Targeting Aberrant PI3K/Akt Activation by PI103 Restores Sensitivity … · Targeting Aberrant...

Cancer Therapy: Preclinical

Targeting Aberrant PI3K/Akt Activation by PI103 Restores Sensitivityto TRAIL-Induced Apoptosis in Neuroblastoma

Daniela Opel1, Ivonne Naumann1,2, Maxi Schneider1, Daniela Bertele1, Klaus-Michael Debatin1, andSimone Fulda2

AbstractPurpose: Because we recently identified Akt activation as a novel poor prognostic indicator in

neuroblastoma, we investigated whether phosphoinositide 30-kinase (PI3K) inhibition sensitizes neuro-

blastoma cells for TRAIL-induced apoptosis.

Experimental Design: The effect of pharmacological or genetic inhibition of PI3K or mTOR was

analyzed on apoptosis induction, clonogenic survival, and activation of apoptosis signaling pathways

in vitro and in a neuroblastoma in vivo model. The functional relevance of individual Bcl-2 family

proteins was examined by knockdown or overexpression experiments.

Results: The PI3K inhibitor PI103 cooperates with TRAIL to synergistically induce apoptosis (combina-

tion index < 0.1), to suppress clonogenic survival, and to reduce tumor growth in a neuroblastoma in vivo

model. Similarly, genetic silencing of PI3K significantly increases TRAIL-mediated apoptosis, whereas

genetic or pharmacological blockage of mTOR fails to potentiate TRAIL-induced apoptosis. Combined

treatment with PI103 and TRAIL enhances cleavage of Bid and the insertion of tBid into mitochondrial

membranes, and reduces phosphorylation of BimEL. Additionally, PI103 decreases expression of Mcl-1,

XIAP, and cFLIP, thereby promoting Bax/Bak activation, mitochondrial perturbations, and caspase-

dependent apoptosis. Knockdown of Bid or Noxa or overexpression of Bcl-2 rescues cells from PI103-

and TRAIL-induced apoptosis, whereas Mcl-1 silencing potentiates apoptosis. Bcl-2 overexpression also

inhibits cleavage of caspase-3, caspase-8, and Bid pointing to a mitochondria-driven feedback amplifica-

tion loop.

Conclusions: PI103 primes neuroblastoma cells for TRAIL-induced apoptosis by shifting the balance

toward proapoptotic Bcl-2 family members and increased mitochondrial apoptosis. Thus, PI3K inhibitors

represent a novel promising approach to enhance the efficacy of TRAIL-based treatment protocols in

neuroblastoma. Clin Cancer Res; 17(10); 3233–47. �2011 AACR.

Introduction

Neuroblastoma represents the most common extracra-nial solid tumor outside the central nervous system inchildhood (1, 2). The prognosis of children older than 1year with stage 4 disease is still very poor with long-termsurvival rates of only 40% despite intensive and multi-modal therapy (3). Therefore, novel strategies are requiredfor the treatment of advanced stage neuroblastoma.Programmed cell death or apoptosis is essential to main-

tain tissue homeostasis (4). In addition, induction of

apoptosis in malignant cells presents a key principle ofcancer therapy (5). There are 2 major pathways that initiateapoptosis and eventually lead to activation of caspases: oneis initiated by death receptors on the cell surface (deathreceptor or extrinsic pathway), whereas the other ismediated by the mitochondria (mitochondrial or intrinsicpathway; ref. 5). The death receptor pathway is typicallystimulated by ligation of death receptors of the TNF recep-tor superfamily by their cognate ligands or agonistic anti-bodies resulting in the formation of the death-inducingsignaling complex (DISC; ref. 6). This in turn leads tocaspase-8 activation, which can directly cleave downstreamcaspases (6). Stimulation of the mitochondrial apoptosispathway culminates in the permeabilization of the outermitochondrial membrane, the release of cytochrome c intothe cytosol, and caspase-3 activation via the apoptosomecomplex (7). Members of the Bcl-2 family proteins arecritical regulators of apoptosis by controlling outer mito-chondrial membrane permeabilization (8). Bcl-2 familyproteins can be divided into antiapoptotic proteins, such asBcl-2 and Mcl-1 or proapoptotic proteins like Bax, Bak,and BH3 domain-onlymolecules (e.g., Bid, Bim, andNoxa;

Authors' Affiliations: 1University Children's Hospital, Ulm and 2Institutefor Experimental Cancer Research in Pediatrics, Goethe-University, Frank-furt, Germany

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

D. Opel and I. Naumann share first authorship for this article.

Corresponding Author: Simone Fulda, Institute for Experimental CancerResearch in Pediatrics, Goethe-University, Frankfurt, Komturstr. 3a, 60528Frankfurt, Germany. Phone: 49-69-67866557; Fax: 49-69-6786659157.E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-10-2530

ClinicalCancer

Research

www.aacrjournals.org 3233

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Published OnlineFirst February 25, 2011; DOI: 10.1158/1078-0432.CCR-10-2530

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ref. 8). The ratio of pro- versus antiapoptotic Bcl-2 proteinsplays a critical role in determining whether a cell will die orsurvive (8).

TRAIL is considered as a promising candidate for cancertherapy, because it directly engages the death receptorpathway of apoptosis (6). Clinical trials show that TRAILor agonistic TRAIL receptor antibodies can be safely admi-nistered alone and in combination with chemotherapeu-tics (6, 9). However, many tumors present with primary oracquired resistance to death receptor–induced apoptosis,which may be due to aberrant activation of survival path-ways (10).

