Published OnlineFirst on January 10, 2011; DOI:10.1158/1078-0432.CCR-10-1993

Transient PI3K Inhibition Induces Apoptosis and Overcomes
HGF-Mediated Resistance to EGFR-TKIs in EGFR Mutant Lung
CancerIvan S. Donev, Wei Wang, Tadaaki Yamada, et al.
Clin Cancer Res Published OnlineFirst January 10, 2011.

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ClinicalCancerResearch

Cancer Therapy: PreclinicalTransient PI3K Inhibition Induces Apoptosis and Overcomes
HGF-Mediated Resistance to EGFR-TKIs in EGFR Mutant Lung Cancer
Ivan S. Donev1, Wei Wang1, Tadaaki Yamada1, Qi Li1, Shinji Takeuchi1, Kunio Matsumoto2,
Takao Yamori3, Yasuhiko Nishioka4, Saburo Sone4, and Seiji Yano1

Abstract
Purpose: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), such as gefitinib
and erlotinib, show favorable response to EGFR mutant lung cancer. However, the responders acquire
resistance almost without exception. We recently reported that hepatocyte growth factor (HGF) induces
EGFR-TKI resistance by activating MET that restores downstream mitogen activated protein kinase (MAPK)/
extracellular signal regulated kinase (ERK)1/2 and phosphoinositide 3-kinase (PI3K)/Akt signaling. The
purpose of this study was to determine whether inhibition of PI3K, a downstream molecule of both EGFR
and MET, could overcome HGF-mediated EGFR-TKI resistance in EGFR mutant lung cancer cells PC-9 and
HCC827.
Experimental Design: We explored therapeutic effect of a class I PI3K inhibitor PI-103 on HGF-induced
EGFR-TKI resistance in vitro and in vivo.
Results: Unlike gefitinib or erlotinib, continuous exposure with PI-103 inhibited proliferation of PC-9
and HCC827 cells, even in the presence of HGF. On the other hand, in gefitinib-resistant xenograft modelby using PC-9 cells mixed with HGF high producing fibroblasts, PI-103 monotherapy did not inhibit tumor
growth. However, PI-103 combined with gefitinib successfully regressed gefitinib-resistant tumor. In vitro
experiments by considering short half-life of PI-103 reveal that transient exposure of PI-103 combined withgefitinib caused sustained inhibition of Akt phosphorylation, but not ERK1/2 phosphorylation, resulting
in induction of tumor cell apoptosis even in the presence of HGF.
Conclusions: These results indicate that transient blockade of PI3K/Akt pathway by PI-103 and gefitinib
could overcome HGF-mediated resistance to EGFR-TKIs by inducing apoptosis in EGFR mutant lungcancer. Clin Cancer Res; 17(8); 2260–9. 2011 AACR.

Introduction
Lung cancer is one of the most prevalent malignancies
and the leading cause of cancer-related death worldwide.
Non–small cell lung cancer (NSCLC) accounts for nearly
80% of lung cancer cases. Substantial efforts are being madeto identify the optimal target for NSCLC therapy. The
tyrosine kinase inhibitors (TKI), such as gefitinib and erlotinib, have been shown to inhibit epidermal growth factor

Authors' Affiliations: 1Division of Medical Oncology; 2Division of Tumor
Dynamics and Regulation, Cancer Research Institute, Kanazawa University, Kanazawa; 3Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo;
4
Department of Respiratory Medicine & Rheumatology, Institute of Health
Biosciences, University of Tokushima Graduate School, Tokushima,Japan
Note: Supplementary data for this article are available at Clinical Cancer
Research Online (http://clincancerres.aacrjournals.org/).
Corresponding Author: Seiji Yano, Division of Medical Oncology, Cancer
Research Institute, Kanazawa University, Kanazawa, Ishikawa 920-0934,
Japan. Phone: þ81-76-265-2780; Fax: þ81-76-234-4524. E-mail:syano@staff.kanazawa-u.ac.jp
doi: 10.1158/1078-0432.CCR-10-1993
2011 American Association for Cancer Research.

2260

receptor (EGFR)-mediated downstream pathways, including mitogen activated protein kinase (MAPK)/extracellularsignal regulated kinase (ERK)1/2 and phosphoinositide 3kinase (PI3K)/Akt, and to show favorable activity in NSCLC
patients with mutant EGFR (1). Recent phase III clinical
trials showed that patients with EGFR mutant NSCLC had
superior outcomes with gefitinib treatment, compared withstandard first-line cytotoxic chemotherapy (2–3). However,the patients develop acquired resistance to EGFR-TKIs
almost without exceptions within a couple of years (4).
In addition, 20% to 25% patients with EGFR activating
mutations show intrinsic resistance to EGFR-TKIs.
Two genetically conferred mechanisms–T790M second
mutation in EGFR (4–5) and the MET gene amplification(6)—have been well reported to induce the acquired resistance to EGFR-TKIs in EGFR mutant lung cancer. Recently,
we identified a third mechanism, hepatocyte growth factor(HGF)-induced resistance. It induces EGFR-TKI resistanceby activating MET that restores phosphorylation of downstream MAPK/ERK1/2 and PI3K/Akt pathways (7–8). This
is not a genetically conferred mechanism and may be
involved in both intrinsic resistance and acquired resistance to EGFR-TKIs in EGFR mutant lung cancer (7).
Although HGF is reported to be produced predominantly

