MINIREVIEW
EGF receptor in relation to tumor development: molecular
basis of responsiveness of cancer cells to EGFR-targeting
tyrosine kinase inhibitors
Kenji Takeuchi and Fumiaki Ito
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan
Introduction
The epidermal growth factor receptor (EGFR) is com-
posed of an extracellular ligand-binding domain, a
transmembrane domain and an intracellular tyrosine
kinase domain. The binding of a ligand to the extracel-
lular domain of the EGFR induces receptor dimeriza-
tion, activation of the intracellular kinase domain and
autophosphorylation of tyrosine residues within the
cytoplasmic domain of the receptor. The tyrosine-
phosphorylated motifs of the EGFR recruit various
adaptors or signaling molecules [1,2]. The EGFR is
able to activate a variety of signaling pathways
through its association with these molecules. Extra-
Keywords
cancer; epidermal growth factor receptor
(EGFR); gefitinib; non-small cell lung cancer
(NSCLC); tyrosine kinase inhibitor (TKI)
Correspondence
K. Takeuchi, Department of Biochemistry,
Faculty of Pharmaceutical Sciences,
Setsunan University, Hirakata, Osaka
573-0101, Japan
Fax: +81 72 866 3117
Tel: +81 72 866 3118
E-mail:

2
-terminal kinase; KIP, kinase inhibitor proteins; MEK,
MAPK ⁄ ERK kinase; MKK, MAPK kinase; MKP-1, mitogen-activated protein kinase phosphatase-1; MOMP, mitochondrial outer membrane
permeabilization; NSCLC, non-small cell lung cancer; Pak1, p21-activated kinase 1; PI3K, phosphatidylinositol 3-kinase; PUMA, p53
up-regulated modulator of apoptosis; RB, retinoblastoma; TKI, tyrosine kinase inhibitor.
316 FEBS Journal 277 (2010) 316–326 ª 2009 The Authors Journal compilation ª 2009 FEBS
cellular signal-regulated kinase (ERK)1 ⁄ 2, which is one
of the three major groups of mitogen-activated protein
kinases (MAPKs) in mammals, is activated by the
EGFR tyrosine kinase and plays an essential role in
cell proliferation. In contrast, EGFR signaling inhibits
the activation of the other two MAPKs, namely p38
MAPK and c-Jun NH
2
-terminal kinase (JNK). Fur-
thermore, the phosphatidylinositol 3-kinase (PI3K) ⁄
Akt pathway, which is activated by the EGFR, has
been implicated in both cell proliferation and survival.
Potential targets of these MAPK and PI3K ⁄ Akt sig-
naling pathways include apoptosis-related molecules
(Bcl-2 family members and Fas) and cell-cycle regula-
tory molecules (e.g. p27
KIP1
; Fig. 1). The EGFR there-
fore plays an important role in both cell proliferation
and survival.
EGFR function is dysregulated in various types of
malignancy [1,2] as a result of gene amplification,
mutations (resulting in a constitutively active EGFR)
or abnormally increased ligand production (reviewed

more, hepatocyte growth factor significantly inhibits
adriamycin-induced apoptosis in the human gastric
adenocarcinoma cell line MKN74 through phosphory-
lation of pro-caspase-9 via the Akt signaling pathway
[14]. Akt also phosphorylates Bad [15], a pro-apoptotic
member of the Bcl-2 family, and the forkhead tran-
scription factor FKHR [16], a pro-apoptotic transcrip-
tion factor. Therefore, the Akt signaling pathway has
emerged as the major mechanism by which growth
factors promote cell survival (reviewed in [17]).
A link between the Akt pathway and gefitinib-
responsiveness was reported by Engelman et al. [18]:
the Akt pathway is down-regulated in response to gefi-
tinib only in NSCLC cell lines that are growth-inhib-
ited by gefitinib. Thus, activated Akt has been
indicated as a molecular determinant of a response to
EGFR-targeting drugs. However, the NSCLC cell line
H3255, harboring the L858R mutation in EGFR exon
Fig. 1. Major signaling pathways
downstream of the activated EGFR
Activation of several signaling cascades
triggered predominately by the ERK1 ⁄ 2
and the PI3K ⁄ Akt pathways results, in turn,
in the inactivation of pro-apoptotic Bcl-2
proteins (e.g. PUMA, Bax, Bim and Bad),
Fas, and CDK inhibitors (e.g. p27
KIP1
,
p21
WAF1