The phosphoinositide 30-kinase (PI3K)/Akt/mTOR path-way plays an important role inmediating prosurvival signals(11). Among its various functions, Akt inhibits apoptosiseither directly by phosphorylating apoptosis-signaling mo-lecules or indirectly by modulating the activity of transcrip-tion factors (12). In neuroblastoma, we recently identifiedPI3K/Akt as a new predictor of poor outcome, indicatingthat this pathway represents a clinically relevant target fortherapeutic intervention (13). We therefore investigatedin this study whether pharmacological inhibition of PI3Kcan be exploited as a novel strategy to restore the sensitivityof neuroblastoma toward TRAIL-induced apoptosis.

Materials and Methods

Cell culture and chemicalsNeuroblastoma cell lines were obtained from the Amer-

ican Type Culture Collection and maintained in Dulbecco’smodified Eagle’s medium (SH-EP), RPMI 1640 (LAN-5), orMEM (CHP-212) medium (Life Technologies, Inc.), supple-mented with 10% fetal calf serum (Biochrom), 1 mmol/Lglutamine (Biochrom), 1% penicillin/streptomycin (Bio-

chrom), and 25 mmol/L HEPES (Biochrom) as describedpreviously (14). N-benzyloxycarbonyl-Val-Ala-Asp-fluoro-methylketone (zVAD.fmk) was purchased from Bachem,PI103 from Alexis Biochemicals, LY294002 from Calbio-chem, and TRAIL was obtained fromR&D Systems. RAD001(everolimus) was kindly provided by Novartis Institute forBioMedical Research (Oncology Basel, Novartis Pharma AG,Basel, Switzerland). All other chemicals were purchased bySigma, unless otherwise indicated.

Determination of apoptosisApoptosis was determined by fluorescence-activated cell

sorting (FACScan; BD Biosciences) analysis of DNA frag-mentation of propidium iodide–stained nuclei asdescribed previously (14). The percentage of specific apop-tosis was calculated as follows: 100 � [experimental apop-tosis (%) � spontaneous apoptosis (%)]/[100% �spontaneous apoptosis (%)].

Colony forming assayTo determine colony formation, 200 cells were seeded in

a 6-well tissue culture plate and allowed to settle overnight.Cells were treated with TRAIL for 24 hours with or without0.6 mmol/L PI103. Next day, medium was exchanged andcolonies were stained after 10 days with 0.75% crystalviolet, 50% ethanol, 0.25% NaCl, and 1.57% formalde-hyde. Colonies were counted and the percentage of surviv-ing colonies relative to solvent-treated controls wascalculated.

Transient RNA interferenceFor transient gene knockdown cells were seeded, allowed

to settle overnight and transfected with 60 pmol of PIK3CAStealth RNAi (PIK3CAHSS10800 4-6; Invitrogene),PIK3CB Stealth RNAi (PIK3CBHSS10800 7-9; Invitro-gene), mTOR Stealth RNAi (FRAP1HSS10382 5-7), StealthRNAi against Bax (BAXHSS14135 4-6; Invitrogene), Bak(NM001188-875;473;613; Invitrogene), Bid (BIDHS-S14147 6-8; Invitrogene), or Stealth RNAi nontargetingcontrol (12935; Invitrogene) by using TransMessengertransfection (301525; Qiagen).

TransductionFor stable gene knockdown, short-hairpin RNA

(shRNA)-targeting Noxa (50-GATCCCCGTAATTATTGA-CACATTTCTTCAAGAGAGAAATGTGTCAATAATTACTTTT-TGGAAA-30 (15) or shRNA-targeting Mcl-1 (GGCAGTC-GCTGGAGATTAT, GATTGTGACTCTCATTTCT) and asequence with no corresponding part in the human gen-ome (GATCATGTAGATACGCTCA) that was used as con-trol were cloned into pRETRO-SUPER as previouslydescribed (16). Stable clones were generated by selectionwith 2 mg/mL puromycin (Clontech). For Bcl-2 over-expression, cells were transduced with murine stem-cellvirus (pMSCV) vector containing mouse Bcl-2 or emptyvector using the packaging cell line PT67 (BD Biosciences).Stable cell lines were selected by 10 mg/mL blasticidin(Invitrogen).

Translational Relevance

Recently, we identified aberrant Akt activation as anovel indicator of poor prognosis in neuroblastoma. Inthis study, we investigated whether the recently devel-oped PI3K inhibitor PI103 decreases the threshold forTRAIL to trigger apoptosis in neuroblastoma. Here, weprovide first evidence that PI103 primes neuroblastomacells for TRAIL-induced apoptosis in a synergistic man-ner in vitro and also in an in vivo model by shifting thebalance toward proapoptotic Bcl-2 family members andactivation of the mitochondrial apoptosis pathway. Thispreclinical evaluation of a rational combination of 2novel classes of targeted drugs, that is, the PI3K inhibitorPI103, and TRAIL, identifies a novel indication forPI103 in TRAIL-based combination therapies. This pro-vides the molecular basis for the design of future clinicalstudies in neuroblastoma and other cancers with aber-rant PI3K/Akt activation and thus has important clinicalimplications.

Opel et al.

