Tài liệu Báo cáo khoa học: EGF receptor in relation to tumor development: molecular basis of responsiveness of cancer cells to EGFR-targeting tyrosine kinase inhibitors docx

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:

(Received 17 July 2009, revised 17
September 2009, accepted 13 October
2009)
doi:10.1111/j.1742-4658.2009.07450.x
The function of the epidermal growth factor receptor (EGFR) is dysregu-
lated in various types of malignancy as a result of gene amplification,
mutations, or abnormally increased ligand production. Therefore, the tyro-
sine kinase activity of the EGFR is a promising therapeutic target. EGFR
tyrosine kinase inhibitors, such as gefitinib (Iressa), show evident anticancer
effects in patients with non-small cell lung cancer. The induction of apop-
tosis has been considered to be the major mechanism for these gefitinib-
mediated anticancer effects. Lung cancer cells harboring mutant EGFRs
become dependent on them for their survival and, consequently, undergo
apoptosis following the inhibition of EGFR tyrosine kinase by gefitinib.
Gefitinib has been shown to inhibit cell survival and growth signaling path-
ways such as the extracellular signal-regulated kinase 1 ⁄ 2 pathway and the
Akt pathway, as a consequence of the inactivation of EGFR. However, the
precise downstream signaling molecules of extracellular signal-regulated
kinase 1 ⁄ 2 and Akt have not yet been elucidated. In this minireview we
have highlighted the effect of tyrosine kinase inhibitors on members of the
Bcl-2 family of proteins, which are downstream signaling molecules and
serve as the determinants that control apoptosis. We also discuss tyrosine
kinase inhibitor-induced apoptosis via c-Jun NH
2
-terminal kinase and p38
mitogen-activated protein kinase.
Abbreviations
BH, Bcl-2 homology domain; Bim, Bcl-2 interacting mediator of cell death; CDK, cyclin-dependent kinase; CRE, cAMP-response element;
CREB, CRE-binding protein; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; HNSCC, head and neck
squamous cell carcinomas; IAP, inhibitor of apoptosis protein; JNK, c-Jun NH

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

in [3]). Moreover, enforced expression of mutant EG-
FRs in transgenic mice promotes the development of
lung carcinomas [4,5]. Therefore, EGFR-tyrosine
kinase is a promising therapeutic target. Small mole-
cules that are active orally against the EGFR [e.g.
gefitinib (Iressa) and erlotinib (Tarceva)] show evident
anticancer effects in patients with non-small cell lung
cancer (NSCLC) [6–8]. Beneficial responsiveness to
these EGFR-targeting tyrosine kinase inhibitors
(TKIs) in patients with NSCLC is closely associated
with EGFR mutations such as del746-750 and L858R
in the kinase domain [9–11]. Lung cancer cells
harboring mutant EGFRs become dependent on them
for their survival and, consequently, undergo apopto-
sis following inhibition of EGFR tyrosine kinase by
gefitinib. Gefitinib has been shown to inhibit cell
growth and survival signaling pathways, such as the
ERK1 ⁄ 2 pathway and the Akt pathway, as a conse-
quence of inactivation of the EGFR [12]. With refer-
ence to Fig. 1, which presents an overview of the
intracellular signaling pathways activated by the
EGFR tyrosine kinase, we will describe some of the
diverse actions of TKIs on cell growth, cell survival
and cell motility.
Akt and ERK1
⁄
2 signaling pathways as
target pathways for TKIs
Akt induces the phosphorylation of pro-caspase-9,
thereby inhibiting its protease activity [13]. Further-