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Transient PI3K Inhibition Overcomes HGF-Mediated ResistanceTranslational Relevance
The acquired resistance to epidermal growth factor
receptor (EGFR) tyrosine kinase inhibitors (TKI) is oneof the most serious problems on the management of
EGFR mutant lung cancer. We recently reported thenovel mechanism that hepatocyte growth factor(HGF) induces EGFR-TKI resistance by activating METthat restores phosphorylation of downstream mitogenactivated protein kinase (MAPK)/extracellular signal
regulated kinase (ERK)1/2 and phosphoinositide 3kinase (PI3K)/Akt pathways.
In this study, we showed that transient blockade of
PI3K/Akt pathway by PI3K inhibitor and gefitinib could
overcome HGF-mediated resistance to EGFR-TKIs by
inducing apoptosis in both in vitro and in vivo models.
Our findings indicate usefulness of double blockade of
EGFR and PI3K, and further postulate the need todevelop PI3K inhibitor analogues with more suitable
pharamcokinetics and metabolic profiles for more successful therapy of EGFR mutant lung cancer.
by stromal cells, it can act both autocrine and paracrine
fashion when inducing resistance to EGFR-TKIs (7, 9).
More recent studies showed that HGF is frequently coexpressed along with the T790M second mutation in EGFR
(10) and MET gene amplification (8) in tumors of patientswith acquired resistance to EGFR-TKIs, indicating theimportance of HGF as therapeutic target for overcoming
resistance to EGFR-TKIs.
Several strategies are available to block HGF-MET–
mediated signaling, including ligand (HGF) blockade,
MET tyrosine kinase inhibition, and inhibition of downstream molecules (PI3K/Akt, MAPK/ERK; ref. 11). PI3Ks
are responsible for the generation of 3-phosphorylated
inositides, including the important second messenger
PtIns(3,4,5)P3 (antiphosphatidylinositol 3,4,5-triphosphate), resulting in activation of signal transduction pathways in many physiologic process (12). PI3Ks are divided
into 3 classes on the basis of their primary structures and invitro substrate specificity (13), with class I PI3Ks being themost well characterized. Class I PI3Ks can be further subdivided into class IA (p110a, p110b, and p110d) and class
IB (p110g) according to their structure and interaction with
p85 and p55 regulatory subunits. Class IA PI3Ks, eachcomposed of a p85 regulatory subunit and a p110 catalytic
subunit, are the most widely involved in cancer (14). The
major effector of PI3K in cancer is Akt, a serine–threonin
kinase that is directly activated in response to PI3K (13–14). Recent studies indicate that the PI3K/Akt pathway
plays crucial roles in resistance to various types of TKIs,
including EGFR TKIs (6, 15–17). Accordingly, a large
numbers of PI3K inhibitors are being developed (18).
We sought to determine whether inhibition of PI3K
signaling pathway could overcome EGFR-TKI resistanceinduced by HGF in EGFR mutant lung cancer. We foundthat transient exposure of class I PI3K inhibitor plus gefi-

www.aacrjournals.org

tinib was sufficient to overcome HGF-mediated resistanceby inducing apoptosis of EGFR mutant lung cancer cells.

Materials and Methods
Cell cultures and reagentsThe EGFR mutant human lung adenocarcinoma celllines, with exon 19 deletion in EGFR PC-9 (delE746_A750) and HCC827 (del E746_A750), were purchased from Immuno-Biological Laboratories Co. andAmerican Type Culture Collection, respectively (19).
The H1975 human lung adenocarcinoma cell line with
EGFR-L858R/T790M double mutation (20) was kindly
provided by Dr. J.D. Minna (University of Texas Southwestern Medical Center, Dallas, TX) and Dr. Y. Sekido(Aichi Cancer Center Research Institute, Nagoya, Japan).
Human lung embryonic fibroblasts, MRC-5, wereobtained from RIKEN Cell Bank. The PC-9, HCC827,
and H1975 cell lines were maintained in RPMI 1640
medium supplemented with 10% FBS and antibiotics.
The MRC-5 (p30–p35) was cultured in 10% FBS DMEM
(Dulbecco’s modied Eagle’s medium). All cells were passaged for less than 3 months before renewal from frozen,
early-passage stocks obtained from the indicated sources.
Cells were regularly screened for mycoplasma with theuse of a MycoAlert Mycoplasma Detection Kit (Lonza).
Gefitinib, erlotinib, PI-103 (PI3K a inhibitor 1), and
GDC-0941 were obtained from AstraZeneca, Chugai
Pharmaceutical Co., Calbiochem, and Selleck Chemicals,
respectively. Human recombinant HGF was prepared as
described previously (21).
Cell proliferation assay
Cell proliferation was measured by the MTT dye reduction method (22). Tumor cells (2  103/100 mL per well)
were plated into each well of 96-well plates in RPMI 1640
with 10% FBS. After 24 hours incubation, several concentrations of gefitinib, erlotinib, PI-103, and/or HGF wereadded to each well, and incubation was continued for afurther 72 or 48 hours. For short exposure to gefitinib and/or PI-103, tumor cells (8  103/800 mL per well) wereincubated in 24-well plates. After 24 hours incubation,
several concentrations of PI-103 and gefitinib were addedfor 1 hour, then washed 2 times with PBS, and thenreplated with fresh medium. Viability was assessed at 48
hours after initial exposure. Cell proliferation was determined with MTT solution (2 mg/mL; Sigma) as described
previously (7). Each experiment was done at least 3 times,
each with triplicate samples.
Determination of drug synergy
Cells were seeded at a density of 2  103 per well of a 96well plate. Concentration ranges were chosen to span thecomplete dose-response range of both drugs. All treatments
were done in quadruplicate. Cell proliferation/viability
was determined after 3 days by using MTT assay. Multiple
drug effect analysis was done by using CalcuSyn Software(Biosoft), which quantitatively describes the interaction

