Journal of Cancer Therapy, 2012, 3, 424-434
http://dx.doi.org/10.4236/jct.2012.324055 Published Online September 2012 (http://www.SciRP.org/journal/jct)
JNK-Mediated FOXO Expression Plays a Critical Role in
EGFR Tyrosine Kinase Inhibitor-Induced BIM Expression
and Apoptosis
Kenji Takeuchi1*, Anh Ho Viet1, Katsumi Kawasaki1, Kazuto Nishio2, Fumiaki Ito1
1Department of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan; 2Department of Genome Bi-
ology, Kinki University School of Medicine, Osaka, Japan.
Received June 26th, 2012; revised July 25th, 2012; accepted September 16th, 2012
ABSTRACT
BIM, a key pro-apoptotic member of the BCL-2 family of proteins, is essential for apoptosis triggered by tyrosine
kinase inhibitors (TKIs) of the epidermal growth factor receptor (EGFR). However, the precise molecular mechanism
by which EGFR-TKIs induce BIM expression has remained unclear. A previous study of ours showed that the active-
tion of c-Jun NH2-terminal kinase (JNK) is critical for the TKI-induced apoptosis in PC-9 cells, a gefitinib-sensitive
human NSCLC cell line. In this study, we therefore examined the effect of JNK activation on BIM expression and fur-
ther investigated the mechanism responsible for TKI-induced apoptosis in PC-9 cells. Northern blotting analysis re-
vealed that the TKI AG1478 induced a substantial increase in the level of BIM mRNA. However, this TKI-induced in-
crease was not observed in dominant-negative JNK overexpressing cell line J12A5 or in the TKI-resistant cell line
HP-5R, in which JNK is not activated in response to AG1478. Therefore, JNK activation was correlated with the
up-regulation of BIM expression. BIM is known to be a downstream target of forkhead box protein O (FOXO) tran-
scription factors. I mmunoblot analysis indicated that th e levels of FOXO1, FOXO3a, and FOXO4 transcr iption factor s
increased after AG1478 treatment of PC-9 cells but that they were not increased in either J12A5 or HP-5R cells, indi-
cating that FOXO was increased in PC-9 cells through JNK activation. FOXO1 knockdown in PC-9 cells decreased
EGFR-TKI-induced BIM expression and apoptosis. These findings provide evidence that JNK activation and subse-
quent increased FOXO expression play a critical role in EGFR-TKI-induced BIM expression and apoptosis.
Keywords: EGFR-TKI; FOXO; BIM; JNK; NSCLC
1. Introduction
Enhanced expression or function of epidermal growth
factor receptor (EGFR) has been documented in a variety
of tumors, including non-small-cell lung cancer (NSC-
LC), breast cancer, and gliomas [1-4]. These alterations
can occur due to increased receptor gene transcription or
amplification or receptor mutations resulting in constitu-
tive activation of the receptor tyrosine kinase [5,6].
Therefore, a variety of approaches to block the EGFR
and its downstream signaling pathways are undergoing
clinical evaluation, including the use of anti-EGFR mo-
noclonal antibodies and small-molecule tyrosine kinase
inhibito rs (T KIs) [7,8].
Gefitinib and erlotinib potently inhibit the tyrosine
kinase activity of EGFR with minimal in hibitory activity
against other tyrosine kinases. Both EGFR-TKIs com-
petitively block the binding of ATP to th e tyrosine kinase
domain of EGFR and are effective therapeutic agents
against NSCLC [7-9]. Various molecular studies have
identified somatic mutations in EGFR as being a major
determinant underlying the dramatic clinical responses to
treatment with gefitinib and erlotinib [10-12]. About
90% of the somatic mutations found in responsive pa-
tients are either small deletions encompassing 5 amino
acids 746 - 750 codons (delE7 46-A750) or missens e mu-
tations resulting in a substitution of leucine with arg inine
at codon 858 (L858R). NSCLC cells with these muta-
tions show a variety of signaling pathways that are acti-
vated through the constitutive activation of EGFR tyro-
sine kinase. Inhibition of both phosphatidylinositol-3’-
OH kinase (PI3K)-AKT and extracellular signal-regu-
lated kinase (ERK) pathways has been reported to be
responsible for EGFR-TKI-induced apoptotic death in
NSCLC cells [13-15].
BCL-2 family proteins are known to determine the
outcome of an apoptotic process initiated by the release
*Corresponding a uthor.

JNK-Mediated FOXO Expression Plays a Critical Role in EGFR Tyrosine Kinase Inhibitor-Induced BIM
Expression and Apoptosis 425
of cytochrome c from the mitochondria [16]. BIM is a
key pro-apoptotic member of the BCL-2 family of pro-
teins and initiates apoptosis signaling by binding to and
antagonizing the functions of th e anti-apoptotic members
of the BCL-2 family [17-19]. Previous studies have
shown that the induction of BIM is essential for apop-
tosis triggered by EGFR-TKI [20-22]. BIM has multiple
phosphorylation sites, and its expression can be down-
regulated throug h ERK-mediated phosp horylation , which
targets it for ubiquitination and proteasome-based degra-
dation [23]. Mitogen-activated protein kinase kinase
(MEK) inhibition cause an increase in the expression and
dephoshor ylation of BIM in NSCLC cells, but to a lesser
extent than that indu ced with gefitinib [21]. On the other
hand, the effect of gefitinib on the PI3K-AKT pathway
may not be required for BIM induction in NSCLC cells.
Therefore, signaling pathways other than MEK-ERK and
PI3K-AKT are involved in the maximal induction of
BIM, and the precise molecular mechanism by which
TKIs induce BIM expression has remained unclear.
The expression level of BIM is regulated at the trans-
cription level as well as by post-translational modifica-
tion. We previously investigated the signaling pathway
by which EGFR-TKI induces apoptosis in PC-9 cells, a
gefitinib-sensitive human NSCLC cell line with a muta-
tion (delE746-A750) in their EGFR and found that the
activation of c-Jun NH2-terminal kinase (JNK) induced
by EGFR-TKI is critical for the TKI-induced apoptosis.
We also found that JNK is activated through a decrease
in the level of mitogen-activated protein kinase phos-
phatase-1 [MKP-1; 24]. In the present study we found
that EGFR-TKI increased JNK-mediated FOXO expres-
sion, thereby stimulating BIM transcription and apop-
tosis.
2. Materials and Methods
2.1. Materials
EGF (ultra-pure) from mouse submaxillary glands was
purchased from Toyobo Co., Ltd. (Osaka, Japan). Fetal
calf serum (FCS) came from PAA Laboratories GmbH
(Pasching, Austria). ALLN (N-acetyl-Leu-Leu-Nle-CHO),
phenylmethylsulfonyl fluoride (PMSF), pepstatin A,
aprotinin, and leupeptin were obtained from Sigma (St
Louis, MO). MG-132 (Z-Leu-Leu-Leu-al) and U0126
(1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)
butadiene) were obtained from Calbiochem (Darmstadt,
Germany). RPMI-1640 medium was from Nissui Phar-
maceutical Co., Ltd. (Tokyo, Japan). Antibodies used
and their sources were as follow: Anti-Bim(C34C5) rab-
bit mAb, anti-Phospho-Bim (Ser69; D7E11) rabbit mAb,
anti-Foxo1 rabbit mAb, anti-Foxo3a rabbit mAb, anti-
Foxo4 rabbit mAb, anti-Phospho-p44/42 MAPK (Erk1/2;
Thr202/Tyr204; E10) mouse mAb, from Cell Signaling
Technology, Inc. (Danvers, MA); anti-α-tubulin antibody
(clone B-5-1-2) and anti-MAP kinase antibody, from
Sigma; anti-OxPhos Complex IV subunit IV, mouse
IgG2a, monoclonal 20E8 (anti-cytochrome oxidase sub-
unit IV) from Life Technologies Corporation (Carlsbad,
CA); anti-(β-actin)(C-11) from Santa Cruz Biotechno-
logy, Inc. (Santa Cruz, CA); swine horseradish peroxi-
dase (HRP)-linked anti-rabbit Ig antibody, from DAKO
(Glostrup, Denmark); and sheep HRP-linked anti-mouse
Ig antibody, from GE Healthcare UK Ltd. (Bucking-
hamshire, England).
