targeting the phosphoinositide 3-kinase p110-a isoform...

Cancer Therapy: Preclinical

Targeting the Phosphoinositide 3-Kinase p110-a IsoformImpairs Cell Proliferation, Survival, and Tumor Growth inSmall Cell Lung Cancer

AnnaWojtalla1, Barbara Fischer3, Nataliya Kotelevets2, Francesco A. Mauri5, Jens Sobek4, Hubert Rehrauer4,Carlos Wotzkow1, Mario P. Tschan1, Michael J. Seckl5, Uwe Zangemeister-Wittke2, and Alexandre Arcaro1,3

AbstractPurpose: The phosphoinositide 3-kinase (PI3K) pathway is fundamental for cell proliferation and

survival and is frequently altered and activated in neoplasia, including carcinomas of the lung. In this study,

we investigated the potential of targeting the catalytic class IA PI3K isoforms in small cell lung cancer (SCLC),

which is the most aggressive of all lung cancer types.

ExperimentalDesign:The expressionof PI3K isoforms inpatient specimenswas analyzed. The effects on

SCLC cell survival and downstream signaling were determined following PI3K isoform inhibition by

selective inhibitors or downregulation by siRNA.

Results:Overexpressionof the PI3K isoformsp110-a andp110-b and the antiapoptotic protein Bcl-2was

shown by immunohistochemistry in primary SCLC tissue samples. Targeting the PI3K p110-a with RNA

interference or selective pharmacologic inhibitors resulted in strongly affected cell proliferation of SCLC

cells in vitro and in vivo, whereas targeting p110-b was less effective. Inhibition of p110-a also resulted

in increased apoptosis and autophagy, which was accompanied by decreased phosphorylation of Akt

and components of the mTOR pathway, such as the ribosomal S6 protein, and the eukaryotic transla-

tion initiation factor 4E-binding protein 1. A DNA microarray analysis revealed that p110-a inhibition

profoundly affected the balance of pro- and antiapoptotic Bcl-2 family proteins. Finally, p110-a inhibition

led to impaired SCLC tumor formation and vascularization in vivo.

Conclusion: Together our data show the key involvement of the PI3K isoformp110-a in the regulationof

multiple tumor-promoting processes in SCLC. Clin Cancer Res; 1–10. �2012 AACR.

IntroductionLung cancer is a major cause of death in the developed

world and the commonest cancer killer in men. Small celllung cancer (SCLC) represents about 13% to 15% of allcases of lung cancer and is strongly associated with cigarettesmoking. Combinatorial chemotherapy regimens with eto-poside and platinum-based agents, as well as radiotherapy,are commonly used for the treatment of patientswith SCLC.However, an initial therapeutic response is usually followed

by disease recurrence in less than 1 year, and therefore theoverall 5-year survival rate is less than 5%. Consequently,novel therapeutic strategies are urgently required for SCLC.In the past years, an increasing number of molecular altera-tions involved in SCLC pathogenesis have been reported,including ectopic expression of neuroendocrine regulatorypeptides, upregulation of antiapoptotic Bcl-2 proteins,overexpression of myc family oncogenes and extracellularmatrix proteins, as well as genetic abnormalities in thetumor suppressor genes TP53 and RB (1, 2). In addition,it has been shown that polypeptide growth factors such ashepatocyte growth factor (HGF), fibroblast growth factor-2(FGF-2), insulin-like growth factor-1 (IGF-1), and stem cellfactor (SCF) control key biologic responses in human SCLCcells, including growth and proliferation, chemoresistance,andmigration (3–6). Downstreamof activated polypeptidegrowth factor receptors, activation of 2 major intracellularsignaling cascades, the phosphoinositide-3 kinase (PI3K)/Akt/mTOR, and the mitogen-activated Erk kinase (MEK)/extracellular signal–regulated kinase (ERK) pathway, havebeen found to be involved in the survival and proliferationof SCLC (3, 5–8). Furthermore, it has been reported thatmTOR and the ribosomal protein S6 kinases (S6K) are

Authors' Affiliations: 1Department of Clinical Research; 2Institute ofPharmacology, University of Bern, Bern; 3Division of Clinical ChemistryandBiochemistry, University Children'sHospital Zurich; 4FunctionalGeno-mics Center Zurich, ETH and University of Zurich, Zurich, Switzerland; and5Lung Cancer Biology Group, Division of Medicine, Imperial College Lon-don, London, United Kingdom

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Alexandre Arcaro, Division of Pediatric Hematol-ogy/Oncology, Department of Clinical Research, University of Bern, Tie-fenaustrasse 120c, Bern 3004, Switzerland. Phone: 41-31-632-32-87; Fax:41-31-308-8028; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-1138

�2012 American Association for Cancer Research.

