1. The target independence. Not all of the glioblastoma cells express EGFR proteins;
therefore, the EGFR inhibitors have no effect on them. A frequent situation encoun-
tered in this type of resistance is the loss of extra-chromosomally encoded EGFR.
Int. J. Mol. Sci. 2023, 24, 11110 14 of 24

It can appear after the use of small molecule therapies. Small circular fragments
of extra-chromosomal DNA act as regulators of dynamic EGFRvIII expression, and
they may be involved in the resistance to inhibition. Studies have shown that after
the treatment of GBM cells with erlotinib, mutant EGFR was reversibly blocked by
producing extra-chromosomal DNA. By seizing the use of erlotinib, the mutations
reappeared, resulting in the upregulation of EGFRvIII [234].
2. The target compensation. The GBM cells activate compensatory pathways that are in-
dependent of EGFR signaling [183], such as the RAF/MEK/MAPK/ERK-, PI3K/Akt-,
and MET-regulated signaling pathways [182,235–238].
Drug resistance may be compensated for by using multi-target therapies that work
to compensate both mechanisms described: targeting truncation mutations for the first
mechanism and targeting compensatory proteins for the second mechanism.
Patient mutations that affect the trafficking of therapeutic antibodies are another
possible mechanism involved in resistance [239,240].
Innate resistance to anti-EGFR drugs is much more prevalent than acquired resistance.
It seems that cancers expressing wild-type EGFR do not respond to TKIs regardless of
the expression level of EGFR [241]. It was hypothesized that EGFR is unimportant for
those types of cancers that are innately resistant to EGFR kinase inhibitors, but this idea
was invalidated by new evidence showing that even in innately resistant cancer types, the
downregulation of EGFR proteins causes severe cell death [157]. In other words, even in
the cancer cells that are innately resistant to EGFR kinase inhibitors, EGFR is indispensable
for survival.

13. Conclusions
A better understanding of RTK signaling pathways, getting to know all of the tools
they provide us, including their genetic, cellular, biochemical, and structural modeling
techniques, can help us to improve patient care. One of the most frequently studied and
used RTKs in the battle against cancer is EGFR. There is still a long way to go to understand
all of the features of the signaling pathways and their ramifications. The focus must be on
the improvement of existent therapy technologies and the development of new ones that
may improve the patient’s quality of life.
Focusing on only one type of EGFR-altered tumor (EGFRvIII deletion) may be insuffi-
cient in the battle against the complex heterogeneity of GBM [163,242]. Bearing in mind
the continuous resistance of GBM to the EGFR inhibition tried so far, we can only hope
that the use of new diagnostic methods for assessing EGFR can help us to find new and
more-effective methods of inhibition.

Author Contributions: Conceptualization, A.D. and L.G.T.; data curation, A.K. and V.S.; formal
analysis, L.G.T. and S.D.; funding acquisition, A.D.; investigation, S.M.B.R., G.V.C. and G.O.; method-
ology, S.M.B.R. and S.D.; project administration, A.D.; resources, A.D. and V.S.; supervision, A.D.
and L.G.T.; visualization, A.K. and S.D.; writing—original draft preparation, S.M.B.R. and L.G.T.;
writing—review and editing, A.-S.S. and A.D. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by Grant PN-III-P4-ID-PCE-2020-1649, UEFISCDI, Romania.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: No new data were created or analyzed in this study. Data sharing is
not applicable to this article.
Conflicts of Interest: The authors declare no conflict of interest.
Int. J. Mol. Sci. 2023, 24, 11110 15 of 24

References
1. Stupp, R.; Mason, W.P.; Van Den Bent, M.J.; Weller, M.; Fisher, B.; Taphoorn, M.J.; Belanger, K.; Brandes, A.A.; Marosi, C.; Bogdahn,
U. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N. Engl. J. Med. 2005, 352, 987–996. [CrossRef]
2. Brandes, A.A.; Bartolotti, M.; Tosoni, A.; Franceschi, E. Nitrosoureas in the management of malignant gliomas. Curr. Neurol.
Neurosci. Rep. 2016, 16, 13. [CrossRef] [PubMed]
3. Lathia, J.D.; Mack, S.C.; Mulkearns-Hubert, E.E.; Valentim, C.L.; Rich, J.N. Cancer stem cells in glioblastoma. Genes Dev. 2015, 29,
1203–1217. [CrossRef] [PubMed]
4. Rodriguez, S.M.B.; Staicu, G.-A.; Sevastre, A.-S.; Baloi, C.; Ciubotaru, V.; Dricu, A.; Tataranu, L.G. Glioblastoma Stem Cells—Useful
Tools in the Battle against Cancer. Int. J. Mol. Sci. 2022, 23, 4602. [CrossRef] [PubMed]
5. Sanai, N.; Alvarez-Buylla, A.; Berger, M.S. Neural stem cells and the origin of gliomas. N. Engl. J. Med. 2005, 353, 811–822.
[CrossRef] [PubMed]
6. Lim, D.A.; Cha, S.; Mayo, M.C.; Chen, M.-H.; Keles, E.; VandenBerg, S.; Berger, M.S. Relationship of glioblastoma multiforme to
neural stem cell regions predicts invasive and multifocal tumor phenotype. Neuro-Oncology 2007, 9, 424–429. [CrossRef]
7. Goffart, N.; Kroonen, J.; Rogister, B. Glioblastoma-initiating cells: Relationship with neural stem cells and the micro-environment.
Cancers 2013, 5, 1049–1071. [CrossRef]
8. Deleanu, R.; Ceafalan, L.C.; Dricu, A. Transcriptomic Crosstalk between Gliomas and Telencephalic Neural Stem and Progenitor
Cells for Defining Heterogeneity and Targeted Signaling Pathways. Int. J. Mol. Sci. 2021, 22, 13211. [CrossRef]
9. Bastien, J.I.; McNeill, K.A.; Fine, H.A. Molecular characterizations of glioblastoma, targeted therapy, and clinical results to date.
Cancer 2015, 121, 502–516. [CrossRef]
10. Rong, Y.; Durden, D.L.; Van Meir, E.G.; Brat, D.J. ‘Pseudopalisading’necrosis in glioblastoma: A familiar morphologic feature that
links vascular pathology, hypoxia, and angiogenesis. J. Neuropathol. Exp. Neurol. 2006, 65, 529–539. [CrossRef]
11. Artene, S.-A.; Turcu-Stiolica, A.; Ciurea, M.E.; Folcuti, C.; Tataranu, L.G.; Alexandru, O.; Purcaru, O.S.; Tache, D.E.; Boldeanu,
M.V.; Silosi, C. Comparative effect of immunotherapy and standard therapy in patients with high grade glioma: A meta-analysis
of published clinical trials. Sci. Rep. 2018, 8, 11800. [CrossRef]
12. Alexandru, O.; Sevastre, A.-S.; Castro, J.; Artene, S.-A.; Tache, D.E.; Purcaru, O.S.; Sfredel, V.; Tataranu, L.G.; Dricu, A. Platelet-
derived growth factor receptor and ionizing radiation in high grade glioma cell lines. Int. J. Mol. Sci. 2019, 20, 4663. [CrossRef]
13. Louis, D.N.; Perry, A.; Reifenberger, G.; Von Deimling, A.; Figarella-Branger, D.; Cavenee, W.K.; Ohgaki, H.; Wiestler, O.D.;
Kleihues, P.; Ellison, D.W. The 2016 World Health Organization classification of tumors of the central nervous system: A summary.
Acta Neuropathol. 2016, 131, 803–820. [CrossRef] [PubMed]
14. Wesseling, P.; Kros, J.M.; Jeuken, J.W. The pathological diagnosis of diffuse gliomas: Towards a smart synthesis of microscopic
and molecular information in a multidisciplinary context. Diagn. Histopathol. 2011, 17, 486–494. [CrossRef]
15. Mao, H.; LeBrun, D.G.; Yang, J.; Zhu, V.F.; Li, M. Deregulated signaling pathways in glioblastoma multiforme: Molecular
mechanisms and therapeutic targets. Cancer Investig. 2012, 30, 48–56. [CrossRef] [PubMed]
16. Boyd, N.H.; Tran, A.N.; Bernstock, J.D.; Etminan, T.; Jones, A.B.; Gillespie, G.Y.; Friedman, G.K.; Hjelmeland, A.B. Glioma stem
cells and their roles within the hypoxic tumor microenvironment. Theranostics 2021, 11, 665. [CrossRef] [PubMed]
17. Burger, P.C. Pathologie Anatomy and CT Correlations in the Glioblastoma Multif orme. Stereotact. Funct. Neurosurg. 1983, 46,
180–187. [CrossRef]
18. Wu, Z.; Dai, L.; Tang, K.; Ma, Y.; Song, B.; Zhang, Y.; Li, J.; Lui, S.; Gong, Q.; Wu, M. Advances in magnetic resonance imaging
contrast agents for glioblastoma-targeting theranostics. Regen. Biomater. 2021, 8, rbab062. [CrossRef]
19. Galldiks, N.; Niyazi, M.; Grosu, A.L.; Kocher, M.; Langen, K.-J.; Law, I.; Minniti, G.; Kim, M.M.; Tsien, C.; Dhermain, F.
Contribution of PET imaging to radiotherapy planning and monitoring in glioma patients-a report of the PET/RANO group.
Neuro-Oncology 2021, 23, 881–893. [CrossRef]
20. Verdugo, E.; Puerto, I.; Medina, M.Á. An update on the molecular biology of glioblastoma, with clinical implications and progress
in its treatment. Cancer Commun. 2022, 42, 1083–1111. [CrossRef]
21. Louis, D.N.; Perry, A.; Wesseling, P.; Brat, D.J.; Cree, I.A.; Figarella-Branger, D.; Hawkins, C.; Ng, H.; Pfister, S.M.; Reifenberger,
G. The 2021 WHO classification of tumors of the central nervous system: A summary. Neuro-Oncology 2021, 23, 1231–1251.
[CrossRef] [PubMed]
22. van den Bent, M.J.; Gao, Y.; Kerkhof, M.; Kros, J.M.; Gorlia, T.; Van Zwieten, K.; Prince, J.; van Duinen, S.; Sillevis Smitt, P.A.;
Taphoorn, M. Changes in the EGFR amplification and EGFRvIII expression between paired primary and recurrent glioblastomas.