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

and p15
INK4b
), and also in the
activation of Pak1.
K. Takeuchi and F. Ito Molecular mechanisms of sensitivity to EGFR-TKIs
FEBS Journal 277 (2010) 316–326 ª 2009 The Authors Journal compilation ª 2009 FEBS 317
21, and PC-9, harboring a deletion (del746-750) in
EGFR exon 19, are highly sensitive to gefitinib; and
this sensitivity to gefitinib is associated with depen-
dence on both Akt and ERK1 ⁄ 2 pathways [10,19].
Apoptosis
The induction of apoptosis has been considered as the
major mechanism for gefitinib-mediated anticancer
effects. Mammals have two distinct, but ultimately
converging, apoptosis signaling pathways: the extrinsic
(also called ‘death receptor’) pathway, which is acti-
vated by death receptors; and the intrinsic (also called
‘mitochondrial’ or ‘Bcl-2-regulated’) pathway [20]. The
intrinsic pathway is characterized by the permeabili-
zation of the outer mitochondrial membrane and the
release of several pro-apoptotic factors into the
cytoplasm. For mitochondrial outer membrane per-
meabilization (MOMP), a coordinated effort between
numerous Bcl-2 proteins must be engaged (reviewed in
[21]). The Bcl-2 proteins can be divided into three
groups according to their function (Fig. 2). Members
of the Bcl-2 protein family are distinguished by the
presence of up to four different Bcl-2 homology
domains (designated BH1–4). The multidomain pro-
apoptotic Bcl-2 proteins, Bax and Bak, contain BH1–3

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

to promote survival signaling by phosphorylating Bad
at Ser136 and Ser112, respectively. These phosphory-
lated residues provide binding sites for 14-3-3 proteins,
which subsequently sequester Bad.
Phosphorylation of Bad-Ser112 via ERK1 ⁄ 2 path-
way (
in Fig. 2) is inhibited by either gefitinib or the
MAPK ⁄ ERK kinase (MEK) inhibitor PD98059 in
mammary epithelial cells and primary cultures of
malignant breast carcinoma [28]. Gefitinib has no
effect on EGF-mediated Bad-Ser112 phosphorylation
in the cells transfected with vectors encoding constitu-
tively active p90Rsk-1. Thus, the EGF induces Bad
phosphorylation through an ERK1 ⁄ 2 pathway involv-
ing p90Rsk-1. It has also been reported that primary
cultures of Bad
) ⁄ )
mammary cancer cells are no longer
sensitive to gefitinib-induced apoptosis, suggesting that
Bad might be an important pro-apoptotic effector
molecule downstream of the EGFR.
Fig. 2. Bcl-2 family proteins as targets of
TKIs.
Molecular mechanisms of sensitivity to EGFR-TKIs K. Takeuchi and F. Ito
318 FEBS Journal 277 (2010) 316–326 ª 2009 The Authors Journal compilation ª 2009 FEBS
PUMA
PUMA was initially identified as a critical mediator of
apoptosis induced by the tumor suppressor p53 [29,30]
and it can be directly activated by p53 through p53-
responsive elements in its promoter region. PUMA can

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-

induced apoptosis [38–40]. Bim knockdown by RNA
interference protects the NSCLC cell line, H3255,
potently against gefitinib, and the level of protection
correlates with the extent of Bim reduction, indicating
that Bim is essential for gefitinib-induced apoptosis of
NSCLC cells. The induction of Bim after treatment
with gefitinib is a consequence of both transcriptional
induction and dephosphorylation. Thus, shutdown of
the EGFR-MEK-ERK signaling cascade by gefitinib
elicits Bim accumulation and causes apoptosis. The
T790M mutation of the EGFR, which renders gefiti-
nib and erlotinib ineffective inhibitors of EGFR
kinase activity, blocks gefitinb-induced up-regulation
of Bim and apoptosis [38]. These experiments point
to an important role for the induction of Bim in
gefitinib-triggered apoptosis of NSCLC cells.
Bax
Bax is a 21-kDa multi-BH domain pro-apoptotic pro-
tein and acts downstream of BH3-only proteins. The
induction of Bax expression can be sufficient to induce
apoptosis and requires no additional death stimulus
[41]. Furthermore, Bax expression is associated with
tumor development [42]. The protein is normally found
in the cytoplasm, where it is heterodimerized to anti-
apoptotic Bcl-2 family members such as Mcl-1 and
Bcl-xL; however, once the cell is exposed to an apop-
totic stimulus, Bax is translocated to the mitochondria
[43] and induces mitochondrial dysfunction, character-
ized by the formation of large pores in the mito-
chondrial membrane [44].

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).