ClinicalCancer

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overexpressed in SCLC cells, comparedwith normal humantype II pneumocytes (6). Therefore, targeting these path-ways with novel selective pharmacologic inhibitors maylead to the development of more effective therapies forSCLC.

The PI3K signaling pathway controls key cellularresponses, such as cell growth and proliferation, survival,migration, and metabolism. Over the last decades, it hasbeen recognized that this intracellular signaling pathway isfrequently activated by genetic and epigenetic alterations inhuman cancer, including lung cancer. The PI3K family ofsignaling enzymes comprises 8 catalytic isoforms, which aresubdivided into 3 classes. The class IA PI3K isoform p110-ais considered to be a validated drug target in human cancer(9–11), in particular because activating mutations in thePIK3CA frequently occur in human cancer. In SCLC, ampli-fication and mutations of the PIK3CA gene were identifiedand the p110-a and p110-b isoforms were found over-expressed in cell lines, in addition to deregulation of thePI3K/Akt/mTOR pathway (12).

Here, we report for the first time that targeting the class IAPI3K isoform p110-a blocks SCLC cell growth and survivalin vitro and in vivo and present evidence that this isoformplays a crucial role in Akt/mTOR pathway activation andBcl-2 family protein expression.

Materials and MethodsAntibodies and reagents

Antibodies: caspase-3, PARP, Akt, PI3K p110-b (SantaCruz Biotechnology), p-AktSer473, p-Bcl2Ser70, Bcl2, Bcl-XL,Bax, Bad, p-4EBP1Thr37/46, p-S6Ser235/236 or p-S6Ser240/244,S6 protein, PI3K p110-a (Cell Signaling Technology),b-actin (Sigma Aldrich), Mcl-1, and NF-kB (Epitomics).Etoposide, chloroquine, z-VAD-FMK, IkB-kinase (IKK)

inhibitor/wedelolactone (Calbiochem). RAD001 was sup-plied by the Novartis Institutes for BioMedical Research(Basel, Switzerland). The PI3K inhibitors PIK75, YM024,TGX221, and PI103 were the kind gift of Prof. ShaunJackson (Australian Centre for Blood Diseases, MonashUniversity, Victoria, Australia).

Cell lines, cell culture, and cell proliferationSCLC cell lines were obtained from the American Type

Culture Collection (Supplementary Table S2). The humanSCLC cell linesH69,H209,H510, and SW2were cultured inRPMI medium containing 10% heat-inactivated fetal calfserum (FCS). Cell viability was analyzed by using MTS(CellTiter 96 AQueous One Solution Cell ProliferationAssay, Promega).

Reverse transcription PCR and TaqMan analysisTotal cellular RNA was extracted using the RNeasy Mini

Kit (Qiagen) according to the manufacturer’s instruction.For each reverse transcription PCR (RT-PCR), 1 mg of totalRNA was used with the High Capacity cDNA Reverse Tran-scription Kit (Applied Biosystems). The reaction conditionswere used according to the manufacturer’s protocol. Taq-Man Gene Expression Assays for the PI3Ks p110-a andp110-b, as well as for VEGFA were done according to themanufacturer’s instructions (Applied Biosystems). As inter-nal control the expression of glyceraldehyde-3-phosphatedehydrogenase (GAPDH) was analyzed in each sample,separately.

ImmunohistochemistryImmunoreactivity was evaluated on commercial tissue

microarray (TMA) sections of SCLC [Biomax LC10010; 2cores: female 9 (22.5%), female age 32–66 years (meanvalue 52.5 years); male 31 (77.5%), male age 34–76 years(mean value 53.0 years); stage I 11, stage II 20, stage IIIa 7,stage IIIb 2; node-negative 12, node-positive 28 (22N1 and6 N2)] using the PI3K p110-a (Cell Signaling Technology,4249), p110-b (Abcam, ab55593), and Bcl-2 (Cell Signal-ing) antibodies in a modification of the antigen retrievaltechnique (13). The antibodies were used at a 1:200 dilu-tion for 1 hour at room temperature and then processedwith Polymer-HRP Kit (BioGenex) with diaminobenzidinedevelopment and Mayer hematoxylin counterstaining.Breast cancer tissue was used as positive external control.Negative controls were obtained by omitting the primaryantibody. A semiquantitative immunohistochemical (IHS)scorewas used, including assessment of both the percentageof positive cells and the intensity of staining. For theintensity, a score of 0 to 3, corresponding to negative, weak,moderate, and strong positivity, was recorded. The range ofpossible scores was thus 0 to 300. IHS and similar semi-quantitative scoring systems have been successfully used forTMA evaluation. Each core was scored individually. Oneobserver scored all cases, which were rechecked randomlyby the same investigator after a period of time.