Clin Cancer Res; 17(10) May 15, 2011 Clinical Cancer Research3234




Western blot analysisWestern blot analysis was carried out as described

previously (14) by using the following antibodies: rabbitanti–phospho-Akt (Ser473), rabbit anti–phospho-S6ribosomal protein (Ser235/236), mouse anti-S6 riboso-mal protein, rabbit anti-mTOR, rabbit anti–PI3K-p110a(1:1,000; Cell Signaling), mouse anti-Akt (1:500), mouseanti-XIAP (1:1,000; clone 28) and rabbit anti–Bcl-XL

(1:1,000; BD Transduction Laboratories), rabbit anti-sur-vivin (1:1,000; R&D Systems), mouse anti–caspase-8(1:1,000; Alexis Biochemicals), mouse anti-cFLIP(1:500; Alexis Biochemicals), rabbit anti–caspase-3, rab-bit anti-Bid and rabbit anti-Bim (1:1,000; Cell Signaling),

rabbit anti–caspase-9, mouse anti–Bcl-2, rabbit anti-Bak,mouse anti–cytochrome c (1:1,000; BD Pharmingen),mouse anti-Noxa (1:1,000; Alexis), rabbit anti–Mcl-1(Stressgen), rabbit anti-BaxNT (1:5,000; Upstate Biotech-nology), mouse anti-OxPhos Complex IV (1:2,000; Invi-trogen), or rabbit anti–PI3K-p110b (1:500; Abcam).Mouse anti–b-actin (1:10,000; Sigma), mouse anti-GAPDH (1:5,000; HyTest), or mouse anti–a-tubulin(1:3,000; Calbiochem) were used as loading controlsfollowed by goat anti-mouse immunoglobulin G (IgG)or goat anti-rabbit IgG conjugated to horseradish perox-idase (1:5,000; Santa Cruz Biotechnology). Enhancedchemiluminescence was used for detection (Amersham

Figure 1. Inhibition of PI3Ksensitizes neuroblastoma cells toTRAIL-induced apoptosis.A, effect of PI103 on PI3K/Akt/mTOR pathway activation inneuroblastoma cells. SH-EP,CHP-212, and LAN-5neuroblastoma cells were treatedfor 24 hours with PI103 atindicated concentrations, 20mmol/L LY294002 (LY) ordimethylsulfoxide (DMSO) assolvent (top) or with PI103 atindicated concentrations andtimes (bottom). Protein expressionlevel and phosphorylation statusof Akt and S6 ribosomal proteinwere analyzed by Westernblotting. b-Actin or a-tubulinserved as loading controls. B andC, PI103 sensitizesneuroblastoma cells to TRAIL-induced apoptosis. B, cells weretreated with TRAIL at indicatedconcentrations in the absence(white bars) or presence of 0.6mmol/L (SH-EP, CHP-212) or 3mmol/L (LAN-5) PI103 (black bars)for 24 hours (SH-EP, CHP-212) orfor 48 hours (LAN-5). Apoptosisinduced by PI103 alone: SH-EPcells, 3.45%; CHP-212 cells, 15%;LAN-5 cells, 3.16%.

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Targeting PI3K/Akt in Neuroblastoma

www.aacrjournals.org Clin Cancer Res; 17(10) May 15, 2011 3235




Bioscience). All Western blots shown are representative ofat least 2 independent experiments. Densitometry analy-sis was done by ImageJ.

Determination of mitochondrial membrane potentialand cytochrome c release

To determine mitochondrial transmembrane potentialcells were incubated with CMXRos (1 mmol/L; MolecularProbes) for 30 minutes at 37�C and immediately analyzedby flow cytometry.

Cytochrome c release was assessed by flow cytometryas previously described (17). To analyze cytochromec and Bid in cytosolic and mitochondrial extracts byWestern blot cells were harvested and washed withPBS. Cells were suspended in lysis buffer [2 mmol/LNaH2PO4, 16 mmol/L Na2HPO4, 150 mmol/L NaCl,500 mmol/L sucrose, 1 mmol/L Dithiothreitol (DTT),Protease Inhibitor (Roche), and 0.5 mg/mL digitonin]for 3 minutes on ice. Unbroken cells, mitochondria,

and nuclei were removed by centrifugation at14,000 rpm for 1 minute at 4�C. The supernatant wascollected as cytosolic fraction and the pellet was resus-pended in lysis buffer [30 mmol/L Tris HCL, 150 mmol/LNaCl, 1% Triton X, 10% glycerol (Invitrogen), ProteaseInhibitor (Roche), 2 mmol/L DTT, 500 mmol/L phenyl-methylsulfonylfluoride] for 2 hours at 4�C, and centri-fuged at 14,000 rpm for 20 minutes at 4�C. Thesupernatant was collected as mitochondrial fraction.Protein expression of cytochrome c or Bid were analyzedby Western blotting. OxPhos Complex IV, a-tubulin,and b-actin were used to control the purity and loadingof the mitochondrial and cytosolic fractions.

Determination of Bax and Bak activationBax and Bak activation was determined by immunopre-

cipitation as previously described (18). Briefly, cells werelysed in CHAPS lysis buffer (10 mmol/L HEPES, pH 7.4;150 mmol/L NaCl; 1% CHAPS). Protein (1 mg) was

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Figure 1. (Continued ). C, SH-EPcells were treated with 0.6 mmol/LPI103 and/or 2 ng/mL TRAIL forindicated times. Apoptosis wasdetermined by FACS analysis ofDNA fragmentation of propidiumiodide–stained nuclei andpercentage of specific apoptosisis shown. Data represent mean þSEM of 3 independentexperiments carried out intriplicate. For statistical analysis,Student's t test was carried outcomparing treatment with PI103versus treatment without PI103.*, P < 0.05; #,P < 0.001.D, inhibition of PI3K cooperateswith TRAIL to suppress colonyformation. SH-EP cells weretreated with PI103 or DMSO and/or 2 ng/mL TRAIL for 24 hours. Atday 10, colonies were stained withcrystal violet and counted. Onerepresentative experiment isshown (left). Colonies are shownas percentage of DMSO-treatedcontrol out of 5 experiments(right). #, P < 0.001.

