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Cancer Therapy: Preclinical
Insulin-Like Growth Factor-1 Receptor Inhibition Induces a
Resistance Mechanism via the Epidermal Growth Factor
Receptor/HER3/AKT Signaling Pathway: Rational Basis for
Cotargeting Insulin-Like Growth Factor-1 Receptor and
Epidermal Growth Factor Receptor in Hepatocellular Carcinoma
Christèle Desbois-Mouthon,
1,2
Aurore Baron,
1,2
Marie-José Blivet-Van Eggelpoël,
1,2
Laetitia Fartoux,
1,2,3
Corinne Venot,
4
Friedhelm Bladt,
4
Chantal Housset,
1,2
and Olivier Rosmorduc
1,2,3
Abstract Purpose: The insulin-like growth factor (IGF) signaling axis is frequently dysregulated in
hepatocellular carcinoma (HCC). Therefore, we investigated whether the specific target-
ing of the IGF type 1 receptor (IGF-1R) might represent a new therapeutic approach for
this tumor.
Experimental Design: Total and phosphorylated levels of IGF-1R were measured in 21
paired samples of human HCCs and adjacent nontumoral livers using ELISA. The anti-
neoplastic potency of a novel anti–IGF-1R antibody, AVE1642, was examined in five hu-
man hepatoma cell lines.
Results: Overexpression of IGF-1R was detected in 33% of HCCs and increased activation
of IGF-1R was observed in 52% of tumors. AVE1642 alone had moderate inhibitory ef-
fects on cell viability. However, its combination with gefitinib, an epidermal growth factor
receptor (EGFR) inhibitor, induced supra-additive effects in all cell lines that were asso-
ciated with cell cycle blockage and inhibition of AKT phosphorylation. The combination
of AVE1642 with rapamycin also induced a synergistic reduction of viability and of AKT
phosphorylation. Of marked interest, AVE1642 alone up-regulated the phosphorylated
and total levels of HER3, the main partner of EGFR, and AVE1642-induced phosphoryla-
tion of HER3 was prevented by gefitinib. Moreover, the down-regulation of HER3 expres-
sion with siRNA reduced AKT phosphorylation and increased cell sensitivity to AVE1642.
Conclusions: These findings indicate that hepatoma cells overcome IGF-1R inhibition
through HER3 activation in an EGFR-dependent mechanism, and that HER3 represents
a critical mediator in acquired resistance to anti-IGF-1R therapy. These results provide a
strong rational for targeting simultaneously EGFR and IGF-1R in clinical trials for HCC].
(Clin Cancer Res 2009;15(17):5445–56)
Hepatocellular carcinoma (HCC) is the fifth most common
malignancy and the third most common cause of mortality
worldwide. Its incidence is increasing in Western countries,
mainly due to hepatitis C virus and excessive alcohol consump-
tion. At the time of diagnosis, most of the patients suffer ad-
vanced tumor disease and a curative treatment is possible
only in a minority of patients and overall survival is poor (1).
In this context, the recent development of molecular therapies
targeting specific receptors and/or signaling proteins has
opened promising perspectives for the treatment of advanced
HCC (2, 3). However, until now, the targeting of vascular en-
dothelial growth factor or epidermal growth factor receptor
(EGFR) has shown disappointing results in HCC treatment
(4) leading to the concept of multitarget therapies. This concept
has been recently validated by the approval of sorafenib, a multi-
kinase inhibitor, as a reference treatment of advanced HCC (5).
Authors' Affiliations:
1
UPMC Univ Paris 06;
2
Institut National de la Santé et de
la Recherche Médicale, UMR_S 938; and
3
AP-HP,Hôpital Saint-Antoine, Service
d'Hépatologie, Paris, France; and
4
Sanofi-aventis, Vitry-sur-Seine, France
Received 11/18/08; revised 6/19/09; accepted 6/22/09; published OnlineFirst
8/25/09.
Grant support: Association pour la Recherche sur le Cancer (O. Rosmorduc),
sanofi-aventis (O. Rosmorduc and C.D. Mouthon), and Académie Nationale
de Médecine (A. Baron).
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Supplementary data for this article are available at Clinical Cancer
Research Online (http://clincancerres.aacrjournals.org/).
Requests for reprints: Christèle Desbois-Mouthon, Institut National de la Santé
et de la Recherche Médicale UMR_S 938, Faculté de Médecine Pierre et Marie
Curie, Site Saint-Antoine, 27 rue Chaligny, 75012, Paris, France. Phone: 33-1-40-
01-13-56; Fax: 33-1-40-01-14-26; E-mail: christele.desbois-mouthon@inserm.fr.
F2009 American Association for Cancer Research.
doi:10.1158/1078-0432.CCR-08-2980
5445 Clin Cancer Res 2009;15(17) September 1, 2009www.aacrjournals.org
Evidence has accumulated showing that the insulin-like
growth factor (IGF) signaling axis is activated in HCC (6, 7):
IGF-II may be overexpressed as a result of loss of promoter-
specific imprinting and reactivation of fetal promoters; increased
amounts of bioactive IGF-II are also the result of a reduced ex-
pression of IGF binding protein and/or inactivation of the
type 2 IGF receptor, which mediates IGF-II degradation. IGF-II
promotes proliferation, survival, and migration in hepatoma
cells through binding to its receptor IGF type 1 receptor (IGF-1R;
refs. 8–10). This receptor as well as its main substrates IRS-1 and
IRS-2 may be also overexpressed in HCC and in experimental
models of liver carcinogenesis (11–14).
The potential interest of targeting IGF-1R–mediated signaling
pathways in HCC has been suggested both in vitro and in vivo:
first, the blockade of IGF-II overexpression impairs HCC devel-
opment in murine models (15, 16), and second, inhibition of
IGF-1R with a monoclonal antibody (mAb; αIR-3;ref.17)or
with tyrosine kinase inhibitors (TKI; AG1024, NVP-AEW541) re-
duces HCC cell proliferation and/or increases apoptosis (9, 18).
