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Research article The Journal of Clinical Investigation http://www.jci.org      Volume 119      Number 5      May 2009  1359 Cannabinoid action induces autophagy- mediated cell death through stimulation of ER stress in human glioma cells María Salazar, 1,2 Arkaitz Carracedo, 1 Íñigo J. Salanueva, 1 Sonia Hernández-Tiedra, 1 Mar Lorente, 1,2 Ainara Egia, 1 Patricia Vázquez, 3 Cristina Blázquez, 1,2 Sofía Torres, 1 Stephane García, 4 Jonathan Nowak, 4 Gian María Fimia, 5 Mauro Piacentini, 5 Francesco Cecconi, 6 Pier Paolo Pandolfi, 7 Luis González-Feria, 8 Juan L. Iovanna, 4 Manuel Guzmán, 1,2 Patricia Boya, 3 and Guillermo Velasco 1,2 1 Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain. 2 Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain. 3 3D Lab (Development, Differentiation, and Degeneration), Department of Cellular and Molecular Physiopathology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain. 4 INSERM U624, Campus de Luminy, Marseille, France. 5 National Institute for Infectious Diseases, IRCCS “L. Spallanzani,” Rome, Italy. 6 Laboratory of Molecular Neuroembryology, IRCCS Fondazione Santa Lucia and Department of Biology, University of Rome “Tor Vergata,” Rome, Italy. 7 Cancer Genetics Program, Beth Israel Deaconess Cancer Center and Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA. 8 Department of Neurosurgery, University Hospital, Tenerife, Spain. Autophagy can promote cell survival or cell death, but the molecular basis underlying its dual role in cancer remains obscure. Here we demonstrate that Δ 9 -tetrahydrocannabinol (THC), the main active component of marijuana, induces human glioma cell death through stimulation of autophagy. Our data indicate that THC induced ceramide accumulation and eukaryotic translation initiation factor 2α (eIF2α) phosphorylation and thereby activated an ER stress response that promoted autophagy via tribbles homolog 3–dependent (TRB3-dependent) inhibition of the Akt/mammalian target of rapamycin complex 1 (mTORC1) axis. We also showed that autophagy is upstream of apoptosis in cannabinoid-induced human and mouse cancer cell death and that activation of this pathway was necessary for the antitumor action of cannabinoids in vivo. These findings describe a mechanism by which THC can promote the autophagic death of human and mouse cancer cells and provide evidence that cannabinoid administration may be an effective therapeutic strategy for targeting human cancers. Introduction Macro-autophagy,  hereafter  referred  to  as  “autophagy,”  is  a  highly conserved cellular process in which cytoplasmic materials  — including organelles — are sequestered into double-membrane  vesicles called autophagosomes and delivered to lysosomes for  degradation or recycling (1). In many cellular settings, triggering  of autophagy relies on the inhibition of mammalian target of rapa- mycin complex 1 (mTORC1), an event that promotes the activa- tion (de-inhibition) of several autophagy proteins (Atgs) involved  in the initial phase of membrane isolation (1). Enlargement of this  complex to form the autophagosome requires the participation of  2 ubiquitin-like conjugation systems. One involves the conjuga- tion of ATG12 to ATG5 and the other of phosphatidylethanol- amine to LC3/ATG8 (1). The final outcome of the activation of the  autophagy program is highly dependent on the cellular context  and the strength and duration of the stress-inducing signals (2–5).  Thus, besides its role in cellular homeostasis, autophagy can be a  form of programmed cell death, designated “type II programmed  cell death,” or play a cytoprotective role, for example in situations  of nutrient starvation (6). Accordingly, autophagy has been pro- posed to play an important role in both tumor progression and  promotion of cancer cell death (2–4), although the molecular  mechanisms responsible for this dual action of autophagy in can- cer have not been elucidated. Δ 9 -Tetrahydrocannabinol (THC), the main active component of  marijuana (7), exerts a wide variety of biological effects by mim- icking endogenous substances — the endocannabinoids — that  bind to and activate specific cannabinoid receptors (8). One of  the most exciting areas of research in the cannabinoid field is the  study of the potential application of cannabinoids as antitumoral  agents (9). Cannabinoid administration has been found to curb  the growth of several types of tumor xenografts in rats and mice  (9, 10). Based on this preclinical evidence, a pilot clinical trial has  been recently run to investigate the antitumoral action of THC on  recurrent gliomas (11). Recent findings have also shown that the  pro-apoptotic and tumor growth–inhibiting activity of cannabi- noids relies on the upregulation of the transcriptional co-activa- tor p8 (12) and its target the pseudo-kinase tribbles homolog 3  (TRB3) (13). However, the mechanisms that promote the activa- tion of this signaling route as well as the targets downstream of  TRB3 that mediate its tumor cell–killing action remain elusive.  In this study we found that ER stress–evoked upregulation of the  p8/TRB3 pathway induced autophagy via inhibition of the Akt/ mTORC1 axis and that activation of autophagy promoted the  apoptotic death of tumor cells. The uncovering of this pathway,  Conflict of interest: The authors have declared that no conflict of interest exists. Nonstandard abbreviations used: Atg, autophagy protein; eIF2α, eukaryotic  translation initiation factor 2α; MEF, mouse embryonic fibroblast; THC, Δ 9 -tetrahy- drocannabinol; mTORC1, mammalian target of rapamycin complex 1; PDI, protein  disulphide isomerase; TRB3, tribbles homolog 3. Citation for this article: J. Clin. Invest. 119:1359–1372 (2009). doi:10.1172/JCI37948. Downloaded from http://www.jci.org on March 12, 2015. http://dx.doi.org/10.1172/JCI37948

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  • Research article

    TheJournalofClinicalInvestigation http://www.jci.org Volume119 Number5 May2009 1359

    Cannabinoid action induces autophagy-mediated cell death through stimulation

    of ER stress in human glioma cellsMara Salazar,1,2 Arkaitz Carracedo,1 igo J. Salanueva,1 Sonia Hernndez-Tiedra,1 Mar Lorente,1,2

    Ainara Egia,1 Patricia Vzquez,3 Cristina Blzquez,1,2 Sofa Torres,1 Stephane Garca,4 Jonathan Nowak,4 Gian Mara Fimia,5 Mauro Piacentini,5 Francesco Cecconi,6 Pier Paolo Pandolfi,7 Luis Gonzlez-Feria,8 Juan L. Iovanna,4 Manuel Guzmn,1,2 Patricia Boya,3 and Guillermo Velasco1,2

    1Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain. 2Centro de Investigacin Biomdica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain.

    33D Lab (Development, Differentiation, and Degeneration), Department of Cellular and Molecular Physiopathology, Centro de Investigaciones Biolgicas, Consejo Superior de Investigaciones Cientficas (CSIC), Madrid, Spain. 4INSERM U624, Campus de Luminy, Marseille, France.

    5National Institute for Infectious Diseases, IRCCS L. Spallanzani, Rome, Italy. 6Laboratory of Molecular Neuroembryology, IRCCS Fondazione Santa Lucia and Department of Biology, University of Rome Tor Vergata, Rome, Italy. 7Cancer Genetics Program,

    Beth Israel Deaconess Cancer Center and Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA. 8Department of Neurosurgery, University Hospital, Tenerife, Spain.