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Donev et al.

between 2 or more drugs (23). This method assigns combination index (CI) values to each drug combination anddefines drug synergy when a CI value is less than 1 or drug
antagonism when a CI value is greater than 1.
Coculture of lung cancer cells with fibroblasts
Cells were cocultured in transwell chambers separated by
8 mm pore filters. Tumor cells (8  103 cells/700 mL) withgefitinib and or PI-103 different doses were placed in thebottom chamber, and fibroblasts (104 cells/300 mL) wereplaced in the top chamber. After 72 hours, the top chamber
was removed and cell proliferation was measured by MTT
assay. For short exposure to PI-103 and/or gefitinib, theproliferation was assessed after 48 hours and drugs wereadministered for only 1 hour in different concentration,and then each well was washed twice with PBS and new
fresh media were added. Each experiment was done at least
3 times, each with triplicate samples.
Assay for RNA interference
Duplexed Stealth RNAi (Invitrogen) against Akt1-1 (50 AUACCGGCAAAGAAGCGAUGCUGCA-30 ), Akt1-2 (50 AACCCUCCUUCACAAUAGCCACGUC-30 ), and Akt1-3(50 -UAGCGUGGCCGCCAGGUCUUGAUGU-30 ) was used
for RNA interference assay. One day before transfection,aliquots of 2  104 tumor cells in 400 mL of antibiotic-free
medium were plated on 24-well plates. After incubation for
24 hours, the cells were transfected with siRNA (50 pmol)or scramble RNA by using Lipofectamine 2000 (1 mL) in
accordance with the manufacturer’s instructions. After 24hours incubation, the cells were washed with PBS andincubated with or without gefitinib (0.1 mmol/L) and/or
rhHGF (recombinant human HGF, 20 ng/mL) for an
additional 72 hours in antibiotic-containing medium. Cell
proliferation was measured by a Cell Counting Kit-8(Dojin) in accordance with the manufacturer’s instructions. Each experiment was done at least in triplicate
and 3 times independently.
Xenograft studies in SCID mice
Suspensions of PC-9 cells (5  106) with MRC-5 (5 106) were injected subcutaneously into the backs of 5week-old female severe combined immunodeficient mice
(SCID). Mice (n ¼ 6 per group) were randomized to: (a)control group, (b) gefitinib only (25 mg/kg/d) orally, (c)PI-103 only prepared in 20% 4-hydroxypropyl b-cyclodextrin (5 mg/kg/d) intraperitoneally, or (d) gefitinib (25 mg/kg/d) and PI-103 (5 mg/kg/d). After 4 days (tumors diameter >4 mm), the treatment was started. The tumor
volume was calculated (mm3 ¼ width2  length/2). All
animal experiments complied with the guidelines for theInstitute for Experimental Animals, Advanced Science
Research Center, Kanazawa University, Kanazawa, Japan(approval no. AP-081088).
Antibodies and Western blotting
For Western blotting analysis, 40 mg of total protein
were resolved by SDS polyacrylamide gel (Bio-Rad) elec-

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Clin Cancer Res; 17(8) April 15, 2011

trophoresis and the proteins were then transferred onto
polyvinylidene difluoride membranes (Bio-Rad). Afterwashing 4 times, membranes were incubated with
Blocking One (Nacalai Tesque, Inc.) for 1 hour at room
temperature and then incubated overnight at 4 C withthe following primary antibodies: anti-MET (25H2),
anti-phospho-MET (anti-p-MET, Y1234/Y1235; 3D7),
anti-p-EGFR (Y1068), anti-ErbB3 (1B2), anti-p-ErbB3(Tyr1289; 21D3), anti-Akt or p-Akt (Ser473), anticleaved caspase-9 (Asp315), anti-cleaved caspase-3(Asp175), anti-cleaved PARP (Asp214) antibodies(1:1,000 dilution, Cell Signaling Technology), antihuman EGFR (1 mg/mL), anti-human/mouse/rat ERK1/ERK-2 (0.2 mg/mL), or anti-p-ERK-1/ERK-2 (T202/
Y204; 0.1 mg/mL) antibodies (R&D Systems). Afterwashing thrice, membranes were incubated for 1 hourat room temperature with species-specific horseradish
peroxidase–conjugated secondary antibodies. Immunoreactive bands were visualized with SuperSignal West
Dura Extended Duration Substrate, an enhanced chemiluminescent substrate (Pierce Biotechnology). Eachexperiment was done at least thrice independently.
TUNEL assay
Terminal deoxynucleotidyl transferase–mediated nickend labeling staining was performed by the Apoptosis
Detection System (Promega). Briefly, the frozen tissue
sections (9 mm thick) were fixed with PBS containing
4% formalin. The slides were washed with PBS and permeabilized with 0.2% Triton X-100. The samples were thenequilibrated, and DNA strand breaks were labeled withfluorescein-12-dUTP (fluorescein-12-2-deoxy-uridine-5triphosphate) by adding nucleotide mixture and terminal
deoxynucleotidyl transferase enzyme. The reaction was
stopped with saline sodium citrate, and the localized green
fluorescence of apoptotic cells was detected by fluorescencemicroscopy (200).
Reverse transcriptase-PCR analysis
Total RNA was isolated from MRC-5 cells treated withvarious concentration of PI-103 for 24 hours, with ISOGEN
RNA extraction. Total RNAs were reversely transcribed byan Omniscript RT Kit (Qiagen) according to the manufacturer’s protocols. The primers for HGF and b-actin
were as follow: HGF forward, 50 -CAGTGTTGAGAAGTTGAATGC-30 , reverse, 50 -GTGTCATTCATAGTATTGTGAG30 , and b-actin forward, 50 -AAGAGAGGCATCCTCACCCT-30 , reverse, 50 -TACATGGCTGGGGTGTTGAA-30 . Polymerase chain reaction was done by Ex Taq Hot Start
Version (Takara). Cycles for HGF and b-actin were 28
and 26, respectively. The bands were visualized by ethidium bromide staining.
Cell apoptosis assay
Cell apoptosis induced by gefitinib was detected with an
Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences
Pharmingen) in accordance with the manufacturer’s protocols as described previously (7). The analysis was done