Opel et al.





Figure 2. Inhibition of PI3K, ratherthan of mTOR, sensitizes SH-EPneuroblastoma cells to TRAIL-induced apoptosis. A, knockdownof PI3K sensitizes SH-EPneuroblastoma cells to TRAIL-induced apoptosis. SH-EP cellswere transfected with siRNAduplex oligoribonucleotidesagainst p100aand p110b, mTOR,or nontargeting siRNA (control).Knockdown of p110a, p110b, andmTOR expression and proteinexpression levels andphosphorylation status of Akt andS6 ribosomal protein wereanalyzed byWestern blotting at 72hours after transfection. a-Tubulinserved as loading control (left).Sevety-two hours aftertransfection, cells were treatedwith 2 ng/mL TRAIL for 24 hours.Apoptosis was determined byFACS analysis of DNAfragmentation of propidiumiodide–stained nuclei andpercentage of specific apoptosisis shown (right). Data representmean þ SEM of 3 independentexperiments carried out intriplicate (*, P < 0.05). B, inhibitionof mTOR by everolimus andrapamycin. SH-EP cells weretreated for 24 hours with0.6 mmol/L PI103, DMSO assolvent, everolimus, or rapamycinat the indicated concentrations.Protein expression level andphosphorylation status of Akt andS6 ribosomal protein wereanalyzed by Western blotting.a-Tubulin served as loadingcontrol. C, inhibition of mTOR failsto enhance TRAIL-inducedapoptosis. SH-EP cells were leftuntreated (med) or were treated for24 hours with 10 nmol/Leverolimus (dark gray bars), 10nmol/L rapamycin (light gray bars),0.6 mmol/L PI103 (black bar), orDMSO as solvent (white bars) incombination with TRAIL at theindicated concentrations.Apoptosis was determined byFACS analysis of DNAfragmentation of propidiumiodide–stained nuclei andpercentage of specific apoptosisis shown. Data represent mean þSEM of at least 3 independentexperiments carried out intriplicate. *, P < 0.05.

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incubated with 2 mgmouse anti-Bax antibody (6A7; Sigma)or anti-Bak antibody (AB-1; Calbiochem) overnight at 4�Cfollowed by addition of 10 mL Dynabeads Pan Mouse IgG(Dako), incubated for 2 hours at 4�C, washed with CHAPSlysis buffer, and were analyzed by Western blotting byusing rabbit anti-BaxNT or rabbit anti-Bak (BD Pharmin-gen) antibody.

Chorioallantoic membrane assayChorioallantoic membrane (CAM) assay was done as

described previously (17). Briefly, 2 � 106 cells wereresuspended in 10 mL serum-free medium and 10 mLMatrigel matrix (BD Biosciences) and implanted on theCAM of fertilized chicken eggs on day 8 of incubation,allowed for 48 hours to form tumors, and treated with1 mmol/L PI103 and/or 10 ng/mL TRAIL for 2 days. Fourdays after seeding tumors were sampled with the sur-rounding CAM, fixed in 4% paraformaldehyde, paraffinembedded, cut in 5 mmol/Lsections, and analyzed byimmunohistochemistry by using 1:1 hematoxylin and0.5% eosin. Images were digitally recorded at a magni-fication of �2 with an AX70 microscope (Olympus),

tumor areas were analyzed with ImageJ digital imagingsoftware.

Statistical analysisStatistical significance was assessed by Student’s t Test

(2-tailed distribution, 2-sample, unequal variance).

Results

Sensitization of neuroblastoma cells to TRAIL-induced apoptosis by the PI3K inhibitor PI103

To analyze the role of PI3K signaling in the regulation ofTRAIL-induced apoptosis in neuroblastoma, we selectedfrom a panel of human neuroblastoma cell lines SH-EP,CHP-212, and LAN-5 neuroblastoma cells, because they allexpress caspase-8, a key component of the death receptorpathway (data not shown). Initially, we investigated theoptimal conditions at which the recently developed class IPI3K inhibitor PI103 (19) blocks PI3K-mediated signalingin these cells. Dose–response and kinetic analysis revealedthat PI103 inhibits PI3K signaling at nanomolar concen-trations rapidly and for a prolonged time up to 48 hours

Time (h)API103TRAIL

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Figure 3. Inhibition of PI3Ksensitizes SH-EP neuroblastomacells to TRAIL-induced caspaseactivation, Bid cleavage, insertionof tBid into mitochondrialmembranes, and mitochondrialouter membrane permeabilization.A, SH-EP cells were treated with0.6 mmol/L PI103 and/or with 2 ng/mLl TRAIL for indicated times.Protein expression of caspase-8,caspase-9, caspase-3, Bid, anda-tubulin was assessed byWestern blot analysis; arrowheadsindicate cleavage fragments (top).SH-EP neuroblastoma cells weretreated with TRAIL in combinationwith 0.6 mmol/L PI103 with orwithout 20 mmol/L zVAD.fmk for24 hours. Apoptosis wasdetermined by FACS analysis ofDNA fragmentation by usingpropidium iodide and percentageof specific apoptosis is shown(bottom). Data represent mean þSEM of 3 independentexperiments carried out intriplicate (#, P < 0.001).

Opel et al.





(Fig. 1A). Importantly, PI103 significantly enhances TRAIL-induced apoptosis in a dose- and time-dependent manner(Fig. 1B and C). Calculation of combination index revealeda synergistic interaction of PI103 and TRAIL (Supplemen-tary Fig. S1). Also, PI103 significantly reduced colonyformation on TRAIL treatment (Fig. 1D), showing an effecton long-term survival. These data show that inhibition ofPI3K by PI103 sensitizes neuroblastoma cells to TRAIL-induced apoptosis.

