Research article

Cannabinoid action induces autophagymediated cell death through stimulation of ER stress in human glioma cells
Mara 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
of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain. 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.
2Centro 1Department

Autophagy can promote cell survival or cell death, but the molecular basis underlying its dual role in cancer remains obscure. Here we demonstrate that 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 2 (eIF2) phosphorylation and thereby activated an ER stress response that promoted autophagy via tribbles homolog 3dependent (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 highlyconservedcellularprocessinwhichcytoplasmicmaterials includingorganellesaresequesteredintodouble-membrane vesiclescalledautophagosomesanddeliveredtolysosomesfor degradationorrecycling(1).Inmanycellularsettings,triggering ofautophagyreliesontheinhibitionofmammaliantargetofrapamycincomplex1(mTORC1),aneventthatpromotestheactivation(de-inhibition)ofseveralautophagyproteins(Atgs)involved intheinitialphaseofmembraneisolation(1).Enlargementofthis complextoformtheautophagosomerequirestheparticipationof 2ubiquitin-likeconjugationsystems.OneinvolvestheconjugationofATG12toATG5andtheotherofphosphatidylethanolaminetoLC3/ATG8(1).Thefinaloutcomeoftheactivationofthe autophagyprogramishighlydependentonthecellularcontext andthestrengthanddurationofthestress-inducingsignals(25). Thus,besidesitsroleincellularhomeostasis,autophagycanbea formofprogrammedcelldeath,designatedtypeIIprogrammed celldeath,orplayacytoprotectiverole,forexampleinsituations
Conflict of interest:Theauthorshavedeclaredthatnoconflictofinterestexists. Nonstandard abbreviations used:Atg,autophagyprotein;eIF2,eukaryotic translationinitiationfactor2;MEF,mouseembryonicfibroblast;THC,9-tetrahydrocannabinol;mTORC1,mammaliantargetofrapamycincomplex1;PDI,protein disulphideisomerase;TRB3,tribbleshomolog3. Citation for this article:J. Clin. Invest.119:13591372(2009).doi:10.1172/JCI37948.

ofnutrientstarvation(6).Accordingly,autophagyhasbeenproposedtoplayanimportantroleinbothtumorprogressionand promotionofcancercelldeath(24),althoughthemolecular mechanismsresponsibleforthisdualactionofautophagyincancerhavenotbeenelucidated. 9-Tetrahydrocannabinol(THC),themainactivecomponentof marijuana(7),exertsawidevarietyofbiologicaleffectsbymimickingendogenoussubstancestheendocannabinoidsthat bindtoandactivatespecificcannabinoidreceptors(8).Oneof themostexcitingareasofresearchinthecannabinoidfieldisthe studyofthepotentialapplicationofcannabinoidsasantitumoral agents(9).Cannabinoidadministrationhasbeenfoundtocurb thegrowthofseveraltypesoftumorxenograftsinratsandmice (9,10).Basedonthispreclinicalevidence,apilotclinicaltrialhas beenrecentlyruntoinvestigatetheantitumoralactionofTHCon recurrentgliomas(11).Recentfindingshavealsoshownthatthe pro-apoptoticandtumorgrowthinhibitingactivityofcannabinoidsreliesontheupregulationofthetranscriptionalco-activatorp8(12)anditstargetthepseudo-kinasetribbleshomolog3 (TRB3)(13).However,themechanismsthatpromotetheactivationofthissignalingrouteaswellasthetargetsdownstreamof TRB3thatmediateitstumorcellkillingactionremainelusive. InthisstudywefoundthatERstressevokedupregulationofthe p8/TRB3pathwayinducedautophagyviainhibitionoftheAkt/ mTORC1axisandthatactivationofautophagypromotedthe apoptoticdeathoftumorcells.Theuncoveringofthispathway,
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Figure 1
Inhibition of autophagy prevents THC-induced cancer cell death. (AC) Effect of THC on U87MG cell morphology. Representative electron 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 vehicletreated (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 fluorescent 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, THCtreated U87MG cells (F and G). THC concentration was 6 M.

whichwebelieveisnovel,forpromotingtumorcelldeathmayhave therapeuticimplicationsinthetreatmentofcancer. Results Autophagy mediates THC-induced cancer cell death.Asafirstapproach togaininsightintothemorphologicalchangesinducedincancercellsbycannabinoidadministration,weperformedelectron microscopyanalysisofU87MGhumanastrocytomacells.Interestingly,doublemembranevacuolarstructureswiththemorphologicalfeaturesofautophagosomeswereobservedinTHC-treated cells(Figure1,AC).TheconversionofthesolubleformofLC3 (LC3-I) to the lipidated and autophagosome-associated form (LC3-II)isconsideredoneofthehallmarksofautophagy(1),and thusweobservedtheoccurrenceofLC3-positivedotsaswellasthe appearanceofLC3-II(Figure1D)incannabinoid-challengedcells. Inaddition,co-incubationwiththelysosomalproteaseinhibitors E64dandpepstatinA,whichblocksthelaststepsofautophagic degradation(14),enhancedTHC-inducedaccumulationofLC3-II (Figure 1E), confirming that cannabinoids induce dynamic autophagyinU87MGcells.Furthermore,incubationwiththecannabinoidreceptor1(CB1)antagonistSR141716preventedTHCinducedLC3lipidationandformationofLC3dots(Figure1D), indicatingthatinductionofautophagybycannabinoidsrelieson CB1receptoractivation. Sinceautophagyhasbeenimplicatedinpromotionandinhibitionofcellsurvival,wenextinvestigateditsparticipationin thecancercelldeathinducingactionofTHC.Pharmacological inhibitionofautophagyatdifferentlevels(SupplementalFigure 1,AC;supplementalmaterialavailableonlinewiththisarticle; doi:10.1172/JCI37948DS1)orselectiveknockdownofATG1(an essentialproteinintheinitiationofautophagy;ref.1)(Figure 1,FandG),ATG5(anessentialproteinintheformationofthe autophagosome;ref.1)(SupplementalFigure1,DF),orAMBRA1 (arecentlyidentifiedbeclin-1interactingproteinthatregulates