Clinical Cancer Research

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Transient PI3K Inhibition Overcomes HGF-Mediated Resistanceto irreversible EGFR-TKI that is expected to overcome
T790M second-mutation–mediated resistance to gefitinib
or erlotinib (25). Interestingly, continuous exposure (72hours) of PI-103 inhibited proliferation of H1975 cells in adose-dependent manner (Supplementary Fig. S1). HGF
slightly stimulated proliferation of H1975 cells andinduced the resistance to irreversible EGFR-TKI, CL387,785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]2-butynamide). However, HGF did not affect the sensitivityof H1975 cells to PI-103. These results suggest that PI-103
has potential to overcome HGF-induced resistance to not
only reversible EGFR-TKIs but also irreversible EGFR TKIs.
Moreover, combined use of PI-103 with CL-387,785
further inhibited proliferation of H1975 cells, irrespectiveof the presence of HGF.
We next examined whether PI-103 sensitized EGFR
mutant lung cancer cells when combined with gefitinibin the presence or absence of HGF. PI-103 inhibited theproliferation of PC-9 and HCC827 cells in a dose-dependent manner. Gefitinib markedly suppressed the proliferation, and HGF induced the resistance to gefitinib.
Surprisingly, PI-103 combined with gefitinib further inhibited the proliferation of PC-9 and HCC827 cells not only inthe absence of HGF but also in the presence of HGF(Fig. 2A). Proliferation data were analyzed by the established method of Chou and Talalay (23) by using CalcuSynsoftware. The resulting CI values were les than 1 over mostof the effect range of the drugs, demonstrating that the
combination of PI-103 and gefitinib inhibited the proliferation synergistically in PC-9 and HCC827 cells. Same
results were reproduced with GDC-0941–a derivative ofPI103 with improved pharmacokinetic and pharmacodynamic properties that are active against all isoforms of classon a FACSCalibur flow cytometer with CellQuest Software(Becton Dickinson).
Statistical analysis
Two-tailed Student’s t test was performed, when noted,
by GraphPad Software. Differences of P < 0.001 wereconsidered statistically different (24).

Results
Continuous exposure of PI-103 effectively suppresses
the in vitro proliferation of EGFR mutant lung cancer
cells in the presence of HGF
Both PC-9 and HCC827 cells were highly sensitive tocontinuous exposure (72 hours) to gefitinib and erlotinib. HGF alone did not affect proliferation of PC-9 cells,
but it slightly stimulated the proliferation of HCC827
cells. Under these experimental conditions, HGF dose
dependently induced resistance to gefitinib and erlotinibof PC-9 and HCC827 cells (Fig. 1), as reported previously(7). Under the same experimental conditions, continuous
exposure (72 hours) of PI-103 inhibited the proliferationof PC-9 and HCC827 cells though the IC50 (half maximal
inhibitory concentration) to PI-103 was higher (0.3mmol/L for HCC827; 0.8 mmol/L for PC-9 cells) thangefitinib (0.01 mmol/L for HCC827; 0.03 mmol/L for PC-9
cells) and erlotinib (0.01 mmol/L for HCC827; 0.02
mmol/L for PC-9 cells). Importantly, HGF did notdecrease the sensitivity of PC-9 and HCC827 cells to
PI-103, suggesting the potential of PI-103 to overcome
HGF-induced resistance to gefitinib and erlotinib in vitro.
We recently reported that HGF induces resistance in lungcancer cells (H1975) with EGFR T790M second mutation

100

60
40
20
0

*

*

*

*

*

0.01 0.03 0.1 0.3 1

3

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40

*

0

*

*

*

*

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3

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0.01 0.03 0.1 0.3 1

HCC827
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*

*

0 0.01 0.03 0.1 0.3

1

3

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120
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20
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*

*

*

3

)
PI-103 (μmol/L)(μ(μ)
Erlotinib (μmol/L)Ge
Gefitinibt b (μ(μmol/L)o/ )Gefitinib (μmol/L)

www.aacrjournals.org

120
100
80

120

120
100
80

0

%Growth%Figure 1. Continuous exposure of
PI-103 suppresses the in vitro
proliferation of EGFR mutant lungcancer cells, irrespective of thepresence of HGF. Tumor cells
were continuously treated withincreasing concentrations of
EGFR-TKI (gefitinib or erlotinib) or
PI-103, with or without HGF, andcell growth was determined after
72 hours by MTT assay. Datashown are the representative of 5
independent experiments. Error
bars indicate SD of triplicate
cultures. *, P < 0.001 (Student's
t test).

%GrowthPC-9

HGF (ng/ml)

*

*

*

0 0.01 0.03 0.1 0.3

1

3

50

140
120
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PI-103
PI 103 (μmol/L)

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Donev et al.