Neuro-Oncology 2015, 17, 935–941. [CrossRef] [PubMed]
23. Brennan, C.W.; Verhaak, R.G.; McKenna, A.; Campos, B.; Noushmehr, H.; Salama, S.R.; Zheng, S.; Chakravarty, D.; Sanborn, J.Z.;
Berman, S.H. The somatic genomic landscape of glioblastoma. Cell 2013, 155, 462–477. [CrossRef]
24. Ceccarelli, M.; Barthel, F.P.; Malta, T.M.; Sabedot, T.S.; Salama, S.R.; Murray, B.A.; Morozova, O.; Newton, Y.; Radenbaugh, A.;
Pagnotta, S.M. Molecular profiling reveals biologically discrete subsets and pathways of progression in diffuse glioma. Cell 2016,
164, 550–563. [CrossRef]
25. Verhaak, R.G.; Hoadley, K.A.; Purdom, E.; Wang, V.; Qi, Y.; Wilkerson, M.D.; Miller, C.R.; Ding, L.; Golub, T.; Mesirov, J.P.
Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA,
IDH1, EGFR, and NF1. Cancer Cell 2010, 17, 98–110. [CrossRef]
Int. J. Mol. Sci. 2023, 24, 11110 16 of 24

26. Noushmehr, H.; Weisenberger, D.J.; Diefes, K.; Phillips, H.S.; Pujara, K.; Berman, B.P.; Pan, F.; Pelloski, C.E.; Sulman, E.P.; Bhat,
K.P. Identification of a CpG island methylator phenotype that defines a distinct subgroup of glioma. Cancer Cell 2010, 17, 510–522.
[CrossRef]
27. Patel, A.P.; Tirosh, I.; Trombetta, J.J.; Shalek, A.K.; Gillespie, S.M.; Wakimoto, H.; Cahill, D.P.; Nahed, B.V.; Curry, W.T.; Martuza,
R.L. Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science 2014, 344, 1396–1401. [CrossRef]
[PubMed]
28. Nakada, M.; Nakada, S.; Demuth, T.; Tran, N.; Hoelzinger, D.; Berens, M. Molecular targets of glioma invasion. Cell. Mol. Life Sci.
2007, 64, 458–478. [CrossRef]
29. Dricu, A. Oncogenic Signalling of Growth Factor Receptors in Cancer: Mechanisms and Therapeutic Opportunities. Int. J. Mol.
Sci. 2022, 23, 7376. [CrossRef]
30. Manning, G.; Whyte, D.B.; Martinez, R.; Hunter, T.; Sudarsanam, S. The protein kinase complement of the human genome. Science
2002, 298, 1912–1934. [CrossRef]
31. Hubbard, S.R. Structural analysis of receptor tyrosine kinases. Prog. Biophys. Mol. Biol. 1999, 71, 343–358. [CrossRef]
32. Robinson, D.R.; Wu, Y.-M.; Lin, S.-F. The protein tyrosine kinase family of the human genome. Oncogene 2000, 19, 5548–5557.
[CrossRef]
33. Schlessinger, J. Cell signaling by receptor tyrosine kinases. Cell 2000, 103, 211–225. [CrossRef] [PubMed]
34. Eskilsson, E.; Røsland, G.V.; Solecki, G.; Wang, Q.; Harter, P.N.; Graziani, G.; Verhaak, R.G.; Winkler, F.; Bjerkvig, R.; Miletic, H.
EGFR heterogeneity and implications for therapeutic intervention in glioblastoma. Neuro-Oncology 2018, 20, 743–752. [CrossRef]
[PubMed]
35. Hunter, T. Tyrosine phosphorylation: Thirty years and counting. Curr. Opin. Cell Biol. 2009, 21, 140–146. [CrossRef] [PubMed]
36. Wehrman, T.; He, X.; Raab, B.; Dukipatti, A.; Blau, H.; Garcia, K.C. Structural and mechanistic insights into nerve growth factor
interactions with the TrkA and p75 receptors. Neuron 2007, 53, 25–38. [CrossRef] [PubMed]
37. Zhang, X.; Gureasko, J.; Shen, K.; Cole, P.A.; Kuriyan, J. An allosteric mechanism for activation of the kinase domain of epidermal
growth factor receptor. Cell 2006, 125, 1137–1149. [CrossRef] [PubMed]
38. Yuzawa, S.; Opatowsky, Y.; Zhang, Z.; Mandiyan, V.; Lax, I.; Schlessinger, J. Structural basis for activation of the receptor tyrosine
kinase KIT by stem cell factor. Cell 2007, 130, 323–334. [CrossRef]
39. Yayon, A.; Klagsbrun, M.; Esko, J.D.; Leder, P.; Ornitz, D.M. Cell surface, heparin-like molecules are required for binding of basic
fibroblast growth factor to its high affinity receptor. Cell 1991, 64, 841–848. [CrossRef]
40. Schlessinger, J.; Plotnikov, A.N.; Ibrahimi, O.A.; Eliseenkova, A.V.; Yeh, B.K.; Yayon, A.; Linhardt, R.J.; Mohammadi, M. Crystal
structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization. Mol. Cell
2000, 6, 743–750. [CrossRef]
41. Chung, I.; Akita, R.; Vandlen, R.; Toomre, D.; Schlessinger, J.; Mellman, I. Spatial control of EGF receptor activation by reversible
dimerization on living cells. Nature 2010, 464, 783–787. [CrossRef] [PubMed]
42. Soos, M.A.; Field, C.; Siddle, K. Purified hybrid insulin/insulin-like growth factor-I receptors bind insulin-like growth factor-I,
but not insulin, with high affinity. Biochem. J. 1993, 290, 419–426. [CrossRef]
43. Pandini, G.; Frasca, F.; Mineo, R.; Sciacca, L.; Vigneri, R.; Belfiore, A. Insulin/insulin-like growth factor I hybrid receptors have
different biological characteristics depending on the insulin receptor isoform involved. J. Biol. Chem. 2002, 277, 39684–39695.
[CrossRef]
44. Shewchuk, L.M.; Hassell, A.M.; Ellis, B.; Holmes, W.; Davis, R.; Horne, E.L.; Kadwell, S.H.; McKee, D.D.; Moore, J.T. Structure
of the Tie2 RTK domain: Self-inhibition by the nucleotide binding loop, activation loop, and C-terminal tail. Structure 2000, 8,
1105–1113. [CrossRef] [PubMed]
45. Wybenga-Groot, L.E.; Baskin, B.; Ong, S.H.; Tong, J.; Pawson, T.; Sicheri, F. Structural basis for autoinhibition of the Ephb2
receptor tyrosine kinase by the unphosphorylated juxtamembrane region. Cell 2001, 106, 745–757. [CrossRef]
46. Brummer, T.; Schmitz-Peiffer, C.; Daly, R.J. Docking proteins. FEBS J. 2010, 277, 4356–4369. [CrossRef] [PubMed]
47. Alexandru, O.; Horescu, C.; Sevastre, A.-S.; Cioc, C.; Baloi, C.; Oprita, A.; Dricu, A. Receptor tyrosine kinase targeting in
glioblastoma: Performance, limitations and future approaches. Contemp. Oncol./Współczesna Onkol. 2020, 24, 55–66. [CrossRef]
48. An, Z.; Aksoy, O.; Zheng, T.; Fan, Q.-W.; Weiss, W.A. Epidermal growth factor receptor and EGFRvIII in glioblastoma: Signaling
pathways and targeted therapies. Oncogene 2018, 37, 1561–1575. [CrossRef]
49. Jorissen, R.N.; Walker, F.; Pouliot, N.; Garrett, T.P.; Ward, C.W.; Burgess, A.W. Epidermal growth factor receptor: Mechanisms of
activation and signalling. EGF Recept. Fam. 2003, 284, 31–53. [CrossRef]
50. Wood, E.R.; Truesdale, A.T.; McDonald, O.B.; Yuan, D.; Hassell, A.; Dickerson, S.H.; Ellis, B.; Pennisi, C.; Horne, E.; Lackey, K. A
unique structure for epidermal growth factor receptor bound to GW572016 (Lapatinib) relationships among protein conformation,
inhibitor off-rate, and receptor activity in tumor cells. Cancer Res. 2004, 64, 6652–6659. [CrossRef]
51. Ferguson, K.M. Structure-based view of epidermal growth factor receptor regulation. Annu. Rev. Biophys. 2008, 37, 353–373.
[CrossRef]
52. Normanno, N.; De Luca, A.; Bianco, C.; Strizzi, L.; Mancino, M.; Maiello, M.R.; Carotenuto, A.; De Feo, G.; Caponigro, F.; Salomon,
D.S. Epidermal growth factor receptor (EGFR) signaling in cancer. Gene 2006, 366, 2–16. [CrossRef] [PubMed]
53. Bogdan, S.; Klämbt, C. Epidermal growth factor receptor signaling. Curr. Biol. 2001, 11, R292–R295. [CrossRef]
Int. J. Mol. Sci. 2023, 24, 11110 17 of 24

54. Hubbard, S.R.; Till, J.H. Protein tyrosine kinase structure and function. Annu. Rev. Biochem. 2000, 69, 373–398. [CrossRef]
[PubMed]
55. Hoelzinger, D.B.; Demuth, T.; Berens, M.E. Autocrine factors that sustain glioma invasion and paracrine biology in the brain
microenvironment. J. Natl. Cancer Inst. 2007, 99, 1583–1593. [CrossRef] [PubMed]
56. Yarden, Y.; Sliwkowski, M.X. Untangling the ErbB signalling network. Nat. Rev. Mol. Cell Biol. 2001, 2, 127–137. [CrossRef]
57. Thomas, R.; Weihua, Z. Rethink of EGFR in cancer with its kinase independent function on board. Front. Oncol. 2019, 9, 800.
[CrossRef]
58. Sigismund, S.; Avanzato, D.; Lanzetti, L. Emerging functions of the EGFR in cancer. Mol. Oncol. 2018, 12, 3–20. [CrossRef]
59. Jureczek, J.; Feldmann, A.; Bergmann, R.; Arndt, C.; Berndt, N.; Koristka, S.; Loureiro, L.R.; Mitwasi, N.; Hoffmann, A.; Kegler,
A. Highly efficient targeting of EGFR-expressing tumor cells with UNiCAR T cells via target modules based on cetuximab® .
OncoTargets Ther. 2020, 13, 5515. [CrossRef] [PubMed]
60. Tanaka, K.; Sasayama, T.; Irino, Y.; Takata, K.; Nagashima, H.; Satoh, N.; Kyotani, K.; Mizowaki, T.; Imahori, T.; Ejima, Y.