Inhibition of PI3K is superior to mTOR inhibition forsensitizing SH-EP neuroblastoma cells to TRAIL-induced apoptosisAs we previously found that activation of Akt, but not of

mTOR correlates with poor prognosis in neuroblastoma,we asked whether mTOR signaling may differentially mod-ulate apoptosis sensitivity compared with PI3K. To addressthis question, we blocked PI3K/Akt/mTOR signaling selec-

tively at the level of PI3K versus mTOR by using RNAinterference (RNAi). Control experiments confirmed thatsilencing of the PI3K subunits p110a and p110b or ofmTOR resulted in selective downregulation of the respec-tive target proteins and to decreased phosphorylation ofAkt (in case of p110a/p110b knockdown) or of S6 ribo-somal protein (in case of mTOR or p110a/p110b knock-down; Fig. 2A). As silencing of mTOR inhibits bothmTORC1 and mTORC2 complexes, the increase in Aktphosphorylation in cells with mTOR knockdown (Fig. 2A)may be due to differential sensitivities of mTORC1 andmTORC2 complexes to incomplete mTOR inhibition.Interestingly, knockdown of p110a/p110b significantlyincreased TRAIL-induced apoptosis (Fig. 2A). The reducedefficacy of genetic versus pharmacological inhibition ofPI3K to confer sensitization to TRAIL-induced apoptosismay be due to incomplete knockdown of p110a/p110b. Bycomparison, silencing of mTOR did not alter sensitivity

Figure 3. (Continued ). B, SH-EPcells were treated with 0.6 mmol/LPI103 and/or 2 ng/mL TRAIL withor without 20 mmol/L zVAD.fmk for4 hours. Protein expression levelof cytochrome c (Cyt C), Bid,cytochrome oxidase (COX) IV,b-actin, and a-tubulin wereassessed byWestern blot analysisin the cytosolic and mitochondrialcompartment of fractionated SH-EP cells. C, SH-EP cells weretransfected with 3 different siRNAduplex oligoribonucleotidesagainst Bid (siBid Seq1–3) or withnontargeting control siRNA(sicontrol). Knockdown of Bidprotein was assessed by Westernblotting; b-actin was used asloading control (left). Seventy-twohours after transfection, cells weretreated with 5 ng/mL TRAIL and/or0.6 mmol/L PI103. Apoptosis wasdetermined by FACS analysis ofDNA fragmentation of propidiumiodide–stained nuclei (right). Datarepresent mean þ SEM of 3independent experiments carriedout in triplicate (#, P < 0.001).

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toward TRAIL (Fig. 2A). Similarly, pharmacological inhibi-tion of mTOR by everolimus and rapamycin failed tosensitize neuroblastoma cells for TRAIL-induced apoptosis,although both inhibitors suppressed phosphorylation ofS6 ribosomal protein at nanomolar concentrations (Fig. 2Band C). Doxorubicin was used as positive control foreverolimus-mediated enhancement of apoptosis in neuro-blastoma cells (20). These findings show that pharmaco-logical or genetic inhibition of PI3K is superior to mTORinhibition to sensitize neuroblastoma cells to TRAIL-mediated apoptosis.

TRAIL and PI103 cooperate to trigger Bid activationand mitochondrial outer membrane permeabilization

To gain insights into the molecular mechanismsmediating apoptosis sensitization by PI3K inhibition, wemonitored caspase cleavage byWestern blot analysis. Com-bined treatment of TRAIL and PI103 enhanced the cleavageof caspase-8, caspase-3, and caspase-9 as well as the con-version of Bid into tBid (Fig. 3A, top). To test the require-ment of caspases, we used the broad-range caspaseinhibitor zVAD.fmk. Apoptosis on treatment with TRAILand PI103 was completely blocked in the presence ofzVAD.fmk (Fig. 3A, bottom), showing that apoptosiswas mediated by caspases.

As tBid links the death receptor to the mitochondrialpathway by translocating tomitochondrial membranes, wethen analyzed tBid in the mitochondrial fraction. Interest-ingly, PI103 and TRAIL cooperated to cause accumulationof tBid at mitochondrial membranes (Fig. 3B). Silencing ofBid via siRNA significantly reduced apoptosis triggered byPI103 and TRAIL and also by treatment with TRAIL alone(Fig. 3C), showing the requirement of tBid for apoptosisinduction. Furthermore, PI103 significantly enhancedTRAIL-induced loss of mitochondrial membrane potential(MMP) and cytochrome c release (Fig. 3D). Translocationof tBid to mitochondrial membranes, loss of MMP, andcytochrome c release were all blocked in the presence ofzVAD.fmk (Fig. 3B, 3D), showing that they occur in acaspase-dependent manner. This set of experiments showsthat PI103 sensitizes neuroblastoma cells to TRAIL-inducedcaspase activation, Bid cleavage, translocation of tBid tomitochondria, and caspase-dependent mitochondrialouter membrane permeabilization.

PI3K inhibition shifts the balance towardproapoptotic Bcl-2 proteins

Next, wemonitored protein expression levels of key apop-tosis regulators byWestern blot analysis. Combination treat-ment of TRAIL and PI103 reduced protein levels of Mcl-1,XIAP, survivin, cFLIPL, and cFLIPS (Fig. 4A; SupplementaryFig. S2). Also, phosphorylation of BimEL was suppressedin cells treated with PI103 and TRAIL as indicated by thedownwardmobility shift of BimEL (Fig. 4A). By comparison,treatment with TRAIL increased the expression of Noxa,whereas expression levels of Bax, Bak, Bcl-2, or Bcl-XL werenot substantially altered (Fig. 4A; Supplementary Fig. S3).