However, we and others recently reported that the combination
of AG1024 with an EGFR TKI (erlotinib, gefitinib) resulted in
synergistic antineoplastic effects in hepatoma cell lines (9, 19),
suggesting that the blockage of either EGFR or IGF-1R may allow
the other receptor to compensate for.
Having shown that IGF-1R was overexpressed or hyperacti-
vated in human HCC tumors, the study was then designed to
examine the antineoplastic effects of cotargeting IGF-1R and
EGFR in HCC cells by using a novel mAb against IGF-1R
(AVE1642), currently in phase I/II clinical trials (20), andgefitinib.
Although AVE1642 alone had moderate inhibitory effects on
cell viability, we observed that its combination with gefitinib ex-
erted supra-additive effects in the five cell lines tested. We char-
acterized the molecular mechanisms of this synergy by showing
that (a) both the total and the phosphorylated levels of HER3
(erbB-3), the main partner of EGFR, were up-regulated in the
presence of AVE1642; (b) gefitinib reduced the phosphoryla-
tion of HER3 induced by AVE1642, which was paralleled by a
marked inhibition of AKT phosphorylation; and (c)thedown-
regulation of HER3 expression using a siRNA strategy also reduced
AKT phosphorylation and significantly increased hepatoma
cell sensitivity to AVE1642. These data highlight an original
compensatory mechanism between IGF-1R and EGFR signaling
pathways in hepatoma cells and provide a strong rational ba-
sis for targeting simultaneously EGFR and IGF-1R in advanced
HCC to prevent the early development of EGFR-mediated
resistance.
Materials and Methods
Reagents. AVE1642, a humanized version of the murine mAb EM164
(21), was provided by sanofi-aventis/Immunogen; cetuximab (Erbitux) was
purchased from Merck KGaA; and gefitinib was provided by AstraZeneca.
Bovine insulin and human recombinant IGF-I and IGF-II were purchased
from Sigma-Aldrich.
Cell culture. HepG2 (HBs
neg
,Rb
wt
,p53
wt
,β-catenin
mut
), Hep3B
(HBs
pos
, undetected Rb, deleted p53, β-catenin
wt
), and HuH7 (HBs
neg
,
Rb
wt
,p53
mut
,β-catenin
wt
) cells were from the American Type Culture Col-
lection.HuH6 (HBs
neg
,p53
wt
,β-catenin
mut
), and PLC/P RF5 (HBs
pos
,Rb
wt
,
p53
mut
, axin
mut
) were provided by Dr Christine Perret (Institut Cochin,
Paris, France). Normal human hepatocytes were isolated from liver tissue
obtained from patients undergoing partial liver resection for liver metasta-
sis, and primary cultures were established as previously described (22).
Pools of HCC cells resistant to gefitinib were generated as previously re-
ported in non–small cell lung carcinoma cell lines (23). Briefly, HuH6
and HepG2 cell lines were continuously exposed to gefitinib (10 μmol/L)
in routine culture medium that was replaced every 4 d. Initially, cell
number was dramatically reduced, and for the next 2 mo, the surviving
cells were passaged approximatively every 15 d. Cell proliferation slowly
increased the next 2 mo and a stable growth rate was reached after a
total of 5 mo.
Western blotting and ELISA. The following antibodies were used to
detect electrotransferred proteins: phospho-Tyr
204
extracellular signal-
regulated kinase (ERK)1/2 (E-4), ERK1 (C-16), polyADP-ribose poly-
merase (H-250), IRS-1 (C-20), insulin receptor (IR, β-subunit, C-19;
all from Santa Cruz Biotechnology, Inc.), phospho-EGFR (Tyr
992
), total
EGFR, phospho-AKT (Ser
473
), total AKT, phospho-IGF-1R (Tyr
1131
)/
IR (Tyr
1146
), total IGF-1R (β-subunit), phospho-HER3 (Tyr1289;
21D3), PTEN (138G6), phospho-tyrosine mouse mAb (P-Tyr-100; all
from Cell Signaling Technology, Inc., IRS-2, total HER3 (clone 2F12;
all from Upstate), and β-actin (clone AC-15; Sigma-Aldrich). Immune
complexes were visualized by enhanced chemiluminescence (Pierce
Biotechnology, Inc.). Total amounts of HER3 were quantified by ELISA
according to manufacturer's instructions (R&D systems, Inc.). For HER3
immunoprecipitation, cell extracts (1,000-1,500 μg) were incubated
overnight at 4°C with 4 μg of anti-HER3 antibody (clone 2F12) together
with 30 μL of protein A/G PLUS-agarose (Santa Cruz Biotechnology,
Inc.). Immunoprecipitates were washed thrice with lysis buffer, re-
suspended in gel loading buffer, and analyzed by Western blotting.
Cell surface protein biotinylation. Biotinylation of surface proteins
was done for 30 min at 4°C using the cell surface protein isolation kit
(Pierce Biotechnology) and following instructions from the manufacturer.
Liver tissue specimens. Tumoral and nontumoral liver tissue speci-
mens were obtained from 21 patients with HCC who underwent partial
hepatectomy. Histologic analyses of nontumoral livers showed cirrhosis
in all cases. Cirrhosis was related to HCV infection (14 cases), to HBV
infection (3 cases), and to alcohol abuse (4 cases). Liver tissue samples
were flash frozen in liquid nitrogen and stored at -80°C until analysis.
This study was done with informed consent of patients in accordance
with the French legislation.
Translational Relevance
Based on a recent phase III study (Llovet 2008),
sorafenib, a multikinase inhibitor, has been approved
in Europe and the United States as the reference treat-
ment for patients with advanced hepatocellular carci-
noma (HCC). However, despite the therapeutic
advance allowed with sorafenib, medical need for
new compounds is still important in this indication.