2.2. Cell Culture
Human non-small-cell lung cancer cell line PC-9 was
obtained from Tokyo Medical University (Tokyo, Japan).
PC-9 cells were maintained in RPMI-1640 medium (con-
taining 10 µg/ml gentamycin) supplemented with 5%
FBS in 5% CO2 at 37˚C in a ful ly h umi di fi e d a tmo sp he re.
Exponentially growing cells were used in all experi-
ments.
2.3. Isolation AG1478-Resistant Cells from PC-9
Cells
PC-9 cells were sequentially treated with AG1478,
namely, at 50 nM for 17 days, 75 nM for 3 days, and
then 100 nM for 1 day. Subsequently, the surviving cells
were plated into the wells of a 96-well microplate at 0.5
cell/well and cloned as AG1478-resistant cell lines, one
of which was named HP-5R. We then examined the ef-
fect of AG1478 on the viability of both parental PC-9
and HP-5R cells.
2.4. Determination of Cell Viability
The anti-proliferative effect of AG1478 on PC-9 cells
was assessed by using a Cell Counting Kit-8 (DOJIN,
Kumamoto, Japan) according to the manufacturer’s in-
structions. The Cell Counting Kit-8 is a co lorimetric me-
thod in which the intensity of the dye is proportional to
the number of the viable cells. Briefly, 200 µL of a sus-
pension of PC-9 cells was seeded into each well of a
96-well plate at a density of 2000 cells/well. After 48 h,
the culture medium was replaced with 100 µL of AG-
1478 solution at various concentrations. After incubation
for 48 h at 37˚C, 5 µL of WST-8 solution was added to
each well; and the cells were then incubated for a further
30 min at 37˚C. Thereafter, the optical density was mea-
sured at 450 nm by using a BIO-RAD microplate reader
model 550 (BIO-RAD, Hercules, CA). Each experiment
was performed by using 6 replicate wells for each drug
concentration and was carried out independently 3 times.

JNK-Mediated FOXO Expression Plays a Critical Role in EGFR Tyrosine Kinase Inhibitor-Induced BIM
Expression and Apoptosis
426
2.5. Preparation of Cellular Lysates and
Immunoblotting
Preparation of cellular lysates and immunoblotting were
performed as described before [25]. Briefly, cells were
lysed with buffer A (20 mM Tris/HCl, pH 7.4, containing
137 mM NaCl, 2 mM EGTA, 5 mM EDTA, 1% Nonidet
P-40, 1% Triton X-100, 100 µg/ml PMSF, 1 µg/ml pep-
statin A, 1 µg/ml p-toluenesulfonyl-L-arginine methyl
ester, 2 µg/ml leupeptin, 1 mM sodium orthovanadate, 50
mM sodium fluoride, and 30 mM Na4P2O7). The lysates
were then incubated on ice for 30 min, and the insoluble
material was cleared by centrifugation. The samples were
normalized for protein content and separated by SDS-
PAGE, after which they were transferred to an Immo-
bilon-P membrane (Millipore, Bedford, MA) for im-
munoblotting with antibodies. The relative amount of
BIM and FOXO1 was analyzed with Image J (NIH)
software.
2.6. siRNA Transfection for Knockdown of
FOXO1
The transfection was performed according to the manu-
facturer’s instruction. Briefly, PC-9 cells were plated at
1.2 × 105 cells in 35-mm dishes and cultured for 24 h.
The cells were then transfected with FOXO1 siRNA oli-
gonucleotide (StealthTM RNAi) or non-target control oli-
gonucleotide (StealthTM RNAi Negative Control Medium
GC Duplex #2) by using Lipofectamine RNAi MAX
(Invitrogen Corp.). After 48 h, the transfected cells were
incubated or not with AG1478 for 30 min (for FOXO1)
or for 3 h (for BIM); and their lysates were prepared and
then used for the detection of FOXO1 and BIM, done by
immunoblotting. Cell survival after siRNA transfection
was assessed by performing the WST-8 assay.
2.7. Northern Blot Analysis
Northern blot analysis was performed as described before
[25]. Briefly, cells were treated with 500 nM AG1478,
and total RNA was obtained by use of Isogen (Nippon
Gene Tokyo, Japan). Fifteen micrograms of RNA was
separated electrophoretically. The RNA was transferred
to a Hybond-N+ membrane (GE Healthcare). The blots
were hybridized with human BIM cDNA that had been
labeled with [32 P] dCTP by use of a Rediprime II DNA
Labeling System (GE Healthcare).
2.8. Mitochondrial Isolation
Mitochondria were isolated from PC-9 cells and J12A5
cells by using a Mitochondria Isolation Kit (Pierce,
Rockford, IL) according to the manufacturer’s instruct-
tions. The mitochondrial fraction was subjected to pro-
tein extraction with 2X Laemmli sample buffer (4% SDS,
20% glycerol, 0.125 M Tris/HCl pH 6.8), followed by
centrifugation at 15,000 × g at room temperature for 10
min, and then assessed for protein content with a BCA
Protein Assay Kit (Pierce). 20 µg of proteins were sub-
jected to electrophoresis and analyzed by immunoblot-
ting for BIM. The equal loading and transfer were en-
sured by reprobing the membranes with anti-CoxIV an-
tibody as a marker of mitochondria.
2.9. Cytoplasmic and Nuclear Extracts
Preparation of cytoplasmic and nuclear extracts was per-
formed essentially as described previously [25]. Briefly,
after having been washed with ice-cold PBS, cells were
lysed at 4˚C by incubating them for 10 min in hypotonic
buffer. After centrifugation, the supernatants were col-
lected as cytoplasmic extracts. Nuclear extracts were
prepared by resuspension of the crude nuclei in high-salt
buffer at 4˚C for 30 min, and the supernatants were then
collected after centrifugation.
3. Results
BIM has been identified as a key apoptotic effector of
gefitinib in sensitive cells with a mutation (delE746-
A750) in their EGFR [20-22]. In this study we used
AG1478 [4-(3-chloroanilino)-6,7-dimethoxquinazoline]
as an EGFR-TKI, which inhibitor has a quin azoline stru-
cture similar to that of gefitinib and erlotinib and studied
the effect of AG1478 on the expression of BIM EL, the
major isoform of BIM, in PC-9 cells. The levels of BIM
in both total and mitochondrial fractions increased in
response to AG1478 in a time-dependent manner (Fig-
ures 1(a) and (b)). We previously demonstrated that AG-
1478 intensively stimulates the phosphorylation of JNK
and that the level of phosphorylated JNK continues to
increase for at least 24 h [24]. So we examined whether
AG1478 induced the expression of BIM through JNK
activation. As shown in Figure 1, AG1478 did not in-
duce BIM expression in the dominant-negative JNK-
overexpressing cell line J12A5, which had no detectable
JNK activity, indicating that activation of JNK was es-
sential for AG1478-induced BIM expression. We also
observed that gefitinib increased the expression of BIM
in PC-9 cells but not in J12A5 cells (data not shown).