Compensatory glutamine metabolism promotes glioblastoma resistance to mTOR inhibitor treatment. J. Clin. Investig. 2015, 125,
1591–1602. [CrossRef] [PubMed]
61. Yang, R.; Li, X.; Wu, Y.; Zhang, G.; Liu, X.; Li, Y.; Bao, Y.; Yang, W.; Cui, H. EGFR activates GDH1 transcription to promote
glutamine metabolism through MEK/ERK/ELK1 pathway in glioblastoma. Oncogene 2020, 39, 2975–2986. [CrossRef] [PubMed]
62. Carpenter, G.; Cohen, S. Epidermal growth factor. Annu. Rev. Biochem. 1979, 48, 193–216. [CrossRef] [PubMed]
63. Maire, C.L.; Ligon, K.L. Molecular pathologic diagnosis of epidermal growth factor receptor. Neuro-Oncology 2014, 16, viii1–viii6.
[CrossRef] [PubMed]
64. Hervieu, A.; Kermorgant, S. The role of PI3K in Met driven cancer: A recap. Front. Mol. Biosci. 2018, 5, 86. [CrossRef]
65. Dawson, J.P.; Berger, M.B.; Lin, C.-C.; Schlessinger, J.; Lemmon, M.A.; Ferguson, K.M. Epidermal growth factor receptor
dimerization and activation require ligand-induced conformational changes in the dimer interface. Mol. Cell. Biol. 2005, 25,
7734–7742. [CrossRef]
66. Lemmon, M.A.; Schlessinger, J. Cell signaling by receptor tyrosine kinases. Cell 2010, 141, 1117–1134. [CrossRef]
67. Kharbanda, A.; Walter, D.M.; Gudiel, A.A.; Schek, N.; Feldser, D.M.; Witze, E.S. Blocking EGFR palmitoylation suppresses PI3K
signaling and mutant KRAS lung tumorigenesis. Sci. Signal. 2020, 13, eaax2364. [CrossRef]
68. Mayer, I.A.; Arteaga, C.L. The PI3K/AKT pathway as a target for cancer treatment. Annu. Rev. Med. 2016, 67, 11–28. [CrossRef]
69. Mattoon, D.R.; Lamothe, B.; Lax, I.; Schlessinger, J. The docking protein Gab1 is the primary mediator of EGF-stimulated
activation of the PI-3K/Akt cell survival pathway. BMC Biol. 2004, 2, 24. [CrossRef]
70. Mulcahy, E.Q.X.; Colón, R.R.; Abounader, R. HGF/MET signaling in malignant brain tumors. Int. J. Mol. Sci. 2020, 21, 7546.
[CrossRef]
71. Kiyatkin, A.; Aksamitiene, E.; Markevich, N.I.; Borisov, N.M.; Hoek, J.B.; Kholodenko, B.N. Scaffolding protein Grb2-associated
binder 1 sustains epidermal growth factor-induced mitogenic and survival signaling by multiple positive feedback loops. J. Biol.
Chem. 2006, 281, 19925–19938. [CrossRef]
72. Oprita, A.; Baloi, S.-C.; Staicu, G.-A.; Alexandru, O.; Tache, D.E.; Danoiu, S.; Micu, E.S.; Sevastre, A.-S. Updated insights on EGFR
signaling pathways in glioma. Int. J. Mol. Sci. 2021, 22, 587. [CrossRef]
73. Pawson, T. Specificity in signal transduction: From phosphotyrosine-SH2 domain interactions to complex cellular systems. Cell
2004, 116, 191–203. [CrossRef]
74. Ward, A.F.; Braun, B.S.; Shannon, K.M. Targeting oncogenic Ras signaling in hematologic malignancies. Blood J. Am. Soc. Hematol.
2012, 120, 3397–3406. [CrossRef]
75. Gao, S.P.; Mark, K.G.; Leslie, K.; Pao, W.; Motoi, N.; Gerald, W.L.; Travis, W.D.; Bornmann, W.; Veach, D.; Clarkson, B. Mutations
in the EGFR kinase domain mediate STAT3 activation via IL-6 production in human lung adenocarcinomas. J. Clin. Investig. 2007,
117, 3846–3856. [CrossRef] [PubMed]
76. Gao, S.P.; Chang, Q.; Mao, N.; Daly, L.A.; Vogel, R.; Chan, T.; Liu, S.H.; Bournazou, E.; Schori, E.; Zhang, H. JAK2 inhibition
sensitizes resistant EGFR-mutant lung adenocarcinoma to tyrosine kinase inhibitors. Sci. Signal. 2016, 9, ra33. [CrossRef]
[PubMed]
77. Padfield, E.; Ellis, H.P.; Kurian, K.M. Current therapeutic advances targeting EGFR and EGFRvIII in glioblastoma. Front. Oncol.
2015, 5, 5. [CrossRef] [PubMed]
78. Östman, A.; Böhmer, F.-D. Regulation of receptor tyrosine kinase signaling by protein tyrosine phosphatases. Trends Cell Biol.
2001, 11, 258–266. [CrossRef]
79. Serban, F.; Artene, S.-A.; Georgescu, A.M.; Purcaru, S.O.; Tache, D.E.; Alexandru, O.; Dricu, A. Epidermal growth factor,
latrophilin, and seven transmembrane domain-containing protein 1 marker, a novel angiogenesis marker. OncoTargets Ther. 2015,
8, 3767–3774.
80. McDonell, L.M.; Kernohan, K.D.; Boycott, K.M.; Sawyer, S.L. Receptor tyrosine kinase mutations in developmental syndromes
and cancer: Two sides of the same coin. Hum. Mol. Genet. 2015, 24, R60–R66. [CrossRef] [PubMed]
81. Vogelstein, B.; Papadopoulos, N.; Velculescu, V.E.; Zhou, S.; Diaz, L.A., Jr.; Kinzler, K.W. Cancer genome landscapes. Science 2013,
339, 1546–1558. [CrossRef]
82. Wang, Z.; Longo, P.A.; Tarrant, M.K.; Kim, K.; Head, S.; Leahy, D.J.; Cole, P.A. Mechanistic insights into the activation of oncogenic
forms of EGF receptor. Nat. Struct. Mol. Biol. 2011, 18, 1388–1393. [CrossRef] [PubMed]
Int. J. Mol. Sci. 2023, 24, 11110 18 of 24

83. Red Brewer, M.; Yun, C.-H.; Lai, D.; Lemmon, M.A.; Eck, M.J.; Pao, W. Mechanism for activation of mutated epidermal growth
factor receptors in lung cancer. Proc. Natl. Acad. Sci. USA 2013, 110, E3595–E3604. [CrossRef] [PubMed]
84. Yun, C.-H.; Boggon, T.J.; Li, Y.; Woo, M.S.; Greulich, H.; Meyerson, M.; Eck, M.J. Structures of lung cancer-derived EGFR mutants
and inhibitor complexes: Mechanism of activation and insights into differential inhibitor sensitivity. Cancer Cell 2007, 11, 217–227.
[CrossRef] [PubMed]
85. Rosell, R.; Carcereny, E.; Gervais, R.; Vergnenegre, A.; Massuti, B.; Felip, E.; Palmero, R.; Garcia-Gomez, R.; Pallares, C.; Sanchez,
J.M. Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive
non-small-cell lung cancer (EURTAC): A multicentre, open-label, randomised phase 3 trial. Lancet Oncol. 2012, 13, 239–246.
[CrossRef]
86. Zhou, C.; Wu, Y.-L.; Chen, G.; Feng, J.; Liu, X.-Q.; Wang, C.; Zhang, S.; Wang, J.; Zhou, S.; Ren, S. Erlotinib versus chemotherapy
as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802):
A multicentre, open-label, randomised, phase 3 study. Lancet Oncol. 2011, 12, 735–742. [CrossRef]
87. Mitsudomi, T.; Morita, S.; Yatabe, Y.; Negoro, S.; Okamoto, I.; Tsurutani, J.; Seto, T.; Satouchi, M.; Tada, H.; Hirashima, T. Gefitinib
versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor
receptor (WJTOG3405): An open label, randomised phase 3 trial. Lancet Oncol. 2010, 11, 121–128. [CrossRef]
88. Maemondo, M.; Inoue, A.; Kobayashi, K.; Sugawara, S.; Oizumi, S.; Isobe, H.; Gemma, A.; Harada, M.; Yoshizawa, H.; Kinoshita,
I. Gefitinib or chemotherapy for non–small-cell lung cancer with mutated EGFR. N. Engl. J. Med. 2010, 362, 2380–2388. [CrossRef]
89. Sequist, L.V.; Yang, J.C.-H.; Yamamoto, N.; O’Byrne, K.; Hirsh, V.; Mok, T.; Geater, S.L.; Orlov, S.; Tsai, C.-M.; Boyer, M. Phase III
study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations. J. Clin.
Oncol. 2013, 31, 3327–3334. [CrossRef]
90. Jänne, P.A.; Yang, J.C.-H.; Kim, D.-W.; Planchard, D.; Ohe, Y.; Ramalingam, S.S.; Ahn, M.-J.; Kim, S.-W.; Su, W.-C.; Horn, L.
AZD9291 in EGFR inhibitor–resistant non–small-cell lung cancer. N. Engl. J. Med. 2015, 372, 1689–1699. [CrossRef]
91. Soria, J.-C.; Ohe, Y.; Vansteenkiste, J.; Reungwetwattana, T.; Chewaskulyong, B.; Lee, K.H.; Dechaphunkul, A.; Imamura, F.;
Nogami, N.; Kurata, T. Osimertinib in untreated EGFR-mutated advanced non–small-cell lung cancer. N. Engl. J. Med. 2018, 378,
113–125. [CrossRef] [PubMed]
92. Lee, J.C.; Vivanco, I.; Beroukhim, R.; Huang, J.H.Y.; Feng, W.L.; DeBiasi, R.M.; Yoshimoto, K.; King, J.C.; Nghiemphu, P.; Yuza, Y.
Epidermal growth factor receptor activation in glioblastoma through novel missense mutations in the extracellular domain. PLoS
Med. 2006, 3, e485. [CrossRef] [PubMed]
93. Arjona, D.; Bello, M.J.; Alonso, M.E.; Aminoso, C.; Isla, A.; De Campos, J.; Sarasa, J.; Gutierrez, M.; Villalobo, A.; Rey, J. Molecular
analysis of the EGFR gene in astrocytic gliomas: mRNA expression, quantitative-PCR analysis of non-homogeneous gene
amplification and DNA sequence alterations. Neuropathol. Appl. Neurobiol. 2005, 31, 384–394. [CrossRef] [PubMed]
94. Danciulescu, O.T.; Folcuti, R.; Dricu, A. Temozolomide and targeted therapy against epidermal growth factor receptor in glioma.