Activation of insulin-like growth factor (IGF) signaling
pathways is strongly implicated in the pathogenesis
of HCC and, based on preclinical data, clinical trials
using IGF type 1 receptor (IGF-1R) inhibitors are cur-
rently ongoing. Here, we show that the specific inhibi-
tion of IGF-1R using a monoclonal antibody induces a
resistance mechanism to this drug in HCC cells via the
activation of the epidermal growth factor receptor
(EGFR)/HER3/AKT pathway, which could be pre-
vented by a combination of EGFR and IGF-1R inhibi-
tors. Therefore, these results together with our
previous data showing cross-talks between EGFR
and IGF-1R in HCC (Desbois-Mouthon 2006) provide
a strong rationale for IGF-1R and EGFR cotargeting
in future clinical trials.
5446Clin Cancer Res 2009;15(17) September 1, 2009 www.aacrjournals.org
Cancer Therapy: Preclinical
Measurements of IGF-1R expression and phosphorylation in human liver
tissue specimens. Liver tissue specimens were homogenized in cell ex-
traction buffer (Biosource, Invitrogen) and centrifuged at 14,000× gfor
15 min at 4°C. The amounts of phosphorylated (Y1135/Y1136) and total
IGF-1R were quantified in supernatants using ELISA following manufac-
turer's instructions (Biosource, Invitrogen). The anti-phospho(Y1135/
Y1136) IGF-1R antibody did not cross-react with phosphorylated IR.
Cell viability. Cells were seeded in 24-well plates (3 × 10
5
cells per
well) and allowed to proliferate for 24 h. Then cells were incubated for
a further 72 h in serum-free or in complete medium [10% fetal bovine
serum (FBS)] containing or not AVE1642, gefitinib, rapamycin, and/or
cetuximab. At the end of the treatment period, cell viability was measured
using the MTT assay.
Flow cytometry analysis of cell cycle and apoptosis. Cells plated in
60-mm dishes were exposed to complete medium containing AVE1642
and/or gefitinib for 48 and 72 h. Both adherent and floating cells were
collected, washed, fixed in ice-cold 70% ethanol at -20°C, and stained
with 20 μg/mL propidium iodide in the presence of 100 μg/mL RNase
A for 30 min at 37°C in the dark. DNA content was analyzed by flow
cytometry (FACS Calibur, Becton Dickinson). Apoptotic cells with hypo-
diploid DNA staining were found in the ≪sub-G
1
≫peak.
Reverse transcription-PCR analysis. Total RNA was extracted using
RNA Plus lysis solution (MP Biomedicals) and reverse-transcribed as pre-
viously described (24). Transcripts coding for IGF-II and β-actin were
analyzed by semiquantitative PCR as previously described (14). HER3
mRNA transcripts were quantified by real-time PCR using previously
published primers (25). Each sample was normalized on the basis of
its 18S mRNA content as reported (24). PCR was done on a LightCycler
instrument (Roche Applied Science).
Immunofluorescence microscopy. Cells seeded on glass coverslips
were incubated with a 1:50 dilution of a mouse monoclonal anti-
HER3 antibody directed against the extracellular domain (Ab-4, clone
H3.90.6, Thermo Fisher Scientific) or of a control IgG (Jackson Immu-
noResearch Laboratories, Inc.) in HBSS for 1 h at 4°C. Cells were then
rinsed and fixed with 4% paraformaldehyde. Cells were blocked and in-
cubated with a 1:200 dilution of FITC-conjugated goat anti-mouse anti-
body [Alexa Fluor 488 F(ab′)
2
; Invitrogen] in 1% bovine serum albumin
in PBS for 1.5 h at room temperature. The slides were counterstained with
4′,6-diamidino-2-phenylindole (Sigma-Aldrich) for nuclei detection.
Fluorescence was visualized using a Leica differentially methylated re-
gion immunofluorescence microscope with DFC300 FX digital camera.
Down-regulation of HER3 expression using a siRNA strategy. HER3
expression was down-regulated by using a mixture of four siRNAs tar-
geting human HER3 (ON-TARGETplus SMARTpool; Dharmacon). Con-
trol siRNA was a pool from Dharmacon (siGENOME nontargeting
siRNA pool). Cells plated in 35-mm dishes (35 × 10
4
cells/dish) or in
24-well plates (15 × 10
4
cells per well) were cultured for 24 h to 40%
confluency and then transfected with 100 nmol/L siRNA using Lipo-
fectamine 2000 (InVitrogen). Twenty-four h later, cells were stimulated
or not with AVE1642 for further 48 to 72 h before being harvested for
protein extraction or analyzed for cell viability in a MTT assay.
Statistical analysis. Results are given as means ± SEM. Statistical
comparison of mean values was done using the Student's ttest.
Results
The IGF-II/IGF-1R signaling axis is dysregulated in human
HCC cell lines and tissue. Five human hepatoma cell lines
(HepG2, Hep3B, HuH7, HuH6, and PLC/PRF5) were used in
the present study. All these cell lines overexpressed IGF-1R
and its substrates IRS-1 and IRS-2 compared with normal human
hepatocytes (Fig. 1A). The heterogeneity of IGF-1R bands be-
tween cell lines has been reported by others (26) and may reflect
differential phosphorylations and glycosylations. IGF-II was
more expressed in Hep3B, HepG2, and HuH7 cells compared
with HuH6 and PLC/PRF5 cells (Fig. 1B). The overexpression
of both IGF-II and IGF-1R suggested the existence of an autocrine
stimulating loop in these cells.
IGF-1R status was examined in frozen tumors. The amounts
of total and phosphorylated IGF-1R were quantified by ELISA
in paired tumoral and nontumoral liver specimens from 21 pa-
tients with HCC. The ratio of phospho-IGF-1R to total IGF-1R
was calculated to evaluate the activation level of IGF-1R. We ob-
served increased expression of IGF-1R in 7 tumors (33%; Fig. 1C,
left) and increased activation of IGF-1R in 11 tumors (52%;
Fig. 1C, right). Of note, phospho-IGF-1R levels did not correlate
well with overall IGF-1R levels. These observations fully justified
the need for subsequent investigations examining the effects of
the specific targeting of IGF-1R in HCC cells.