    Autophagycanpromotecellsurvivalorcelldeath,butthemolecularbasisunderlyingitsdualroleincancerremainsobscure.Herewedemonstratethat9-tetrahydrocannabinol(THC),themainactivecomponentofmarijuana,induceshumangliomacelldeaththroughstimulationofautophagy.OurdataindicatethatTHCinducedceramideaccumulationandeukaryotictranslationinitiationfactor2(eIF2)phosphorylationandtherebyactivatedanERstressresponsethatpromotedautophagyviatribbleshomolog3dependent(TRB3-dependent)inhibitionoftheAkt/mammaliantargetofrapamycincomplex1(mTORC1)axis.Wealsoshowedthatautophagyisupstreamofapoptosisincannabinoid-inducedhumanandmousecancercelldeathandthatactivationofthispathwaywasnecessaryfortheantitumoractionofcannabinoidsinvivo.ThesefindingsdescribeamechanismbywhichTHCcanpromotetheautophagicdeathofhumanandmousecancercellsandprovideevidencethatcannabinoidadministrationmaybeaneffectivetherapeuticstrategyfortargetinghumancancers.

    IntroductionMacro-autophagy, hereafter referred to as autophagy, is ahighlyconservedcellularprocessinwhichcytoplasmicmaterialsincludingorganellesaresequesteredintodouble-membranevesiclescalledautophagosomesanddeliveredtolysosomesfordegradationorrecycling(1).Inmanycellularsettings,triggeringofautophagyreliesontheinhibitionofmammaliantargetofrapa-mycincomplex1(mTORC1),aneventthatpromotestheactiva-tion(de-inhibition)ofseveralautophagyproteins(Atgs)involvedintheinitialphaseofmembraneisolation(1).Enlargementofthiscomplextoformtheautophagosomerequirestheparticipationof2ubiquitin-likeconjugationsystems.Oneinvolvestheconjuga-tionofATG12toATG5andtheotherofphosphatidylethanol-aminetoLC3/ATG8(1).Thefinaloutcomeoftheactivationoftheautophagyprogramishighlydependentonthecellularcontextandthestrengthanddurationofthestress-inducingsignals(25).Thus,besidesitsroleincellularhomeostasis,autophagycanbeaformofprogrammedcelldeath,designatedtypeIIprogrammedcelldeath,orplayacytoprotectiverole,forexampleinsituations

    ofnutrientstarvation(6).Accordingly,autophagyhasbeenpro-posedtoplayanimportantroleinbothtumorprogressionandpromotionofcancercelldeath(24),althoughthemolecularmechanismsresponsibleforthisdualactionofautophagyincan-cerhavenotbeenelucidated.

    9-Tetrahydrocannabinol(THC),themainactivecomponentofmarijuana(7),exertsawidevarietyofbiologicaleffectsbymim-ickingendogenoussubstancestheendocannabinoidsthatbindtoandactivatespecificcannabinoidreceptors(8).Oneofthemostexcitingareasofresearchinthecannabinoidfieldisthestudyofthepotentialapplicationofcannabinoidsasantitumoralagents(9).Cannabinoidadministrationhasbeenfoundtocurbthegrowthofseveraltypesoftumorxenograftsinratsandmice(9,10).Basedonthispreclinicalevidence,apilotclinicaltrialhasbeenrecentlyruntoinvestigatetheantitumoralactionofTHConrecurrentgliomas(11).Recentfindingshavealsoshownthatthepro-apoptoticandtumorgrowthinhibitingactivityofcannabi-noidsreliesontheupregulationofthetranscriptionalco-activa-torp8(12)anditstargetthepseudo-kinasetribbleshomolog3(TRB3)(13).However,themechanismsthatpromotetheactiva-tionofthissignalingrouteaswellasthetargetsdownstreamofTRB3thatmediateitstumorcellkillingactionremainelusive.InthisstudywefoundthatERstressevokedupregulationofthep8/TRB3pathwayinducedautophagyviainhibitionoftheAkt/mTORC1axisandthatactivationofautophagypromotedtheapoptoticdeathoftumorcells.Theuncoveringofthispathway,

    Conflictofinterest:Theauthorshavedeclaredthatnoconflictofinterestexists.

    Nonstandardabbreviationsused:Atg,autophagyprotein;eIF2,eukaryotictranslationinitiationfactor2;MEF,mouseembryonicfibroblast;THC,9-tetrahy-drocannabinol;mTORC1,mammaliantargetofrapamycincomplex1;PDI,proteindisulphideisomerase;TRB3,tribbleshomolog3.

    Citationforthisarticle:J. Clin. Invest.119:13591372(2009).doi:10.1172/JCI37948.

    Downloaded from http://www.jci.org on March 12, 2015. http://dx.doi.org/10.1172/JCI37948

  • research article

    1360 TheJournalofClinicalInvestigation http://www.jci.org Volume119 Number5 May2009

    Downloaded from http://www.jci.org on March 12, 2015. http://dx.doi.org/10.1172/JCI37948

  • research article

    TheJournalofClinicalInvestigation http://www.jci.org Volume119 Number5 May2009 1361

    whichwebelieveisnovel,forpromotingtumorcelldeathmayhavetherapeuticimplicationsinthetreatmentofcancer.

    ResultsAutophagy mediates THC-induced cancer cell death.Asafirstapproachtogaininsightintothemorphologicalchangesinducedincan-cercellsbycannabinoidadministration,weperformedelectronmicroscopyanalysisofU87MGhumanastrocytomacells.Inter-estingly,doublemembranevacuolarstructureswiththemorpho-logicalfeaturesofautophagosomeswereobservedinTHC-treatedcells(Figure1,AC).TheconversionofthesolubleformofLC3(LC3-I) to the lipidatedandautophagosome-associated form(LC3-II)isconsideredoneofthehallmarksofautophagy(1),andthusweobservedtheoccurrenceofLC3-positivedotsaswellastheappearanceofLC3-II(Figure1D)incannabinoid-challengedcells.Inaddition,co-incubationwiththelysosomalproteaseinhibitorsE64dandpepstatinA,whichblocksthelaststepsofautophagicdegradation(14),enhancedTHC-inducedaccumulationofLC3-II(Figure 1E), confirming that cannabinoids induce dynamicautophagyinU87MGcells.Furthermore,incubationwiththecan-nabinoidreceptor1(CB1)antagonistSR141716preventedTHC-inducedLC3lipidationandformationofLC3dots(Figure1D),indicatingthatinductionofautophagybycannabinoidsreliesonCB1receptoractivation.Sinceautophagyhasbeenimplicatedinpromotionandinhi-

    bitionofcellsurvival,wenextinvestigateditsparticipationinthecancercelldeathinducingactionofTHC.Pharmacologicalinhibitionofautophagyatdifferentlevels(SupplementalFigure1,AC;supplementalmaterialavailableonlinewiththisarticle;doi:10.1172/JCI37948DS1)orselectiveknockdownofATG1(anessentialproteinintheinitiationofautophagy;ref.1)(Figure1,FandG),ATG5(anessentialproteinintheformationoftheautophagosome;ref.1)(SupplementalFigure1,DF),orAMBRA1(arecentlyidentifiedbeclin-1interactingproteinthatregulates

    autophagy;ref.15)(SupplementalFigure1,DF)stronglyreducedcannabinoid-inducedautophagyandcelldeath.Moreover,trans-formedAtg5-deficientmouseembryonicfibroblasts(MEFs),whicharedefectiveinautophagy(16),weremoreresistantthantheirwild-typecounterpartstoTHC-inducedcelldeath(Figure1H)anddidnotundergoautophagyuponcannabinoidtreatment(Figure1I).Takentogether,thesefindingsdemonstratethatautophagyplaysaprominentroleinTHC-inducedcancercelldeath.