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PC-9

HCC827
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%Growth%Growth

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PI-103103 (1 μmol/L)
l/ )

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_

I PI3Ks and now used in clinical trials in patients with solid
tumors (26; Supplementary Fig. 2). This synergy was also
observed in a coculture system where HGF high producing
fibroblasts, MRC-5, induced gefitinib resistance. While PI103 did not inhibit HGF expression in MRC-5 cells (Supplementary Fig. S3), PI-103 synergistically inhibited theproliferation of PC-9 and HCC827 cells, irrespective of thepresence of MRC-5 cells, when combined with gefitinib(Fig. 2B). These results suggest that PI3K inhibitor combined with gefitinib may be more beneficial than either
monotherapy.
PI-103 with or without gefitinib suppresses PI3K/Akt
pathway even in the presence of HGF
To explore the molecular mechanism by which PI-103
combined with gefitinib showed greater antiproliferative
effect, we examined the phosphorylation of MET, EGFR,
ErbB3, and their downstream pathways (PI3K/Akt and
ERK1/2) by Western blotting (Fig. 3A). PC-9 and
HCC827 cells expressed EGFR, ErbB3, and MET proteins,
and these molecules were phosphorylated at various
levels. These receptors and downstream molecules, suchas Akt and ERK1/2, were also phosphorylated. While HGF
alone did not affect phosphorylation of EGFR or ErbB3, it
stimulated phosphorylation of MET and thereby activated
Akt and ERK1/2. Although gefitinib inhibited phosphorylation of Akt and ERK1/2 in the absence of HGF, it failed
to inhibited Akt and ERK1/2 phosphorylation in the presence of HGF. Importantly, PI-103 did not affect the phosphorylation of ERK1/2 or upstream molecules such as MET,EGFR, and ErbB3, but did inhibit the phosphotylation of
Akt, regardless of presence of HGF. In addition, PI-103

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Clin Cancer Res; 17(8) April 15, 2011

_

+

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+

+
_

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+
_

_

+
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+

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+

Figure 2. PI-103 combined withgefitinib overcomes HGF-induced
gefitinib resistance in vitro. A,
tumor cells were continuously
treated with various
concentrations of gefitinib and PI103, with or without HGF, and cell
growth was determined after 48
hours by MTT assay. B, tumor
cells were cocultured with humanlung fibroblasts (MRC-5) and were
continuously treated withindicated concentrations ofgefitinib, and/or PI-103. Cell
growth was determined after 48
hours by MTT assay. Data shown
are the representative of 3
independent experiments. Error
bars indicate SD of triplicate
cultures.

combined with gefitinib inhibited phosphorylation of both
Akt and ERK1/2 in the absence of HGF. The combination of
PI-103 and gefitinib inhibited Akt phosphorylation, even in
the presence of HGF, but failed to inhibit ERK1/2 phosphorylation. These results confirm our previous observations (7) and further suggest that PI-103 overcomes thisresistance by inhibiting phosphorylation of downstream
PI3K/Akt. The importance of PI3K/Akt as a target of PI-103
was further supported the evidence obtained in experimentswith siRNA for Akt. Treatment with siAkt1 alone knocked
down the Akt expression in PC-9 cells (Fig. 3B) and resulted
in inhibition of cell proliferation by 20% (Fig. 3C). Notably,
the treatment with siAkt1 reversed HGF-induced gefitinib
resistance in combination with gefitinib (Fig. 3C).
PI-103 combined with gefitinib overcomes HGFinduced gefitinib resistance in vivo
We recently established an in vivo model by inoculating
PC-9 cells premixed with HGF high producing MRC-5
cells and showed that gefitinib resistance that was abrogated by HGF-MET inhibition (9). By using this model,
we next evaluated whether PI-103 overcomes HGFinduced resistance to gefitinib in vivo. Consistent withprevious observations, we found that treatment withgefitinib alone prevented the enlargement of tumors
produced by the mixture of PC-9 cells and MRC-5 cells,
but did not cause tumor regression. Since gefitinib
induces tumor shrinkage of PC-9 tumors (9), our results
suggest that MRC-5 cells induced gefitinib resistance invivo. Under these experimental conditions, treatment
with PI-103 alone did not inhibit tumor growth, whereas
combined treatment with PI-103 and gefitinib dramati-

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Transient PI3K Inhibition Overcomes HGF-Mediated Resistance

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MET

GAPDH

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p-ERK1/2
ERK1/2
p-Akt

ScramblesiAkt1-2*

80*P < 0.001

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0
GAPDH

Geﬁnib
HGF

_
_

+
_

_
+

+
+

Figure 3. PI-103 with or without gefitinib suppresses PI3K/Akt pathway even in the presence of HGF. A, tumor cells were treated with or without gefitinib(1 mmol/L), PI-103 (1 mmol/L), and/or HGF (20 ng/mL) for 1 hour. Then, cells were lyzed, and the indicated proteins were detected by immunoblotting.
Data shown are the representative of 3 independent experiments. B, PC-9 cells were treated with 3 different siRNAs specific for Akt1 or scramble
siRNA. C, resultant cells were treated with gefitinib (0.1 mmol/L) and or HGF (20 ng/mL) for 48 hours. Then, MTT assay was performed. GAPDH, glyceraldehyde
3-phosphate dehydrogenase.