autophagy;ref.15)(SupplementalFigure1,DF)stronglyreduced cannabinoid-inducedautophagyandcelldeath.Moreover,transformedAtg5-deficientmouseembryonicfibroblasts(MEFs),which aredefectiveinautophagy(16),weremoreresistantthantheir wild-typecounterpartstoTHC-inducedcelldeath(Figure1H)and didnotundergoautophagyuponcannabinoidtreatment(Figure 1I).Takentogether,thesefindingsdemonstratethatautophagy playsaprominentroleinTHC-inducedcancercelldeath. THC induces autophagy via ER stressdependent upregulation of p8 and TRB3.Inadditiontothepresenceofautophagosomes,electron microscopyanalysisofcannabinoid-treatedcellsrevealedthepresenceofnumerouscellswithdilatedER(Figure2A).Inlinewith thisobservation,immunostainingoftheERluminalmarkerproteindisulphideisomerase(PDI)showedastrikingdilationinthe ERofTHC-treatedU87MGcells(Figure2B),aneventthatwas associatedwithanincreasedphosphorylationofthesubunitof eukaryotictranslationinitiationfactor2(eIF2),ahallmarkofthe ERstressresponse(17)(Figure2C).Inaddition,THC-inducedER dilationandeIF2phosphorylationwerepreventedbypharmacologicalblockadeoftheCB1receptor(Figure2,BandC). Time-courseanalysisofPDIandLC3immunostaining,eIF2 phosphorylation,andLC3lipidationofcannabinoid-treatedcells revealedthatERstressoccurredearlierthanautophagy(Figure2, DandE).Ofinterest,cannabinoidadministrationproducedsimilaractivationofERstressandautophagy,aswellascelldeath,in otherhumanastrocytomacelllines(SupplementalFigure2,AF), aprimarycultureofhumangliomacells(SupplementalFigure 2,GI),andseveralhumancancercelllinesofdifferentorigin, includingpancreaticcancer(SupplementalFigure2,JL),breast cancer,andhepatoma(datanotshown).However,neitherERdilationnoreIF2 phosphorylationorautophagywasevidentinnormal,nontransformedprimaryastrocytes(SupplementalFigure3), whichareresistanttocannabinoid-inducedcelldeath(13). WenextinvestigatedwhetheractivationofERstressisinvolved intheinductionofautophagyinresponsetocannabinoidtreatmentofcancercells.WehavepreviouslyshownthatTHC-induced accumulationofdenovosynthesizedceramide,aneventthat occursintheER(18),leadstoupregulationofthestress-regulated proteinp8anditsERstressrelateddownstreamtargets,ATF4, CHOP,andTRB3,toinducecancercelldeath(13).Ofimportance, incubationwithISP-1(aselectiveinhibitorofserinepalmitoyltransferase,theenzymethatcatalyzesthefirststepofsphingolipidbiosynthesis;ref.18)preventedceramideaccumulation(SupplementalFigure4A);THC-inducedERdilation(Supplemental Figure4B);eIF2phosphorylation(Figure3A);p8,ATF4,CHOP, andTRB3upregulation(SupplementalFigure4C);andautophagy (Figure3B),supportingthatceramideaccumulationisinvolvedin cannabinoid-triggeredERstressandautophagy.Wealsoverified bymeansofRNAinterferencethatCaCMKKwhichhadbeen previouslyimplicatedinactivatingautophagyinresponsetoER stressassociatedcalciumrelease(19)wasnotinvolvedinTHCinducedautophagyandcelldeath(datanotshown).AsphosphorylationofeIF2 onSer51attenuatesgeneralproteinsynthesis whileenhancingtheexpressionofseveralERstressresponsegenes (17),weusedcellsderivedfromeIF2S51Aknockinmicetotest whethereIF2phosphorylationregulatestheexpressionofp8and itsdownstreamtargets.Inagreementwiththishypothesis,THC treatment(whichpromotedceramideaccumulationinbothwildtypeandeIF2S51AimmortalizedMEFs;SupplementalFigure 5A)triggeredp8,ATF4,CHOP,andTRB3upregulation(Figure
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Figure 2
ER 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 vehicletreated 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.