Cleavage of Bax into p18 Bax occurs in response to
various stimuli, such as interferon-a [48] and chemo-
therapeutic agents [49]. p18 Bax fragment is as efficient
as full-length Bax in promoting cytochrome c release
[49,50] and more potent than full-length Bax in induc-
ing apoptotic cell death [51]. It is also suggested that
an increase in gefitinib-induced expression of total Bax
is caused by the decreased degradation of Bax. As
ERK1 ⁄ 2 and Akt are significantly inhibited in gefiti-
nib-treated HAG-1 cells, simultaneous inhibition of
these pathways by gefitinib may lead to the accumula-
tion of Bax and subsequent apoptosis. As described
below, gefitinib initiates the intrinsic pathway of apop-
K. Takeuchi and F. Ito Molecular mechanisms of sensitivity to EGFR-TKIs
FEBS Journal 277 (2010) 316–326 ª 2009 The Authors Journal compilation ª 2009 FEBS 319
tosis through p38a-dependent Bax activation in intesti-
nal epithelial cells (pathway
in Fig. 2). Accordingly,
inhibition of Akt-dependent Bax phosphorylation at
Ser184, generation of the p18 Bax fragment and
p38a-dependent Bax activation are proposed for Bax
activation. Further studies are needed to understand
the precise mechanism by which gefitinib induces
apoptosis in a Bax-dependent manner.
Inhibitor of apoptosis protein family
Induction of MOMP following apoptotic stimuli
results in the release of cytochrome c and subsequent
activation of caspase-9 and caspase-3, which are
cystein proteases that cleave vital cellular targets and
cause apoptosis. Caspases can be inhibited by members

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

-to-S phase transition of the cell-cycle by phos-
phorylating the retinoblastoma (RB) protein. Activa-
tion of cyclin–CDK complexes is counterbalanced by
CDK inhibitors, including those of the kinase inhibitor
proteins (KIP) family and the INK4 family (Fig. 3).
The KIP family consists of p27
KIP1
, p21
WAF1 ⁄ CIP1
and
p57
KIP2
; and the INK4 family consists of p15
INK4b
,
p16
INK4a
, p18
INK4c
and p19
INK4d
.
AG1478, which, like gefitinib and erlotinib, acts as a
specific inhibitor of the EGFR tyrosine kinase, has
been shown to result in a dose-dependent up-regula-
tion of p27
KIP1
and in hypophosphorylation of the RB
protein in human epidermoid carcinoma cell line A431
cells [56]. These changes are temporally associated with

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

INK4b
is increased by MEK inhibi-
tors, but not by Akt inhibitors, the induction of
Fig. 3. The cell-cycle is arrested at the G
1
phase by TKI-induced CDK inhibitors EGFR-
TKIs result in the up-regulation of CDK inhib-
itors, including KIP family members and
INK4 family members Members of the KIP
family can inhibit the catalytic activity of
CDK2, 4 and 6 Members of the INK4 family
are specific inhibitors of the cyclin
D–CDK4 ⁄ 6 complex It is not yet known
if EGFR-TKIs can stop G
2
transition
and G
2
⁄ M cell-cycle progression by
up-regulation of KIP family members.
Molecular mechanisms of sensitivity to EGFR-TKIs K. Takeuchi and F. Ito
320 FEBS Journal 277 (2010) 316–326 ª 2009 The Authors Journal compilation ª 2009 FEBS
p15
INK4b
by inhibition of the ERK1 ⁄ 2 pathway is
associated with the antiproliferative effects of gefitinib.
Inhibition of cell motility
The EGFR can transmit signals for re-organization of
the cytoskeleton, the formation of lamellipodia, mem-
brane ruffling and changes in cell morphology.

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

of apoptosis in intestinal epithelial cells. Instead, p38
MAPK phosphorylation is associated with mitochon-
drial translocation of Bax and subsequent induction of
apoptosis following EGFR inhibition (pathway
in
Fig. 2). Furthermore, p38a, one of the four p38 iso-
forms, is required for Bax activation and apoptosis.
Because activation of p38 by UVB irradiation in
human keratinocytes results in induction of a confor-
mational change in Bax and its translocation to mito-
chondria [63], p38 may be an important upstream
molecule of Bax activation in response to a variety of
apoptosis-inducing stimuli.
JNK and mitogen-activated protein kinase
phosphatase-1
The activity of JNK, one of the MAPKs, is tightly
controlled by both protein kinases, such as MAPK
kinase 4 (MKK4) or MAPK kinase 7 (MKK7) and
protein phosphatases such as MAPK phosphatase
(MKP). Mitogen-activated protein kinase phosphatase-
1 (MKP-1) is a dual-specificity protein phosphatase,
which can dephosphorylate both phosphothreonine
and phosphotyrosine residues and subsequently block
the activities of MAPKs [64]. Although MKP-1 was
initially characterized as an ERK-specific phosphatase
[65], subsequent studies have determined that MKP-1
preferentially acts on JNK and p38 MAPK in response
to various stresses [66].
MKP-1 has been correlated with tumorigenesis.
Several observations have indicated that MKP-1 is

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).