    THC induces autophagy via ER stressdependent upregulation of p8 and TRB3.Inadditiontothepresenceofautophagosomes,electronmicroscopyanalysisofcannabinoid-treatedcellsrevealedthepres-enceofnumerouscellswithdilatedER(Figure2A).Inlinewiththisobservation,immunostainingoftheERluminalmarkerpro-teindisulphideisomerase(PDI)showedastrikingdilationintheERofTHC-treatedU87MGcells(Figure2B),aneventthatwasassociatedwithanincreasedphosphorylationofthesubunitofeukaryotictranslationinitiationfactor2(eIF2),ahallmarkoftheERstressresponse(17)(Figure2C).Inaddition,THC-inducedERdilationandeIF2phosphorylationwerepreventedbypharmaco-logicalblockadeoftheCB1receptor(Figure2,BandC).Time-courseanalysisofPDIandLC3immunostaining,eIF2

    phosphorylation,andLC3lipidationofcannabinoid-treatedcellsrevealedthatERstressoccurredearlierthanautophagy(Figure2,DandE).Ofinterest,cannabinoidadministrationproducedsimi-laractivationofERstressandautophagy,aswellascelldeath,inotherhumanastrocytomacelllines(SupplementalFigure2,AF),aprimarycultureofhumangliomacells(SupplementalFigure2,GI),andseveralhumancancercelllinesofdifferentorigin,includingpancreaticcancer(SupplementalFigure2,JL),breastcancer,andhepatoma(datanotshown).However,neitherERdila-tionnoreIF2 phosphorylationorautophagywasevidentinnor-mal,nontransformedprimaryastrocytes(SupplementalFigure3),whichareresistanttocannabinoid-inducedcelldeath(13).WenextinvestigatedwhetheractivationofERstressisinvolved

    intheinductionofautophagyinresponsetocannabinoidtreat-mentofcancercells.WehavepreviouslyshownthatTHC-inducedaccumulationofdenovosynthesizedceramide,aneventthatoccursintheER(18),leadstoupregulationofthestress-regulatedproteinp8anditsERstressrelateddownstreamtargets,ATF4,CHOP,andTRB3,toinducecancercelldeath(13).Ofimportance,incubationwithISP-1(aselectiveinhibitorofserinepalmitoyl-transferase,theenzymethatcatalyzesthefirststepofsphingo-lipidbiosynthesis;ref.18)preventedceramideaccumulation(Sup-plementalFigure4A);THC-inducedERdilation(SupplementalFigure4B);eIF2phosphorylation(Figure3A);p8,ATF4,CHOP,andTRB3upregulation(SupplementalFigure4C);andautophagy(Figure3B),supportingthatceramideaccumulationisinvolvedincannabinoid-triggeredERstressandautophagy.WealsoverifiedbymeansofRNAinterferencethatCaCMKKwhichhadbeenpreviouslyimplicatedinactivatingautophagyinresponsetoERstressassociatedcalciumrelease(19)wasnotinvolvedinTHC-inducedautophagyandcelldeath(datanotshown).Asphosphor-ylationofeIF2 onSer51attenuatesgeneralproteinsynthesiswhileenhancingtheexpressionofseveralERstressresponsegenes(17),weusedcellsderivedfromeIF2S51AknockinmicetotestwhethereIF2phosphorylationregulatestheexpressionofp8anditsdownstreamtargets.Inagreementwiththishypothesis,THCtreatment(whichpromotedceramideaccumulationinbothwild-typeandeIF2S51AimmortalizedMEFs;SupplementalFigure5A)triggeredp8,ATF4,CHOP,andTRB3upregulation(Figure

    Figure 1Inhibition of autophagy prevents THC-induced cancer cell death. (AC) Effect of THC on U87MG cell morphology. Representative elec-tron microscopy photomicrographs are shown (6 h). Scale bars: 500 nm. Note the presence of early (A, open arrows, and B) and late (A, filled arrows, and C) autophagosomes in THC-treated but not vehicle-treated (veh-treated) cells. (D) Top: Effect of SR141716 (SR1; 1 M) and THC on LC3 immunostaining (green) in U87MG cells (18 h; n = 3). The percentage of cells with LC3 dots relative to the total cell number is shown in the corner of each panel (mean SD). Scale bar: 20 m. Bottom: Effect of SR1 and THC on LC3 lipidation in U87MG cells (18 h; n = 3). (E) Effect of E64d (10 M) and pepstatin A (PA; 10 g/ml) on THC-induced LC3 lipidation in U87MG cells (18 h; n = 3). (F and G) Effect of THC treatment and transfection with control siRNAs (siC) or ATG1-selective siRNAs (siATG1) on cell viability (F; mean SD; n = 3), LC3 immunostaining (G, left panels; 18 h; percentage of cells with LC3 dots relative to the total number of cells cotransfected with a red fluo-rescent control siRNA, mean SD; n = 3; scale bar: 20 m), and LC3 lipidation (G, right panel; 18 h; n = 3) in U87MG cells. (H and I) Effect of THC on cell viability (H; mean SD; n = 3), LC3 immunostaining (I, left panels; 18 h; percentage of cells with LC3 dots relative to the total cell number, mean SD; n = 3; scale bar: 20 m), and LC3 lipidation (I, right panel; 18 h; n = 3) in Atg5+/+ and Atg5/ RasV12/T-large antigen MEFs. *P < 0.05 and **P < 0.01 compared with THC-treated U87MG (D) and Atg5+/+ (H and I) cells and compared with siC-transfected, THC-treated U87MG cells (F and G). THC concentration was 6 M.

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  • research article

    1362 TheJournalofClinicalInvestigation http://www.jci.org Volume119 Number5 May2009

    3C)aswellasautophagy(SupplementalFigure5B)inwild-typecellsbutnotintheireIF2S51Acounterparts.Wesubsequentlyaskedwhetherp8anditsdownstreamtar-

    getsregulateautophagy.Knockdownofp8orTRB3preventedTHC-inducedautophagy(Figure3,DandE)butnotERdilation(SupplementalFigure4D)inU87MGcells.Furthermore,THCinducedautophagy inp8+/+butnotp8-deficienttransformedMEFs(Figure3FandSupplementalFigure5C).Altogether,thesefindingsrevealthatTHCinducesautophagyofcancercellsvia

    activationofanERstresstriggeredsignalingroutethatinvolvesstimulationofceramidesynthesisdenovo,eIF2phosphoryla-tion,andp8andTRB3upregulation.

    THC inhibits Akt and mTORC1 via TRB3.InhibitionofmTORC1isconsideredakeystepintheearlytriggeringofautophagy(6).Wethereforetestedwhethercannabinoid-inducedupregulationofthep8pathwayleadstoautophagyviainhibitionofthiscom-plex.THCtreatmentofU87MGcellsreducedthephosphorylationofp70S6kinase(awell-establishedmTORC1substrate)andthe

    Figure 2ER stress precedes autophagy in cannabinoid action. (A) Effect of THC on U87MG cell morphology. Note the presence of the dilated ER in THC- but not vehicle-treated cells (6 h). Arrows point to the ER. Scale bars: 500 nm. (B) Effect of SR1 (1 M) and THC on PDI immunostaining (red) in U87MG cells (8 h; n = 3). The percentage of cells with PDI dots relative to the total cell number is shown in the corner of each panel (mean SD). Scale bar: 20 m. (C) Effect of SR1 (1 M) on THC-induced eIF2 phosphorylation of U87MG cells (3 h; OD relative to vehicle-treated cells, mean SD; n = 3). (D) Effect of THC on PDI (red) and LC3 (green) immunostaining in U87MG cells (n = 3). The percentage of cells with PDI or LC3 dots relative to total cell number at each time point (mean SD) is shown. Scale bar: 20 m. (E) Effect of THC on eIF2 phosphorylation and LC3 lipidation in U87MG cells (n = 3). **P < 0.01 compared with THC-treated (B) or vehicle-treated (C and D) cells.