Int. J. Clin. Exp. Med. 2016, 9, 15249–15261.
95. van den Bent, M.J.; Brandes, A.A.; Rampling, R.; Kouwenhoven, M.C.; Kros, J.M.; Carpentier, A.F.; Clement, P.M.; Frenay, M.;
Campone, M.; Baurain, J.-F. Randomized phase II trial of erlotinib versus temozolomide or carmustine in recurrent glioblastoma:
EORTC brain tumor group study 26034. J. Clin. Oncol. 2009, 27, 1268. [CrossRef]
96. Franceschi, E.; Cavallo, G.; Lonardi, S.; Magrini, E.; Tosoni, A.; Grosso, D.; Scopece, L.; Blatt, V.; Urbini, B.; Pession, A. Gefitinib in
patients with progressive high-grade gliomas: A multicentre phase II study by Gruppo Italiano Cooperativo di Neuro-Oncologia
(GICNO). Br. J. Cancer 2007, 96, 1047–1051. [CrossRef]
97. Vivanco, I.; Robins, H.I.; Rohle, D.; Campos, C.; Grommes, C.; Nghiemphu, P.L.; Kubek, S.; Oldrini, B.; Chheda, M.G.; Yannuzzi,
N. Differential sensitivity of glioma-versus lung cancer–specific EGFR mutations to EGFR kinase inhibitors. Cancer Discov. 2012,
2, 458–471. [CrossRef]
98. Lopez-Gines, C.; Gil-Benso, R.; Ferrer-Luna, R.; Benito, R.; Serna, E.; Gonzalez-Darder, J.; Quilis, V.; Monleon, D.; Celda, B.;
Cerdá-Nicolas, M. New pattern of EGFR amplification in glioblastoma and the relationship of gene copy number with gene
expression profile. Mod. Pathol. 2010, 23, 856–865. [CrossRef]
99. Ohgaki, H.; Kleihues, P. Genetic pathways to primary and secondary glioblastoma. Am. J. Pathol. 2007, 170, 1445–1453. [CrossRef]
100. Carraway, K.L.; Sweeney, C. EGF receptor activation by heterologous mechanisms. Cancer Cell 2002, 1, 405–406. [CrossRef]
101. Ludes-Meyers, J.H.; Subler, M.A.; Shivakumar, C.V.; Munoz, R.M.; Jiang, P.; Bigger, J.E.; Brown, D.R.; Deb, S.P.; Deb, S.
Transcriptional activation of the human epidermal growth factor receptor promoter by human p53. Mol. Cell. Biol. 1996, 16,
6009–6019. [CrossRef] [PubMed]
102. Reznik, T.E.; Sang, Y.; Ma, Y.; Abounader, R.; Rosen, E.M.; Xia, S.; Laterra, J. Transcription-dependent epidermal growth factor
receptor activation by hepatocyte growth factor. Mol. Cancer Res. 2008, 6, 139–150. [CrossRef] [PubMed]
103. Hanawa, M.; Suzuki, S.; Dobashi, Y.; Yamane, T.; Kono, K.; Enomoto, N.; Ooi, A. EGFR protein overexpression and gene
amplification in squamous cell carcinomas of the esophagus. Int. J. Cancer 2006, 118, 1173–1180. [CrossRef] [PubMed]
104. Sun, T.; Aceto, N.; Meerbrey, K.L.; Kessler, J.D.; Zhou, C.; Migliaccio, I.; Nguyen, D.X.; Pavlova, N.N.; Botero, M.; Huang, J.
Activation of multiple proto-oncogenic tyrosine kinases in breast cancer via loss of the PTPN12 phosphatase. Cell 2011, 144,
703–718. [CrossRef]
105. Albertson, D.G. Gene amplification in cancer. Trend. Genet. 2006, 22, 447–455. [CrossRef]
Int. J. Mol. Sci. 2023, 24, 11110 19 of 24

106. Albertson, D.G.; Collins, C.; McCormick, F.; Gray, J.W. Chromosome aberrations in solid tumors. Nat. Genet. 2003, 34, 369–376.
[CrossRef]
107. Stransky, N.; Cerami, E.; Schalm, S.; Kim, J.L.; Lengauer, C. The landscape of kinase fusions in cancer. Nat. Commun. 2014, 5, 4846.
[CrossRef]
108. Network, C.G.A.R. Comprehensive molecular profiling of lung adenocarcinoma. Nature 2014, 511, 543.
109. Mistry, A.R.; Felix, C.A.; Whitmarsh, R.J.; Mason, A.; Reiter, A.; Cassinat, B.; Parry, A.; Walz, C.; Wiemels, J.L.; Segal, M.R. DNA
topoisomerase II in therapy-related acute promyelocytic leukemia. N. Engl. J. Med. 2005, 352, 1529–1538. [CrossRef]
110. Ito, T.; Seyama, T.; Iwamoto, K.S.; Hayashi, T.; Mizuno, T.; Tsuyama, N.; Dohi, K.; Nakamura, N.; Akiyama, M. In vitro irradiation
is able to cause RET oncogene rearrangement. Cancer Res. 1993, 53, 2940–2943.
111. Mizuno, T.; Kyoizumi, S.; Suzuki, T.; Iwamoto, K.; Seyama, T. Continued expression of a tissue specific activated oncogene in the
early steps of radiation-induced human thyroid carcinogenesis. Oncogene 1997, 15, 1455–1460. [CrossRef]
112. Tsai, A.G.; Lieber, M.R. Mechanisms of chromosomal rearrangement in the human genome. BMC Genom. 2010, 11, S1. [CrossRef]
[PubMed]
113. Konduri, K.; Gallant, J.-N.; Chae, Y.K.; Giles, F.J.; Gitlitz, B.J.; Gowen, K.; Ichihara, E.; Owonikoko, T.K.; Peddareddigari, V.;
Ramalingam, S.S. EGFR Fusions as Novel Therapeutic Targets in Lung CancerTherapeutically Targetable EGFR Fusions in Lung
Cancer. Cancer Discov. 2016, 6, 601–611. [CrossRef] [PubMed]
114. Lovly, C.M.; Gupta, A.; Lipson, D.; Otto, G.; Brennan, T.; Chung, C.T.; Borinstein, S.C.; Ross, J.S.; Stephens, P.J.; Miller, V.A.
Inflammatory myofibroblastic tumors harbor multiple potentially actionable kinase fusions. Cancer Discov. 2014, 4, 889–895.
[CrossRef] [PubMed]
115. Singh, A.B.; Harris, R.C. Autocrine, paracrine and juxtacrine signaling by EGFR ligands. Cell. Signal. 2005, 17, 1183–1193.
[CrossRef]
116. Walsh, J.H.; Karnes, W.; Cuttitta, F.; Walker, A. Autocrine growth factors and solid tumor malignancy. West. J. Med. 1991, 155, 152.
117. Ciardiello, F.; Tortora, G. A novel approach in the treatment of cancer: Targeting the epidermal growth factor receptor. Clin.
Cancer Res. 2001, 7, 2958–2970.
118. Singh, B.; Carpenter, G.; Coffey, R.J. EGF receptor ligands: Recent advances. F1000Research 2016, 5. [CrossRef]
119. Ramnarain, D.B.; Park, S.; Lee, D.Y.; Hatanpaa, K.J.; Scoggin, S.O.; Otu, H.; Libermann, T.A.; Raisanen, J.M.; Ashfaq, R.; Wong, E.T.
Differential gene expression analysis reveals generation of an autocrine loop by a mutant epidermal growth factor receptor in
glioma cells. Cancer Res. 2006, 66, 867–874. [CrossRef]
120. Du, Z.; Lovly, C.M. Mechanisms of receptor tyrosine kinase activation in cancer. Mol. Cancer 2018, 17, 58. [CrossRef] [PubMed]
121. Chen, H.-Y.; Brady, D.C.; Villanueva, J. Double trouble: Kinase domain duplication as a new path to drug resistance. Pigment Cell
Melanoma Res. 2016, 29, 493. [CrossRef] [PubMed]
122. Gallant, J.-N.; Sheehan, J.H.; Shaver, T.M.; Bailey, M.; Lipson, D.; Chandramohan, R.; Brewer, M.R.; York, S.J.; Kris, M.G.; Pietenpol,
J.A. EGFR Kinase Domain Duplication (EGFR-KDD) Is a Novel Oncogenic Driver in Lung Cancer That Is Clinically Responsive to
AfatinibEGFR-KDD as a Therapeutic Target in Lung and Other Cancers. Cancer Discov. 2015, 5, 1155–1163. [CrossRef] [PubMed]
123. Idbaih, A.; Aimard, J.; Boisselier, B.; Marie, Y.; Paris, S.; Criniere, E.; Carvalho Silva, R.; Laigle-Donadey, F.; Rousseau, A.; Mokhtari,
K. Epidermal growth factor receptor extracellular domain mutations in primary glioblastoma. Neuropathol. Appl. Neurobiol. 2009,
35, 208–213. [CrossRef] [PubMed]
124. Ekstrand, A.J.; James, C.D.; Cavenee, W.K.; Seliger, B.; Pettersson, R.F.; Collins, V.P. Genes for epidermal growth factor receptor,
transforming growth factor α, and epidermal growth factor and their expression in human gliomas in vivo. Cancer Res. 1991, 51,
2164–2172.
125. Gullick, W.J.; Marsden, J.J.; Whittle, N.; Ward, B.; Bobrow, L.; Waterfield, M.D. Expression of epidermal growth factor receptors
on human cervical, ovarian, and vulval carcinomas. Cancer Res. 1986, 46, 285–292. [PubMed]
126. Inal, C.; Yilmaz, E.; Piperdi, B.; Perez-Soler, R.; Cheng, H. Emerging treatment for advanced lung cancer with EGFR mutation.
Expert Opin. Emerg. Drugs 2015, 20, 597–612. [CrossRef]
127. Siegelin, M.D.; Borczuk, A.C. Epidermal growth factor receptor mutations in lung adenocarcinoma. Lab. Investig. 2014, 94,
129–137. [CrossRef]
128. De Luca, A.; Normanno, N. Predictive biomarkers to tyrosine kinase inhibitors for the epidermal growth factor receptor in
non-small-cell lung cancer. Curr. Drug Targets 2010, 11, 851–864. [CrossRef]
129. Li, A.R.; Chitale, D.; Riely, G.J.; Pao, W.; Miller, V.A.; Zakowski, M.F.; Rusch, V.; Kris, M.G.; Ladanyi, M. EGFR mutations in lung
adenocarcinomas: Clinical testing experience and relationship to EGFR gene copy number and immunohistochemical expression.