JNK activity in cells is controlled by mitogen-activated
protein kinase phosphatase-1 (MKP-1). To confirm a role
of JNK in the increased expression of BIM after AG1478
treatment, we used pcMKP-1-transfected cell lines
(M1A4 and M1B2), which were isolated as clones over-
expressing MKP-1. A previous study of ours showed that
JNK activation in M1A4 an d M1B2 cells is not observed
in response to AG1478 treatment [24]. As shown in Fig-

JNK-Mediated FOXO Expression Plays a Critical Role in EGFR Tyrosine Kinase Inhibitor-Induced BIM
Expression and Apoptosis
427
(a)
(b)
(c)
Figure 1. AG1478-induced BIM expression mediated by JNK activation. Cells were treated with 500 nM AG1478 for the in-
dicated time periods, and extracts from the total cell (a) (c) and mitochondorial (b) fractions were prepared. The extracts
were then electrophoresed on 12.5% SDS-PAGE gels for the detection of BIM as described under “Experimental proce-
dures”. The
 -tubulin or CoxIV level was examined as a control for the equal loading of the samples. Relative signal intensity
in figure B represents the ratio of the BIM signal to the CoxIV signal in each sample relative to control shown as 1. Similar
results were obtained from 3 separate experiments.
ure 1(c), AG1478 did not induce BIM expression in both
M1A4 and M1B2 cell lines. This result indicates that
activation of JNK is essential for AG1478-induced BIM
expression.
(a)
(b)
(c)
BIM is known to have multiple phosphorylation sites
and its expression to be down-regulated through ERK-
mediated phosphorylation, which targets it for ubiquity-
nation and subsequent degradation in proteasomes [26,
27]. Thus, BIM expression may be regulated in PC-9
cells by multiple stimuli, including the JNK and ERK
pathways. So we next determined the effect of AG1478
on BIM phosphorylation at its serine 69, which phos-
phorylation is known to occur in response to activation of
the ERK pathway. AG1478 decreased BIM phosphoryla-
tion and concomitantly increased BIM expression (Fig-
ure 2(a)). To study the causal relationship between BIM
phosphorylation and its expression level, we examined
the effect of U0126, a mitogen-activated protein kinase
kinase (MEK) inhibitor, on BIM expression. U0126 in-
hibited the phosphorylation of ERK, but its activity to
increase BIM expression was low (Figure 2(b)). Since
ERK-dependent phosphorylation has been reported to
antagonize the proteasomal degradation of BIM, we next
examined the effect of the proteasome inhibitors MG-132
and ALLN on BIM expression (Figure 2(c)). In the
presence of either of these inhibitors, the BIM expression
level was significantly lower than that in AG1478-treated
Figure 2. Effect of MEK inhibitor and proteasome inhibi-
tors on BIM expression PC-9 cells were treated with 500
nM AG1478, 5 M U0126 or proteasome inhibitor ALLN
(50M) or MG-132 (1M) for the indicated time periods.
The cells were then harvested, and equal aliquots of protein
extracts (40 g protein per lane) were analyzed on 12.5%
SDS-PAGE gels for the detection of phospho-Ser69-BIM,
BIM, and phospho-ERK1/2. The  -tubulin level was exam-
ined as a control for the equal loading of the samples. Simi-
lar results were obtained from 3 separate experiments.

JNK-Mediated FOXO Expression Plays a Critical Role in EGFR Tyrosine Kinase Inhibitor-Induced BIM
Expression and Apoptosis
428
cells (Figure 2(c)). These results indicate that AG1478
increased BIM expression mainly through JNK activa-
tion rather than by inhibition of the ERK pathway, lead-
ing to decreased phosphorylation and proteasomal deg-
radation of BIM .
In order to investigate if the induction of BIM was
regulated at the transcription step by JNK, we treated
PC-9 cells with AG1478 over a time course of 3 h, and
then prepared total RNA from these cells to determine
the expression of BIM mRNA by Northern blotting.
AG1478 induced a substantial increase in BIM mRNA,
indicating that AG1478 increased BIM transcription (Fi-
gure 3(a)). Further, no increase in BIM mRNA was ob-
served in the dominant-negative JNK-overexpressing
J12A5 cells (Figure 3(b)). It thus appears that BIM tran s-
cription was up-regulated b y JNK activation in AG1478-
treated PC-9 cells.
Previous data from other cell systems have suggested
that BIM is a downstream target of FOXO transcription
factors [28-30]. To explore if FOXO could be involved
in the induction of BIM expression in response to AG-
1478, we carried out immunoblot analysis to study the
effect of the inhibitor on the expression of FOXO pro-
teins. The levels of FOXO1, FOXO3a, and FOXO4
transcription factors increased within 15 min, and the
FOXO1 level peaked at 30 - 60 min after AG1478 treat-
ment (Figure 4(a)). FOXO1 expression in total and nu-
clear fractions was increased in response to AG1478
treatment in PC-9 cells, but they were only slightly in-
creased in J12A5 cells (Figures 4(b) and (c)). Ther efore,
the level of FOXO1 was increased in the nuclei of PC-9
cells through JNK activation.
To examine whether FOXO1 was involved in the BIM
expression increased by AG1478, we transfected PC-9
cells with either negative control short-interfering RNA
(RNAi NC) or FOXO1 siRNAs (RNAi1 and RNAi2). As
shown in Figure 5(a), an increase in the FOXO1 level
was observed 30 min after the start of AG1478 treatment
of PC-9 cells that had been transfected with RNAi NC,
but no increase was detected in those cells transfected
with FOXO1 RNAi 1 or 2. We next studied the effect of
AG1478 on BIM expression in PC-9 cells that had been
transfected with FOXO1 siRNAs. A small increase in
BIM expression by AG1478 was observed even in the
FOXO1 knockdown PC-9 cells, but its increase was far
less than that in RNAi NC-treated cells (Figure 5(b)).
Our data also showed that FOXO1 knockdown in PC-9
cells resulted in decreased apoptosis in response to
AG1478, as determined by cell viability (Figure 5(c)).
Thus, not only FOXO1 but also other FOXO subgroup
members may be involved in EGFR-TKI-induced BIM
expression and cell death.
Despite the dramatic efficacy of gefitinib and erlotinib
(a)
(b)
Figure 3. AG1478 increased BIM transcription through
JNK pathway. PC9 (a) and J12A5 cells (b) were treated
with 500 nM AG1478 for the indicated time periods, after
which Northern blot analysis of BIM mRNA was carried
out as described under “Experimental procedures”. The
lower panel shows β-actin mRNA to demonstrate equal
loading of the samples. Similar results were obtained from 3
independent experiments.
(a)
(b)
(c)
Figure 4. AG1478-induced expression of transcription fac-
tor FOXO in PC-9 cells. (a) PC-9 cells were incubated with
500 nM AG1478 for the indicated time periods. The cells
were then harvested, and equal aliquots of cell extracts (40
µg protein per lane) were examined by immunoblotting
using antibodies specific for the FOXO1, FOXO3a, and
FOXO4. The membrane was subsequently reprobed with
an
 -tubulin antibody; (b) Cellular lysates were prepared
from PC-9 and J12A5 cells. The lysates were analyzed by
SDS/PAGE and immunoblotting with specific antibody
against FOXO1 (upper panel) or
 -tubulin (lower panel); (c)
PC-9 and J12A5 cells were treated with 500 nM AG1478 for
the indicated time periods. Nuclear extracts were prepared
from the cells and used for immunoblot analysis of FOXO1.