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    TheJournalofClinicalInvestigation http://www.jci.org Volume119 Number5 May2009 1363

    Figure 3THC induces autophagy via ER stressevoked p8 and TRB3 upregulation. (A and B) Effect of ISP-1 (1 M) on THC-induced eIF2 phosphoryla-tion (A; 3 h; n = 3) and LC3 immunostaining (B, left panels; 18 h; percentage of cells with LC3 dots relative to the total cell number, mean SD; n = 3; scale bar: 20 m) in U87MG cells. sip8, p8-selective siRNA; siTRB3, TRB3-selective siRNA. (C) Effect of THC on p8, ATF4, CHOP, and TRB3 mRNA levels of eIF2 WT and eIF2 S51A MEFs as determined by real-time quantitative PCR (8 h; n = 3). Numbers indicate the mean fold increase SD relative to vehicle-treated eIF2 WT MEFs. (D) Top: Analysis of p8 and TRB3 mRNA levels. Results from a representative RT-PCR experiment are shown. The numbers indicate gene expression levels as determined by real-time quantitative PCR (mean fold change SD relative to siC-transfected cells; n = 5). Bottom: Effect of THC on LC3 immunostaining (green) of U87MG cells transfected with siC, sip8, or siTRB3 (18 h; n = 4). The percentage of cells with LC3 dots relative to cells cotransfected with a red fluorescent control siRNA is shown in each panel (mean SD). Scale bar: 20 m. (E) Effect of THC on LC3 lipidation in U87MG cells transfected with siC, sip8, or siTRB3 (18 h; n = 6). (F) Effect of THC on LC3 lipidation (top; 18 h; n = 5) and LC3 immunostaining (bottom; 18 h; percentage of cells with LC3 dots relative to the total cell number, mean SD; n = 4; scale bar: 40 m) in p8+/+ or p8/ MEFs. *P < 0.05 and **P < 0.01 compared with THC-treated U87MG (B), eIF2 WT (C), or p8+/+ (F) cells and compared with siC-transfected, THC-treated U87MG cells (D).

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    Figure 4THC inhibits the Akt/mTORC1 pathway via TRB3. (A) Effect of THC on p70S6K and S6 phosphorylation of U87MG cells (n = 6). (B) Effect of THC on cell viability (left panel; 24 h; mean SD; n = 6) and LC3 lipidation (right panel; 18 h; n = 4) in Tsc2+/+ and Tsc2/ MEFs. (C) Effect of THC on Akt, TSC2, PRAS40, p70S6K, and S6 phosphorylation of U87MG cells (18 h; OD relative to vehicle-treated cells, mean SD; n = 7). (D) Effect of THC on cell viability (left panel; 24 h; mean SD; n = 4) and LC3 lipidation (right panel; 18 h; n = 4) of pBABE and myristoylated Akt (myr-Akt) MEFs. (E) Effect of THC on Akt co-immunoprecipitation with TRB3 in U87MG cell extracts (8 h; OD relative to vehicle-treated cells, mean SD; n = 9; input: TRB3). (F and G) Effect of THC on Akt, TSC2, PRAS40, p70S6K, and S6 phosphorylation and LC3 lipidation (G only) of siC- and siTRB3-transfected (F; 18 h; OD relative to vehicle-treated siC-transfected U87MG cells, mean SD; n = 7; upper panel shows an analysis of TRB3 mRNA levels) and EGFP (Ad-EGFP) or rat TRB3 (Ad-TRB3) adenoviral vectorinfected (G; 18 h; OD relative to vehicle-treated Ad-EGFPinfected U87MG cells, mean SD; n = 4; upper panel shows an analysis of rTRB3 mRNA levels) U87MG cells. (H) Effect of THC on Akt, p70S6K, and S6 phosphorylation of p8+/+ and p8/ MEFs (n = 7). *P < 0.05 and **P < 0.01 compared with THC-treated Tsc2+/+ (B) and pBABE (D) MEFs and compared with vehicle-treated (C and E), vehicle-treated siC-transfected (F), or Ad-EGFPinfected (G) U87MG cells.

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    TheJournalofClinicalInvestigation http://www.jci.org Volume119 Number5 May2009 1365

    ribosomalproteinS6(awell-establishedp70S6kinasesubstrate)(Figure4,AandC),indicatingthatmTORC1isinhibitedincan-nabinoid-challengedcells.Inaddition,thecannabinoid-induceddecreaseinp70S6kinaseandS6phosphorylation,autophagy,andcelldeathwerenotevidentinTsc2/cells,inwhichmTORC1isconstitutivelyactive(20)(Figure4BandSupplementalFigure6,AandB),furthersupportingamajorroleformTORC1inhibitionintheinductionofautophagiccelldeathbycannabinoids.TheproteinkinaseAktpositivelyregulatestheactivityofthe

    mTORC1complexbyphosphorylatingandinhibitingTSC2andPRAS40(awell-establishedAktsubstratewithinthemTORC1complex).Thus,AktinhibitiondecreasesmTORC1activityandpromotesautophagy(20).Inlinewiththisidea,THCdecreasedthephosphorylationofAkt,TSC2,andPRAS40aswellasp70S6kinaseandS6(Figure4C).ThisinhibitionoftheAkt/mTORC1pathwaywasabrogatedbyincubationwithaCB1receptorantago-nist(SupplementalFigure6C)oraceramidesynthesisinhibitor(SupplementalFigure6D).Likewise,cellsoverexpressingamyris-toylated(constitutivelyactive)formofAktwereresistanttoTHC-inducedmTORC1inhibition,autophagy,andcelldeath(Figure4DandSupplementalFigure6,EandF),furthersupportingthatTHCinducesautophagyviaAktinhibition.SinceTRB3hasbeenshowntodirectlyinteractwithandinhibit

    Akt(21,22),weinvestigatedwhetherupregulationofTRB3wasresponsible forTHC-inducedAkt/mTORC1 inhibition. Sev-eralobservationssupportthatthisisindeedthecase:(a)THCincreasedtheamountofAktcoimmunoprecipitatedwithTRB3fromU87MGextracts(Figure4E),(b)knockdownofTRB3pre-ventedtheeffectofTHConAkt,TSC2,PRAS-40,p70S6kinase,andS6phosphorylation(Figure4F),and(c)TRB3overexpressiondecreasedAkt,TSC2,PRAS40,p70S6kinase,andS6phosphoryla-tion,enhancedtheinhibitoryeffectofTHConthephosphoryla-tionoftheseproteins,andpromotedautophagy(Figure4G).Inlinewiththeseobservations,THCfailedtoinhibitAkt,p70S6kinase,andS6phosphorylationofeIF2S51Aknockinorp8-deficientMEFs,inwhichTRB3didnotbecomeupregulateduponcannabinoidtreatment(Figure4HandSupplementalFigure6,GandH).Altogether,thesedatademonstratethatupregulationofp8andTRB3induceautophagyoftumorcellsviainhibitionoftheAkt/mTORC1pathway.