J. Mol. Diagn. 2008, 10, 242–248. [CrossRef]
130. Jung, M.J.; Woo, C.G.; Lee, S.; Chin, S.; Kim, H.K.; Kwak, J.J.; Koh, E.S.; Lee, B.; Jang, K.-T.; Moon, A. Gene copy number variation
and protein overexpression of EGFR and HER2 in distal extrahepatic cholangiocarcinoma. Pathology 2017, 49, 582–588. [CrossRef]
131. Birkman, E.-M.; Ålgars, A.; Lintunen, M.; Ristamäki, R.; Sundström, J.; Carpén, O. EGFR gene amplification is relatively common
and associates with outcome in intestinal adenocarcinoma of the stomach, gastro-oesophageal junction and distal oesophagus.
BMC Cancer 2016, 16, 406. [CrossRef] [PubMed]
132. Libermann, T.A.; Nusbaum, H.R.; Razon, N.; Kris, R.; LAX, I.; Soreq, H.; Whittle, N.; Waterfield, M.D.; Ullrich, A.; Schlessinger, J.
Amplification and overexpression of the EGF receptor gene in primary human glioblastomas. J. Cell Sci. 1985, 1985, 161–172.
[CrossRef] [PubMed]
Int. J. Mol. Sci. 2023, 24, 11110 20 of 24

133. Alexandru, O.; Purcaru, S.O.; Tataranu, L.G.; Lucan, L.; Castro, J.; Folcuţi, C.; Artene, S.-A.; Tuţă, C.; Dricu, A. The influence of
EGFR inactivation on the radiation response in high grade glioma. Int. J. Mol. Sci. 2018, 19, 229. [CrossRef]
134. Ekstrand, A.J.; Sugawa, N.; James, C.D.; Collins, V.P. Amplified and rearranged epidermal growth factor receptor genes in human
glioblastomas reveal deletions of sequences encoding portions of the N-and/or C-terminal tails. Proc. Natl. Acad. Sci. USA 1992,
89, 4309–4313. [CrossRef] [PubMed]
135. Yamazaki, H.; Fukui, Y.; Ueyama, Y.; Tamaoki, N.; Kawamoto, T.; Taniguchi, S.; Shibuya, M. Amplification of the structurally and
functionally altered epidermal growth factor receptor gene (c-erbB) in human brain tumors. Mol. Cell. Biol. 1988, 8, 1816–1820.
136. Ohgaki, H.; Kleihues, P. Genetic alterations and signaling pathways in the evolution of gliomas. Cancer Sci. 2009, 100, 2235–2241.
[CrossRef]
137. Kita, D.; Yonekawa, Y.; Weller, M.; Ohgaki, H. PIK3CA alterations in primary (de novo) and secondary glioblastomas. Acta
Neuropathol. 2007, 113, 295–302. [CrossRef]
138. Ohgaki, H.; Dessen, P.; Jourde, B.; Horstmann, S.; Nishikawa, T.; Di Patre, P.-L.; Burkhard, C.; Schüler, D.; Probst-Hensch, N.M.;
Maiorka, P.C. Genetic pathways to glioblastoma: A population-based study. Cancer Res. 2004, 64, 6892–6899. [CrossRef]
139. Yu, H.A.; Arcila, M.E.; Rekhtman, N.; Sima, C.S.; Zakowski, M.F.; Pao, W.; Kris, M.G.; Miller, V.A.; Ladanyi, M.; Riely, G.J.
Analysis of Tumor Specimens at the Time of Acquired Resistance to EGFR-TKI Therapy in 155 Patients with EGFR-Mutant Lung
CancersMechanisms of Acquired Resistance to EGFR-TKI Therapy. Clin. Cancer Res. 2013, 19, 2240–2247. [CrossRef]
140. Campo, M.; Gerber, D.; Gainor, J.F.; Heist, R.S.; Temel, J.S.; Shaw, A.T.; Fidias, P.; Muzikansky, A.; Engelman, J.A.; Sequist, L.V.
Acquired resistance to first-line afatinib and the challenges of prearranged progression biopsies. J. Thorac. Oncol. 2016, 11,
2022–2026. [CrossRef]
141. Barker II, F.G.; Simmons, M.L.; Chang, S.M.; Prados, M.D.; Larson, D.A.; Sneed, P.K.; Wara, W.M.; Berger, M.S.; Chen, P.; Israel,
M.A. EGFR overexpression and radiation response in glioblastoma multiforme. Int. J. Radiat. Oncol. Biol. Phys. 2001, 51, 410–418.
[CrossRef]
142. Feldkamp, M.M.; Lala, P.; Lau, N.; Roncari, L.; Guha, A. Expression of activated epidermal growth factor receptors, Ras-guanosine
triphosphate, and mitogen-activated protein kinase in human glioblastoma multiforme specimens. Neurosurg.-Baltim. 1999, 45,
1442–1453. [CrossRef]
143. Shinojima, N.; Tada, K.; Shiraishi, S.; Kamiryo, T.; Kochi, M.; Nakamura, H.; Makino, K.; Saya, H.; Hirano, H.; Kuratsu, J.-I.
Prognostic value of epidermal growth factor receptor in patients with glioblastoma multiforme. Cancer Res. 2003, 63, 6962–6970.
[PubMed]
144. Xu, N.; Fang, W.; Mu, L.; Tang, Y.; Gao, L.; Ren, S.; Cao, D.; Zhou, L.; Zhang, A.; Liu, D. Overexpression of wildtype EGFR is
tumorigenic and denotes a therapeutic target in non-small cell lung cancer. Oncotarget 2016, 7, 3884. [CrossRef] [PubMed]
145. Velu, T.J.; Beguinot, L.; Vass, W.C.; Willingham, M.C.; Merlino, G.T.; Pastan, I.; Lowy, D.R. Epidermal-growth-factor-dependent
transformation by a human EGF receptor proto-oncogene. Science 1987, 238, 1408–1410. [CrossRef]
146. Greulich, H.; Chen, T.-H.; Feng, W.; Jänne, P.A.; Alvarez, J.V.; Zappaterra, M.; Bulmer, S.E.; Frank, D.A.; Hahn, W.C.; Sellers, W.R.
Oncogenic transformation by inhibitor-sensitive and-resistant EGFR mutants. PLoS Med. 2005, 2, e313. [CrossRef] [PubMed]
147. Gonzalez-Conchas, G.A.; Rodriguez-Romo, L.; Hernandez-Barajas, D.; Gonzalez-Guerrero, J.F.; Rodriguez-Fernandez, I.A.;
Verdines-Perez, A.; Templeton, A.J.; Ocana, A.; Seruga, B.; Tannock, I.F. Epidermal growth factor receptor overexpression and
outcomes in early breast cancer: A systematic review and a meta-analysis. Cancer Treat. Rev. 2018, 62, 1–8. [CrossRef]
148. Carlsson, J.; Wester, K.; De La Torre, M.; Malmström, P.-U.; Gårdmark, T. EGFR-expression in primary urinary bladder cancer and
corresponding metastases and the relation to HER2-expression. On the possibility to target these receptors with radionuclides.
Radiol. Oncol. 2015, 49, 50–58. [CrossRef]
149. Biernat, W.; Huang, H.; Yokoo, H.; Kleihues, P.; Ohgaki, H. Predominant expression of mutant EGFR (EGFRvIII) is rare in primary
glioblastomas. Brain Pathol. 2004, 14, 131–136. [CrossRef] [PubMed]
150. Costa, V.; Fregnani, E.R.; Fonseca, F.P.; Alves, F.A.; Pinto, C.A.L.; Kaminagakura, E. EGFR is not amplified in ameloblastoma. Oral
Surg. Oral Med. Oral Pathol. Oral Radiol. 2018, 125, 454–458. [CrossRef] [PubMed]
151. Matsuda, N.; Lim, B.; Wang, X.; Ueno, N.T. Early clinical development of epidermal growth factor receptor targeted therapy in
breast cancer. Expert Opin. Investig. Drugs 2017, 26, 463–479. [CrossRef] [PubMed]
152. Thorne, A.H.; Zanca, C.; Furnari, F. Epidermal growth factor receptor targeting and challenges in glioblastoma. Neuro-Oncology
2016, 18, 914–918. [CrossRef] [PubMed]
153. Sacco, A.G.; Worden, F.P. Molecularly targeted therapy for the treatment of head and neck cancer: A review of the ErbB family
inhibitors. OncoTargets Ther. 2016, 9, 1927–1943.
154. Sieghart, W.; Pinter, M.; Dauser, B.; Rohr-Udilova, N.; Piguet, A.-C.; Prager, G.; Hayden, H.; Dienes, H.-P.; Dufour, J.-F.; Peck-
Radosavljevic, M. Erlotinib and sorafenib in an orthotopic rat model of hepatocellular carcinoma. J. Hepatol. 2012, 57, 592–599.
[CrossRef]
155. Ioannou, N.; Dalgleish, A.; Seddon, A.; Mackintosh, D.; Guertler, U.; Solca, F.; Modjtahedi, H. Anti-tumour activity of afatinib, an
irreversible ErbB family blocker, in human pancreatic tumour cells. Br. J. Cancer 2011, 105, 1554–1562. [CrossRef]
156. Weihua, Z.; Tsan, R.; Huang, W.-C.; Wu, Q.; Chiu, C.-H.; Fidler, I.J.; Hung, M.-C. Survival of cancer cells is maintained by EGFR
independent of its kinase activity. Cancer Cell 2008, 13, 385–393. [CrossRef]
Int. J. Mol. Sci. 2023, 24, 11110 21 of 24

157. Tsuchihashi, K.; Okazaki, S.; Ohmura, M.; Ishikawa, M.; Sampetrean, O.; Onishi, N.; Wakimoto, H.; Yoshikawa, M.; Seishima, R.;
Iwasaki, Y. The EGF Receptor Promotes the Malignant Potential of Glioma by Regulating Amino Acid Transport System xc (−)
EGFR Regulates xCT in Glioma. Cancer Res. 2016, 76, 2954–2963. [CrossRef]
158. Nagy, P.; Arndt-Jovin, D.J.; Jovin, T.M. Small interfering RNAs suppress the expression of endogenous and GFP-fused epidermal
growth factor receptor (erbB1) and induce apoptosis in erbB1-overexpressing cells. Exp. Cell Res. 2003, 285, 39–49. [CrossRef]
159. Kang, C.-S.; Pu, P.-Y.; Li, Y.-H.; Zhang, Z.-Y.; Qiu, M.-Z.; Huang, Q.; Wang, G.-X. An in vitro study on the suppressive effect
of glioma cell growth induced by plasmid-based small interference RNA (siRNA) targeting human epidermal growth factor
receptor. J. Neuro-Oncol. 2005, 74, 267–273. [CrossRef]
160. Chen, G.; Kronenberger, P.; Teugels, E.; Umelo, I.A.; De Greve, J. Effect of siRNAs targeting the EGFR T790M mutation in a
non-small cell lung cancer cell line resistant to EGFR tyrosine kinase inhibitors and combination with various agents. Biochem.