The
 -actin level was examined as a control for the equal
loading of the samples. Similar results were obtained from 3
independent experiments.

JNK-Mediated FOXO Expression Plays a Critical Role in EGFR Tyrosine Kinase Inhibitor-Induced BIM
Expression and Apoptosis 429
(a)
(b)
(c)
Figure 5. Transcription factor FOXO1 required for AG1478-
induced BIM expression and apoptosis. A and B. PC-9 cells
were transiently transfected with 10 nM FOXO1 siRNA
oligonucletides (RNAi1 and RNAi2) or negative control
siRNA oligonucleotides (RNAi NC). At 24 h post siRNA
transfection, the cells were then incubated with or without
500 nM AG1478 for an additional 30 min for the detection
of FOXO1 (a) or for an extra 3h for the detection of BIM
(b). Cell lysates were examined for FOXO1 or BIM ex-
pression by immunoblotting (c). At 24 h post siRNA trans-
fection, PC-9 cells were incubated with or without 500 nM
AG1478 for 48 h. The viability of cells was determined by
conducting WST-8 assays. The reading obtained for AG-
1478-untreated cells was considered as 100% viability. The
data are shown as the mean ± standard deviation (SD) (n =
6). The indicated p-values were calculated by using the
Welch two-sample t-test. *p < 0.01 for the indicated com-
parisons. Similar results were obtained from 3 separate ex-
periments.
MKP-1 and JNK in these HP-5R cells (Figures 6(c) and
(d)). Therefore, AG1478-induced apoptosis was associ-
ated with the JNK activation. Further, AG1478 treatment
did not up-regulate the expression of BIM and FOXO1 in
HP-5R cells, in contrast to their up-regulation in PC-9
cells (Figures 6(e) and (f)). These results support our
conclusion that JNK activation and subsequent increased
FOXO expression play a role in EGFR-TKI-induced
BIM expression.
4. Discussion
EGFR-TKIs induce apoptosis in NSCLC with EGFR-
activating mutations. Recently, it was shown that BIM,
one of the most potent pro-apoptotic members, is essen-
tial for apoptosis triggered by EGFR-TKIs in NSCLC
with EGFR-activating mutations [20-22]. In this study,
we first showed that TKI AG1478 increased the ex-
pression of BIM in NSCLC PC-9 cells, which possess
one of the most common EGFR-activating mutations, i.e.,
deletion of exon 19. On the other hand, AG1478 did not
affect the expression of other pro-apoptotic (MCL-1,
BAX, and BAD) or anti-apoptotic members (BCL-2 and
BCL-xL; unpu bl i shed data).
Upon activation by growth factors, ERK phosphory-
lates BIM at its serine 69; and this phosphorylation tar-
gets BIM for ubiquitination and proteasome-dependent
degradation [26,27]. We therefore assumed that a de-
crease in BIM phosphorylation by ERK would result in
the accumulation of BIM in AG1478-treated PC-9 cells.
Indeed, AG1478 decreased BIM phosphorylation at its
serine 69 and increased BIM expression (Figure 2(a)).
However, although U0126, a MEK inhibitor, inhibited
the phosphorylation of ERK, it could not increase BIM
expression (Figure 2(b)). Moreover, no or only a little
increase in BIM expression was found in th e presence of
proteasome inhibitors MG-132 and ALLN (Figure 2(c)).
Thus, signaling pathways other than MEK-ERK were
involved to achieve maximal BIM induction, supporting
a previous finding that MEK-ERK inhibition was not
able to cause BIM up-regulation to the same extent seen
with gefitinib [22].
for the treatment of NSCLC patients with EGFR muta-
tions, all patients ulti mately develop resistance to both of
these TKIs. To analyze the mechanism responsible for
this acquired resistance to TKIs, we have been isolating
AG1478-resistant cell lines from PC-9 cells up to the
present. So we used one of these isolated cell lines,
termed HP-5R, for such analysis. As shown in Figure 6,
HP-5R was resistant to AG1478 at least up to 200 nM.
Since AG1478 treatment inhibited EGFR tyrosine phos-
phorylation in HP-5R cells, as in PC-9 cells (data not
shown), no secondary EGFR mutatio n such as T790M or
D761Y was associated with the TKI resistance of HP-5R,
indicating that intracellular sign aling pathways o th er than
EGFR had changed in HP-5R cells. Interestingly, AG-
1478 decreased the expression of the MKP-1 protein and
concomitantly stimulated the phosphorylation of JNK in
PC-9 cells, but it had no effect on the expression of
The expression level of BIM is regulated not only by
post-translational modification but also at the transcript-
tional level [31-34]. Our Northern blot analysis revealed
that AG1478 induced a substantial increase in the level
of BIM mRNA, indicating that AG1478 up-regulated
BIM transcription in PC-9 cells. Cragg et al. previously
reported that the effect of gefitinib on the PI3K-AKT
pathway was not required for full BIM induction in NS-
CLC cells [21]. It thus appears that neither MEK-ERK
nor PI3K-AKT was a major signal in TKI-induced up-
regulation of BIM expr ession. In th is study, we ex amined
whether JNK activation was associated with BIM up-

JNK-Mediated FOXO Expression Plays a Critical Role in EGFR Tyrosine Kinase Inhibitor-Induced BIM
Expression and Apoptosis
430
(a)
(b)
(c)
(d)
(e)
(f)
Figure 6. Effects of AG1478 on the expression of MKP-1, activated JNK, BIM, and FOXO1 in AG1478-resistant cell line
HP-5R. (a) PC-9 and HP-5R cells were seeded into a 96-well microplate, and treated with 20 or 200 nM AG1478 for 48 h. The
viability of cells was determined by conducting WST-8 assays. The value of untreated cells was considered as 100% viability.
The data presented are the mean ± SD (n = 6); (b) PC-9 and HP-5R cells were seeded at a density of 3 × 105 cells per 60-mm
dish and then treated with 500 nM AG1478. The phase-contrast photomicrographs were taken after a 48-h incubation with
AG1478. Scale bar, 100 µm; (c) PC-9 and HP-5R cells were treated with 500 nM AG1478 for the indicated periods of time.
Total protein (40 µg protein per lane) was subjected to immunoblotting, and the membranes were hybridized with an anti-
body against MKP-1 (upper panel). Equal loading of the samples was checked by using an antibody against -tubulin (lower
panel); (d) Cell lysates were analyzed on SDS-PAGE gels for phospho-JNK content (upper panel), as well as for JNK content
(lower panel); (e) and (f) Cell lysates were analyzed for BIM content (e); as well as for FOXO1 content (f). The equal loading
of the samples was checked by using an antibody against -tubulin (each lower panel). Relative signal intensity represents the
ratio of the FOXO1 signal to the α-tubulin signal in each sample relative to control shown as 1. Similar results were obtained
from 3 separate experiments.
regulation, because our previous report showed that the
activation of JNK induced by EGFR-TKI was critical for
the TKI-induced apoptosis [24] . Our present resul t showed
that the TKI-induced increase in BIM mRNA was not
observed in the dominant-negative JNK-over-expressing
cell line J12A5. Further, the TKI-resistant cell line HP-
5R, in which JNK is not activated in response to AG1478,
did not show up-regulation of BIM. Therefore, JNK ac-
tivation was correlated with the up-regulation of BIM
expression.