cally regressed the tumors (Fig. 4A and B). Similar results
were reproduced by GDC-0941 that is now used inclinical trials in patients with solid tumors (Supplementary Fig. S4). These combined treatments did not cause
obvious side effects, such as weight loss of the mice.
We further examined apoptotic cells in the tumors treated with gefitinib and/or PI-103. Although there were few
apoptotic cells in control-treated (Fig. 4C) or PI-103–treated tumors (Fig. 4D), discernible numbers of apoptotic
cells were detected in gefitinib-treated tumors (Fig. 4E).
However, more apoptotic cells were found in tumors
treated with both PI-103 and gefitinib (Fig. 4F; Fig. 4A
and B). Immunoblots with these tumors revealed thattreatment with PI-103 with or without gefitinib did notaffect the phosphorylation of ERK1/2. On the other hand,
PI-103 alone or in combination with gefitinib inhibited Akt
phosphorylation in the tumor. Most notably, PI-103 combined with gefitinib induced cleaved caspase-3, the effector
caspase that mediates death signaling (Fig. 4G). These
results strongly suggested the importance of PI3K/Akt asa target of this combined therapy.
Short exposure of PI-103 combined with gefitinib
further inhibits Akt mediating signal andproliferation of EGFR mutant lung cancer cells
Our finding that PI-103 monotherapy did not inhibit
tumor growth, whereas PI-103 overcame synergistically
HGF (MRC-5)-induced gefitinib resistance when combinedwith gefitinib, was unexpected. Although PI-103 has been

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reported to have very rapid tissue distribution and tissue
clearance in vivo (half-life of PI-103 in the major organs is0.7–1.3 hours; ref. 12), we hypothesized that rapid tissue
clearance of PI-103 might be responsible for its insufficienttherapeutic effect in vivo. To mimic pharmacodynamics of
PI-103 in vivo, we exposed PC-9 and HCC827 cells to PI103 transiently for 1 hour, washed the cells, and incubatedthe resultant cultures in fresh medium for 48 hours(Fig. 5A). Transient exposure to PI-103 resulted only inapproximately 15% inhibition of the proliferation of PC-9
or HCC827 cells, whereas transient exposure to gefitinib
resulted in higher inhibition of proliferation (>30%).
Importantly, transient exposure to both PI-103 and gefitinib inhibited the proliferation of these 2 cell lines, reaching
IC50 (Fig. 5B). Analysis using CalcuSyn software (23)indicates that the effect was synergistic. These phenomena
were also observed when EGFR mutant cancer cells were
cocultured with MRC-5 cells (Fig. 5C) to induce gefitinib
resistance, representing the therapeutic efficacy seen in vivo
model. In contrast, transient exposure to PI-103 and/or
gefitinib did not inhibit proliferation of MRC-5 cells(Fig. 5D).
We further evaluated the kinetics of PI3K/Akt phosphorylation after transient exposure of PC-9 cells to PI-103 andgefitinib (Fig. 6A). As shown earlier (Fig. 3), 1 hour treatment with either PI-103 or gefitinib completely inhibited
Akt phosphorylation in the absence of HGF. We found outthat in the absence of HGF, gefitinib, alone or combinedwith PI-103, inhibited Akt phosphorylation for up to

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Donev et al.

B

A

Tumor Volumee (mm3)

1,000

Control

PI-103Geﬁnib

100

*

*

*

*

*

7
9
11 13 15
Days aer inoculaon

17

19

Control
PI-103G
Geﬁnibﬁib
Geﬁnib+PI-103

10

Control

G

C

D

E

F

Geﬁnib+PI-1035

PI-1030Geﬁnib

1

p-Akt
Akt
p-ERK1/2
ERK1/2Cleavedcaspase-3
GAPDH

1 hour; subsequently, however, Akt phosphorylation in
PC-9 cells treated with PI-103 alone started to recover. In
the presence of HGF, gefitinib did not inhibit Akt phosphorylation, whereas Akt phosphorylation in PC-9 cells
treated with PI-103 alone recovered by 1 hour after washing. However, cells treated with PI-103 plus gefitinib
showed inhibition of Akt phosphorylation 1 hour after
washing (Fig. 6A). More importantly, transient exposure to
PI-103 plus gefitinib, but not either alone, resulted in the
induction of cleaved caspase-9 and caspase-3, the initiatorand effector caspases that mediate death signaling andcleaved PARP (Fig. 6A). Flow cytometry analyses with
Annexin V further confirmed that transient exposure with
PI-103 plus gefitinib induced apoptosis of HGF-treated PC9 cells (Fig. 6B). These findings indicate that transient
exposure to PI-103 combined with gefitinib is sufficientfor inducing death signaling even in the presence of HGF,
supporting the results observed in vivo experiments.

Discussion
Accumulating evidence indicate that HGF-MET axis isconsiderable therapeutic target for several solid tumors.
HGF can act as autocrine growth factors for glioblastoma,
thyroid cancer, and gastric cancer. Moreover, HGF stimulates the invasion and dissemination of various types ofcancers (27), and inducing EGFR-TKI resistance in EGFR
mutant lung cancer. In contrast to MET amplification–induced resistance, restoring the PI3K/Akt pathway andmediated by ErbB3 as an adaptor, HGF activates normal

2266

Clin Cancer Res; 17(8) April 15, 2011

Geﬁnib+PI-103

Figure 4. PI-103 combined withgefitinib overcomes HGF-induced
gefitinib resistance in vivo. A, PC-9
cells (5  106) mixed with MRC-5
cells (5  106) were inoculated
subcutaneously into SCID mice on
day 0. Mice received oral gefitinib(25 mg/kg/d) and/or
intraperitoneal PI-103 (5 mg/kg/d),
starting on day 4. The tumor size
was measured every 2 days andtumor volumes were calculated as
described in Materials andMethods. Data shown are therepresentative of 2 independent
experiments. Error bars indicate
standard errors of 6 mice. *, P <0.001 (Student's t test). B,
macroscopic appearances ofrepresentative tumors harvested
on day 19 are shown. Apoptotic
cells were stained by TUNEL
method as described in Materials
and Methods. C, control. D, PI103 alone. E, gefitinib alone. F,
gefitinib þ PI-103. G, tumors were
harvested 1 hour after treatment
on day 6. Tumor lysates wereanalyzed by immunoblotting with
the indicated antibodies.