Serial sections of paraffin-embedded chorioallantoicmembrane (CAM) tumors were dewaxed in xylol and

Translational RelevanceSmall cell lung cancer (SCLC) is a common form of

lung cancer and is associated with very unsatisfactorysurvival rates. Novel therapies are thus urgently requiredand will arise from a better understanding of the diseasebiology.Wewere interested in gaining further insight intothe potential of targeting phosphoinositide 3-kinase iso-forms in SCLC. In this report, we show that the catalyticp110-a isoform is overexpressed in a subset of primarySCLC samples. The growthof SCLC cellswas impairedontargeting p110-a using RNA interference or specific phar-macologic inhibitors. Inhibition of p110-a also inducedapoptosis and autophagy, which was paralleled by adecrease in the expression levels of antiapoptotic Bcl-2family proteins. Importantly, SCLC tumors treated withp110-a inhibitors displayed reduced proliferation andenhanced apoptosis in vivo. Together, the results pre-sented in this study show that specific p110-a inhibitorsmay in the future represent new drugs for SCLC.

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rehydrated through graded dilutions of ethanol. Antigenrecoverywas conductedwith citrate buffer (10mmol/L) in amicrowave (800 W) for 7 minutes. Endogenous peroxidaseactivity was blocked with 3% H2O2. Sections were incubat-ed in a humid chamber at 4�C overnight with or without(negative controls) active caspase-3 (Abcam, ab2302; recog-nizes the cleaved active p17 fragment) and Ki67 (NovusBiologicals, NB110-57147) antibodies. Biotin-conjugatedgoat anti-rabbit (Dako, E0432), and avidin–biotin–horse-radish peroxidase (HRP) complex (Vector Laboratories,Vectastain ABC Kit, PK6100) were used. Bound peroxidasewas detected with 3,30-diaminobenzidine substrate. Sec-tions were subsequently counterstained with hematoxylinand mounted in Aquatex (Merck).

Transient transfection and stable transductionof SCLCcellsSCLC cells were transiently transfected with either

nontargeting siRNA or SMARTpool siRNA duplexes tar-geting the PI3K p110-a or PI3K p110-b, using the AmaxaNucleofector system (Amaxa Biosystems), according tothe manufacturer’s protocol. Briefly, 1.5 � 106 SCLC cellsgrown in RPMI/10% FCS were transfected with 6 mL of20 mmol/L siRNA in 100 mL of Nucleofector Solution Vusing the program T-16 on the Amaxa Nucleofector. Fol-lowing transfection, cells were transferred into RPMI/10% FCS overnight, before they were used for experi-ments.Lentiviral vectors expressing short hairpin RNA (shRNA)

constructs specifically targeting the PI3K p110-a (Sigma-Aldrich) were tested for stable knockdown efficiency. Theconstructs shPI3KC_2892 and shPI3KC_3433 were chosenfor further experiments as they most efficiently silencedp110-a. The nontargeting vector SHC002 was used as acontrol. Lentivirus production and transduction of cells wasdone as described earlier (14). The vectors contain a puro-mycin resistance gene and stably transduced cell popula-tions were selected for 2 weeks using puromycin at theconcentration 1.5 mg/mL.

In vivo experimentsThe effect of the PI3K inhibitor PIK75 on the growth

and SCLC tumor formation in vivo were assessed on theCAM of chick embryos (15). Briefly, 3 � 106 H69 SCLCcells were placed in RPMI containing 50% BD MatrigelMatrix (BD Biosciences) on the CAM on embryonic day 9.Increasing concentrations of the p110-a inhibitors PIK75were deposited with the SCLC cells. Controls were treatedwith the solvent of the corresponding drug. CAMs wereexamined for vessel formation under a stereomicroscope.Tumor size and density of vessels per area around thetumor were determined using the software Vessel_tracer(16). The application of Matrigel without/with PIK75 onthe CAM was used as a negative control (SupplementaryFig. S3).

SDS-PAGE and Western blottingThe assays were done as described earlier (3).