Biophys. Res. Commun. 2013, 431, 623–629. [CrossRef]
161. Francis, J.M.; Zhang, C.-Z.; Maire, C.L.; Jung, J.; Manzo, V.E.; Adalsteinsson, V.A.; Homer, H.; Haidar, S.; Blumenstiel, B.;
Pedamallu, C.S. EGFR Variant Heterogeneity in Glioblastoma Resolved through Single-Nucleus SequencingSingle-Nucleus
Sequencing of GBM Mutational Heterogeneity. Cancer Discov. 2014, 4, 956–971. [CrossRef]
162. Snuderl, M.; Fazlollahi, L.; Le, L.P.; Nitta, M.; Zhelyazkova, B.H.; Davidson, C.J.; Akhavanfard, S.; Cahill, D.P.; Aldape, K.D.;
Betensky, R.A. Mosaic amplification of multiple receptor tyrosine kinase genes in glioblastoma. Cancer Cell 2011, 20, 810–817.
[CrossRef] [PubMed]
163. Mellinghoff, I.K.; Wang, M.Y.; Vivanco, I.; Haas-Kogan, D.A.; Zhu, S.; Dia, E.Q.; Lu, K.V.; Yoshimoto, K.; Huang, J.H.; Chute,
D.J. Molecular determinants of the response of glioblastomas to EGFR kinase inhibitors. N. Engl. J. Med. 2005, 353, 2012–2024.
[CrossRef]
164. Wen, P.Y.; Chang, S.M.; Lamborn, K.R.; Kuhn, J.G.; Norden, A.D.; Cloughesy, T.F.; Robins, H.I.; Lieberman, F.S.; Gilbert, M.R.;
Mehta, M.P. Phase I/II study of erlotinib and temsirolimus for patients with recurrent malignant gliomas: North American Brain
Tumor Consortium trial 04-02. Neuro-Oncology 2014, 16, 567–578. [CrossRef] [PubMed]
165. Pelloski, C.E.; Ballman, K.V.; Furth, A.F.; Zhang, L.; Lin, E.; Sulman, E.P.; Bhat, K.; McDonald, J.M.; Yung, W.A.; Colman, H.
Epidermal growth factor receptor variant III status defines clinically distinct subtypes of glioblastoma. J. Clin. Oncol. 2007, 25,
2288–2294. [CrossRef]
166. Nishikawa, R.; Sugiyama, T.; Narita, Y.; Furnari, F.; Cavenee, W.K.; Matsutani, M. Immunohistochemical analysis of the mutant
epidermal growth factor, ∆EGFR, in glioblastoma. Brain Tumor Pathol. 2004, 21, 53–56. [CrossRef] [PubMed]
167. Frederick, L.; Wang, X.-Y.; Eley, G.; James, C.D. Diversity and frequency of epidermal growth factor receptor mutations in human
glioblastomas. Cancer Res. 2000, 60, 1383–1387.
168. Cho, J.; Pastorino, S.; Zeng, Q.; Xu, X.; Johnson, W.; Vandenberg, S.; Verhaak, R.; Cherniack, A.D.; Watanabe, H.; Dutt, A.
Glioblastoma-Derived Epidermal Growth Factor Receptor Carboxyl-Terminal Deletion Mutants Are Transforming and Are
Sensitive to EGFR-Directed TherapiesStudy of GBM-Derived EGFR C-Terminal Deletion Mutants. Cancer Res. 2011, 71, 7587–7596.
[CrossRef]
169. Frattini, V.; Trifonov, V.; Chan, J.M.; Castano, A.; Lia, M.; Abate, F.; Keir, S.T.; Ji, A.X.; Zoppoli, P.; Niola, F. The integrated
landscape of driver genomic alterations in glioblastoma. Nat. Genet. 2013, 45, 1141–1149. [CrossRef]
170. Shah, N.; Lankerovich, M.; Lee, H.; Yoon, J.-G.; Schroeder, B.; Foltz, G. Exploration of the gene fusion landscape of glioblastoma
using transcriptome sequencing and copy number data. BMC Genom. 2013, 14, 818. [CrossRef]
171. Hu, Y.; Dingerdissen, H.; Gupta, S.; Kahsay, R.; Shanker, V.; Wan, Q.; Yan, C.; Mazumder, R. Identification of key differentially
expressed MicroRNAs in cancer patients through pan-cancer analysis. Comput. Biol. Med. 2018, 103, 183–197. [CrossRef]
[PubMed]
172. Donzelli, S.; Cioce, M.; Muti, P.; Strano, S.; Yarden, Y.; Blandino, G. MicroRNAs: Non-coding fine tuners of receptor tyrosine
kinase signalling in cancer. In Seminars in Cell & Developmental Biology; Academic Press: Cambridge, MA, USA, 2016; pp. 133–142.
173. Khan, A.Q.; Ahmed, E.I.; Elareer, N.R.; Junejo, K.; Steinhoff, M.; Uddin, S. Role of miRNA-regulated cancer stem cells in the
pathogenesis of human malignancies. Cells 2019, 8, 840. [CrossRef] [PubMed]
174. Gu, J.; Lu, Z.; Ji, C.; Chen, Y.; Liu, Y.; Lei, Z.; Wang, L.; Zhang, H.-T.; Li, X. Melatonin inhibits proliferation and invasion via
repression of miRNA-155 in glioma cells. Biomed. Pharmacother. 2017, 93, 969–975. [CrossRef]
175. Xiong, W.; Ran, J.; Jiang, R.; Guo, P.; Shi, X.; Li, H.; Lv, X.; Li, J.; Chen, D. miRNA-320a inhibits glioma cell invasion and migration
by directly targeting aquaporin 4. Oncol. Rep. 2018, 39, 1939–1947. [CrossRef]
176. Acunzo, M.; Romano, G.; Wernicke, D.; Croce, C.M. MicroRNA and cancer–a brief overview. Adv. Biol. Regul. 2015, 57, 1–9.
[CrossRef]
177. Rao, S.A.M.; Arimappamagan, A.; Pandey, P.; Santosh, V.; Hegde, A.S.; Chandramouli, B.A.; Somasundaram, K. miR-219-5p
inhibits receptor tyrosine kinase pathway by targeting EGFR in glioblastoma. PLoS ONE 2013, 8, e63164. [CrossRef]
178. Zhang, Y.; Kim, J.; Mueller, A.; Dey, B.; Yang, Y.; Lee, D.; Hachmann, J.; Finderle, S.; Park, D.; Christensen, J. Multiple receptor
tyrosine kinases converge on microRNA-134 to control KRAS, STAT5B, and glioblastoma. Cell Death Differ. 2014, 21, 720–734.
[CrossRef] [PubMed]
179. Zhang, K.-L.; Han, L.; Chen, L.-Y.; Shi, Z.-D.; Yang, M.; Ren, Y.; Chen, L.-C.; Zhang, J.-X.; Pu, P.-Y.; Kang, C.-S. Blockage of a
miR-21/EGFR regulatory feedback loop augments anti-EGFR therapy in glioblastomas. Cancer Lett. 2014, 342, 139–149. [CrossRef]
Int. J. Mol. Sci. 2023, 24, 11110 22 of 24

180. Stommel, J.M.; Kimmelman, A.C.; Ying, H.; Nabioullin, R.; Ponugoti, A.H.; Wiedemeyer, R.; Stegh, A.H.; Bradner, J.E.; Ligon,
K.L.; Brennan, C. Coactivation of receptor tyrosine kinases affects the response of tumor cells to targeted therapies. Science 2007,
318, 287–290. [CrossRef]
181. Huang, P.H.; Cavenee, W.K.; Furnari, F.B.; White, F.M. Uncovering therapeutic targets for glioblastoma: A systems biology
approach. Cell Cycle 2007, 6, 2750–2754. [CrossRef]
182. Arteaga, C.L.; Engelman, J.A. ERBB receptors: From oncogene discovery to basic science to mechanism-based cancer therapeutics.
Cancer Cell 2014, 25, 282–303. [CrossRef] [PubMed]
183. Yamaoka, T.; Ohba, M.; Ohmori, T. Molecular-targeted therapies for epidermal growth factor receptor and its resistance
mechanisms. Int. J. Mol. Sci. 2017, 18, 2420. [CrossRef] [PubMed]
184. Kwak, E.L.; Bang, Y.-J.; Camidge, D.R.; Shaw, A.T.; Solomon, B.; Maki, R.G.; Ou, S.-H.I.; Dezube, B.J.; Jänne, P.A.; Costa, D.B.
Anaplastic lymphoma kinase inhibition in non–small-cell lung cancer. N. Engl. J. Med. 2010, 363, 1693–1703. [CrossRef]
185. Mok, T.S.; Wu, Y.-L.; Thongprasert, S.; Yang, C.-H.; Chu, D.-T.; Saijo, N.; Sunpaweravong, P.; Han, B.; Margono, B.; Ichinose, Y.
Gefitinib or carboplatin–paclitaxel in pulmonary adenocarcinoma. N. Engl. J. Med. 2009, 361, 947–957. [CrossRef]
186. Li, D.; Ambrogio, L.; Shimamura, T.; Kubo, S.; Takahashi, M.; Chirieac, L.; Padera, R.; Shapiro, G.; Baum, A.; Himmelsbach, F.
BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models. Oncogene 2008, 27, 4702–4711.