As we observed a decline of cFLIPL and cFLIPS expressionon treatment with TRAIL and PI103 (Fig. 4A), we analyzedformation of the DISC as one of the earliest signaling eventon TRAIL receptor ligation and TRAIL receptor surfaceexpression. However, we found no detectable changes inthe recruitment of the adaptor molecule Fas-associatedprotein with Death Domain (FADD) or caspase-8 to sti-mulated TRAIL receptors within the first 4 hours on addi-tion of TRAIL or in surface expression of agonistic TRAILreceptors (Supplementary Fig. S4).

As Mcl-1 and Noxa are markedly downregulated andupregulated, respectively, on treatment with PI103 andTRAIL (Fig. 4A), we next explored their functional relevanceby using RNAi-mediated silencing. Knockdown of Noxasignificantly reduced, whereas silencing of Mcl-1 signifi-cantly increased apoptosis on combination treatment withTRAIL and PI103 (Fig. 4B). Also, apoptosis induced bytreatment with TRAIL alone was enhanced and reducedwhen Mcl-1 and Noxa were silenced, respectively (Fig. 4B).Also, knockdown ofMcl-1 led to increased PI103-mediatedapoptosis (Fig. 4B).

As activation of Bax and Bak presents a central eventin mitochondrial outer membrane permeabilization, wenext analyzed their activation status by using conforma-tion-specific antibodies. Notably, PI103 enhanced theTRAIL-mediated activation of Bax and Bak (Fig. 4C;

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Figure 3. (Continued ). D, SH-EP cells were treated with 0.6 mmol/L PI103and/or 2 ng/mL TRAIL for indicated times. Loss of MMP (top) andcytochrome c release (bottom) were assessed by flow cytometry. Datarepresent mean þ SEM of at least 3 independent experiments carried outin triplicate. *, P < 0.05; #, P < 0.001.

Opel et al.





Supplementary Fig. S5). Simultaneous knockdown of Baxand Bak significantly reduced TRAIL- and PI103-mediatedcell death (Fig. 4C), indicating that Bax and Bak areinvolved in PI103-conferred sensitization to TRAIL-induced apoptosis. Together, this set of experimentsindicates that changes in the expression or activation ofpro- and antiapoptotic Bcl-2 proteins present critical mole-

cular events for the PI103-mediated sensitization ofneuroblastoma cells toward TRAIL.

Overexpression of Bcl-2 prevents the synergisticinduction of apoptosis by PI103 and TRAIL

To further investigate whether the mitochondrial path-way is required for the synergistic interaction of TRAIL and

Figure 4. PI3K inhibition shift thebalance toward proapoptotic Bcl-2 proteins and promotes Bax/Bak-dependent apoptosis. A, SH-EPcells were treated with 0.6 mmol/LPI103 or DMSO and/or 2 ng/mLTRAIL for indicated times. Proteinexpression of phospho-Akt, Akt,phospho-S6, S6, cFLIP, XIAP,Mcl-1, survivin, Bax, Bak, BimEL,and Noxa were determined byWestern blotting. GAPDH andb-actin served as loading controls.The relative phosphorylation ofAkt was densitometricallydetermined and is expressed inpercentage of phospho-Akt/Aktratio, with DMSO set as 100%.Changes of expression level ofMcl-1, Noxa, cFLIP, survivin, andXIAP are densitometricallydetermined and are expressed inpercentage of protein/loadingcontrol ratio, with DMSO set as100%.

p-Akt

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ratio survivin/loading100 60 106 67 100 70 119 84 100 54 118 51 100 33 58 15

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PI103, we overexpressed Bcl-2 to interfere with mitochon-drial perturbations. Ectopic expression of Bcl-2 profoundlyreduced apoptosis on combination treatment with PI103and TRAIL and alsowith TRAIL alone (Fig. 5A). In addition,

Bcl-2 overexpression inhibited TRAIL-induced Bax activa-tion, loss of MMP, as well as cleavage of caspase-8 into p43/p41 and p18 active fragments, cleavage of Bid, and proces-sing of the caspase-3 p20 fragment into the p17/p12 active

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Figure 4. (Continued ). B, SH-EPcells were stably transduced witha vector containing shRNA againstMcl-1, Noxa, or control shRNA byusing pRETRO-SUPER vector.Knockdown of Mcl-1 or Noxaexpression was controlled byWestern blotting; a-tubulin servedas loading control (left).Transduced cells were treatedwith 0.6 mmol/L PI103 and/or 2 ng/mL TRAIL for 24 hours. Apoptosiswas determined by FACS analysisof DNA fragmentation ofpropidium iodide–stained nucleiand percentage of specificapoptosis is shown. Datarepresent mean þ SEM of at least2 independent experimentscarried out in triplicate (#, P<0.001). C, SH-EP cells weretreated with 0.6 mmol/L PI103 orDMSO with or without 2 ng/mLTRAIL for 4 hours. Bax- and Bak-activation were analyzed byimmunoprecipitation of proteinlysates by using activeconformation-specific antibodiesfor Bax and Bak and Westernblotting. D, SH-EP cells weretransfected with siRNA duplexoligoribonucleotides against Baxand Bak (black bars) or withnontargeting control siRNA (whitebars). Knockdown of Bax and Bakprotein expression was assessedbyWestern blotting; a-tubulin wasused as loading control (left).Seventy-two hours aftertransfection, cells were treatedwith 2 ng/mL TRAIL and/or 0.6mmol/L PI103. Apoptosis wasdetermined by FACS analysis ofDNA fragmentation of propidiumiodide–stained nuclei (right). Datarepresent mean þ SEM of 3independent experiments carriedout in triplicate (*, P < 0.05).