The BIM gene is a direct target of FOXO; and the
5’-UTR of the human BIM gene contains a forkhead-
binding site, which is required for regulated expression
of BIM following withdrawal of growth factor [23,28, 35,
36]. FOXO subfamily includes FOXO1/FKHR, FOX-
O3a/FKHRL1, and FOXO4/AFX [37-40]; and their ac-
tivities are negatively regulated by multiple signaling
pathways, such as PI3K-AKT, ERK, and IKK [41-44].
Our result showed that the levels of FOXO1, FOXO3a
and FOXO4 in the PC-9 cells increased after AG1478
treatment. It thus appears that AG1478 affected FOXO
expression through one or some of these multip le signal-
ing pathways. AKT has been shown to control the active-
ties of FOXO through their phosphorylation, and this
phosphorylation triggers their export from the nucleus
and promotes binding of these transcription factors to the
14-3-3 chaperone proteins in the cytosol [45,46]. Indeed,
our immunoblot analysis indicated that FOXO1 and
FOXO3a were phosphorylated at their Akt-targeted sites
(threonine 24 and serine 256 in FOXO1 and threonine 32
in FOXO3a) and that their phosphorylation level was
decreased in a time-dependent manner after AG1478
treatment (unpublished data). However, the PI3K inhibit-
tor LY294002 had little effect on the induction of BIM
and FOXO in response to AG1478, although it had in-
hibitory activity toward the phosphorylation of FOXO at
its AKT-targeted sites (unpublished data). Therefore, the
PI3K-AKT-FOXO pathway was not critical for BIM in-
duction in AG1478-treated PC-9 cells.
It has been reported that growth-factor deprivation

JNK-Mediated FOXO Expression Plays a Critical Role in EGFR Tyrosine Kinase Inhibitor-Induced BIM
Expression and Apoptosis 431
causes FOXO factors to reside in the nucleus, where they
are active as transcription factors, thereby resulting in
pro-apoptotic signaling via the induction of TRAIL, FasL
or BIM [41]. We showed that up-regulation of FOXO1
occurred in PC-9 cells after AG1478 treatment, but not in
the dominant-negative JNK overexpressing J12A5 cells
or in the TKI-resistant HP-5R cells, indicating that JNK
activation played a role in the up-regulation of FOXO1.
Recently, Wang et al. indicated that nuclear translocation
of FOXO3a can be regulated by UV irradiation through a
pathway from JNK through decreased ERK and Akt ac-
tivities [47]. It was also earlier shown that the micro-
tubule-targeting compound paclitaxel enhances not only
the nuclear relocation of FOXO3a through JNK-de-
pendent inhibition of the PI3K-AKT signaling pathway
but also FOXO3a expression [48]. Essera et al. indicated
that treatment with H2O2 activates Ral-mediated JNK-
dependent phosphorylation of FOXO4 at its threonine
447 and 451, thereby causing the nuclear translocation
and transcriptional activation of FOXO4 [49]. Taken
together, the data in dicate it to be likely that JNK active-
tion was essential for the up-regu lation of FOXO in PC-9
cells.
AG1478 increased BIM expression through activation
of the JNK-FOXO signaling pathway, subsequently re-
sulting in cell death. This scenario was substantiated by
the knockdown experiment on FOXO1, because its
knockdown led to a significant reduction in both the BIM
expression and cell death (Figures 5(b) and (c)). How-
ever, FOXO1 knockdown did not completely abolish
either the AG1478-induced BIM expression or the cell
death. It has been reported that FOXO isoforms regulate
distinct but overlapping sets of genes, indicating unique
roles of FOXO isoforms in a variety of cells [50]. Thus,
to define the exact role of FOXO transcription factors in
PC-9 cells, a knockdown experiment on FOXO3a or
double knockdown of FOXO1 and FOXO3a will be re-
quired. We previously reported that the inhibition of both
survivin downregulation and BIM induction attenuate
gefitinib-induced ap optosis to a greater ex ten t than do th e
inhibition of either pathway alone [51]. So, both survivin
downregulation and BIM induction contribute independ-
ently to gefitinib-induced apoptosis. In addition to the
knockdown of FOXO isoforms, inhibition of survivin
downregulation may be necessary to protect cells fully
from AG1478.
In a previous paper, we showed that JNK, which is ac-
tivated through a decrease in the MKP level, is critical
for TKI-induced apoptosis [24]. Our present study has
expanded this finding and d emonstrated that JNK active-
tion stimulated BIM transcription through increasing
FOXO1 expression. In conclusion, we propose that this
MKP-JNK-FOXO-BIM pathway contributed to the in-
duction of apoptosis of PC-9 cells by TKI.
Despite the dramatic efficacy of gefitinib and erlotinib
in NSCLC patients with EGFR mutations, all patients
ultimately develop resistance to both TKIs. A secondary
mutation in EGFR (T790M) and amplification of MET
have been identified as major mechanisms of acquired
resistance to TKIs [52-54]. We showed herein that TKIs
induced activation of MKP-JNK-FOXO-BIM pathway.
Alterations in this pathway may cause resistance to ge-
fitinib or erlotinib in NSCLC patients.
5. Acknowledgements
This work was supported in part by a grant-in-aid for
scientific research from the Ministry of Education, Cul-
ture, Sports, Science, and Technology of Japan (#2059-
0077 and #23590098). We thank Mr. T. Shinya, Mr. T.
Imoto, Miss A. Nagai, and Miss S. Arasaki for their
technical assistance.
REFERENCES
[1] D. S. Salomon, R. Brandt, F. Ciardiello and N. Normanno,
“Epidermal Growth Factor-Related Peptides and Their
Receptors in Human Malignancies,” Critical Reviews in
Oncology/Hematology, Vol. 19, No. 3, 1995, pp. 183-
232. doi:10.1016/1040-8428(94)00144-I
[2] S. B. Fox, K. Smith, J. Hollyer, M. Greenall, D. Hastrich
and A. L. Harris, “The Epidermal Growth Factor Recep-
tor as a Prognostic Marker: Results of 370 Patients and
Review of 3009 Patients,” Breast Cancer Research and
Treatment, Vol. 29, No. 1, 1994, pp. 41-49.
doi:10.1007/BF00666180
[3] T. A. Libermann, H. R. Nusbaum, N. Razon, R. Kris, I.
Lax, H. Soreq, N. Whittle, M. D. Waterfield, A. Ullrich
and J. Schlessinger, “Amplification, Enhanced Expression
and Possible Rearrangement of EGF Receptor Gene in
Primary Human Brain Tumours of Glial Origin,” Nature,
Vol. 313, No. 5998, 1985, pp. 144-147.
doi:10.1038/313144a0
[4] A. J. Wong, S. H. Bigner, D. D. Bigner, K. W. Kinzler, S.
R. Hamilton and B. Vogelstein, “Increased Expression of
the Epidermal Growth Factor Receptor Gene in M alig nant
Gliomas Is Invariably Associated with Gene Amplifica-
tion,” Proceedings of the National Academy of Sciences
of the United States of America, Vol. 84, No. 19, 1987, pp.