domains and only induce MOMP following apoptotic
stimuli, resulting in the release of cytochrome c, activa-
tion of the caspase cascade and cellular destruction
[22]. To prevent cell death, Bax and Bak are bound
and inhibited by the anti-apoptotic members of the
Bcl-2 protein family (Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and
A1), which contain four BH domains [22]. The third
subgroup, the BH3-only proteins, are structurally
diverse and contain only one conserved domain (BH3).
Often, the BH3-only proteins are subdivided into
direct activators [Bid and Bcl-2 interacting mediator of
cell death (Bim)] and de-repressors [Bad, Bik, Bmf,
NOXA, and p53 up-regulated modulator of apoptosis
(PUMA)]. These de-repressors initiate apoptosis signal-
ing by binding and antagonizing the anti-apoptotic
Bcl-2 family members, thereby causing activation of
Bax and Bak [23]. Regulation of Bcl-2 family members
can occur by a number of mechanisms, including
up-regulation of synthesis, enhancement of degrad-
ation and phosphorylation. In the event that cancer
cells undergo apoptosis in response to gefitinib, inhibi-
tion of Akt- and ERK1 ⁄ 2-dependent pathways eventu-
ally change the expression level of one or more of
these Bcl-2 family members.
Bad
Bad is one of the ‘death-promoting’ members of the
Bcl-2 family, and its pro-apoptotic activity is regulated
primarily by phosphorylation at several sites [24]. Acti-
vated Akt [13,25] and ERK1 ⁄ 2-p90 ribosomal S6
kinase-1 (p90Rsk-1) [26,27] pathways have been shown

also induce p53-independent apoptosis in response to a
wide variety of stimuli [31]. Therefore, PUMA is a crit-
ical mediator of both p53-dependent and p53-indepen-
dent apoptosis and mediates apoptosis through the
Bcl-2 family proteins Bax ⁄ Bak [32]. PUMA is induced
by gefitinib, independently of p53, in head and neck
squamous cell carcinomas (HNSCC) [33]. This BH3-
only protein functions as a critical mediator of gefiti-
nib-induced apoptosis, and in the Akt pathway and
p73, p53 family proteins serve as key regulators of
PUMA induction after EGFR inhibition (pathway
in Fig. 2). Overexpression of EGFR is found in more
than 80% of HNSCC. Thus, TKIs have emerged as
promising treatments, not only for NSCLC but also
for HNSCC.
Bim
Bim is a member of the BH3-only proteins [34].
Under conditions that promote cell growth, Bim is
bound to dynein light chain (LC8) of the microtubu-
lar motor complex and is sequestered away from
other Bcl-2 family members [35]. Following a pro-
apoptotic stimulus, however, Bim is localized to the
mitochondria, where it initiates the mitochondrial cell
death pathway by directly activating Bax ⁄ Bak [36].
Bim expression is regulated by both transcriptional
and post-transcriptional levels (pathway
in Fig. 2).
Phosphorylation of Bim by ERK1 ⁄ 2 targets Bim for
degradation by the ubiquitin-proteasome system [37].
Bim has recently been reported to mediate gefitinib-

Stimulation of the Akt pathway inhibits Bax translo-
cation from the cytoplasm to the mitochondria and
promotes survival [45]. Anti-apoptotic stimuli lead to
the activation of Akt and to Ser184 phosphorylation
of Bax [46]. This phosphorylation promotes the seques-
tration of Bax in the cytoplasm and increases the abil-
ity of Bax to heterodimerize with the anti-apoptotic
Bcl-2 family members Mcl-1 and Bcl-xL, thereby
inhibiting activation of apoptosis signals. Gefitinib is
known to induce apoptosis through shutdown of Akt
signaling. However, it has not been demonstrated
whether this shutdown transmits the apoptotic signal
via inhibition of Bax phosphorylation.
Regulation of Bax also occurs by mechanisms other
than phosphorylation. Gefitinib inhibits growth of
human gallbladder adenocarcinoma cells (HAG-1) by
arresting the cells in the G
0
⁄ G
1
phase [47]. This arrest
is accompanied by depression of cyclin D1 mRNA as
well as by the accumulation of p27 protein. However,
when HAG-1 cells are treated with gefitinib for more
than 72 h, the apoptotic population increases. Corre-
spondingly, gefitinib up-regulates expression of total
Bax, with a subsequent increase in p18 Bax that has
been shown to be generated through the cleavage of
full-length Bax during apoptosis (pathway
in Fig. 2).