Opel et al.





fragments (Fig. 5B–D). Likewise, inhibition of caspases byzVAD.fmk inhibited Bax activation (Fig. 5B) and Bid clea-vage, its translocation to mitochondrial membranes, andloss of MMP (Fig. 3B andD). Together, these findings pointto a mitochondria-controlled feedback amplification loopfrom caspase-3 to caspase-8 activation, Bid cleavage, andmitochondrial outer membrane permeabilization.

PI103 cooperates with TRAIL to suppressneuroblastoma growth in vivoFinally, we investigated the antitumor activity of PI103

and TRAIL in vivo, using the CAM assay, an established invivo tumor model for neuroblastoma (17, 18, 21, 22), forexample. Neuroblastoma cells were seeded on the CAM ofchicken embryos, allowed to settle and to initiate tumors,and then treated with TRAIL in the presence or absence of

PI103. Importantly, PI103 and TRAIL acted in concert tosignificantly suppress tumor growth of neuroblastomacompared with either agent alone (Fig. 6). This shows thatPI103 cooperates with TRAIL to suppress neuroblastomagrowth in an in vivo model of neuroblastoma.

Discussion

We recently identified aberrant PI3K/Akt activation as anovel indicator of poor prognosis in neuroblastoma (13).Therefore, we investigated whether therapeutic targeting ofPI3K/Akt by the recently developed PI3K inhibitor PI103could restore apoptosis sensitivity in neuroblastoma. Here,we report for the first time that inhibition of PI3K is anefficient strategy to prime neuroblastoma cells to TRAIL-induced apoptosis. This conclusion is supported by several

Figure 5. Bcl-2 overexpressionprevents the synergistic inductionof apoptosis by TRAIL and PI103.A, SH-EP cells were transducedwith mouse Bcl-2 (mBcl-2) orpMSCV empty vector (EV).Overexpression of mBcl-2 wasassessed by Western blotting anda-tubulin served as loadingcontrol (left). Transduced cellswere treated for 24 hours with0.6 mmol/L PI103 and/or 5 ng/mLTRAIL (right). Apoptosis wasdetermined by FACS analysis ofDNA fragmentation of propidiumiodide–stained nuclei. Datarepresent mean þ SEM of 2independent experiments carriedout in triplicate (*, P < 0.05, #,P < 0.001). B–D, transduced cellswere treated for 4 hours with0.6 mmol/L PI103, and/or 2 ng/mLTRAIL. B, Bax activation wasanalyzed by immunoprecipitationof protein lysates by using theactive conformation-specificantibodies for Bax and Bak andWestern blotting.

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independent lines of evidence. First, pharmacological orgenetic inhibition of PI3K synergistically enhances TRAIL-induced apoptosis as shown by calculation of combinationindex. Second, data obtained in several neuroblastoma celllines underscore the generality of this finding. Third, thecombination treatment of PI103 and TRAIL is superior tosingle agents in suppressing long-term survival in colonyforming assays and cooperates to reduce tumor growth inan in vivomodel of neuroblastoma, underlining the clinicalrelevance of our findings.

Mechanistically, our data show for the first time thatPI3K inhibition acts in concert with TRAIL to cleave Bidinto tBid and to trigger accumulation of tBid at mitochon-drial membranes, an event that contributes to apoptosisinduction as shown by knockdown experiments (Fig. 6B).Interestingly, PI103 enhances TRAIL-induced cleavage ofcaspase-8 and Bid in a mitochondrial feedback amplifica-tion loop, which likely involves caspase-3–mediated acti-vation of caspase-8 and Bid, because Bcl-2 overexpressionblocks the PI103-mediated enhancement of TRAIL-induced cleavage of caspase-3, caspase-8, and Bid. Bycomparison, PI103 has no effect on the recruitment ofcaspase-8 to the TRAIL DISC. This caspase-3–driven mito-chondrial amplification loop is further facilitated by thePI103-mediated reduction of XIAP expression. To this end,Akt has been reported to protect XIAP from proteasomaldegradation via phosphorylation (23).

Furthermore, we found that PI103 reduces phosphoryla-tion of BimEL and Mcl-1 expression. The reduced phos-phorylation of BimEL by PI103 is consistent with the Akt-mediated phosphorylation of BimEL, which enhances itsproteasomal degradation (24). Mcl-1 downregulation byPI103 may be the result of reduced Mcl-1 transcription onPI3K/Akt inhibition (25). In addition, the rapid down-regulation of Mcl-1 points to PI103-mediated posttransla-tional events, for example, Mcl-1 degradation viainhibition of GSK3b phosphorylation on PI3K inhibition(26). In addition, Noxa exerts its proapoptotic function byantagonizing the antiapoptotic effect ofMcl-1 (27). The keyinvolvement of Mcl-1 and Noxa in this PI103-mediatedsensitization to TRAIL-induced apoptosis is supported bydata showing that knockdown of Noxa rescues SH-EPneuroblastoma cells from apoptosis, whereas knockdownof Mcl-1 leads to increased cell death. These alterations inthe expression or activation of tBid, Noxa, and Mcl-1 shiftthe balance toward proapoptotic Bcl-2 proteins, whichenhances TRAIL-induced Bax activation andmitochondrialouter membrane permeabilization in the presence ofPI103. Additionally, PI103 promotes Bax activation bypreventing the Akt-mediated inhibitory phosphorylationof Bax (28, 29). The key role of the mitochondrial pathwayis underscored by experiments carried out in Bcl-2 over-expressing cells, which are almost completely resistant toPI103- and TRAIL-induced apoptosis.