    THC-induced autophagy promotes the apoptotic death of cancer cells.While analyzing themechanismof cannabinoid cell-killingaction,weobservedthatincubationwiththepan-caspaseinhibi-torZVAD-fmkpreventedcelldeathtothesameextentasgenetic(Figure5A)orpharmacological(SupplementalFigure7)inhibi-tionofautophagy.Furthermore,Bax/Bakdoubleknockout(DKO)immortalizedMEFs,whichareprotectedagainstmitochondrialapoptosis(23),wereresistanttoTHC-inducedcelldeathandapoptosis(Figure5B)butunderwenteIF2phosphorylationandautophagy(Figure5C)uponTHCtreatment.Wethereforeinvestigatedwhethercannabinoid-inducedautophagypromotedtheapoptoticdeathofcancercells.Time-courseanalysisofLC3andactivecaspase-3immunostaininginU87MGcellsrevealedthatautophagyprecededtheappearanceofapoptoticfeaturesinTHC-treatedcells(Figure5D).Inaddition,selectiveknockdownofATG1(Figure5D)aswellasofAMBRA1orATG5(Supplemen-talFigure8)preventedTHC-inducedcaspase-3activation.More-over,unliketheirwild-typecounterparts,Atg5-deficientimmor-talizedMEFsdidnotundergophosphatidylserinetranslocationtotheouterleafletoftheplasmamembrane(Figure5E),loss

    ofmitochondrialmembranepotential(Figure5F),orincreasedproductionofreactiveoxygenspecies(SupplementalFigure9)inresponsetocannabinoidtreatment.Thesefindingsindicatethatactivationoftheautophagy-mediatedcelldeathpathwayoccursupstreamofapoptosisincannabinoidantitumoralaction.

    Activation of autophagy is necessary for cannabinoid antitumoral action in vivo.Todeterminetheinvivorelevanceofourfindings,wefirstinvestigatedwhetherTHCpromotestheactivationoftheabove-describedautophagy-mediatedcelldeathpathwayinU87MGcellderivedtumorxenografts,inwhichwehaverecentlyshownthatcannabinoidtreatmentreducestumorgrowth(specifically,THCadministrationfor14daysdecreasedtumorgrowthby50%;ref.13).Analysisofthesetumorsrevealedthatcannabinoidadminis-trationincreasesTRB3expressionanddecreasesS6phosphoryla-tion(Figure6A).Likewise,formationofLC3dotsaswellasincreaseinLC3-IIandactivecaspase-3immunostainingwereobservedinTHC-treated,butnotvehicle-treated,tumors(Figure6B).Tofurther investigatewhetheractivationofthep8pathway

    mediatescannabinoidantitumoralaction,wealsoanalyzedtumorsderivedfromp8+/+andp8/RasV12/E1A-transformedMEFs(inthiscase,THCadministrationfor8daysdecreasedby45%thegrowthofp8+/+tumorsbuthadnosignificanteffectonp8/tumors;ref.13).THCtreatmentincreasedTRB3expression,decreasedS6phos-phorylation,andincreasedautophagyaswellasTUNELandactivecaspase-3immunostaininginp8+/+butnotp8/tumors(Figure6CandSupplementalFigure10).Moreover,THCtreatmentenhancedthenumberofcellswithLC3dotsandTUNEL-positivenucleiinp8+/+butnotinp8/tumors(Figure6C).Inordertoverifytheimportanceofautophagyforcannabinoid

    antitumoralaction,wenextgeneratedtumorswithAtg5+/+andAtg5/RasV12/T-largeantigentransformedMEFs.THCadminis-trationreducedbymorethan80%thegrowthoftumorsderivedfromwild-typecellsbuthadnosignificanteffectonthosetumorsgeneratedbyautophagy-deficientcells(Figure7A).Furthermore,cannabinoidadministrationincreasedautophagy,TUNEL(Fig-ure7B),andactivecaspase-3immunostaining(SupplementalFig-ure11)inAtg5+/+butnotAtg5/tumors.Likewise,cannabinoidadministrationincreasedthenumberofcellswithLC3dotsandTUNEL-positivenucleiinAtg5+/+butnotAtg5/tumors(Figure7B).Takentogether,thesefindingsdemonstratethatactivationoftheautophagy-mediatedcelldeathpathwayisindispensableforcannabinoidantitumoralaction.Finally,weanalyzedthetumorsof2patientsenrolledinaclinical

    trialaimedatinvestigatingtheeffectofTHConrecurrentglioblas-tomamultiforme.ThepatientsweresubjectedtointracranialTHCadministration,andbiopsiesweretakenbeforeandafterthetreat-ment(11).Inthe2patients,cannabinoidinoculationincreasedTRB3immunostaininganddecreasedS6phosphorylation(Figure8A).Interestingly,thenumberofcellswithautophagicphenotype(Figure8B)aswellaswithactivecaspase-3immunostaining(Fig-ure8C)wasincreasedinthetumorsamplesobtainedafterTHCtreatment.Althoughthesestudieswereonlyconductedinspeci-mensfrom2patients,theyareinlinewiththepreclinicalevidenceshownaboveandsuggestthatcannabinoidadministrationmightalsotriggerautophagy-mediatedcelldeathinhumantumors.

    DiscussionInthisstudyweshowthatcannabinoids,anewfamilyofpotentialantitumoralagents,induceautophagyofcancercellsandthatthisprocessmediatesthecelldeathpromotingactivityofthesecom-

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    Figure 5Autophagy is upstream of apoptosis in cannabinoid-induced cancer cell death. (A) Effect of THC and the pan-caspase inhibitor ZVAD (10 M) on the viability of Atg5+/+ and Atg5/ MEFs (36 h; percentage of viable cells relative to the corresponding Atg5+/+ vehicle-treated cells, mean SD; n = 3). (B) Effect of THC on the apoptosis of Bax/Bak WT and Bax/Bak DKO MEFs as determined by cytofluorometric analysis of Annexin V/propidium iodide (PI) (24 h; mean SD; n = 3). The mean SD percentage of Annexin Vpositive/PI-positive and Annexin Vpositive, PI-nega-tive cells is shown in the upper and lower corners, respectively. (C) Effect of THC on eIF2 phosphorylation (3 h; n = 3) and LC3 lipidation (18 h; n = 4) of Bax/Bak WT and DKO MEFs. (D) Left: Effect of THC on autophagy and apoptosis of U87MG cells transfected with siC or siATG1. Green bars, cells with LC3 dots; red bars, active caspase-3positive cells; white bars, cells with both LC3 dots and active caspase-3 staining. Data correspond to the percentage of cells with LC3 dots (green bars), active caspase-3positive cells (red bars), and cells with LC3 dots and active caspse-3 staining (white bars) relative to the total number of transfected cells at each time point (mean SD; n = 3). Right: Representative photomicrographs (36 h; scale bar: 20 m). (E and F) Effect of THC on apoptosis (E; 24 h; n = 3) and loss of mitochondrial membrane potential as determined by DiOC6(3) staining (F; 24 h; n = 4) of Atg5+/+ and Atg5/ MEFs. In E, the mean SD percentage of Annexin Vpositive/PI-positive and Annexin Vpositive, PI-negative cells is shown in the upper and lower corners, respectively. **P < 0.01 compared with THC-treated Atg5+/+ (A, E, and F) and Bax/Bak WT (B) MEFs and from THC-treated, siC-transfected cells (D).