of the inhibitor of apoptosis protein (IAP) family such
as cIAP-1, cIAP-2, X-linked IAP and survivin. Recent
studies have suggested that activation of the PI3K ⁄ Akt
pathway by EGFR signaling causes up-regulation of
survivin expression [52]. The levels of cIAP-2 are
down-regulated by gefitinib or erlotinib in intestinal
epithelial cells [53]. Furthermore, the expression of
cIAP-1 and of X-linked IAP is reduced by AG1478 in
squamous cell carcinoma cell lines NA and Ca9-22
[54]. As small interfering RNA (siRNA)-based deple-
tion of IAP increases apoptosis in response to gefitinib,
IAPs might be a molecular target for the induction of
apoptosis by TKIs.
Inhibition of cell proliferation
EGFR signaling activates a variety of pathways such
as those for cell survival, cell proliferation, cell motil-
ity, angiogenesis and expression of extracellular matrix
proteins [55]. Accordingly, TKIs against EGFR exert
not only apoptosis-inducing action but also other
divergent actions. For instance, EGFR inhibition leads
to the induction of cell-cycle arrest at the G
1
-S bound-
ary [56]. Cell-cycle regulation is important in growth
control, and therefore deregulation of the cell-cycle
machinery has been implicated in carcinogenesis [57].
Cyclins and cyclin-dependent kinases (CDKs), in asso-
ciation with each other, play key roles in promoting
the G
1

recruitment of tumor cells in the G
1
phase and a
marked reduction in the proportion of cells in the S
phase. The G
1
arrest and up-regulation of p27
KIP1
resulting from EGFR blockade are caused by the
interruption of PI3K signals. In addition to p27
KIP1
,
p21
WAF1 ⁄ CIP1
is involved in gefitinib-induced growth
inhibition in HNSCC [58]. Another group of cell-cycle
regulatory molecules – those of the INK4 family – has
also been implicated in gefitinib-induced inhibition of
cell growth. Gefitinib up-regulates p15
INK4b
in human
immortalized keratinocyte HaCaT cells and results in
RB hypophosphorylation and G
1
arrest [59]. More-
over, mouse embryo fibroblasts lacking p15
INK4b
are
resistant to the growth-inhibitory effects of gefitinib.
As the level of p15

Accordingly, dysregulation of the EGFR contributes
to the progression, invasion and maintenance of the
malignant phenotype. In keratinocyte and cutaneous
squamous cancer cells, gefitinib blocks EGF-induced
cytoskeleton remodeling and in vitro invasiveness, as
well as cell growth [60]. Gefitinib also effectively inhib-
its ERK1 ⁄ 2 activation and p21-activated kinase 1
(Pak1) activity (see Fig. 1). Pak1 is a serine ⁄ threonine
kinase and is a critical component of many growth fac-
tor receptor-mediated signal transduction pathways,
leading to directional cell motility and cell invasiveness
[61]. Because deregulation of EGFR signaling is com-
monly associated with stimulation of ERK1 ⁄ 2 and
Pak1 pathways, gefitinib might lead to inhibition of
invasiveness of human cancer cells through the inhibi-
tion of ERK1 ⁄ 2 and Pak1. The use of gefitinib in cells
with activated ERK1 ⁄ 2 or Pak1 pathways might
potentially lead to beneficial anti-cancer activity
through the inhibition of not only cell survival but also
cell invasiveness.
p38, JNK and Fas as target molecules
of gefitinib
p38
As described above, the treatment of intestinal epithe-
lial cells with gefitinib results in a dramatic increase in
apoptosis and activation of the intrinsic apoptotic
pathway via trafficking of activated Bax to the mito-
chondria [62]. Akt is known to phosphorylate Bax and
to prohibit its mitochondrial translocation. However,
the Akt pathway plays a minor role in the induction