DNA microarrayThe cDNAmicroarray analysis was conducted at the Func-

tional Genomic Center of the University of Zurich (Zurich,Switzerland). Gene expression data were obtained by hybrid-izing Human Genome U133 Plus 2.0 Affymetrix Gene Chipsarrays, on which more than 54,000 transcripts were repre-sented. Each experiment represented a group of 3 indepen-dent biologic replicates. Raw data generated by the GCOSSoftware (Affymetrix) were processed by using the log scalerobust multi-array analysis method (17) and further statisti-cally analyzed by using the software R and applying Student ttest. The GeneGO MetaCore (GeneGO) was used to definefunctional annotations for the selected genes, thus assigningthem to ontologic categories for association with relevantbiologic processes and pathways. The accession number ofthe data in Gene Expression Omnibus is GSE40564.

ResultsExpression of the PI3K p110-a and p110-b and theantiapoptotic protein Bcl-2 in SCLC patient samples

Our previous work in SCLC cell lines had revealed thatclass IA PI3K isoforms are overexpressed in comparisonwithtype II pneumocytes, which are one of the precursors ofSCLC. To investigate whether this finding could be con-firmed in primary tumors, a SCLC TMA was stained withantibodies specific for p110-a or p110-b (Fig. 1A). Thisanalysis revealed that, while normal lung tissue did notexpress detectable levels of either PI3K isoforms, p110-aand p110-b displayed enhanced expression in subsets ofprimary SCLC (Fig. 1A andB). Around25%of SCLC tumorsshowed overexpression of p110-a, whereas 18% of caseswere positive for p110-b (Fig. 1B). In general, SCLC tumorsdid not display concomitant overexpression of p110-a andp110-b, which was only present in 5% of the tumors (datanot shown). In addition, the expression of the antiapoptoticprotein Bcl-2 was assessed in SCLC patient samples. TheIHC staining of a TMA with a specific antibody revealed ahigh Bcl-2 overexpression in approximately 90% (35 of 39cases) of the SCLC patient samples (Fig. 1C and D), com-pared with normal lung tissue, which did not displaydetectable levels of Bcl-2.

Class IA PI3K p110-a inhibition or silencing blocks cellproliferation and Akt/mTOR signaling

Previous work had shown that targeting the PI3K/Akt/mTOR pathway by using small-molecule inhibitorsimpaired SCLC proliferation in vitro and in vivo. Therefore,we investigated the impact of isoform-specific inhibitors ofclass IA PI3K isoforms on the proliferation of SCLC celllines. The potent p110-a inhibitor PIK75 (IC50 in vitro¼ 7.8� 1.7 nmol/L; ref. 18) significantly impaired the prolifer-ation of 4 SCLC cell lines with IC50 values in the range of 50to 100 nmol/L (Fig. 2A). The p110-a inhibitor YM024,which is much less potent against the enzyme in vitro (IC50

in vitro¼ 0.3 mmol/L), also impaired the proliferation of theSCLC cell line panel, but at higher concentrations (Fig. 2A).The potent p110-b inhibitor TGX-221 (IC50 in vitro¼ 8.5�

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0.9 nmol/L; ref. 18) only partially impaired the prolifera-tion of SCLC cell lines, but at high concentrations (Fig. 2B).In contrast, the p110-d inhibitor IC87114 did not signifi-cantly impair the proliferation of the cell lines under study(Fig. 2B). We also evaluated the impact of the dual p110-a/mTOR inhibitor PI103 in the panel of SCLC cell lines.PI103 displayed antiproliferative activity against the SCLCpanel in vitro, with IC50 values in the range of 100 to 500nmol/L (Supplementary Fig. S1A).

The impact of the selective PI3K inhibitors on the acti-vation status of the Akt/mTOR pathway in SCLC cell lineswas then investigated by Western blot analysis (Fig. 2C).PIK75 and PI103 strongly affected the activation status ofAkt and the phosphorylation of the mTOR downstreamtargets ribosomal S6 protein and eukaryotic translation

initiation factor 4E-binding protein 1 (4E-BP1; Fig. 2C;Supplementary Fig. S1B). YM024 also impaired Akt activa-tion and partially inhibited mTOR pathway activation (Fig.2C; Supplementary Fig. S1B). In contrast, TGX221 was lesseffective at blocking the activation of the Akt/mTOR path-way (Fig. 2C; Supplementary Fig. S1B).