The anti–IGF-1R antibody AVE1642 specifically inhibits IGF
but not insulin signaling in hepatoma cells. As a first step, experi-
ments were conducted in HepG2, HuH7, and Hep3B cells to en-
sure the specificity of the anti–IGF-1R antibody AVE1642 against
IGF signaling. IGF-1R/IR phosphorylation was examined by
Western blotting using a dual phospho-IGF-1R (Y1131)/IR
(Y1146) antibody. Similar results were obtained in the three cell
lines but, for the clarity of the presentation, only data obtained in
Hep3B cells are shown in Fig. 2A. In control cells, IGF-I, IGF-II,
and insulin (5 × 10
-9
mol/L; 15 min) increased the phosphoryla-
tion of both IGF-1R/IR and Akt. In the presence of AVE1642
(0.5 μg/mL), the stimulatory effects of IGF-I and IGF-II were
markedly inhibited, whereas those of insulin were fully main-
tained. In addition, we observed that long-term treatment (48
and 72 h) with AVE1642 decreased IGF-1R but not IR expression
in HepG2 and Hep3B (Fig. 2B). Therefore, besides its neutralizing
effect on IGF-1R, AVE1642 can also promote IGF-1R down-
regulation and degradation in hepatoma cells as previously re-
ported in other cancer cell types (21, 27). Altogether, these results
show that AVE1642 specifically inhibits IGF but not insulin in-
duction of receptor and AKT phosphorylation.
AVE1642 exerted a moderate antitumoral effect in hepatoma
cells. The blockade of IGF-1R by AVE1642 may impact both
autocrine and serum-induced cell growth. To examine this
point, cell viability was examined in cells cultured for 72 h in
serum-free medium or in complete medium (10% FBS) con-
taining or not AVE1642 (0.5 μg/mL). In serum-free conditions
(Fig. 2C, top), AVE1642 decreased by 41% the viability in
HepG2 cells, whereas its effects were less potent in Hep3B,
PLC/PRF5, and HuH7 cells (24.5%, 17.9%, and 12% decrease,
respectively). AVE1642 was without effect in HuH6 cells. In
complete medium (Fig. 2C, bottom), similar results were ob-
tained, HepG2 cells being the most sensitive cell line (45% in-
hibition). Of note, even higher concentrations of AVE1642 (up
to 2 μg/mL) did not further inhibit hepatoma cell viability (data
not shown). AVE1642 was without any significant effect on via-
bility in normal hepatocytes (Fig. 2D). These findings show that
AVE1642 by itself has an inhibitory effect, even if moderate, on
both autocrine and serum-induced cell growth in almost all hep-
atoma cell lines.
Combining AVE1642 to gefitinib induces a synergistic inhibi-
tion of proliferation in hepatoma cell lines. Our previous study
showing cross-talks between EGFR and IGF-1R (9) led us to ex-
amine whether the antineoplastic effect of AVE1642 might be
influenced by EGFR in hepatoma cells. To this aim, cell lines
were cultured in complete medium and treated with AVE1642
(0.5 μg/mL) in presence or absence of gefitinib. A 2-μmol/L con-
centration of gefitinib was used for these experiments because in
5447 Clin Cancer Res 2009;15(17) September 1, 2009www.aacrjournals.org
IGF-1R Inhibition Restores Gefitinib Sensitivity in HCC
preliminary studies, this concentration was found to be submax-
imal on cell viability (data not shown). In addition, this concen-
tration approaches the serum concentrations reported in patients
orally treated with gefitinib (400-600 mg/day). We observed
that the combination of AVE1642 with gefitinib induced a
supra-additive inhibition of cell growth in all cell lines, even in
HuH6 cells in which AVE1642 exhibited no effect by its own
(Fig. 3A). Similar synergistic effects were obtained when
AVE1642 was combined with a submaximal dose of cetuximab,
an EGFR-neutralizing antibody (Fig. 3B).
To discriminate whether the synergistic reduction in cell via-
bility induced by AVE1642 plus gefitinib resulted from reduced
proliferation and/or to increased cell death, we did flow cyto-
metric analyses. Forty-eight-hour treatment with the AVE1642/
gefitinib combination decreased the percentage of HepG2 cells
in the S phase and arrested cells in the G
0
-G
1
phase of the cell
cycle (Fig. 3C). No increased rate of apoptosis (% of sub-G
1
cells
after 48 and 72 hours of incubation) was evidenced in these ex-
perimental conditions (data not shown; Fig. 3C). In addition,
cell incubation with a nontoxic dose (10 μmol/L) of the pan-
caspase inhibitor ZVAD-fmk did not alter the synergistic effect
of AVE1642 combined to gefitinib on cell viability (data not
shown). Altogether, these findings indicate that the combination
of AVE1642 and gefitinib synergistically inhibits hepatoma
cell viability through cell cycle inhibition without apoptosis
induction.