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    Figure 6THC activates the autophagic cell death pathway in vivo. (A) Effect of peritumoral THC administration on TRB3 and p-S6 immunostaining in U87MG tumors. TRB3- or p-S6stained area normalized to the total number of nuclei in each section; numbers indicate the mean fold change SD; 18 sections were counted for each of 3 dissected tumors for each condi-tion. Scale bar: 50 m. (B) Left: Effect of peritumoral THC administration on LC3 and active caspase-3 immunostaining in U87MG tumors. Arrows point to cells with LC3 dots. The numbers indicate the percentage of active caspase-3posi-tive cells relative to the total number of nuclei in each sec-tion SD. Ten sections were counted for each of 3 dissected tumors for each condition. Scale bars: 20 m. Right: Effect of peritumoral THC administration on LC3 lipidation in U87MG tumors. Representative samples from 1 vehicle-treated and 1 THC-treated tumor are shown. Numbers indicate the LC3-I and LC3-II OD values relative to vehicle-treated tumors (mean SD). n = 3. (C) Left: Effect of THC administration on LC3 immunostaining (green) and TUNEL (red) in RasV12/E1A p8+/+ and p8/ tumor xenografts. Arrows point to cells with LC3 dots and TUNEL-positive nuclei. Right: Bar graph shows the percentage of TUNEL-positive nuclei or cells with TUNEL-positive nuclei and LC3 dots relative to the total number of nuclei in each section (mean SD). Eighteen sections were counted from 3 dissected tumors for each condition. Scale bars: 50 m. Inset shows the magnification of 1 selected cell (arrows point to LC3 dots; scale bar: 10 m). *P < 0.05 and **P < 0.01 compared with vehicle-treated tumors.

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    1368 TheJournalofClinicalInvestigation http://www.jci.org Volume119 Number5 May2009

    pounds.Severalobservationsstronglysupportthisidea:(a)THCinducedautophagyandcelldeathindifferenttypesofcancercellsbutnotinnontransformedastrocytes,whichareresistanttocan-nabinoidkillingaction,(b)pharmacologicalorgeneticinhibitionofautophagypreventedTHC-inducedcelldeath,(c)autophagy-deficienttumorswereresistanttoTHCgrowth-inhibitingaction,and(d)THCadministrationactivatedtheautophagiccelldeathpathwayin3differentmodelsoftumorxenograftsaswellasin2humantumorsamples.Dependingonthecellularcontextandthestrengthandduration

    ofthetriggeringstimulus,autophagyisinvolvedinthepromotionorinhibitionofcancercellsurvival(4,5,24,25).However,themolec-ularbasesofthisdualroleofautophagyincancerremainunknown.

    Datapresentedheredemonstratethatinductionofautophagybycannabinoidsleadstocancercelldeathandidentifythesignalingrouteresponsiblefortheactivationofthiscellularprocess.Thus,ourfindingssuggestthatTHCviaactivationoftheCB1recep-torandstimulationofceramidesynthesisdenovoactivatesanearlyERstressresponsethatleadstoincreasedphosphorylationofeIF2onSer51.ExperimentsperformedwitheIF2S51Amutantcellshaveshownthatphosphorylationofthisresidue,whichisknowntoattenuategeneralproteintranslationwhileenhancingtheexpressionofseveralgenesrelatedwiththeERstressresponse(17),isrequiredfortheupregulationofthestressproteinp8anditsERstressrelateddownstreamtargetsATF4,CHOP,andTRB3aswellasfortheinductionofautophagybycannabinoids.Furthermore,

    Figure 7Autophagy is essential for cannabinoid antitumoral action. (A) Effect of peritumoral THC administration on the growth of Atg5+/+ (upper panel) and Atg5/ (lower panel) RasV12/T-large antigen MEF tumor xenografts generated in nude mice (mean SD; n = 7 for each condition). Photo-graphs show representative images of vehicle- and THC-treated tumors. (B) Left: Effect of THC administration on LC3 immunostaining (green) and apoptosis as determined by TUNEL (red) in Atg5+/+ and Atg5/ MEF tumor xenografts. Representative images from 1 vehicle-treated and 1 THC-treated Atg5+/+ and Atg5/ tumors are shown. Right: Bar graphs show the percentage of TUNEL-positive nuclei and cells with TUNEL-positive nuclei and LC3 dots relative to the total number of nuclei in each section (mean SD). Eighteen sections were counted from 3 dissected tumors for each condition (vehicle-treated and THC-treated). Scale bar: 50 m. (C) Schematic of the proposed mechanism of THC-induced cell death (see text for details). **P < 0.01 compared with vehicle-treated tumors.

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    wedemonstratethattheupregulationofp8andTRB3,whichhasbeenpreviouslyimplicatedincannabinoid-evokedcelldeath(13),isacrucialeventinthetriggeringofautophagy.Ceramideaccumula-tionhasbeenproposedtoinduceERstress(26,27)andautoph-

    agy(28),andeIF2phosphorylationhasbeenimplicatedintheinductionofautophagyinresponsetodifferentsituations(2931).However,themolecularmechanismsresponsiblefortheseactionshavenotbeenclarified.Findingspresentedherenowsuggestthat

    Figure 8THC administration promotes autophagy in glioblastomas of 2 patients. Analysis of different parameters in 2 patients with glioblastoma mul-tiforme before and after intracranial THC treatment (it was estimated that doses of 610 M were reached at the site of administration). (A) TRB3 and p-S6 immunostaining. Representative photomicrographs are shown. Numbers indicate the TRB3- or p-S6stained area normalized to the total number of nuclei in each section (mean fold change SD) relative to the corresponding pre-treatment sample. Fifteen sections were counted for each tumor and each condition (before and after treatment). Scale bar: 50 m. (B) Representative photomicrographs of LC3 diamino-benzidine immunostaining. The mean percentage of cells with LC3 dots SD relative to the total number of nuclei in each section is noted in the corner of each panel. Ten sections were counted from each biopsy for each condition. Arrows point to cells with LC3 dots. Scale bar: 20 m. (C) Representative photomicrographs of active caspase-3 diaminobenzidine immunostaining. Numbers indicate the percentage of cells with active caspase-3 staining SD relative to the total number of nuclei in each section. Ten sections were counted from each biopsy for each condition. Arrows point to cells with active caspase-3 staining. Scale bar: 20 m. *P < 0.05 and **P < 0.01 compared with before treatment.

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    upregulationofthep8-TRB3pathwayconstitutesamechanismbywhichdenovosynthesizedceramideandeIF2phosphorylationpromoteautophagy,thusidentifyingwhatwebelieveisanovelcon-nectionbetweenERstressandautophagy.Ourdataalsodemonstratethattheautophagy-promotingactiv-

    ityofthep8-regulatedpathwayisbasedonitsabilitytoinhibittheAkt/mTORC1axis.RegulationofmTORC1largelyreliesontheactivityoftheprosurvivalkinaseAkt,whoseinhibitionleadstomTORC1inactivationand,inturn,toautophagy(20).Ourfind-ingsrevealthatTHCupregulatesTRB3,promotingitsinteractionwithAktandleadingtodecreasedphosphorylationofthiskinaseaswellasofitsdirectsubstratesTSC2andPRAS40,whichtrig-gersmTORC1inhibitionandinductionofautophagy.TRB3hasbeenpreviouslyshowntoinhibitAkt(21,22),althoughtheprecisecontributionofthispseudo-kinasetotheregulationofAktactiv-ityindifferentcellularcontextsisunclear(32).Herewedemon-stratethatTRB3inhibitionoftheAkt/mTORC1axisisessentialforcannabinoid-inducedautophagyofcancercells.Moreover,weshowthatthispathwayisessentialforcannabinoidantitumoralaction.Thus,THCadministrationleadstoTRB3upregulation,mTORC1inhibition,inductionofautophagy,andreductionoftumorgrowthindifferentmodelsoftumorxenografts,butnotinp8-deficienttumorsthataredefectiveintheupregulationofthep8/TRB3pathway.Furthermore,activationofthispathwaywasalsoevidentin2gliomapatientsthathadbeentreatedwithTHC.TheseresultsthusuncoveraroleforTRB3thatmaybeofgreatimportanceintheregulationofcancercelldeath.Autophagyhasbeenproposedtoprotectfromapoptosis,act