To validate our findings with pharmacologic inhibitors,we used RNA interference (RNAi) to downregulate theexpression of p110-aor p110-b in SCLC cell lines. Transientdownregulation of p110-a strongly impaired the activationof Akt and phosphorylation of the ribosomal S6 protein,whereas p110-b silencing was ineffective (Fig. 3A). In addi-tion, p110-a silencing induced a significant decrease in theproliferation of SCLC cells (40% reduction), whereas p110-b downregulation was less effective (25% reduction; Fig.

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Figure 1. Expression of the PI3K p110-a and p110-b, and the antiapoptotic protein Bcl-2, in SCLC patient samples. A, immunostaining with PI3K p110-a andp110-b showing negative SCLC staining and representative p110-a and p110-b SCLC positive staining, compared with normal lung tissue. B, analysis of theIHC staining PI3K p110-a and p110-b in SCLC patient samples. C, immunostaining with Bcl-2 showing negative and representative Bcl-2 intermediate andstrongly positive SCLC staining. D, the mean IHC staining scores Bcl-2 in 20 paraffin-embedded specimens each of normal lung tissue and of 40 SCLCparaffin-embedded specimens. IHC scores ¼ percentage of positive cells � staining intensity (for details see Materials and Methods).

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3B). We also used lentiviral delivery of shRNAs targetingp110-a in SCLC cell lines. Also, this approach resulted instable silencing of p110-a, robust affected PI3K down-stream signaling (Fig. 3C), and SCLC cell proliferation waspartially impaired by shRNA targeting of p110-a (Fig. 3D).Together these results show that targeting p110-a selec-

tively impairs cell proliferation and activation of the Akt/mTOR pathway in SCLC cell lines in vitro.

Class IA PI3K p110-a inhibition induces apoptosis andautophagy in SCLC cell linesPrevious work had shown that targeting the PI3K/Akt/

mTOR pathway by using small-molecule inhibitors (7, 19)induced apoptosis in SCLC cell lines. Therefore, we inves-tigated the impact of isoform-specific inhibitors of class IAPI3K isoforms on the survival of SCLC cell lines. Treatmentof a panel of SCLC cell lines with PIK75 or YM024 inducedcleavage of PARP and reduction of procaspase-3, which aremarkers of apoptosis induction (Fig. 4A), whereas TGX221was less efficient at inducing apoptosis (Fig. 4A). Theinduction of apoptosis upon p110-a inhibition seemed to

be stronger in the SCLC cell lines H69 andH209, comparedwith the effects observed in H510 (intermediate) and SW2(low; Fig. 4A). The caspase inhibitor zVAD-FMKwas able torescue 20%of thePIK75-induced apoptosis inH69 cells and10% of the TGX221-induced apoptosis (P < 0.01; Fig. 4B).Because the strongly affected cell viability in SCLC cellsupon p110-a inhibition could not only be explained bythe apoptotic cell response, we further investigated othercell death mechanisms, such as autophagy. Although theinhibition of p110-a with PIK75 or YM024 induced anincreased conversion of LC3-I to LC3-II, which is indica-tive for autophagic activity, a comparable response couldnot be observed in cells in which p110-b was inhibited(Fig. 4C; Supplementary Fig. S2). ATG5, another proteinused as a typical marker for autophagy, was also expressedin SCLC cells. Compared with the vehicle-treated control,enhanced ATG5 expression was not, or only slightly,observed in PIK75-treated H69 cells (Fig. 4C). Interesting-ly, at later time points, a decrease in ATG5 expressionwas observed (data not shown). Chloroquine is knownto inhibit autophagic activity due to deacidification of

Figure 2. PI3K inhibition blocks cellviability and PI3K downstreamsignaling. A and B, the SCLC celllines H69, H209, H510, and SW2were incubated with increasingconcentrations of the class I PI3Kinhibitors PIK75, YM024 (A),TGX221, and IC87114 (B) in serum-containingmedium. Cell viability wasassessed using the MTS assay after3 days. The data are mean with SDfrom 4 replicates and at least 3independent experiments. C, H69cells were incubated with increasingconcentrations of the PI3K p110-ainhibitors PIK75, YM024, and PI103(PI3K p110-a/mTOR inhibitor) andthe PI3K p110-b inhibitor TGX221.After 24 hours, the cells wereharvested and whole-cell lysatesanalyzedbySDS-PAGEandWesternblotting for the proteins indicated.