MET receptor and induces the resistance that restores PI3K/
Akt pathway mediated by Gab1/2 as an adaptor (7–8).
HGF also accelerates the expansion of preexisting clones
with MET gene amplification and facilitates the inductionof EGFR-TKI resistance in a population of EGFR mutant
lung cancer (8). In addition, HGF frequently detected in
EGFR-TKI–resistant tumors with EGFR-T790M second
mutation and may induce the resistance to irreversible
EGFR-TKIs (25). These observations highlight an important role of HGF ligand in controlling of tumor progression
and drug sensitivity.
We have shown here that the combination of gefitinib
and class I PI3K inhibitors, PI-103 and GDC-0941, overcame HGF-mediated gefitinib resistance in EGFR mutant
lung cancer cells. Although HGF restored ERK1/2 andPI3K/Akt phosphorylation via MET activation even in
the presence of gefitinib, transient exposure of PI-103 plus
gefitinib efficiently inhibited PI3K/Akt phosphorylation,induced death signaling, and caused apoptosis of PC-9
cells. The combined treatment with PI-103 and gefitinib
did not inhibit phosphorylation of ERK1/2 (Fig. 3) or
STAT3 (data not shown). Recently, it was shown thattransient potent inhibition of BCR-ABL kinase activity is
associated with maximal clinical benefit in patients withchronic myelogenous leukemia (CML; ref. 28). Our results
illustrate the possibility that transient double blockade of
EGFR and PI3K may be useful for controlling HGF-induced
resistance to EGFR-TKIs in EGFR mutant lung cancer.
A large number of PI3K inhibitors are developed and arebeing evaluated in the preclinical and clinical trials (18,

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Transient PI3K Inhibition Overcomes HGF-Mediated Resistance

A

PC-9

ProtocolB

wash

MediumGeﬁnib(1 μmol/L)

PC-9

48 h

2h

Fresh medium

MTT

C%Growth%Growth
%

80
60

80
60

40

40

20

20
0
0

0.3

1

PI-103 (μmol/L)
MRC-5

D

HCC827
120

0
_

0.3
_

1
_

0

0.3

1

+

+

+

MRC-5

120
100
%Growth

100
80
60

80
60

40

40

20

20

0
PI-103 (μmol/L)

MediumGeﬁnib(1 μmol/L)

100

100

0
PI-103 (μmol/L)

Assay

PC-9

120

120

0

0.3

29). PI3K inhibitors have been found to induce G0/G1 cellarrest rather than apoptosis, and primarily causing stasis oftumor growth in vivo without substantial tumor shrinkage.
For example, intraperitoneal administration of high dosesof PI-103 (30–70 mg/kg) resulted in growth inhibition
rather than regression in a range of human tumor xenografts (12, 30). Moreover, in EGFR mutant or k-ras mutant
lung cancer models, tumor regression associating with
apoptosis was observed only when PI3K/Akt pathway
and MEK/MAPK pathway were simultaneously blocked(24, 31). Since these 2 pathways collaborate with eachother to maintain cell survival, simultaneous blockade ofboth pathways is necessary to induce apoptosis. Surprisingly, our in vitro experiments revealed that though transient exposure of PI-103 plus gefitinib failed to inhibit
ERK1/2 phosphorylation, it caused sustained PI3K/Akt
inhibition, induced proapoptotic molecules, such as
cleaved caspase-3 and caspase-9 and PARP, and thereby
induced PC-9 cell apoptosis even in the presence of HGF.
The mechanism by which combined use of PI-103 andgefitinib induces apoptosis of PC-9 cells, even in the presence of HGF, is not fully understood at present. One
possible explanation is that double blockade of PI3K/Akt
signaling pathway at upstream (EGFR level) and downstream (PI3K/Akt level) is efficient for inducing the apoptosis. The other possibility is that combined use of PI-103
and gefitinib inhibited the unknown pathway(s) that are
responsible for survival of EGFR mutant lung cancer cells.
While the combined therapy did not inhibit phosphorylation of STAT3 (data not shown) in PC-9 cells, we cannot

www.aacrjournals.org

1h±PI 103Geﬁnib ±PI-103%GrowthFigure 5. Short exposure of PI103 combined with gefitinib
effectively suppressed theproliferation of PC-9 and HCC827,
but not MRC-5 cells. A, protocol.
B, tumor cells were incubated withdifferent concentrations of PI-103
and/or gefitinib for 1 hour, washedtwice with PBS, and incubated in
fresh medium for 48 hours. C,
tumor cells were incubated with
MRC-5 cells and different
concentrations of PI-103 and/or
gefitinib for 1 hour, washed twicewith PBS, and coincubated with
MRC-5 cells in fresh medium for
48 hours. D, MRC-5 cells weretreated with different
concentrations of PI-103 and/or
gefitinib for 1 hour, washed twicewith PBS, and further incubated in
fresh medium for 48 hours. The
cell growth was determined by
MTT assay. Data shown are therepresentative of 3 independent
experiments. Error bars indicate
SD of triplicate cultures.