6899-6903. doi:10.1073/pnas.84.19.6899
[5] A. W. Burgess, H. S. Cho, C. Eigenbrot, K. M. Ferguson,
T. P. Garrett, D. J. Leahy, M. A. Lemmon, M. X. Sli-
wkowski, C. W. Ward and S. Yokoyama, “An Open-
and-Shut Case? Recent Insights into the Activation of
EGF/ErbB Receptors,” Molecular Cell, Vol. 12, No. 3,
2003, pp. 541-552. doi:10.1016/S1097-2765(03)00350-2
[6] A. Citri and Y. Yarden, “EGF-ERBB Signalling: Towards
the Systems Level,” Nature Reviews Molecular Cell Bi-
ology, Vol. 7, No. 7, 2006, pp. 505-516.
doi:10.1038/nrm1962

JNK-Mediated FOXO Expression Plays a Critical Role in EGFR Tyrosine Kinase Inhibitor-Induced BIM
Expression and Apoptosis
432
[7] R. S. Herbst and P. A. Bunn Jr., “Targeting the Epidermal
Growth Factor Receptor in Non-Small Cell Lung Can-
cer,” Clinical Cancer Research, Vol. 9, No. 16, 2003, pp.
5813-5824.
[8] A. F. Gazdar, H. Shigematsu, J. Herz and J. D. Minna,
“Mutations and Addiction to EGFR: The Achilles ‘Heal’
of Lung Cancers?” Trends in Molecular Medicine, Vol.
10, No. 10, 2004, pp. 481-486.
doi:10.1016/j.molmed.2004.08.008
[9] K. Nakagawa, T. Tamura, S. Negoro, S. Kudoh, N. Ya-
mamoto, N. Yamamoto, K. Takeda, H. Swaisland, I. Na-
katani, M. Hirose, R. P. Dong and M. Fukuoka, “Phase I
Pharmacokinetic Trial of the Selective Oral Epidermal
Growth Factor Receptor Tyrosine Kinase Inhibitor Gefit-
inib (‘Iressa’, ZD1839) in Japanese Patients with Solid
Malignant Tumors,” Annals of Oncology, Vol. 14, No. 6,
2003, pp. 922-930. doi:10.1093/annonc/mdg250
[10] T. J. Lynch, D. W. Bell, R. Sordella, S. Gurubhagavatula,
R. A. Okimoto, B. W. Brannigan, P. L. Harris, S. M.
Haserlat, J. G. Supko, F. G. Haluska, D. N. Louis, D. C.
Christiani, J. Settleman and D. A. Haber, “Activating
Mutations in the Epidermal Growth Factor Receptor Un-
derlying Responsiveness of Non-Small-Cell Lung Cancer
to Gefitinib,” The New England Journal of Medicine, Vol.
350, No. 21, 2004, pp. 2129-2139.
doi:10.1056/NEJMoa040938
[11] J. G. Paez, P. A. Jänne, J. C. Lee, S. Tracy, H. Greulich, S.
Gabriel, P. Herman, F. J. Kaye, N. Lindeman, T. J. Bog-
gon, K. Naoki, H. Sasaki, Y. Fujii, M. J. Eck, W. R. Sell-
ers, B. E. Johnson and M. Meyerson, “EGFR Mutations in
Lung Cancer: Correlation with Clinical Response to Ge-
fitinib Therapy,” Science, Vol. 304, No. 5676, 2004, pp.
1497-1500. doi:10.1126/science.1099314
[12] W. Pao, V. Miller, M. Zakowski, J. Doherty, K. Politi, I.
Sarkaria, B. Singh, R. Heelan, V. Rusch, L. Fulton, E.
Mardis, D. Kupfer, R. Wilson, M. Kris and H. Varmus,
“EGF Receptor Gene Mutations Are Common in Lung
Cancers from ‘Never Smokers’ and Are Associated with
Sensitivity of Tumors to Gefitinib and Erlotinib,” Pro-
ceedings of the National Academy of Sciences of the
United States of America, Vol. 101, No. 36, 2004, pp.
13306-13311. doi:10.1073/pnas.0405220101
[13] M. L. Janmaat, F. A. Kruyt, J. A. Rodriguez and G. Gi-
accone, “Response to Epidermal Growth Factor Receptor
Inhibitors in Non-Small Cell Lung Cancer Cells: Limited
Antiproliferative Effects and Absence of Apoptosis Asso-
ciated with Persistent Activity of Extracellular Sig-
nal-Regulated Kinase or Akt Kinase Pathways,” Clinical
Cancer Research, Vol. 9, No. 6, 2003, pp. 2316-2326.
[14] M. Ono, A. Hirata, T. Kometa ni, M. Miyagawa, S. Ueda,
H. Kinoshita, T. Fujii and M. Kuwano, “Sensitivity to
Gefitinib (Iressa, ZD1839) in Non-Small Cell Lung Can-
cer Cell Lines Correlates with Dependence on the Epi-
dermal Growth Factor (EGF) Receptor/Extracellular Sig-
nal-Regulated Kinase 1/2 and EGF Receptor/Akt Pathway
for Proliferation,” Molecular Cancer Therapeutics, Vol. 3,
No. 4, 2004, pp. 465-472.
[15] K. Takeuchi and F. Ito, “EGF Receptor in Relation to
Tumor Development: Molecular Basis of Responsiveness
of Cancer Cells to EGFR-Targeting Tyrosine Kinase In-
hibitors,” The FEBS Journal, Vol. 277, No .2, 2010, pp.
316-326.
[16] J. E. Chipuk, L. Bouchier-Hayes and D. R. Green, “Mi-
tochondrial Outer Membrane Permeabilization during
Apoptosis: The Innocent Bystander Scenario,” Cell Death
and Differentiation, Vol. 13, No. 8, 2006, pp. 1396-1402.
doi:10.1038/sj.cdd.4401963
[17] L. O’Connor, A. Strasser, L. A. O’Reilly, G. Hausmann, J.
M. Adams, S. Cory and D. C. Huang, “Bim: A Novel
Member of the Bcl-2 Family That Promotes Apoptosis,”
The EMBO Journal, Vol. 17, No. 2, 1998, pp. 384-395.
doi:10.1093/emboj/17.2.384
[18] H. Puthalakath, D. C. Huang, L. A. O’Reilly, S. M. King
and A. Strasser, “The Proapoptotic Activity of the Bcl-2
Family Member Bim Is Regulated by Interaction with the
Dynein Motor Complex,” Molecular Cell, Vo. 3, No. 3,
1999, pp. 287-296. doi:10.1016/S1097-2765(00)80456-6
[19] M. Marani, T. Tenev, D. Hancock, J. Downward and N. R.
Lemoine, “Identification of Novel Isoforms of the BH3
Domain Protein Bim Which Directly Activate Bax to
Trigger Apoptosis,” Molecular and Cellular Biology, Vol.
22, No. 11, 2002, pp. 3577-3589.
doi:10.1128/MCB.22.11.3577-3589.2002
[20] D. B. Costa, B. Halmos, A. Kumar, S. T. Schumer, M. S.
Huberman, T. J. Boggon, D. G. Tenen and S. Kobayashi,
“BIM Mediates EGFR Tyrosine Kinase Inhibitor-Induced
Apoptosis in Lung Cancers with Oncogenic EGFR Muta-
tions,” PLoS Medicine, Vol. 4, No. 10, 2007, pp. 1669-
1679. doi:10.1371/journal.pmed.0040315
[21] M. S. Cragg, J. Kuroda, H. Puthalakath, D. C. Huang and
A. Strasser, “Gefitinib-Induced Killing of NSCLC Cell
Lines Expressing Mutant EGFR Requires BIM and Can
Be Enhanced by BH3 Mimetics,” PLoS Medicine, Vol. 4,
No. 10, 2007, pp. 1681-1689.
doi:10.1371/journal.pmed.0040316
[22] Y. Gong, R. Somwar, K. Politi, M. Balak, J. Chmielecki,
X. Jiang and W. Pao, “Induction of BIM Is Essential for
Apoptosis Triggered by EGFR Kinase Inhibitors in Mu-
tant EGFR-Dependent Lung Adenocarcinomas,” PLoS
Medicine, Vol. 4, No. 10, 2007, pp. 1655-1668.
doi:10.1371/journal.pmed.0040294
[23] R. Ley, K. Balmanno, K. Hadfield, C. Weston and S. J.