The synergistic effect of AVE1642 combined with gefitinib routes
through the down-regulation of AKT signaling. As a next step,
we analyzed the molecular mechanisms whereby AVE1642 and
gefitinib had a synergistic inhibitory effect on hepatoma cell
proliferation. Cells were treated for 48 hours in the presence
of AVE1642 combined or not with gefitinib and then stimulated
for 20 minutes with 10% FBS. Similar results were obtained in
the five cell lines. When used alone, AVE1642 slightly decreased
serum-induced AKT phosphorylation (by about 1.5- to 2-fold),
whereas gefitinib had no effect (Fig. 4A). When AVE1642 was
combined with gefinitib, serum-induced AKT phosphorylation
was markedly reduced (by about 3- to 5-fold) compared with
the effects of each drug used alone. In contrast, the combination
of drugs had no effect on serum-induced ERK phosphorylation
(Fig. 4A). This strongly suggested that the inhibition of AKT sig-
naling rather than the inhibition of ERK signaling contributed
to the growth inhibitory effect of AVE1642 combined with
gefitinib. Down-regulation of AKT phosphorylation upon treat-
ment with the AVE1642/gefitinib combinationdid not result from
the modulation of PTEN expression (Fig. 4A). The combination
Fig. 1. Expression of components of
the IGF-II/IGF-1R signaling axis in
human HCC cell lines and tissue
specimens. A, whole-cell extracts
obtained from normal human
hepatocytes and HCC cell lines were
analyzed by Western blotting for
IGF-1R, IRS-1, and IRS-2 expressions.
The membrane was reblotted with
an AKT antibody to ensure equivalent
loading. B, total RNA (2 μg) extracted
from the five hepatoma cell lines were
analyzed by semiquantitative reverse
transcription-PCR using human IGF-II
primers. β-Actin PCR products were
run in parallel to ensure that equivalent
amounts of cDNA were amplified.
C, the levels of phosphorylated
(Y1135/Y1136) and total IGF-1R were
examined in 21 paired tumoral
(tumor) and nontumoral (adjacent)
liver specimens using ELISA.
5448Clin Cancer Res 2009;15(17) September 1, 2009 www.aacrjournals.org
Cancer Therapy: Preclinical
of AVE1642 with rapamycin (1 nmol/L), a specific inhibitor of
mammalian target of rapamycin (a downstream target of AKT),
also induced a synergistic inhibition of cell viability in hepatoma
cells (Fig. 4B). Previous studies conducted in diverse cancer cell
types (28–30) have shown that rapamycin triggers a rapid feed-
back mechanism resulting in AKT activation and which may
be dependent on IGF-1R signaling. We examined whether such
a mechanism also occurred in HCC cells and, if so, whether
AVE1642 had the potential to block it. Rapamycin alone
(1 and 10 nmol/L) markedly increased the phospho-AKT content
in HCC cell lines and this effect was abolished by AVE1642
(Fig. 4C). These findings indicate that rapamycin up-regulates
the AKT pathway in HCC cells through an IGF-1R–dependent
pathway.
AVE1642 increases HER3 phosphorylation and expression in
hepatoma cell lines. AKT activation by EGFR is due to the ability
of this receptor to dimerize with and to transphosphorylate the
kinase-inactive receptor HER3 (ErbB-3), leading to the recruitment
Fig. 2. Effects of the anti–IGF-1R
antibody AVE1642 on IGF and insulin
signaling in hepatoma cells. A, Hep3B
cells were treated for 1 h with or
without AVE1642 (0.5 μg/mL) and then
stimulated for 15 min with IGF-I, IGF-II,
or insulin (10
-9
mol/L). Whole-cell
extracts were analyzed by Western
blotting for levels of phosphorylated
and total IGF-1R/IR and AKT. B, HepG2
and Hep3B cells were treated with
AVE1642 (0.5 μg/mL) for 48 and
72 h and total expression levels of
IGF-1R and IR were assessed by
Western blotting. Blots are
representative of two independent
experiments. C, HCC cell lines were
treated for 72 h with or without AVE1642
(0.5 μg/mL) in serum-free (top)orin
complete (bottom) medium and then
cell viability was measured. D, normal
hepatocytes were treated for 72 h with
increasing concentrations of AVE1642
(0-2 μg/mL) and cell viability was
measured. Columns, means of three to
four independent experiments done in
quadruplet; bars, SEM. *, P< 0.05; **,
P< 0.002; ***, P< 0.001 compared with
untreated cells.
5449 Clin Cancer Res 2009;15(17) September 1, 2009www.aacrjournals.org
IGF-1R Inhibition Restores Gefitinib Sensitivity in HCC
of the p85 regulatory subunit of phosphoinositide 3-kinase
(PI3K) to HER3 and subsequent activation of PI3K (31). Because
AKT phosphorylation was almost completely abrogated in the
presence of AVE1642 and gefitinib but not in the presence of
AVE1642 alone, we examined by Western blot analyses whether
the EGFR/HER3/AKT pathway may be modulated by AVE1642.
All hepatoma cell lines expressed EGFR and HER3, whereas HER-2
(ErbB-2) expression was barely detected (data not shown). HepG2
Fig. 3. Antitumoral effects of AVE1642
alone or in combination with gefitinib in
hepatoma cell lines. A, cell lines were
treated for 72 h with or without
AVE1642 (AVE; 0.5 μg/mL) and/or
gefitinib (gef;2μmol/L) in complete
medium, and then cell viability was
measured. B, the same experiments
were done with cetuximab (cetux;
1-2 μg/mL) instead of gefitinib.
Columns, means of three independent
experiments done in quadruplet; bars,
SEM. Black lines, the additivity
threshold. C, HepG2 cells were treated
for 48 h with or without AVE1642
(0.5 μg/mL) and/or gefitinib (2 μmol/L)
and analyzed for cell cycle repartition
by flow cytometry. A representative
experiment out of two is shown.