Although MKP-1 expression has been implicated in
cancer and in TKI sensitivity, the mechanism by which
EGFR activation controls the MKP-1 expression level
is unclear. MKP-1 is encoded by an early response
gene, which is transiently induced by mitogens and
K. Takeuchi and F. Ito Molecular mechanisms of sensitivity to EGFR-TKIs
FEBS Journal 277 (2010) 316–326 ª 2009 The Authors Journal compilation ª 2009 FEBS 321
stress signals such as serum, growth factors, cytokines,
UV irradiation, heat shock, hypoxia and anticancer
drugs [68] through both transcriptional [70,71] and
post-translational [72,73] mechanisms. As depicted in
Fig. 3, the human mkp-1 gene contains four exons and
three introns coding for an inducible mRNA that is
approximately 2.4 kb long [74]. The pro-
moter ⁄ enhancer region of the mkp-1 gene contains
multiple activator protein 2 (AP2), trans-acting tran-
scription factor 1 (SP1) and cAMP-response element
(CRE) sites, but only one site for each of activator
protein 1 (AP1), neurofibromin 1 (NF1) and TATA
box [74]. ERK1 ⁄ 2 can phosphorylate Ser133 of the
CRE-binding protein (CREB) through p90Rsk-2, and
Ser133 phosphorylation is required for CREB-medi-
ated transcription [75]. These results suggest that
EGFR signaling can induce the transcription of mkp-1
via phosphorylation of CREB. However, induction of
MKP-1 in mouse embryo fibroblasts following treat-
ment with arsenite and irradiation with UVC is pre-
dominantly mediated by the p38 MAPK pathway.
Both p38 MAPK and ERK have been implicated in
the transcriptional induction of MKP-1, and each may

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

not a caspase-9 inhibitor, are capable of restoring cell
viability. Thus, Fas appears to play a major role in the
initiation of gefitinib-induced apoptosis through activa-
tion of the caspase-8 ⁄ caspase-3 cascade. Treatment of
A549 cells with gefitinib results in the translocation of
p53 from the cytosol to the nucleus. Moreover, inhibi-
tion of p53 using antisense oligonucleotide causes
down-regulation of Fas and a significant decrease in
gefitinib-induced apoptosis. p53 may thus play a role
in determining gefitinib sensitivity by regulating Fas
expression in NSCLC.
Conclusions
Important regulators of cell survival and apoptosis are
the Bcl-2 family of proteins. Members of this family,
such as Bcl-2 and Bcl-xL, can inhibit apoptosis,
Fig. 4. Regulation of MKP-1 expression by
EGFR signaling MKP-1 expression is
controlled through both transcription and
post-translation steps ERK1 ⁄ 2, JNK and p38
MAPK can activate transcription of the
mkp-1 gene The promoter ⁄ enhancer region
of the mkp-1 gene has the potential to bind
many transcription factors AP1, activator
protein 1; CAD, phosphatase catalytic
domain; CRE, cAMP-responsive element;
NF1, neurofibromin 1; SP1, trans-acting
transcription factor 1.
Molecular mechanisms of sensitivity to EGFR-TKIs K. Takeuchi and F. Ito
322 FEBS Journal 277 (2010) 316–326 ª 2009 The Authors Journal compilation ª 2009 FEBS
whereas others promote apoptosis. The balance

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

to identify additional mechanisms of resistance and to
overcome acquired resistance to TKIs. Research on
the signaling routes from the EGFR to Bcl-2 family
members will provide critical information to augment
the efficacy of TKIs and to identify patients who will
have a positive response to TKIs.
Acknowledgements
This work was supported, in part, by a grant-in-aid
for scientific research from the Ministry of Education,
Culture, Sports, Science, and Technology of Japan,
and by funding from the Fugaku Trust for Medical
Research.
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