1

0
PI-103 (μmol/L)

0

0

0.3

1

0.3

1

Geﬁnib (μmol/L)

0

1

0

0

1

1

rule out the involvement of other unknown pathway(s).
Further experiments are warranted to clarify the mechanisms in future.
PI-103 is a class I PI3K inhibitor that reported favorable
antitumor activity without any obvious side effects in
preclinical animal models (12, 32). Pharmacokinetically,
PI-103 is metabolized to form glucuronide and is cleared
rapidly from plasma, with metabolysis of more than 70%
PI-103 after 30 minutes of incubation with human andmouse microsomes (12). This is consistent with our results,
showing that Akt phosphorylation in PC-9 cells treatedwith PI-103 alone was recovered by 1 hour (Fig. 6). PI-103
is not in clinical trials and work is now in progress to
optimize its pharmacokinetics properties by structuralmodification (13). While continuous exposure for 72
hours of PI-103 inhibited the proliferation of PC-9 and
HCC827 cells, transient exposure for 1 hour of PI-103
failed to do so in vitro condition. In vivo treatment with
PI-103 (5 mg/kg intraperitoneally, once a day) also failed
to inhibit the growth of PC-9 cells mixed with HGF highproducing fibroblasts (MRC-5). Collectively, the insufficient effect of PI-103 monotherapy may be, at least in part,due to short half-life and rapid metabolism of this drug invivo. GDC-0941 (5 mg/kg pharmacokinetics of oral, once aday) also failed to inhibit the growth of PC-9 cells mixed
with MRC-5 cells. Though GDC-0941 is a derivative ofPI103 with improved pharmacokinetic and pharmacodynamic properties, intratumoral concentration of GDC0941 might not be enough to inhibit tumor growth in
our in vivo experimental conditions. However, GDC-0941,

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Donev et al.

A

B

Wash
PC-9
cells

Wash
PC-9
cells

1h

1h

Geﬁnib± PI-103

Fresh medium± HGF

Harvest1h

48 h

Geﬁnib±PI-103Fresh medium
+ HGF
HGF

Geﬁnib
PI-103
HGF

_
_
_

+
_
_

_
+
_

+
+
_

_
_

+
_

+

+

_
+
+

HGF+PI-103

+
+
+

1.85%

p-Akt

1.28%

PI

Akt

Annexin V

Cleavedcaspase-9
Cleavedcaspase-3
CleavedPARP

HGF+Geﬁnib

GAPDH

HGF+Geﬁnib+ PI-103

2.01%

8.50%

Annexin V

like PI-103, successfully reversed the resistance when combined with gefitinib (Supplementary Fig. S4). Since we did
not use maximum tolerated dose of these PI3K inhibitors(70–150 mg/kg; refs. 12, 26), our findings suggest that
suboptimal dose of PI3K inhibitors may overcome HGFinduced resistance if combined with EGFR-TKIs.
In this study, we showed the possibility that double
blockade of PI3K/Akt signaling pathway at upstream(EGFR level) and downstream (PI3K/Akt level) may be
useful for overcoming HGF-induced resistance to EGFRTKIs in EGFR mutant lung cancer cells. This concept canalso be applicable to circumvent HGF-induced resistance toirreversible EGFR-TKIs (25). Moreover, cancer cell populations were recently shown to exhibit reversible tolerance to
EGFR-TKIs by maintaining a phenotypical distinct subpopulation of cells that can protect the overall populationfrom eradication by EGFR-TKIs (33). This reversible tolerance is mediated by activation of insulin like growth factor(IGF)-1 receptor (IGF-1R), but can be overcome by IGF-1R
inhibitor combined with gefitinib ((35). Since PI3K/Akt isinvolved in IGF-1R–mediating signaling, the combined useof PI3K inhibitor may also protect against the emergence ofreversibly tolerant subpopulations and may potently era-

Figure 6. Apoptosis and time
kinetics of phosphorylated Akt
and proapoptotic molecules after
short exposure to PI-103 and/or
gefitinib. A, PC-9 cells wereincubated (1 mmol/L), PI-103(1 mmol/L), and/or HGF(20 ng/mL) for 1 hour. Then, theresultant cultures were incubatedin fresh medium for 1 hour. Then,the cells were lyzed, and theindicated proteins were detectedby immunoblotting. B, PC-9 cells
were incubated with HGF (20 ng/mL) and PI-103 (1 mmol/L), and/or
gefitinib (1 mmol/L), for 1 hour, andthen washed 2 times with PBS.
The resultant cultures wereincubated in fresh medium for 24hours. The apoptotic cells were
determined by Annexin V assay kit
according to manufactory
protocol. Values shown arepercentage of apoptotic cells.
FL1-H and FL2-H, heights offluorescence intensity.

dicate EGFR mutant lung cancer. Further investigations in
PI3K/Akt signaling pathway are warranted for developing
more successful compounds with better activity and safety
for EGFR mutant lung cancer patients.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.

Acknowledgements
We thank Dr. J.D. Minna and Dr. Y. Sekido for kindly providing H1975
cells.

Grant Support
This study was supported by Grants-in-Aid of Cancer Research from the
Ministry of Education, Science, Sports, and Culture of Japan (S. Yano,
21390256 and 22112010A01).
The costs of publication of this article were defrayed in part by thepayment 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.
Received July 26, 2010; revised November 24, 2010; accepted December
18, 2010; published OnlineFirst January 10, 2011.

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