Cook, “Activation of the ERK1/2 Signaling Pathway
Promotes Phosphorylation and Proteasome-Dependent
Degradation of the BH3-Only Protein, Bim,” Journal of
Biological Chemistry, Vol. 278, No. 21, 2003, pp. 18811-
18816. doi:10.1074/jbc.M301010200
[24] K. Takeuchi, T. Shin-ya, K. Nishio and F. Ito, “Mitogen-
Activated Protein Kinase Phosphatase-1 Modulated JNK
Activation Is Critical for Apoptosis Induced by Inhibitor
of Epidermal Growth Factor Receptor-Tyrosine Kinase,”
The FEBS Journal, Vol. 276, No. 5, 2009, pp. 1255-1265.
doi:10.1111/j.1742-4658.2008.06861.x
[25] K. Takeuchi, Y. Motoda and F. Ito, “Role of Transcrip-
tion Factor Activator Protein 1 (AP1) in Epidermal
Growth Factor-Mediated Protection against Apoptosis
Induced by a DNA-Damaging Agent,” The FEBS Journal,

JNK-Mediated FOXO Expression Plays a Critical Role in EGFR Tyrosine Kinase Inhibitor-Induced BIM
Expression and Apoptosis 433
Vol. 273, No. 16, 2006, pp. 3743-3755.
doi:10.1111/j.1742-4658.2006.05377.x
[26] F. Luciano, A . Jacque l, P . Co losetti, M. Herrant, S. Cagnol,
G. Pages and P. Auberger, “Phosphorylation of Bim-EL
by Erk1/2 on Serine 69 Promotes Its Degradation via the
Proteasome Pathway and Regulates Its Proapoptotic Fun-
ction,” Oncogene, Vol. 22, No. 43, 2003, pp. 6785-6793.
doi:10.1038/sj.onc.1206792
[27] R. Ley, K. E. Ewings, K. Hadfield and S. J. Cook,
“Regulatory Phosphorylation of Bim: Sorting Out the
ERK from the JNK,” Cell Death and Differentiation, Vol.
12, No. 8, 2005, pp. 1008-1014.
doi:10.1038/sj.cdd.4401688
[28] P. F. Dijkers, R. H. Medema, J. W. Lammers, L. Koen-
derman and P. J. Coffer, “Expression of the Pro-Apop-
totic Bcl-2 Family Member Bim Is Regulated by the
Forkhead Transcription Factor FKHR-L1,” Current Bi-
ology, Vol. 10, No. 19, 2000, pp. 1201-1204.
doi:10.1016/S0960-9822(00)00728-4
[29] E. L. Greer and A. Brunet, “FOXO Transcription Factors
at the Interface between Longevity and Tumor Suppres-
sion,” Oncogene, Vol. 24, No. 50, 2005, pp. 7410-7425.
doi:10.1038/sj.onc.1209086
[30] Z. Fu and D. J. Tindall, “FOXOs, Cancer and Regulation
of Apoptosis,” Oncogene, Vol. 27, No. 16, 2008, pp.
2312-2319. doi:10.1038/onc.2008.24
[31] M. Stahl, P. F. Dijkers, G. J. Kops, S. M. Lens, P. J. Cof-
fer, B. M. Burgering and R. H. Medema, “The Forkhead
Transcription Factor FoxO Regulates Transcription of
p27Kip1 and Bim in Response to IL-2,” Journal of Im-
munology, Vol. 168, No. 10, 2002, pp. 5024-5031.
[32] J. Gilley, P. J. Coffer and J. Ham, “FOXO Transcription
Factors Directly Activate Bim Gene Expression and
Promote Apoptosis in Sympathetic Neurons,” Journal of
Cell Biology, Vol. 162, No. 4, 2003, pp. 613-622.
doi:10.1083/jcb.200303026
[33] A. Sunters, S. Fernández de Mattos, M. Stahl, J. J.
Brosens, G. Zoumpoulidou, C. A. Saunders, P. J. Coffer,
R. H. Medema, R. C. Coombes and E. W. Lam, “FoxO3a
Transcriptional Regulation of Bim Controls Apoptosis in
Paclitaxel-Treated Breast Cancer Cell Lines,” The Jour-
nal of Biological Chemistry, Vol. 278, No. 50, 2003, pp.
49795-49805. doi:10.1074/jbc.M309523200
[34] A. Essafi, S. Fernández de Mattos, Y. A. Hassen, I.
Soeiro, G. J. Mufti, N. S. Thomas, R. H. Medema and E.
W. Lam, “Direct Transcriptional Regulation of Bim by
FoxO3a Mediates STI571-Induced Apoptosis in Bcr-
Abl-Expressing Cells,” Oncogene, Vol. 24, No. 14, 2005,
pp. 2317-2329. doi:10.1038/sj.onc.1208421
[35] R. Ley, K. E. Ewings, K. Hadfield, E. Howes, K. Bal-
manno and S. J. Cook, “Extracellular Signal-Regulated
Kinases 1/2 Are Serum-Stimulated ‘Bim(EL) Kinases’
That Bind to the BH3-Only Protein Bim(EL) Causing Its
Phosphorylation and Turnover,” The Journal of Biologi-
cal Chemistry, Vol. 279, No. 10, 2004, pp. 8837-8847.
doi:10.1074/jbc.M311578200
[36] R. Ley, K. Hadfi eld, E. Howe s and S. J. Cook, “ Id e nt i fi ca -
tion of a DEF-Type Docking Domain for Extracellular
Signal-Regulated Kinases 1/2 That Directs Phosphoryla-
tion and Turnover of the BH3-Only Protein BimEL,” The
Journal of Biological Chemistry, Vol. 280, No. 18, 2005,
pp. 17657-17663. doi:10.1074/jbc.M412342200
[37] N. Galili, R. J. Davis, W. J. Fredericks, S. Mukhopadhyay,
F. J. Rauscher III, B. S. Emanuel, G. Rovera and F. G.
Barr, “Fusion of a Fork Head Domain Gene to PAX3 in
the Solid Tumour Alveolar Rhabdomyosarcoma,” Nature
Genetics, Vol. 5, No. 3, 1993, pp. 230-235.
doi:10.1038/ng1193-230
[38] M. J. Anderson, C. S. Viars, S. Czekay, W. K. Cavenee
and K. C. Arden, “Cloning and Characterization of Three
Human Forkhead Genes That Comprise an FKHR-Like
Gene Subfamily,” Genomics, Vol. 47, No. 2, 1998, pp.
187-199. doi:10.1006/geno.1997.5122
[39] J. Hillion, M. Le Coniat, P. Jonveaux, R. Berger and O. A.
Bernard, “AF6q21, a Novel Partner of the MLL Gene in
t(6;11)(q21;q23), Defines a Forkhead Transcriptional
Factor Subfamily,” Blood, Vol. 90, No. 9, 1997, pp. 3714-
3719.
[40] A. Borkhardt, R. Repp, O. A. Haas, T. Leis, J. Harbott, J.
Kreuder, J. Hammermann, T. Henn and F. Lampert, “Clo-
ning and Characterization o f AFX, the Gene That Fuses to
MLL in Acute Leukemias with a t(X;11)(q13;q23),” On-
cogene, Vol. 14, No. 2, 1997, pp. 195-202.