    asanapoptosis-alternativepathwaytoinducecelldeath,oracttogetherwithapoptosisasacombinedmechanismforcelldeath(6,33).However,verylittleisknownabouttheroleoftheinterplaybetweenthese2cellularprocessesinthecontroloftumorgrowthinresponsetoanticanceragents.Ourresultsnowclearlydemon-stratethatinductionofautophagyisinvolvedinthemechanismbywhichcannabinoidspromotetheactivationofthemitochon-drialpro-apoptoticpathway.Thus,neithertumorsinwhichthep8-regulatedpathwayhasbeenablated(andinwhich,therefore,THCtreatmentdoesnotinduceautophagy)nortumorsintrin-sicallydeficientinautophagyundergoapoptosisinresponsetoTHC,andsotheyareresistanttoTHCantitumoralaction.Thesefindingsrevealthatautophagyisrequiredfortheactivationofapoptosisinresponsetocannabinoidtreatmentinvivo.ItisworthnotingthattheconcentrationsofTHCusedinthis

    studyareinthesamerangeasthoseadministeredintracraniallytothepatientsinwhichweobservedactivationoftheautophagy-mediatedcelldeathpathway(11)andcouldbethusconsideredclinicallyrelevant.Ofinterest,intraperitonealadministrationofTHCtoU87MGtumorxenograftsproducesasimilardecreaseintumorgrowth(thatoccursinconcertwithincreasedautophagyandapoptosis)tothatobservedwhenthecannabinoidisadminis-teredperitumorally(ourunpublishedobservations).Consideringthatnosignsoftoxicitywereobservedintheclinicaltrialpatients(11)orintumor-bearinganimalstreatedintracranially,peritumor-ally,orintraperitoneallywithTHC(refs.34and35anddatanotshown),andthatnooverttoxiceffectshavebeenreportedinotherclinicaltrialsofcannabinoiduseincancerpatientsforvariousapplications(e.g.,inhibitionofnausea,vomiting,andpain)andusingdifferentroutesofadministration(e.g.,oral,oro-mucosal)(9,36),ourfindingssupportthatsafe,therapeuticallyefficaciousdosesofTHCmaybereachedincancerpatients.

    Insummary,inthisstudyweidentifywhatwebelieveisanewroutethatlinkstheERstressresponsetotheactivationofautoph-agyandpromotestheapoptoticdeathoftumorcells(Figure7C).Theidentificationofthispathwaywillhelptounderstandthemoleculareventsthatleadtoactivationofautophagy-mediatedcelldeathbyanticancerdrugsandmaycontributetothedesignofnewtherapeuticstrategiesforinhibitingtumorgrowth.

    MethodsCell culture and viability.Corticalastrocyteswerepreparedfrom24-hour-oldmiceaspreviouslydescribed(13).PrimaryculturesofbraintumorcellswerepreparedandculturedasdescribedintheSupplementalMethods.U87MG,T98G,U373MG,andMiaPaCa2cells,p8+/+andp8/RasV12/E1AMEFs,Atg5+/+andAtg5/T-largeantigenMEFs(providedbyNoboruMizushima,TokyoMedicalandDentalUniversity,Tokyo,Japan),Bax/Bakwild-typeandBax/BakDKOT-largeantigenMEFs(providedbyLucaScorrano,DulbeccoTelethonInstitute,Milan,Italy,andPatriziaAgosti-nis,CatholicUniversityofLeuven,Leuven,Belgium),eIF2S51SWTandeIF2S51AT-largeantigenMEFs(providedbyRichardKaufman,Uni-versityofMichigan,AnnArbor,Michigan,USA,andCesardeHaroandJuanJ.Berlanga,CentrodeBiologaMolecularSeveroOchoa,AutonomaUniversity,Madrid,Spain),Tsc2+/+andTsc2/p53/MEFs,emptyvector(pBABE)andpBABE-myr-AktMEFs,andAtg5+/+andAtg5/RasV12/T-largeantigenMEFswereculturedinDMEMcontaining10%FBSandtrans-ferredtomediumcontaining0.5%FBS(exceptRasV12/E1A-transformedMEFs,whichweretransferredtomediumcontaining2%FBS)18hbeforeperformingthedifferenttreatments.p8+/+andp8/RasV12/E1AMEFsaswellasAtg5+/+andAtg5/RasV12/T-largeantigenMEFscorrespondtoapolyclonalmixofatleast20differentselectedclones.Unlessotherwiseindicated,THCwasusedatafinalconcentrationof5M.CellviabilitywasdeterminedbytheMTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-liumbromide]test(Sigma-Aldrich).

    Flow cytometry.Briefly,cells(approximately5105cellsperassay)weretrypsinized,dividedin2tubes,washed,andcollectedbycentrifugationat1,500gfor5min.Onealiquotwasincubatedfor10minat37CwithAnnexinVFITC(BDBiosciences).Propidiumiodide(1g/ml)wasaddedjustbeforecytofluorometricanalysis.Theotheraliquotwassimultane-ouslylabeledwith3,3-dihexyloxacarbocyanineiodide(DiOC6[3],40nM;Invitrogen)andhydroethidium(5M;Invitrogen)for10minutesat37C,followedbycytofluorometricanalysis.Cells(10,000)wererecordedineachanalysis.FluorescenceintensitywasanalyzedinanEPICSXLflowcytometer(BeckmanCoulter).

    Western blot.Westernblotanalysiswasperformedfollowingstandardprocedures.AlistoftheantibodiesusedcanbefoundinSupplemen-talMethods.DensitometricanalysiswasperformedwithQuantityOnesoftware(Bio-Rad).

    Transfections.U87MGcells (75%confluent)were transfectedwithsiRNAduplexesusingtheDharmaFECT1Transfectionreagent(Dhar-macon).Cellsweretrypsinizedandseeded24haftertransfection,atadensityof5,000cells/cm2.Transfectionefficiencywasgreaterthan70%asmonitoredwithacontrolfluorescent(red)siRNA(siGLORISC-FreesiRNA;Dharmacon).Inimmunofluorescenceexperiments,controlandselectivesiRNAswereusedina1:5ratio,andcellswithredspotswerescoredastransfected.

    Infections with adenoviral vectors.U87MGcells(75%confluent)weretrans-ducedfor1hwithsupernatantsobtainedfromHEK293cellsinfectedwithadenoviralvectorscarryingEGFP(providedbyJavierG.Castro,HospitalInfantilUniversitarioNioJess,Madrid,Spain),ratHA-taggedTRB3(donatedbyPatrickIynedjian,UniversityofGeneva,Geneva,Switzerland)(32),orhumanEGFP-LC3(providedbyAvivaTolkovskyandChristoph

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    Goemans,UniversityofCambridge,Cambridge,UnitedKingdom).Infec-tionefficiencywasgreaterthan80%asdeterminedbyEGFPfluorescence.

    RNA interference.Double-strandedRNAduplexeswerepurchasedfromDharmacon.AlistofsequencescanbefoundintheSupplementalMethods.

    RT-PCR analysis. RNAwasisolatedusingTrizolReagent(Invitrogen).cDNAwasobtainedwithTranscriptorReverse transcriptase (RocheAppliedScience).PrimersandamplificationconditionscanbefoundintheSupplementalMethods.

    Real-time quantitative PCR.cDNAwasobtainedusingTranscriptor(RocheAppliedScience).Real-timequantitativePCRassayswereperformedusingtheFastStartUniversalProbeMastermixwithRox(RocheAppliedSci-ence),andprobeswereobtainedfromtheUniversalProbeLibrarySet(RocheAppliedScience).PrimersequencescanbefoundintheSupple-mentalMethods.Amplificationswererunina7900HT-FastReal-TimePCRSystem(AppliedBiosystems).Eachvaluewasadjustedbyusing18SRNAlevelsasareference.