[CrossRef] [PubMed]
187. Kwak, E.L.; Sordella, R.; Bell, D.W.; Godin-Heymann, N.; Okimoto, R.A.; Brannigan, B.W.; Harris, P.L.; Driscoll, D.R.; Fidias, P.;
Lynch, T.J. Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib. Proc. Natl. Acad. Sci. USA
2005, 102, 7665–7670. [CrossRef] [PubMed]
188. Finlay, M.R.V.; Anderton, M.; Ashton, S.; Ballard, P.; Bethel, P.A.; Box, M.R.; Bradbury, R.H.; Brown, S.J.; Butterworth, S.; Campbell,
A. Discovery of a Potent and Selective EGFR Inhibitor (AZD9291) of Both Sensitizing and T790M Resistance Mutations That Spares the
Wild Type Form of the Receptor; ACS Publications: Washington, DC, USA, 2014.
189. Janjigian, Y.Y.; Smit, E.F.; Groen, H.J.; Horn, L.; Gettinger, S.; Camidge, D.R.; Riely, G.J.; Wang, B.; Fu, Y.; Chand, V.K. Dual
Inhibition of EGFR with Afatinib and Cetuximab in Kinase Inhibitor–Resistant EGFR-Mutant Lung Cancer with and without
T790M MutationsDual EGFR Inhibition in TKI-Resistant, EGFR-Mutant NSCLC. Cancer Discov. 2014, 4, 1036–1045. [CrossRef]
190. Zhou, W.; Ercan, D.; Chen, L.; Yun, C.-H.; Li, D.; Capelletti, M.; Cortot, A.B.; Chirieac, L.; Iacob, R.E.; Padera, R. Novel
mutant-selective EGFR kinase inhibitors against EGFR T790M. Nature 2009, 462, 1070–1074. [CrossRef]
191. Lau, S.C.; Chooback, N.; Ho, C.; Melosky, B. Outcome differences between first-and second-generation EGFR inhibitors in
advanced EGFR mutated NSCLC in a large population-based cohort. Clin. Lung Cancer 2019, 20, e576–e583. [CrossRef]
192. Walter, A.O.; Sjin, R.T.T.; Haringsma, H.J.; Ohashi, K.; Sun, J.; Lee, K.; Dubrovskiy, A.; Labenski, M.; Zhu, Z.; Wang, Z. Discovery of
a Mutant-Selective Covalent Inhibitor of EGFR that Overcomes T790M-Mediated Resistance in NSCLCDevelopment of Covalent
EGFRT790M Inhibitor in NSCLC. Cancer Discov. 2013, 3, 1404–1415. [CrossRef]
193. Wang, S.; Song, Y.; Liu, D. EAI045: The fourth-generation EGFR inhibitor overcoming T790M and C797S resistance. Cancer Lett.
2017, 385, 51–54. [CrossRef] [PubMed]
194. Wee, P.; Wang, Z. Epidermal growth factor receptor cell proliferation signaling pathways. Cancers 2017, 9, 52. [CrossRef]
195. Morelli, M.; Cascone, T.; Troiani, T.; De Vita, F.; Orditura, M.; Laus, G.; Eckhardt, S.; Pepe, S.; Tortora, G.; Ciardiello, F. Sequence-
dependent antiproliferative effects of cytotoxic drugs and epidermal growth factor receptor inhibitors. Ann. Oncol. 2005, 16,
iv61–iv68. [CrossRef] [PubMed]
196. Jutten, B.; Rouschop, K. EGFR signaling and autophagy dependence for growth, survival, and therapy resistance. Cell Cycle 2014,
13, 42–51. [CrossRef]
197. Henson, E.; Chen, Y.; Gibson, S. EGFR family members’ regulation of autophagy is at a crossroads of cell survival and death in
cancer. Cancers 2017, 9, 27. [CrossRef] [PubMed]
198. Ren, J.; Bollu, L.R.; Su, F.; Gao, G.; Xu, L.; Huang, W.C.; Hung, M.C.; Weihua, Z. EGFR–SGLT1 interaction does not respond to
EGFR modulators, but inhibition of SGLT1 sensitizes prostate cancer cells to EGFR tyrosine kinase inhibitors. Prostate 2013, 73,
1453–1461. [CrossRef] [PubMed]
199. Hegi, M.E.; Diserens, A.-C.; Bady, P.; Kamoshima, Y.; Kouwenhoven, M.C.; Delorenzi, M.; Lambiv, W.L.; Hamou, M.-F.; Matter,
M.S.; Koch, A. Pathway analysis of glioblastoma tissue after preoperative treatment with the EGFR tyrosine kinase inhibitor
gefitinib—A phase II trial. Mol. Cancer Ther. 2011, 10, 1102–1112. [CrossRef]
200. Uhm, J.H.; Ballman, K.V.; Wu, W.; Giannini, C.; Krauss, J.; Buckner, J.C.; James, C.; Scheithauer, B.W.; Behrens, R.J.; Flynn, P.J.
Phase II evaluation of gefitinib in patients with newly diagnosed Grade 4 astrocytoma: Mayo/North Central Cancer Treatment
Group Study N0074. Int. J. Radiat. Oncol. Biol. Phys. 2011, 80, 347–353. [CrossRef]
201. Raizer, J.J.; Abrey, L.E.; Lassman, A.B.; Chang, S.M.; Lamborn, K.R.; Kuhn, J.G.; Yung, W.A.; Gilbert, M.R.; Aldape, K.A.; Wen,
P.Y. A phase II trial of erlotinib in patients with recurrent malignant gliomas and nonprogressive glioblastoma multiforme
postradiation therapy. Neuro-Oncology 2010, 12, 95–103. [CrossRef]
202. Haas-Kogan, D.A.; Prados, M.D.; Tihan, T.; Eberhard, D.A.; Jelluma, N.; Arvold, N.D.; Baumber, R.; Lamborn, K.R.; Kapadia, A.;
Malec, M. Epidermal growth factor receptor, protein kinase B/Akt, and glioma response to erlotinib. J. Natl. Cancer Inst. 2005, 97,
880–887. [CrossRef]
Int. J. Mol. Sci. 2023, 24, 11110 23 of 24

203. Thiessen, B.; Stewart, C.; Tsao, M.; Kamel-Reid, S.; Schaiquevich, P.; Mason, W.; Easaw, J.; Belanger, K.; Forsyth, P.; McIntosh,
L. A phase I/II trial of GW572016 (lapatinib) in recurrent glioblastoma multiforme: Clinical outcomes, pharmacokinetics and
molecular correlation. Cancer Chemother. Pharmacol. 2010, 65, 353–361. [CrossRef]
204. Reardon, D.A.; Nabors, L.B.; Mason, W.P.; Perry, J.R.; Shapiro, W.; Kavan, P.; Mathieu, D.; Phuphanich, S.; Cseh, A.; Fu, Y. Phase
I/randomized phase II study of afatinib, an irreversible ErbB family blocker, with or without protracted temozolomide in adults
with recurrent glioblastoma. Neuro-Oncology 2015, 17, 430–439. [CrossRef]
205. Kizilbash, S.H.; Gupta, S.K.; Parrish, K.E.; Laramy, J.K.; Kim, M.; Gampa, G.; Carlson, B.L.; Bakken, K.K.; Mladek, A.C.; Schroeder,
M.A. In Vivo Efficacy of Tesevatinib in EGFR-Amplified Patient-Derived Xenograft Glioblastoma Models May Be Limited by
Tissue Binding and Compensatory SignalingTesevatinib Brain PK and Efficacy in GBM PDX Models. Mol. Cancer Ther. 2021, 20,
1009–1018. [CrossRef]
206. Sepúlveda, J.M.; Sánchez-Gómez, P.; Vaz Salgado, M.Á.; Gargini, R.; Balañá, C. Dacomitinib: An investigational drug for the
treatment of glioblastoma. Expert Opin. Investig. Drugs 2018, 27, 823–829. [CrossRef] [PubMed]
207. Napier, T.S.; Udayakumar, N.; Jani, A.H.; Hartman, Y.E.; Houson, H.A.; Moore, L.; Amm, H.M.; van den Berg, N.S.; Sorace, A.G.;
Warram, J.M. Comparison of Panitumumab-IRDye800CW and 5-Aminolevulinic Acid to Provide Optical Contrast in a Model of
Glioblastoma MultiformePanitumumab-IRDye800CW versus 5-ALA for Optical Contrast. Mol. Cancer Ther. 2020, 19, 1922–1929.
[CrossRef]
208. Wang, Q.; Ni, J.; Jiang, T.; Choi, H.G.; Zhang, T.; Gray, N.; Zhao, J.J. CM93, a novel covalent small molecule inhibitor targeting
lung cancer with mutant EGFR. BioRxiv 2020. [CrossRef]
209. Ni, J.; Yang, Y.; Wang, Q.; Bergholz, J.S.; Jiang, T.; Roberts, T.M.; Gray, N.S.; Zhao, J.J. Targeting EGFR in glioblastoma with a novel
brain-penetrant small molecule EGFR-TKI. BioRxiv 2021. [CrossRef]
210. Erasca. Erasca Granted FDA Orphan Drug Designation for CNS-Penetrant EGFR Inhibitor ERAS-801 for the Treatment of
Malignant Glioma. Available online: https://investors.erasca.com/news-releases/news-release-details/erasca-granted-fda-
orphan-drug-designation-cns-penetrant-egfr (accessed on 20 February 2023).
211. Conage-Pough, J.E.; Stopka, S.A.; Oh, J.-H.; Mladek, A.C.; Burgenske, D.M.; Regan, M.S.; Baquer, G.; Decker, P.A.; Carlson, B.L.;
Bakken, K.K. WSD-0922, a novel brain-penetrant inhibitor of epidermal growth factor receptor, promotes survival in glioblastoma
mouse models. Neuro-Oncol. Adv. 2023, 5, vdad066. [CrossRef]
212. Mateos, M.E.; López-Laso, E.; Izquierdo, L.; Pérez-Navero, J.L.; García, S.; Garzás, C. Response to nimotuzumab in a child with a
progressive diffuse intrinsic pontine glioma. Pediatr. Int. 2011, 53, 261–263. [CrossRef]
213. Lam, C.; Bouffet, E.; Bartels, U. Nimotuzumab in pediatric glioma. Future Oncol. 2009, 5, 1349–1361. [CrossRef] [PubMed]
214. Belda-Iniesta, C.; de Castro Carpeño, J.; Saenz, E.C.; Gutiérrez, M.; Perona, R.; González-Barón, M. Long term responses with
cetuximab therapy in glioblastoma multiforme. Cancer Biol. Ther. 2006, 5, 912–914. [CrossRef]
215. Eller, J.L.; Longo, S.L.; Kyle, M.M.; Bassano, D.; Hicklin, D.J.; Canute, G.W. Anti-epidermal growth factor receptor monoclonal
antibody cetuximab augments radiation effects in glioblastoma multiforme in vitro and in vivo. Neurosurgery 2005, 56, 155–162.