overexpressed in human tumors. Constitutive expression
levels of MKP-1 in NSCLC cell lines are higher than
those found in normal cells under basal growth condi-
tions [67]. Overexpression of MKP-1 has been reported
to protect cells against apoptosis induced by UV irradia-
tion, Fas ligand, cisplatin, paclitaxel, proteasome inhibi-
tors or radiation therapy [68]. These observations have
established that MKP-1 plays an important role in
resistance against many types of stresses, including anti-
cancer drugs, in various cell lines. MKP-1 may be a
rational target to enhance anticancer drug activity.
Our recent results have shown that the activation of
JNK induced by EGFR-TKI AG1478 is critical for
the apoptotic action of AG1478 against the NSCLC
cell line PC-9 [69]. Various types of stimuli activate
JNK through phosphorylation by the dual-specificity
JNK kinases; but JNK kinases MKK4 and MKK7 are
not activated by AG1478 treatment. In contrast, JNK
phosphatase (i.e. MKP-1) is constitutively expressed in
PC-9 cells and its expression level is reduced by
AG1478. Furthermore, the inhibition of JNK
activation by ectopic expression of MKP-1 or a domi-
nant-negative form of JNK strongly suppresses
AG1478-induced apoptosis. Thus, JNK, which is acti-
vated through the decrease in the MKP-1 level, is criti-
cal for the apoptotic action of AG1478 against PC-9
cells. Interestingly, AG1478 has no inhibitory activity
towards MKP-1 expression in some resistant cell lines
isolated from gefitinib-sensitive PC-9 cells (unpublished
data of T. Shin-ya, K. Takeuchi and F. Ito).

use a different set of transcription factors to enhance
MKP-1 expression.
Several lines of evidence suggest that phosphoryla-
tion of MKP-1 protein plays an important role in the
stabilization of MKP-1 (Fig. 4). ERK1 ⁄ 2 reduces
MKP-1 degradation by phosphorylating the Ser359
and Ser364 residues of MKP-1 [72]. ERK1 ⁄ 2 is also
responsible for the degradation of MKP-1 via the
phosphorylation of Ser296 and Ser323 residues [76].
Once phosphorylated, Skp2 (also called SCF
Skp2
of
Skp1 ⁄ Cul1 ⁄ F-box protein Skp2; ubiquitin-protein iso-
peptide ligase E3) targets MKP-1 for degradation via
the ubiquitin proteasomal pathway [73]. In addition to
the transcriptional and post-translational control
described here, it is suggested that transcription of the
mkp-1 gene is also controlled at the level of transcrip-
tional elongation [71]. The mechanism responsible for
the regulation of MKP-1 expression is complex, and
both transcriptional down-regulation and degradation
of MKP-1 may be effects observed in cells having an
apoptotic response to EGFR-TKI AG1478.
Fas
Exposure of the human NSCLC cell line, A549, to gef-
itinib causes a marked increase in the expression of
Fas protein and in the activation of caspases 2, 3 and
8 [77]. Co-treatment of cells with Fas antagonist anti-
body significantly blocks gefitinib-induced apoptosis.
Furthermore, caspase-8 and caspase-3 inhibitors, but

between these pro-apoptotic and anti-apoptotic Bcl-2
family members determines the cellular fate (i.e.
survival or apoptosis). In cancer cells that undergo
apoptosis in response to TKIs, shutdown of ERK1 ⁄ 2
and PI3K ⁄ Akt signaling pathways following the inhi-
bition of EGFR activation ultimately results in the
disruption of the balance between pro-apoptotic and
anti-apoptotic Bcl-2 proteins and subsequent apopto-
sis. Bcl-2 family proteins are key molecules for regulat-
ing the permeabilization of the outer mitochondrial
membrane and thus represent pivotal components in
TKI-dependent apoptosis signaling. TKIs change the
transcription level of Bcl-2 family genes and the phos-
phorylation state of their proteins, thereby changing
the amount and localization of Bcl-2 family members.
However, each type of cancer has its own way of
disrupting the balance of the networks of signaling
cascades following TKI treatment. Therefore, under-
standing how Bcl-2 family members are regulated in
each type of cancer is critical for understanding how
TKIs cause apoptosis in each of them.
It is now clear that TKIs are unlikely to provide
cures for the majority of patients with NSCLC.
Despite the initial dramatic efficacy of gefitinib and
erlotinib in NSCLC patients with EGFR mutations,
all patients ultimately develop resistance to TKIs. A
secondary mutation in the EGFR (T790M) and the
amplification of hepatocyte growth factor receptors
have been identified as major mechanisms of acquired
resistance to TKIs [78]. However, it is still important

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