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lysosomes, followed by accumulation of ineffective auto-phagic vesicles. In H69 cells treated with the PI3K inhibi-tors PIK75 and TGX221, chloroquine was able to rescue20% (and 10%, respectively) of the autophagy-induceddecrease in cell viability (Fig. 4D), suggesting additiveroles, or cross-talk, between autophagy and apoptosis inp110-a–induced cell death.

Inhibition of the PI3K p110-a impairs SCLC tumorformation and vascularization in vivo

Together the data collected fromour experiments and theknown literature indicated an important role for p110-a inSCLC cell growth and survival processes in vitro and in vivo.To further investigate its impact on tumor formation andmaintenance, an in vivo assay was used to grow vehicle- andPIK75-treated H69 cells on the CAM of chick embryos.Tumor formation was clearly impaired upon PIK75 treat-ment, whichwas shown by strongly reduced tumor size andtumor weight compared with the control treatment (Fig.5A). In addition, IHC staining of PIK75-treated tumorsections showed a decreasing expression of the proliferationmarker Ki67 and a rising expression of the apoptoticmarkercleaved caspase-3 with increasing PIK75 concentrations(Fig. 5C and D). Finally, p110-a inhibition was able topartially prevent the vascularization on theCAMaround thetumorigenic area, as measured by the vessel density (Fig.5B). Another indication of impaired tumor vascularizationupon p110-a inhibition is the reduced VEGFA expressionobserved in the DNA microarray and quantitative RT-PCR

(qRT-PCR) analysis of PIK75-treated H69 cells (data notshown).

DNA microarray analysis of SCLC cells treated withisoform-specific PI3K inhibitors

To investigate whether the class IA PI3K isoform p110-acontrols the expression of specific gene subsets in SCLC,we conducted DNA microarray analysis in H69 cells treatedwith either vehicle, PIK75 targeting p110-a, or TGX221targeting p110-b (Fig. 6A). The efficacy of thedownregulationof the Akt/mTOR pathway by the respective inhibitors wasshown by Western blot analysis, as well as qRT-PCR forVEGFA expression (Fig. 2C and data not shown). Inhibitingp110-a significantly affected the expression of 3,411 genes(P value 0.01; FC � 1.5), whereas inhibiting p110-b result-ed in significant changes (P value 0.01; FC� 1.5) in 4 genes,suggesting an important role of p110-a, but not p110-b, forthe regulation of the expression of a subset of genes in SCLC(Fig. 6A). We next sought to investigate which genes wereselectively affected by p110-a inhibition, in comparison withp110-b. Among these genes, antiapoptotic proteins of theBcl-2 family of proteins were found to be more significantlydownregulated in SCLC cells treated with p110-a inhibitor,than in the case of the p110-b inhibitor (data not shown).

Theproteins of theBcl-2 family are downstream targetsof p110-a

To validate the Bcl-2 family proteins as targets of p110-ain SCLC cell lines, we used antibody arrays andWestern blot

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Figure 3. Silencing of p110-a butnot p110-b affects cell viability andPI3K downstream signalingactivation. A and C, H69 cells weretransiently transfected with siRNAconstructs targeting p110-a orp110-b, or nontargeting scrambledcontrol (A) or p110-a was stablysilenced by the lentiviral delivery ofshRNA constructs (C). Cell lysateswere analyzed by SDS-PAGE andWestern blottingwith antibodies forthe proteins indicated. B and D, cellviability of H69 cells transiently (B)or stably (D) transfected withconstructs targeting p110-a orp110-b was assessed using theMTS assay after 2 and 3 days. Anontargeting construct was usedas control.

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analysis to confirm the results obtained by DNA microar-rays. An analysis using antibody arrays revealed impairedAkt pathway activation and downregulation of antiapopto-tic Bcl-2 family proteins (data not shown). Downregulationof the expression and impaired activation of the antiapop-totic Bcl-2 family proteins could indeed be shown at theprotein level upon p110-a inhibition (Fig. 6B). As wasalready observed previously for the apoptotic response, theSCLC cell lines under study responded varyingly to p110-ainhibition. In particular, the cell lines H69 and H209displayed a strongly decreased expressionof theBcl-2 family

members Bcl-2, Bcl-XL, and Mcl-1, whereas SW2 cellsseemed to bemore resistant. In addition, p110-a inhibitioninduced the levels of the proapoptotic family members Badand Bax (Fig. 6B). In contrast to the inhibitors of p110-a,TGX221 had little effect on the expression levels of Bcl-2family proteins (Fig. 6B). The antiapoptotic Bcl-2 familyproteins Bcl-2 and Bcl-XL are known targets of the NF-kBtranscriptional network, which controls different cellresponses including immune response, cell proliferation,and survival (20). To gain insight into the transcriptionalnetworks affected by silencing of p110-a in SCLC cells, we