5450Clin Cancer Res 2009;15(17) September 1, 2009 www.aacrjournals.org
Cancer Therapy: Preclinical
and HuH7 cell stimulation with 10% FBS induced the tyrosine
phosphorylation of both EGFR and HER3 and these effects were
inhibited by gefitinib (Fig. 5A). In the presence of AVE1642 alone,
the phosphorylation of EGFR and more especially that of HER3
were further increased. The stimulatory effects of AVE1642
on both EGFR and HER3 were strongly inhibited by gefitinib
(Fig. 5A). AVE1642 increased HER3 protein expression, although
it did not modify the amounts of EGFR (Fig. 5A). These find-
ings were also observed in Hep3B and PLC/PRF5 cells. Increased
tyrosine phosphorylation of HER3 parallel to increased expres-
sion of HER3 was also seen in HER3 immunoprecipitates ob-
tained from HCC cells treated with AVE1642 (Supplementary
Fig. S1). An accurate measurement of HER3 protein amounts
done by ELISA showed that AVE1642 increased HER3 by 1.5-,
2.7-, and 4.6-fold in HepG2, HuH7, and PLC/PRF5 cells, respec-
tively (Fig. 5B). Immunofluorescence microscopy (Fig. 5C) and
surface protein biotinylation (Fig. 5D) showed that AVE1642 in-
creased the amount of membrane-associated HER3. No increase
of HER3 expression was observed in normal hepatocytes treated
with increasing doses of AVE1642 (Supplementary Fig. S2). Alto-
gether, these findings show that, in all tested hepatoma cells, the
specific targeting of IGF-1R stimulates a cross-talk toward the
EGFR signaling pathway via the up-regulation of the total
HER3 cellular content. Interestingly, a reciprocal up-regulation
of IGF-1R signaling consecutive to EGFR inhibition was not ob-
served in HCC cell lines after short-term and long-term treat-
ments with gefitinib. Thus, a 48-hour treatment with gefitinib
(5 and 10 μmol/L) modified neither the phosphorylated nor
Fig. 4. Effects of AVE1642 combined
with gefitinib on AKT and ERK signaling
in hepatoma cells. A, serum-starved
HepG2 and HuH7 cells were treated
for 48 h with or without AVE1642
(0.5 μg/mL) and/or gefitinib (2 μmol/L)
and then stimulated with 10% FBS for
20 min. Whole-cell extracts were
analyzed by Western blotting for
phosphorylated and/or total AKT, ERKs,
and PTEN. B, HepG2, Hep3B, and HuH7
were treated for 72 h with or without
AVE1642 (0.5 μg/mL) and/or rapamycin
(rapa; 1 nmol/L) in complete medium,
and cell viability was measured.
Columns, mean of three independent
experiments done in quadruplet; bars,
SEM. Black lines, additivity threshold.
C, Hep3B and HuH7 cells were
pretreated for 2 h with or without
AVE1642 (0.5 μg/mL) and then
incubated or not with rapamycin (1 and
10 nmol/L; 3 h). Whole-cell extracts
were analyzed by Western blotting for
phosphorylated and/or total AKT levels.
Blots are representative of two
independent experiments.
5451 Clin Cancer Res 2009;15(17) September 1, 2009www.aacrjournals.org
IGF-1R Inhibition Restores Gefitinib Sensitivity in HCC
the total levels of IGF-1R (Supplementary Fig. S3A). Moreover,
HepG2 and HuH6 cells continuously exposed to 10 μmol/L
gefinitib for 5 months (resistant cells) showed no increased levels
of either phosphorylated or total IGF-1R (Supplementary
Fig. S3B) nor elevated sensitivity to growth inhibition by
AVE1642 (Supplementary Fig. S3C) compared with the untreat-
ed counterparts (parental cells). Rather, a decreased expression of
IGF-1R together with a decreased IGF-1R phosphorylation was
observed in HepG2 cells chronically incubated with gefitinib
(Supplementary Fig. S3B).
To further characterize the mechanisms whereby AVE1642
increased HER3 expression in HCC cells, we compared HER3
mRNA expression in control and AVE1642-treated cells by quan-
titative real-time PCR. AVE1642 increased the levels of HER3
transcripts by 1.6-, 1.9-, and 3.8-fold in HepG2, HuH7, and
PLC/PRF5 cells, respectively (Fig. 6A). Given that rapamycin
synergized with AVE1642 to reduce cell viability (Fig. 4B), we ex-
amined whether rapamycin could affect AVE1642 induction of
HER3 expression and phosphorylation. In HepG2 (Supplemen-
tary Fig. S4), HuH7, and Hep3B cells (data not shown), the stim-
ulatory effect of AVE1642 on HER3 remained unchanged in the
presence of rapamycin suggesting that AVE1642 increases HER3
expression and phosphorylation through a mammalian target of
rapamycin (mTOR)-independent pathway.
HER3 limits the antitumoral action of AVE1642 in hepatoma
cells. We examined whether the up-regulation of HER3 ex-
pression interfered with the antitumoral effect of AVE1642. To
address this issue, HepG2 and PLC/PRF5 cells were transiently
Fig. 5. Effects of AVE1642 on EGFR/HER3 signaling in hepatoma cells. A, serum-starved HepG2 and HuH7 cells were treated for 48 h with or without
AVE1642 (0.5 μg/mL) and/or gefitinib (2 μmol/L) and then stimulated with 10% FBS for 20 min. Whole-cell extracts were analyzed by Western blotting for
phosphorylated and total expression levels of EGFR and HER3. B, serum-starved HepG2, HuH7, and PLC/PRF5 cells were treated for 48 h with or without
AVE1642 (0.5 μg/mL) and the levels of total HER3 were quantified for by ELISA. Columns, mean of three independent experiments; bars, SEM. C, HepG2 and
HuH7 cells treated or not with AVE1642 for 24 h were incubated with an anti-HER3 antibody at 4°C to minimize internalization and to enhance cell surface
labeling of receptors. Cells were then fixed and receptor-antibody complexes were visualized with a secondary antibody conjugated with FITC. Parallel
experiments conducted with a control IgG showed no labeling (data not shown). D, cells treated or not with AVE1642 for 24 h were processed for cell surface
protein biotinylation. Biotinylated proteins were purified and analyzed for HER3 expression by Western blot analysis. E-cadherin detection was used as a
loading control.
5452Clin Cancer Res 2009;15(17) September 1, 2009 www.aacrjournals.org
Cancer Therapy: Preclinical
transfected with HER3-specific siRNAs or with control siRNAs.