[CrossRef]
216. Neyns, B.; Sadones, J.; Joosens, E.a.; Bouttens, F.; Verbeke, L.; Baurain, J.-F.; D’Hondt, L.; Strauven, T.; Chaskis, C.; In’t Veld, P.
Stratified phase II trial of cetuximab in patients with recurrent high-grade glioma. Ann. Oncol. 2009, 20, 1596–1603. [CrossRef]
217. Choi, B.D.; Kuan, C.-T.; Cai, M.; Archer, G.E.; Mitchell, D.A.; Gedeon, P.C.; Sanchez-Perez, L.; Pastan, I.; Bigner, D.D.; Sampson,
J.H. Systemic administration of a bispecific antibody targeting EGFRvIII successfully treats intracerebral glioma. Proc. Natl. Acad.
Sci. USA 2013, 110, 270–275. [CrossRef]
218. Orellana, L.; Thorne, A.H.; Lema, R.; Gustavsson, J.; Parisian, A.D.; Hospital, A.; Cordeiro, T.N.; Bernado, P.; Scott, A.M.; Brun-
Heath, I. Oncogenic mutations at the EGFR ectodomain structurally converge to remove a steric hindrance on a kinase-coupled
cryptic epitope. Proc. Natl. Acad. Sci. USA 2019, 116, 10009–10018. [CrossRef]
219. Johns, T.G.; McKay, M.J.; Cvrljevic, A.N.; Gan, H.K.; Taylor, C.; Xu, H.; Smyth, F.E.; Scott, A.M. MAb 806 enhances the efficacy of
ionizing radiation in glioma xenografts expressing the de2-7 epidermal growth factor receptor. Int. J. Radiat. Oncol. Biol. Phys.
2010, 78, 572–578. [CrossRef] [PubMed]
220. Johnson, L.A.; Scholler, J.; Ohkuri, T.; Kosaka, A.; Patel, P.R.; McGettigan, S.E.; Nace, A.K.; Dentchev, T.; Thekkat, P.; Loew, A.
Rational development and characterization of humanized anti–EGFR variant III chimeric antigen receptor T cells for glioblastoma.
Sci. Transl. Med. 2015, 7, 275ra222. [CrossRef]
221. Chen, M.; Sun, R.; Shi, B.; Wang, Y.; Di, S.; Luo, H.; Sun, Y.; Li, Z.; Zhou, M.; Jiang, H. Antitumor efficacy of chimeric antigen
receptor T cells against EGFRvIII-expressing glioblastoma in C57BL/6 mice. Biomed. Pharmacother. 2019, 113, 108734. [CrossRef]
[PubMed]
222. O’Rourke, D.M.; Nasrallah, M.P.; Desai, A.; Melenhorst, J.J.; Mansfield, K.; Morrissette, J.J.; Martinez-Lage, M.; Brem, S.; Maloney,
E.; Shen, A. A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive
resistance in patients with recurrent glioblastoma. Sci. Transl. Med. 2017, 9, eaaa0984. [CrossRef]
223. Goff, S.L.; Morgan, R.A.; Yang, J.C.; Sherry, R.M.; Robbins, P.F.; Restifo, N.P.; Feldman, S.A.; Lu, Y.-C.; Lu, L.; Zheng, Z. Pilot
trial of adoptive transfer of chimeric antigen receptor transduced T cells targeting EGFRvIII in patients with glioblastoma. J.
Immunother. 2019, 42, 126. [CrossRef] [PubMed]
Int. J. Mol. Sci. 2023, 24, 11110 24 of 24

224. Binder, D.C.; Ladomersky, E.; Lenzen, A.; Zhai, L.; Lauing, K.L.; Otto-Meyer, S.D.; Lukas, R.V.; Wainwright, D.A. Lessons learned
from rindopepimut treatment in patients with EGFRvIII-expressing glioblastoma. Transl. Cancer Res. 2018, 7, S510. [CrossRef]
[PubMed]
225. Schuster, J.; Lai, R.K.; Recht, L.D.; Reardon, D.A.; Paleologos, N.A.; Groves, M.D.; Mrugala, M.M.; Jensen, R.; Baehring, J.M.; Sloan,
A. A phase II, multicenter trial of rindopepimut (CDX-110) in newly diagnosed glioblastoma: The ACT III study. Neuro-Oncology
2015, 17, 854–861. [CrossRef]
226. Weller, M.; Butowski, N.; Tran, D.D.; Recht, L.D.; Lim, M.; Hirte, H.; Ashby, L.; Mechtler, L.; Goldlust, S.A.; Iwamoto, F.
Rindopepimut with temozolomide for patients with newly diagnosed, EGFRvIII-expressing glioblastoma (ACT IV): A randomised,
double-blind, international phase 3 trial. Lancet Oncol. 2017, 18, 1373–1385. [CrossRef]
227. Iurlaro, R.; Waldhauer, I.; Planas-Rigol, E.; Bonfill-Teixidor, E.; Arias, A.; Nicolini, V.; Freimoser-Grundschober, A.; Cuartas, I.;
Martínez-Moreno, A.; Martínez-Ricarte, F. A novel EGFRvIII T-cell bispecific antibody for the treatment of glioblastoma. Mol.
Cancer Ther. 2022, 21, 1499–1509. [CrossRef]
228. Li, L.; Quang, T.S.; Gracely, E.J.; Kim, J.H.; Emrich, J.G.; Yaeger, T.E.; Jenrette, J.M.; Cohen, S.C.; Black, P.; Brady, L.W. A phase II
study of anti–epidermal growth factor receptor radioimmunotherapy in the treatment of glioblastoma multiforme. J. Neurosurg.
2010, 113, 192–198. [CrossRef] [PubMed]
229. Karim, R.; Palazzo, C.; Evrard, B.; Piel, G. Nanocarriers for the treatment of glioblastoma multiforme: Current state-of-the-art. J.
Control. Release 2016, 227, 23–37. [CrossRef] [PubMed]
230. Mortensen, J.H.; Jeppesen, M.; Pilgaard, L.; Agger, R.; Duroux, M.; Zachar, V.; Moos, T. Targeted antiepidermal growth factor
receptor (cetuximab) immunoliposomes enhance cellular uptake in vitro and exhibit increased accumulation in an intracranial
model of glioblastoma multiforme. J. Drug Deliv. 2013, 2013, 209205. [CrossRef]
231. Read, J.; Ingram, A.; Al Saleh, H.A.; Platko, K.; Gabriel, K.; Kapoor, A.; Pinthus, J.; Majeed, F.; Qureshi, T.; Al-Nedawi, K. Nuclear
transportation of exogenous epidermal growth factor receptor and androgen receptor via extracellular vesicles. Eur. J. Cancer
2017, 70, 62–74. [CrossRef]
232. Ricklefs, F.; Mineo, M.; Rooj, A.K.; Nakano, I.; Charest, A.; Weissleder, R.; Breakefield, X.O.; Chiocca, E.A.; Godlewski, J.;
Bronisz, A. Extracellular Vesicles from High-Grade Glioma Exchange Diverse Pro-oncogenic Signals That Maintain Intratumoral
HeterogeneityTumor Heterogeneity Is Enhanced by Extracellular Vesicles. Cancer Res. 2016, 76, 2876–2881. [CrossRef]
233. Saleem, H.; Abdul, U.K.; Küçükosmanoglu, A.; Houweling, M.; Cornelissen, F.M.; Heiland, D.H.; Hegi, M.E.; Kouwenhoven,
M.C.; Bailey, D.; Würdinger, T. The TICking clock of EGFR therapy resistance in glioblastoma: Target Independence or target
Compensation. Drug Resist. Updat. 2019, 43, 29–37. [CrossRef]
234. Nathanson, D.A.; Gini, B.; Mottahedeh, J.; Visnyei, K.; Koga, T.; Gomez, G.; Eskin, A.; Hwang, K.; Wang, J.; Masui, K. Targeted
therapy resistance mediated by dynamic regulation of extrachromosomal mutant EGFR DNA. Science 2014, 343, 72–76. [CrossRef]
235. Husain, H.; Scur, M.; Murtuza, A.; Bui, N.; Woodward, B.; Kurzrock, R. Strategies to overcome bypass mechanisms mediating
clinical resistance to EGFR tyrosine kinase inhibition in lung cancer. Mol. Cancer Ther. 2017, 16, 265–272. [CrossRef] [PubMed]
236. Lim, S.M.; Syn, N.L.; Cho, B.C.; Soo, R.A. Acquired resistance to EGFR targeted therapy in non-small cell lung cancer: Mechanisms
and therapeutic strategies. Cancer Treat. Rev. 2018, 65, 1–10. [CrossRef] [PubMed]
237. Camidge, D.R.; Pao, W.; Sequist, L.V. Acquired resistance to TKIs in solid tumours: Learning from lung cancer. Nat. Rev. Clin.
Oncol. 2014, 11, 473–481. [CrossRef] [PubMed]
238. Cortot, A.B.; Jänne, P.A. Molecular mechanisms of resistance in epidermal growth factor receptor-mutant lung adenocarcinomas.
Eur. Respir. Rev. 2014, 23, 356–366. [CrossRef]
239. Jones, S.; King, P.J.; Antonescu, C.N.; Sugiyama, M.G.; Bhamra, A.; Surinova, S.; Angelopoulos, N.; Kragh, M.; Pedersen, M.W.;
Hartley, J.A. Targeting of EGFR by a combination of antibodies mediates unconventional EGFR trafficking and degradation. Sci.
Rep. 2020, 10, 663. [CrossRef]
240. Guo, G.; Narayan, R.N.; Horton, L.; Patel, T.R.; Habib, A.A. The role of EGFR-Met interactions in the pathogenesis of glioblastoma
and resistance to treatment. Curr. Cancer Drug Targets 2017, 17, 297–302. [CrossRef]
241. Wen, Y.; Grandis, J.R. Emerging drugs for head and neck cancer. Expert Opin. Emerg. Drugs 2015, 20, 313–329. [CrossRef]
242. Del Vecchio, C.; Giacomini, C.; Vogel, H.; Jensen, K.; Florio, T.; Merlo, A.; Pollack, J.; Wong, A. EGFRvIII gene rearrangement is an
early event in glioblastoma tumorigenesis and expression defines a hierarchy modulated by epigenetic mechanisms. Oncogene
2013, 32, 2670–2681. [CrossRef]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.