[41] A. Brunet, A. Bonni, M. J. Zigmond, M. Z. Lin, P. Juo, L.
S. Hu, M. J. Anderson, K. C. Arden, J. Blenis and M. E.
Greenberg, “Akt Promotes Cell Survival by Phosphory-
lating and Inhibiting a Forkhead Transcription Factor,”
Cell, Vol. 96, No. 6, 1999, pp. 857-868.
doi:10.1016/S0092-8674(00)80595-4
[42] D. R. Plas and C. B. Thompson, “Akt Activation P ro mot es
Degradation of Tuberin and FOXO3a via the Protea-
some,” The Journal of Biological Chemistry, Vol. 278,
No. 14, 2003, pp. 12361-12366.
doi:10.1074/jbc.M213069200
[43] J. Y. Yang, C. S. Zong, W. Xia, H. Yamaguchi, Q. Ding,
X. Xie, J. Y. Lang, C. C. Lai, C. J. Chang, W. C. Huang,
H. Huang, H. P. Kuo, D. F. Lee, L. Y. Li, H. C. Lien, X.
Cheng, K. J. Chang, C. D. Hsiao, F. J. Tsai, C. H. Tsai, A.
A. Sahin, W. J. Muller, G. B. Mills, D. Yu, G. N. Horto-
bagyi and M. C. Hung, “ERK Promotes Tumorigenesis
by Inhibiting FOXO3a via MDM2-Mediated Degrada-
tion,” Nature Cell Biology, Vol. 10, No. 2, 2008, pp. 138-
148. doi:10.1038/ncb1676
[44] M. C. Hu, D. F. Lee, W. Xia, L. S. Golfman, F. Ou-Yang,
J. Y. Yang, Y. Zou, S. Bao, N. Hanada, H. Saso, R. Ko-
bayashi and M. C. Hung, “IkappaB Kinase Promotes
Tumorigenesis through Inhibition of Forkhead FOXO3a,”
Cell, Vol. 117, No. 2, 2004, pp. 225-237.
doi:10.1016/S0092-8674(04)00302-2
[45] A. Brunet, F. Kanai, J. Stehn, J. Xu, D. Sarbassova, J. V.
Frangioni, S. N. Dalal, J. A. DeCaprio, M. E. Greenberg
and M. B. Yaffe, “14-3-3 Transits to the Nucleus and Par-
ticipates in Dynamic Nucleocytoplasmic Transport,” The
Journal of Cell Biology, Vol. 156, No. 5, 2002, pp. 817-
828. doi:10.1083/jcb.200112059
[46] L. P. Van Der Heide, M. F. Hoekman and M. P. Smidt,

JNK-Mediated FOXO Expression Plays a Critical Role in EGFR Tyrosine Kinase Inhibitor-Induced BIM
Expression and Apoptosis
434
“The Ins and Outs of FoxO Shuttling: Mechanisms of
FoxO Translocation and Transcriptional Regulation,” The
Biochemical Journal, Vol. 380, No. 2, 2004, pp. 297-309.
doi:10.1042/BJ20040167
[47] X. Wang, W. R. Chen and D. Xing, “A Pathway from
JNK through Decreased ERK and Akt Activities for
FOXO3a Nuclear Translocation in Response to UV Irra-
diation,” Journal of Cellular Physiology, Vol. 227, No. 3,
2012, pp. 1168-1178. doi:10.1002/jcp.22839
[48] A. Sunters, P. A. Madureira, K. M. Pomeranz, M. Aubert,
J. J. Brosens, S. J. Cook, B. M. Burgering, R. C. Coom-
bes and E. W. Lam, “Paclitaxel-Induced Nuclear Translo-
cation of FOXO3a in Breast Cancer Cells Is Mediated by
c-Jun NH2-Terminal Kinase and Akt,” Cancer Research,
Vol. 66, No. 1, 2006, pp. 212-220.
doi:10.1158/0008-5472.CAN-05-1997
[49] M. A. Essers, S. Weijzen, A. M. de Vries-Smits, I. Saar-
loos, N. D. de Ruiter, J. L. Bos and B. M. Burgering,
“FOXO Transcription Factor Activation by Oxidative
Stress Mediated by the Small GTPa se Ral and JNK,” The
EMBO Journal, Vol. 23, No. 24, 2004, pp. 4802-4812.
doi:10.1038/sj.emboj.7600476
[50] M. Potente, C. Urbich, K. Sasaki, W. K. Hofmann, C.
Heeschen, A. Aicher, R. Kollipara, R. A. DePinho, A. M.
Zeiher and S. Dimmeler, “Involvement of Foxo Tra nsc rip-
tion Factors in Angiogenesis and Postnatal Neovasculari-
zation,” The Journal of Clinical Investigation, Vol. 115,
No. 9, 2005, pp. 2382-2392. doi:10.1172/JCI23126
[51] K. Okamoto, I. Okamoto, W. Okamoto, K. Tanaka, K.
Takezawa, K. Kuwata, H. Yamaguchi, K. Nishio and K.
Nakagawa, “Role of Survivin in EGFR Inhibitor-Induced
Apoptosis in Non-Small Cell Lung Cancers Positive for
EGFR Mutations,” Cancer Research, Vol. 70, No. 24,
2010, pp. 10402-10410.
doi:10.1158/0008-5472.CAN-10-2438
[52] W. Pao, V. A. Miller, K. A. Politi, G. J. Riely, R. Somwar,
M. F. Zakowski, M. G. Kris and H. Varmus, “Acquired
Resistance of Lung Adenocarcinomas to Gefitinib or Er-
lotinib Is Associated with a Second Mutation in the
EGFR Kinase Domain,” PLoS Medicine, Vol. 2, No. 3,
2005, pp. 225-235. doi:10.1371/journal.pmed.0020073
[53] J. Bean, C. Brennan, J. Y. Shih, G. Riely, A. Viale, L.
Wang, D. Chitale, N. Motoi, J. Szoke, S. Broderick, M.
Balak, W. C. Chang, C. J. Yu, A. Gazdar, H. Pass, V.
Rusch, W. Gerald, S. F. Huang, P. C. Yang, V. Miller, M.
Ladanyi, C. H. Yang and W. Pao, “MET Amplification
Occurs with or without T790M Mutations in EGFR Mu-
tant Lung Tumors with Acquired Resistance to Gefitinib
or Erlotinib,” Proceedings of the National Academy of
Sciences of the United States of America, Vol. 104, No. 5,
2007, pp. 20932-20937.
doi:10.1073/pnas.0710370104
[54] J. A. Engelman, K. Zejnullahu, T. Mitsudomi, Y. Song, C.
Hyland, J. O. Park, N. Lindeman, C. M. Gale, X. Zhao, J.
Christensen, T. Kosaka, A. J. Holmes, A. M. Rogers, F.
Cappuzzo, T. Mok, C. Lee, B. E. Johnson, L. C. Cantley
and P. A. Jänne, “MET Amplification Leads to Gefitinib
Resistance in Lung Cancer by Activating ERBB3 Signal-
ing,” Science, Vol. 316, No. 5827, 2007, pp. 1039-1043.
doi:10.1126/science.1141478
Abbreviations
EGFR: epidermal growth factor receptor;
PI3K: phosphatidylinositol-3’-OH kinase;
JNK: c-Jun NH2-terminal kinase;
ERK: extracellular signal-regulated kinase;
MKP-1: mit ogen-act i vated protein kinase ph osphat ase-1;
NSCLC: non-small-cell lung cancer;
FOXO: forkhead bo x proteins O.