As shown in Fig. 6B, HER3-specific siRNAs induced a partial
knock-down of HER3 expression (approximately 50% and
80% reduction in HepG2 and PLC/PRF5 cells, respectively) both
in unstimulated and in AVE1642-treated cells compared with
cells transfected with the control siRNAs. HER3 down-regulation
was accompanied by a subsequent decrease in the phosphoryla-
tion state of HER3. AKT phosphorylation was also reduced but
only in cells treated with AVE1642. When transfected cells were
analyzed in an MTT assay, we observed that the antitumoral
effect of AVE1642 was significantly increased in cells in which
HER3 was down-regulated compared with control cells (Fig. 6C).
This result confirms the major role of HER3 in the regulation of
hepatoma cell sensitivity to the anti–IGF-1R antibody. Taken as a
whole, our findings show that hepatoma cell lines may adapt
to IGF-1R inhibition by inducing early compensatory inputs
leading to the activation of an EGFR/HER3/AKT-dependent
pathway (Fig. 6D).
Discussion
The dysregulation of the IGF signaling axis, notably via an
increased IGF-II availability, has been well documented in hu-
man HCC (6). However, data concerning the IGF-1R status in
HCC tumors are limited and conflictory. A study analyzing 15
paired samples of HCC and adjacent nontumoral liver tissue
showed no significant increase of IGF-1R mRNA expression in
tumors (32). A recent study showed by Western blot analysis
Fig. 6. Contribution of HER3 to hepatoma cell sensitivity to AVE1642. A, serum-starved HepG2, HuH7, and PLC/PRF5 cells were treated for 48 h with or
without AVE1642 (0.5 μg/mL), and HER3 mRNA transcripts were quantified by real-time PCR. Band C, HepG2 and PLC/PRF5 cells were transiently transfected
with HER3 siRNA or with control (ctl) siRNA and analyzed for HER3 and AKT expression by Western blotting after 48 h of treatment with AVE1642 (B), and for
cell viability after 72 h of treatment with AVE1642 (C). Representative blots of three independent experiments are shown. Columns, means of three
independent experiments; bars, SEM. *, P< 0.001 compared with cells transfected with the control siRNA and treated with AVE1642. D, proposed
mechanism for the synergistic antitumoral effect of AVE1642 combined with gefitinib. Left, IGF-1R is a strong activator of the AKT pathway in hepatoma cells,
which may contribute to the proliferative effect of IGF-II. In contrast, EGFR is a poor activator of AKT in these cells, maybe due to a nonefficient coupling
between EGFR and HER3. As a result, hepatoma cells are poorly sensitive to gefitinib. Right, in the presence of AVE1642, IGF-1R is blocked and an escape
mechanism is induced, resulting in the up-regulation of HER3 expression. EGFR, which is also activated consecutively to IGF-1R inhibition probably by
ligand-dependent mechanism, homodimerizes with and trans-phosphorylated HER3, thus restoring an efficient coupling to the PI3K/AKT pathway. Under
this condition, hepatoma cell sensitivity to gefitinib is increased.
5453 Clin Cancer Res 2009;15(17) September 1, 2009www.aacrjournals.org
IGF-1R Inhibition Restores Gefitinib Sensitivity in HCC
that IGF-1R was frequently overexpressed in a subgroup of
tumors associated with a low copy number of HBV (7 of 9 tu-
mors; ref. 12). In addition, some HCC tumors associated with
hepatitis C virus and increased proliferation presented frequent-
ly with IGF-1R phosphorylation by immunohistochemistry
analysis (11 of 23 tumors; ref. 33). In our panel of HCC, ELISA
measurements revealed that the levels of IGF-1R expression and
activation were increased in approximately 30% and 50% of tu-
mors compared with matched cirrhotic liver tissues, respectively.
As we did not find a clear correlation between total and phos-
phorylated levels of IGF-1R, our data suggest that both phos-
phorylated and total levels of IGF-1R might be predictive of
IGF-1R signaling dependency and of potential sensitivity to
IGF-1R targeting in HCC cells.
Two classes of IGF-1R inhibitors are currently under clinical
development for cancer therapy: (a) mAbs such as AVE1642
that specifically bind to the extracellular domain of IGF-1R,
block ligand/receptor interaction, and promote IGF-1R down-
regulation and degradation and (b) small-molecule TKIs.
However, due to the high sequence homology between IGF-1R
and IR (84%), these latter may be not fully specific for IGF-1R.
AVE1642 is the humanized version of the murine mAb EM164,
which has been shown to potently and specifically inhibit IGF-
1–stimulated proliferation in different cancer cell lines including
breast, lung, colon, and myeloma cells (21, 27). Accordingly, we
observed that, in hepatoma cells, AVE1642 inhibited signaling in
response to exogenously added IGF-I, IGF-II, but not to insulin.
In MTT assays, the antitumoral potency of AVE1642 was compa-
rable in the presence or absence of serum, especially in cell lines
with strong IGF-II production (i.e, HepG2, HuH7, and Hep3B),
suggesting that autocrine IGF-II is an important driver for
hepatoma cell growth. The strongest effect of AVE1642 on cell
viability was observed in HepG2 cells, whereas HuH6 was the
most resistant cell line. There was no correlation between cell
sensitivity to AVE1642 and IGF-II mRNA expression in serum-
free conditions because AVE1642 induced the same inhibition
of cell viability in PLC/PRF5 (low IGF-II mRNA expression levels)
and HuH7 (high IGF-II mRNA expression levels) cells. Other-
wise, it has been shown that the antiproliferative effect of
AVE1642 was not correlated to IGF-1R expression in a panel
of cancer cells.