    Immunoprecipitation.U87MGcellswerelysedinHEPESlysisbuffer(seeSupplementalMethodsforbuffercomposition).Lysate(14mg)waspre-clearedbyincubatingwith520lofproteinGSepharoseconjugatedtopre-immuneIgG.Thelysateextractswerethenincubatedwith520lofproteinGSepharoseconjugatedto520goftheanti-TRB3anti-bodyorpre-immuneIgG.TRB3antibody(aminoterminalend,ab50516;Abcam)wascovalentlyconjugatedtoproteinGSepharoseusingdimethylpimelimidate. Immunoprecipitationswerecarriedoutfor1hat4Conarotatorywheel.Theimmunoprecipitateswerewashed4timeswithHEPESlysisbuffer,followedby2washeswithHEPESkinasebuffer.Theimmunoprecipitateswereresuspendedin30lofsamplebuffer(notcon-taining2-mercaptoethanol)andfilteredthrougha0.22-mSpin-Xfilter,and2-mercaptoethanolwasaddedtoaconcentrationof1%(vol/vol).Sam-plesweresubjectedtoelectrophoresisandimmunoblotanalysis.

    Ceramide levels. Ceramidelevelsweredeterminedaspreviouslydescribed(37).Confocal laser scanning microscopy. Standardprotocols for immuno-

    fluorescencemicroscopywereused(seeSupplementalMethodsfortheantibodiesused).ToquantifythepercentageofcellswithLC3orPDIdots,atleast200cellsperconditionwerecountedinrandomlyselectedfields.Inallcases,onlythosecellswith4ormoreprominentdotsofeitherLC3orPDIwerescoredpositively.

    In vivo treatments.TumorsderivedfromU87MGcellsandp8+/+andp8/MEFswere inducedandtreatedaspreviouslydescribed(13).TumorsderivedfromAtg5+/+orAtg5/RasV12/T-largeantigenMEFs(seeSupplemen-talMethodsfortheprocedureusedtogeneratethesecells)wereinducedinnudemicebysubcutaneousinjectionof107cellsinPBSsupplementedwith0.1%glucose.Tumorswereallowedtogrowuntilanaveragevolumeof200250mm3,andanimalswereassignedrandomlytothedifferentgroups.Atthispoint,vehicleorTHC(15mg/kg/d)in100lofPBSsupplementedwith5mg/mlBSAwasadministereddailyinasingleperitumoralinjection.Tumorsweremeasuredwithanexternalcaliper,andvolumewascalculatedas(4/3)(width/2)2(length/2).AllproceduresinvolvinganimalswereperformedwiththeapprovaloftheComplutenseUniversityAnimalExperi-mentationCommitteeaccordingtoSpanishofficialregulations.

    Human tumor samples.Tumorbiopsieswereobtainedfrom2recurrentglioblastomamultiformepatientswhohadbeentreatedwithTHC.Thecharacteristicsofthepatientsandtheclinicalstudyhavebeendescribedindetailelsewhere(11).Briefly,THCdissolvedin30mlofphysiologicalsalinesolutionplus0.5%(wt/vol)humanserumalbuminwasadministeredintratumorallytothepatients.Patient1receivedatotalof1.46mgofTHCfor30days,whilepatient2receivedatotalof1.29mgofTHCfor26days(itwasestimatedthatdosesof610MTHCwerereachedatthesiteofadministration;ref.11).Sampleswerefixedinformalin,embeddedinpar-affin,andusedforimmunomicroscopy.

    Immunomicroscopy of tumor samples.Samples fromtumorxenograftsweredissected,Tissue-Tek(Sakura)embedded,frozen,and,beforethestainingprocedureswereperformed,fixedinacetonefor10minatroomtemperature.Samplesfromhumantumorsweresubjectedtodeparaf-finization,rehydration,andantigenretrievalbeforethestainingproce-dureswereperformed.Standardprotocolsforimmunofluorescenceorimmunohistochemistrymicroscopywereused(seeSupplementalMeth-ods).NucleiwerecounterstainedwithTOTO-3iodide(U87MGandhumantumorsamples;Invitrogen)orHoechst33342(MEFtumors;Invitrogen).FluorescenceimageswereacquiredusingMetamorph-Offline6.2software(UniversalImaging)andZeissAxioplan2Microscope.

    TUNEL.Tumorsampleswerefixed,blocked,andpermeabilized,andTUNELwasperformedaspreviouslydescribed(13).

    Electron microscopy.Ultrastructuralanalysisofvehicle-andTHC-treatedcellswasassessedbyconventionalembeddingintheepoxy-resinEML-812(TaabLaboratories).Ultrathin(20-to30-nm-thick)sectionsofthesam-pleswereobtainedusingaLeica-Reichert-Jungultramicrotomeandthenstainedwithsaturateduranylacetateleadcitratebystandardprocedures.UltrathinsectionswereanalyzedinaJEOL1200-EXIItransmissionelec-tronmicroscopeoperatingat100kV.

    Statistics.StatisticalanalysiswasperformedbyANOVAwithapost-hocanalysisusingtheStudent-Neuman-Keulstest.Differenceswereconsid-eredsignificantwhenthePvaluewaslessthan0.05.

    AcknowledgmentsThisworkwassupportedbygrantsfromtheSpanishMinistryofEducationandScience(MEC)(HF2005/0021,toG.Velasco;SAF2006/00918, toM.Guzmn;andBFU2006-00508, toP.Boya),Santander-ComplutensePR34/07-15856,toG.Velasco),ComunidaddeMadrid (S-SAL/0261/2006, toM.Guzmn),andLaLiguecontreleCancerandCanceropolePACA(toJ.L.Iovanna).M.Salazarwas the recipientofa fellowship fromtheMEC.A.CarracedowastherecipientoffellowshipsfromGobiernoVasco,theFederationofEuropeanBiochemicalSoci-eties,andtheEuropeanMolecularBiologyOrganization.M.LorenteandP.BoyahaveaJuandelaCiervaandaRamnyCajalcontractfromtheMEC,respectively.S.Hernndez-TiedrahasatechniciancontractfromtheSpanishMinistryofEduca-tionandtheFondoSocialEuropeo.TheauthorsthankDarioAlessi (UniversityofDundee,Dundee,UnitedKingdom)fordonatinganti-PRAS40antibodiesandfortechnicalsupportforimmunoprecipitationexperiments;GemmaFabris,JosefinaCasas,andEvaDalmau(InstitutodeInvestigacionesQumicasyAmbientales,Barcelona,Spain)foranalyzingceramidesam-ples;JosLizcano,JosBayascas,MaraM.Caffarel,andPatriziaAgostinisfortheirexperimentalsuggestions;andothermem-bersofourlaboratoryfortheircontinualsupport.

    Received forpublicationNovember3,2008, andaccepted inrevisedformFebruary11,2009.

    Addresscorrespondenceto:GuillermoVelasco,DepartmentofBio-chemistryandMolecularBiologyI,SchoolofBiology,ComplutenseUniversity,c/JosAntonioNovaiss/n,28040Madrid,Spain.Phone:34-913944668;Fax:34-913944672;E-mail:[email protected].

    ArkaitzCarracedoandAinaraEgiaspresentaddress is:Can-cerGeneticsProgram,BethIsraelDeaconessCancerCenterandDepartmentofMedicine,BethIsraelDeaconessMedicalCenter,HarvardMedicalSchool,Boston,Massachusetts,USA.

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  • research article

    1372 TheJournalofClinicalInvestigation http://www.jci.org Volume119 Number5 May2009

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