Figure 4. PI3K inhibition increasesapoptosis and autophagy in SCLC. Aand C, H69, H209, H510, and SW2cells grown in serum-containingmedium were incubated withincreasing concentrations of thePI3K p110-a inhibitors PIK75 andYM024, the PI3K p110-a inhibitorTGX221, PI103 (PI3K p110-a/mTORinhibitor), RAD001, or etoposide/cisplatin. After 24 hours, the cellswere harvested and cell lysatesanalyzedbySDS-PAGEandWesternblotting for the proteins indicated. Band D, H69 cells grown in serum-containing medium were incubatedwith the PI3K inhibitors PIK75 (0.05mmol/L) and TGX221 (10 mmol/L),rapamycin (0.1 mg/mL), or etoposide(10mmol/L) in absenceor presenceofzVAD-FMK (50 mmol/L) orchloroquine (20 mmol/L). Cellproliferation was assessed using theMTS assay after 12 hours. The dataare mean of 4 replicates and3 independently carried outexperiments. ��, P < 0.01; �, P < 0.05.

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conducted a biostatistical analysis of the gene expressiondata, using GeneGo. The transcriptional networks that weremost significantly altered comprised HNF4a, SP1, and c-Myc, the estrogen receptor (ER) and also NF-kB (Supple-mentary Table S1). We could indeed observe a deregulationof NF-kB expression in H69 cells treated with PIK75 andYM024, whereas p110-b inhibition did not have any effect(Fig. 6C). These results suggestedNF-kB to be a downstreamtarget of PI3K p110-a signaling in SCLC, and therefore, weassumed that targeting NF-kB may have an effect on theexpression of Bcl-2 and Bcl-XL. This hypothesis was con-firmed by the observation that the expression levels of Bcl-2were indeed decreased in H69 cells treated with wedelo-lactone, an inhibitor of the IKK (Fig. 6D).

DiscussionThe PI3K/Akt/mTOR pathway has been shown to play a

key role in SCLC cell proliferation, survival, chemoresis-tance, andmigration.Mutations in PIK3CA and gene ampli-fication were reported in primary SCLC, as well as increasedexpression of PIK3CA at themRNA and protein level. Broadspecificity PI3K/mTOR inhibitors have shown antitumoractivity in SCLC models in vitro and in vivo (21). In thisreport, we have investigated the impact of isoform-specificinhibitors and RNAi targeting class IA PI3K isoforms onSCLC cell responses. In general, agents targeting p110-areduced SCLC cell proliferation in vitro, impaired the SCLCtumor formation, which was accompanied by affectedvascularization in vivo and decreased the activation statusof classical PI3K downstream targets, such as Akt, mTOR,and S6K. Targeting p110-a by RNAi or isoform-specific

inhibitors had more pronounced effects on SCLC cellresponses than in the case of p110-b or p110-d, indicatinga selective role for p110-a in SCLC. In view of theseobservations, we hypothesized that p110-a may controlthe expression of a selective subset of genes implicated inSCLC cell proliferation and/or survival. The comparativeDNA microarray analysis of SCLC cell lines in which eitherp110-a or p110-b were inhibited by selective compoundsidentified such a gene subset. The Bcl-2 family of proteinswas validated as a downstream target of p110-a by acombination of approaches. The observation that Bcl-2expression was elevated in primary SCLC, in comparisonwith normal lung tissue further supports thismodel, in viewof the overexpression of p110-a. Importantly, the Bcl-2family of proteins has been previously shown to play acrucial role in the survival of SCLC cell lines in vitro and invivo (5, 22–26). The p110-a inhibitors induced increases inboth SCLC apoptosis and autophagy, which is consistentwith Bcl-2 family proteins being a target of p110-a. Bcl-2family proteins are key regulators of both apoptosis andautophagy (27) and their reduced expression upon inhibi-tion of the p110-a/NF-kB pathway may play an essentialrole in the effects of the p110-a inhibitors in SCLC.