In contrast to PI3K inhibition, we found that genetic orpharmacological inhibition of mTOR does not confersensitivity to TRAIL-induced apoptosis. This result is inaccordance with our recent study showing that phosphor-ylation of S6 ribosomal protein does not correlate withpoor prognosis in neuroblastoma, whereas phosphoryla-tion of Akt does correlate (13). In glioblastoma, we simi-larly found that mTOR inhibition does not enhance TRAIL-or doxorubicin-induced apoptosis (20, 30). By compari-son, mTOR inhibitors have been reported to suppresstumor growth and angiogenesis in neuroblastoma cellswith MYCN amplification (31) and to induce apoptosistogether with chemotherapeutic agents (20, 32),implying a context-dependent role of mTOR signaling in

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Figure 5. (Continued ). C, MMP was assessed by flow cytometry.D, protein expression of caspase-8, caspase-3, Bid, murine Bcl-2(mBcl-2), and b-actin was assessed by Western blotting. Arrowheadsindicate cleavage fragments.

Opel et al.





neuroblastoma. In addition to neuroblastoma, PI3K inhi-bition has previously been reported to prime glioblastoma,leukemia, or colon carcinoma cells for TRAIL-inducedapoptosis (20, 33–35).Our findings have several important implications.

First, our study translates our recent identification ofPI3K/Akt as a potential therapeutic target in neuroblastoma(13) into the development of a novel strategy to primeneuroblastoma for TRAIL-induced apoptosis. Therapeuticintervention of aberrant PI3K/Akt activation in cancers,including neuroblastoma, is currently an area of high inter-est. So far, PI103 is described as a chemosensitizer incombination with anticancer drugs, for example, in T-cellacute lymphoblastic leukemia (36), glioblastoma (30, 37),or chronic lymphocytic leukemia (38). Our study identifiesa novel indication for PI103 by showing that PI103 sensi-

tizes for TRAIL-induced apoptosis. As TRAIL receptor ago-nists are currently evaluated as single agents in early clinicaltrials including childhood cancer (www.clintrials.org),TRAIL-based combination therapies, for example, by theaddition of a PI3K inhibitor, become relevant to maximizethe antitumor activity of TRAIL. This approach will likelybe effective in a subpopulation of neuroblastoma, becausecaspase-8 is frequently silenced by epigenetic mechanisms inneuroblastoma (18, 39–41).

Second, our data provide novel insights into the signaltransduction pathways, which are regulated by PI3K inhi-bitors and which can be exploited to prime cancer cells forTRAIL-induced apoptosis. Thus, beyond neuroblastomathese results will likely have an important impact on thedevelopment of combination therapies with PI3K inhibi-tors also in other types of cancer.

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Figure 6. PI103 cooperates with TRAIL to suppress neuroblastoma growth in vivo. A, SH-EP cells were seeded on the CAM of chicken embryos,allowed for 48 hours to form tumors and treated with 1 mmol/L PI103 and/or 10 ng/mL TRAIL for 2 days. Tumor growth was analyzed by hematoxylinand eosin–stained paraffin sections of the CAM. Shown are tumor area (left) and representative pictures of hematoxylin and eosin–stained sections ofthe CAM (right; bar, 1,000 mmol/L). Error bars, mean þ SD of 6 samples per group;. *, P < 0.05. Similar results were obtained in 2 independentexperiments. B, schematic overview of the mechanisms responsible for the cooperative induction of apoptosis by PI103 and TRAIL. PI103inhibits Akt phosphorylation, resulting in downregulation of Mcl-1 and cFLIP and decreased phosphorylation of Bim. PI103 and TRAIL cooperateto trigger cleavage of Bid into tBid and its insertion into mitochondrial membranes, Bax/Bak activation, mitochondrial outer membranepermeabilization (MOMP), caspase-3 activation, and apoptosis. A caspase-3–driven feedback amplification loop promotes caspase-8 and Bid cleavage.", upregulation/activation; #, downregulation; ee, inhibition. See text for more details.






In conclusion, this preclinical evaluation of a rationalcombination of 2 novel classes of targeted drugs, that is,the PI3K inhibitor PI103 and TRAIL, in relevant preclinicalin vitro and in vivo models of neuroblastoma providesthe molecular basis for the design of new combinationtherapies for the treatment of neuroblastoma. This strategymay help to overcome apoptosis resistance of neuro-blastoma and other cancers with aberrant activation ofPI3K/Akt.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank C.A. Schmitt (Berlin, Germany) for kindly providing mouseBcl-2 vector and the Novartis Institute for BioMedical Research for kindlyproviding everolimus.

Grant Support

This work has been partially supported by grants from the DeutscheForschungsgemeinschaft, Deutsche Krebshilfe, Novartis Stiftung f€ur therapeu-tische Forschung, Kind-Philipp-Stiftung, European Community (ApopTrain,APO-SYS), and IAP6/18 (to S. Fulda).

Received September 20, 2010; revised February 2, 2011; acceptedFebruary 3, 2011; published OnlineFirst February 25, 2011.

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2011;17:3233-3247. Published OnlineFirst February 25, 2011.Clin Cancer Res Daniela Opel, Ivonne Naumann, Maxi Schneider, et al. Sensitivity to TRAIL-Induced Apoptosis in NeuroblastomaTargeting Aberrant PI3K/Akt Activation by PI103 Restores

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