5
We observed that the combination of AVE1642 with gefitinib
or with cetuximab induced a supra-additive inhibition of viability
in all cell lines compared with drugs alone. These findings suggest
that ligand-dependent activation of EGFR limits the antineoplasic
action of AVE1642 in hepatoma cells, irrespective of their pheno-
type (i.e, wild-type or mutated p53, β-catenin, and/or Rb), their
viral status (HBs
neg
or HBs
pos
antigen), or their own sensitivity
to AVE1642. We show that the synergy between AVE1642 and
anti-EGFR therapy is a general phenomenon and not a peculiar-
ity of a few cell lines. The synergy between AVE1642 and gefitinib
led mainly to a cytostatic effect with growth arrest in G
0
-G
1
without apoptosis. The analysis of the underlying mechanisms
revealed that AVE1642 increased the amounts of total and phos-
phorylated HER3. Therefore, our findings indicate that, follow-
ing blockade of IGF-1R signaling, hepatoma cells are able to
switch from IGF-1R to EGFR dependency to maintain cell growth
and AKT phosphorylation. During the preparation of this article,
an adaptive up-regulation of the EGFR pathway in response to
IGF-1R inhibition with small TKIs has been reported in ovarian
and colon cancer cells (34, 35). A reciprocal regulation seems not
to occur in hepatoma cells because we did not observe any stim-
ulating effect of gefitinib on IGF-1R signaling after short-term
and even long-term exposures to this drug. Such a regulation
had been reported in breast, prostate, and non–small cell
lung cancer cells in which EGFR TKIs induced a significant
activation of IGF-1R signaling through different mechanisms:
up-regulation of IGF-1R activity (36), increased availability of
IRS-1 (37), formation of EGFR/IGF-1R heterodimers (38), or loss
of IGF binding protein expression (39).
HER3 is a peculiar member of the EGFR family because it
lacks a functional intrinsic tyrosine kinase activity and is able
to directly bind PI3K and activate the donwstream AKT pathway
(31). HER3 functions through heterodimerization with EGFR
or HER-2. In EGFR/HER3 and HER-2/HER3 heterodimers, the
transphosphorylation of HER3 allows the recruitment of the
p85 regulatory subunit of PI3K. A growing body of literature
has recently emerged showing that HER3 is a crucial sensor of
sensitivity of cancer cells to EGFR TKIs. First, there is a strong cor-
relation between the amounts of phosphorylated and total HER3
and response to gefitinib or to erlotinib in lung, pancreas, and
liver cancer cells (40–43). Second, it has been shown that cancer
cells that rely mainly on the EGFR/HER3 pathway for AKT acti-
vation displayed exquisite sensitivity to gefitinib and erlotinib
(44, 45). In this context, the increased expression of phosphory-
lated HER3 observed in hepatoma cells exposed to AVE1642
might be the main mechanism accounting for the synergy be-
tween AVE1642 and gefitinib in these cells. Arguments for this
hypothesis are the following: first, all hepatoma cell lines that
express wild-type EGFR (9) were poorly sensitive to gefitinib
alone in terms of inhibition of cell viability (IC
50
, approximately
8-10 μmol/L in all cell lines; data not shown) and of AKT phos-
phorylation; second, gefitinib, by preventing AVE1642-induced
phosphorylation of HER3, led to the down-regulation of AKT phos-
phorylation; and third, counter-acting the effect of AVE1642 on
HER3 expression using siRNAs sensitized hepatoma cells to
AVE1642. Of note, there was no correlation between basal levels
of HER3 and cell response to the bitherapy AVE1642/gefitinib.
Moreover, HER3 expression did not correlate with IGF-II ex-
pression (data not shown), suggesting that baselines of HER3 and
IGF-II are not predictive markers of HCC response to the bitherapy.
AVE1642 synergized not only with gefitinib but also with
rapamycin to reduce cell viability. In both combinations, a
marked down-regulation of AKT phosphorylation was observed
but the underlying mechanisms seemed to be different. Indeed,
contrasting with what was observed in the presence of gefitinib,
rapamycin did not prevent the induction of HER3 phosphory-
lation by AVE1642. Nevertheless, AVE1642 had the potential to
impede the induction of AKT phosphorylation by rapamycin.
These latter findings corroborate with those obtained in other
cancer cell types showing that in HCC cells rapamycin is able to
up-regulate the AKT pathway through an IGF-1R-dependent
pathway (28–30). The inhibitory effect of AVE1642 on this pro-
cess may contribute to the antitumoral effect of the rapamycin/
AVE1642 combination in HCC cells.
It is highly probable that the up-regulation of HER3 expres-
sion consecutive to IGF-1R inhibition may be responsible for
the increase of HER3 phosphorylation. Indeed, this could favor
the formation of EGFR/HER3 heterodimers and the subsequent
5
Unpublished results.
5454Clin Cancer Res 2009;15(17) September 1, 2009 www.aacrjournals.org
Cancer Therapy: Preclinical
trans-phosphorylation of HER3 by EGFR. This could also be en-
hanced by the fact that AVE1642 promoted a slight but repro-
ducible increase of EGFR phosphorylation. Otherwise,
AVE1642 did not seem to modulate the availability of specific
ligands for HER3 because HER3 neutralization with a specific
antibody was without effect on cell response to AVE1642 (data
not shown).
In summary, we present evidence for the interest of targeting
IGF-1R in HCC because this receptor may be overexpressed
and/or activated in tumors. However, we also show that the
antitumoral efficiency of a specific antibody against IGF-1R is
counteracted in HCC cells due to its ability to stimulate the
EGFR/HER3/AKT signaling pathway. These results provide
therefore a strong rational for IGF-1R and EGFR cotargeting
in human HCC to maximize antitumoral effect and to prevent
the early development of resistance. Clinical studies are current-
ly ongoing to validate this combinatory approach.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank the Tumorothèque Cancerest, hôpital Saint-Antoine, Paris
(Pr Jean-François Fléjou, Pr Dominique Wendum), for providing us with
human biopsies, Dr Christine Perret for providing us with cell lines and to
Roland Delelo for human hepatocytes, and cytometry platform (IFR65) for
fluorescence-activated cell sorting analysis.
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