We have previously evaluated the mTOR inhibitor ever-olimus in SCLC cell lines and found that it was effective in asubset of SCLC cell lines characterized by activation of theAkt/mTOR pathway and low expression levels of antiapop-totic Bcl-2 family proteins (24). In view of the resultsobtained with isoform-selective inhibitors of p110-a, it canbe speculated that these agents may bemore potent, as theyinduce a downregulation of antiapoptotic Bcl-2 family

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Figure 5. PI3K inhibition reducestumor formation andvascularization in vivo. A, H69 cellswere treated with vehicle ordifferent concentrations of PIK75and applied on the CAM of chickembryos. On day 13, pictures andtumors were taken to analyzetumor size and weight (A) andvessel density (B). B, analysis of thevessel density reduction uponPI3Kp110-a inhibition in vehicle- orPIK75-treated H69 tumors. C,immunostaining with Ki67 andcleaved caspase-3 comparing theexpression in vehicle- and PIK75-treated SCLC tumor sections. D,analysis of Ki67 or caspase-3positively stained tumor cellscomparing vehicle- and PIK75-treated H69 tumor sections.Positively stained cells (%) ¼number of positive cells/totalnumber of cells � 100. ��, P <0.001; ��, P < 0.01; �, P < 0.05.

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proteins and of the activity of the Akt/mTOR pathway. Thismay be particularly relevant for SCLC tumors bearing acti-vating mutations in the PIK3CA gene.Interestingly, our previous work in neuroblastoma has

shown that the class IA PI3K isoform p110-d contributes tocell proliferation andsurvival by controlling theactivationofthe mTOR pathway and the expression levels of antiapop-totic Bcl-2 family proteins. Overexpression of p110-d wasfound in a subset of primary neuroblastoma and cell lines,and p110-d was essential for signal transduction by receptortyrosinekinases, suchas the IGF-1RandEGF receptor (EGFR;ref. 28). In contrast, in SCLC cell lines, p110-a seems to playa more important role in the activation of the Akt/mTORpathway, which may explain the impact of agents targetingthis isoform on SCLC proliferation and survival. A previousreporthas shownthat the activityof anyclass IA PI3K isoformcanmaintaincell survival (29). Therefore, the relative impor-tance of class IA PI3K isoforms in selected cancer types maybe, in part, attributed to differences in expression levels.

Thus, targeting PI3K p110-a signaling may represent anattractive novel approach to develop novel therapies forSCLC. Indeed, different pharmacologic inhibitors of thisisoform now exist, which will soon enter clinical trials inoncology, and could, in the future, be developed as newdrugs for SCLC.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: A. Wojtalla, B. Fischer, N. Kotelevets, U. Zange-meister-Wittke, A. ArcaroDevelopment of methodology: A. Wojtalla, N. Kotelevets, U. Zangemeis-ter-Wittke, A. ArcaroAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): A. Wojtalla, B. Fischer, N. Kotelevets, J. Sobek, C.Wotzkow, M.P. Tschan, M.J. Seckl, U. Zangemeister-Wittke, A. ArcaroAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): A.Wojtalla, B. Fischer, N. Kotelevets, F.A.Mauri, J. Sobek, H. Rehrauer, A. Arcaro

Figure 6. The proteins of the Bcl-2family are downstream targets ofp110-a. A, gene expression analysisby DNAmicroarray: heatmap of geneexpression changes caused byp110-a inhibition. B–D, H69, H209,and SW2 (B) or H69 (C and D) cellsgrown in serum-containing mediumwere incubated with increasingconcentrations of the PI3K p110-ainhibitors PIK75 and YM024, thePI3K p110-b inhibitor TGX221,RAD001, and etoposide/cisplatin orthe IKK inhibitor wedelolactone (D).After 24 hours, the cells wereharvested and cell lysates analyzedby SDS-PAGE and Western blottingfor the proteins indicated.

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Writing, review,and/orrevisionof themanuscript:A.Wojtalla, B. Fischer,C. Wotzkow, M.P. Tschan, M.J. Seckl, U. Zangemeister-Wittke, A. ArcaroAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): A. Wojtalla, C. WotzkowStudy supervision: N. Kotelevets, A. Arcaro

Grant SupportThis study was supported by Association for International Cancer

Research, Wilhelm Sander-Stiftung, Novartis Stiftung f€ur Medizinisch-Bio-

logische Forschung, and Department of Clinical Research of the Universityof Bern.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 10, 2012; revised October 12, 2012; accepted October 21,2012; published OnlineFirst November 21, 2012.

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Published OnlineFirst November 21, 2012.Clin Cancer Res Anna Wojtalla, Barbara Fischer, Nataliya Kotelevets, et al. Small Cell Lung CancerImpairs Cell Proliferation, Survival, and Tumor Growth in

IsoformαTargeting the Phosphoinositide 3-Kinase p110-

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