3C)aswellasautophagy(SupplementalFigure5B)inwild-type cellsbutnotintheireIF2S51Acounterparts. Wesubsequentlyaskedwhetherp8anditsdownstreamtargetsregulateautophagy.Knockdownofp8orTRB3prevented THC-inducedautophagy(Figure3,DandE)butnotERdilation (SupplementalFigure4D)inU87MGcells.Furthermore,THC inducedautophagyinp8+/+ but not p8-deficient transformed MEFs(Figure3FandSupplementalFigure5C).Altogether,these findingsrevealthatTHCinducesautophagyofcancercellsvia
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activationofanERstresstriggeredsignalingroutethatinvolves stimulationofceramidesynthesisdenovo,eIF2phosphorylation,andp8andTRB3upregulation. THC inhibits Akt and mTORC1 via TRB3.InhibitionofmTORC1 isconsideredakeystepintheearlytriggeringofautophagy(6). Wethereforetestedwhethercannabinoid-inducedupregulation ofthep8pathwayleadstoautophagyviainhibitionofthiscomplex.THCtreatmentofU87MGcellsreducedthephosphorylation ofp70S6kinase(awell-establishedmTORC1substrate)andthe

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Figure 3
THC induces autophagy via ER stressevoked p8 and TRB3 upregulation. (A and B) Effect of ISP-1 (1 M) on THC-induced eIF2 phosphorylation (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 RTPCR 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 4
THC 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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ribosomalproteinS6(awell-establishedp70S6kinasesubstrate) (Figure4,AandC),indicatingthatmTORC1isinhibitedincannabinoid-challengedcells.Inaddition,thecannabinoid-induced decreaseinp70S6kinaseandS6phosphorylation,autophagy,and celldeathwerenotevidentinTsc2/cells,inwhichmTORC1is constitutivelyactive(20)(Figure4BandSupplementalFigure6,A andB),furthersupportingamajorroleformTORC1inhibitionin theinductionofautophagiccelldeathbycannabinoids. TheproteinkinaseAktpositivelyregulatestheactivityofthe mTORC1complexbyphosphorylatingandinhibitingTSC2and PRAS40(awell-establishedAktsubstratewithinthemTORC1 complex).Thus,AktinhibitiondecreasesmTORC1activityand promotesautophagy(20).Inlinewiththisidea,THCdecreased thephosphorylationofAkt,TSC2,andPRAS40aswellasp70S6 kinaseandS6(Figure4C).ThisinhibitionoftheAkt/mTORC1 pathwaywasabrogatedbyincubationwithaCB1receptorantagonist(SupplementalFigure6C)oraceramidesynthesisinhibitor (SupplementalFigure6D).Likewise,cellsoverexpressingamyristoylated(constitutivelyactive)formofAktwereresistanttoTHCinducedmTORC1inhibition,autophagy,andcelldeath(Figure 4DandSupplementalFigure6,EandF),furthersupportingthat THCinducesautophagyviaAktinhibition. SinceTRB3hasbeenshowntodirectlyinteractwithandinhibit Akt(21,22),weinvestigatedwhetherupregulationofTRB3was responsible for THC-induced Akt/mTORC1 inhibition. Severalobservationssupportthatthisisindeedthecase:(a)THC increasedtheamountofAktcoimmunoprecipitatedwithTRB3 fromU87MGextracts(Figure4E),(b)knockdownofTRB3preventedtheeffectofTHConAkt,TSC2,PRAS-40,p70S6kinase, andS6phosphorylation(Figure4F),and(c)TRB3overexpression decreasedAkt,TSC2,PRAS40,p70S6kinase,andS6phosphorylation,enhancedtheinhibitoryeffectofTHConthephosphorylationoftheseproteins,andpromotedautophagy(Figure4G).In linewiththeseobservations,THCfailedtoinhibitAkt,p70S6 kinase,andS6phosphorylationofeIF2S51Aknockinorp8deficientMEFs,inwhichTRB3didnotbecomeupregulatedupon cannabinoidtreatment(Figure4HandSupplementalFigure6,G andH).Altogether,thesedatademonstratethatupregulationof p8andTRB3induceautophagyoftumorcellsviainhibitionofthe Akt/mTORC1pathway. THC-induced autophagy promotes the apoptotic death of cancer cells. While analyzing the mechanism of cannabinoid cell-killing action,weobservedthatincubationwiththepan-caspaseinhibitorZVAD-fmkpreventedcelldeathtothesameextentasgenetic (Figure5A)orpharmacological(SupplementalFigure7)inhibitionofautophagy.Furthermore,Bax/Bakdoubleknockout(DKO) immortalizedMEFs,whichareprotectedagainstmitochondrial apoptosis(23),wereresistanttoTHC-inducedcelldeathand apoptosis(Figure5B)butunderwenteIF2phosphorylation andautophagy(Figure5C)uponTHCtreatment.Wetherefore investigatedwhethercannabinoid-inducedautophagypromoted theapoptoticdeathofcancercells.Time-courseanalysisofLC3 andactivecaspase-3immunostaininginU87MGcellsrevealed thatautophagyprecededtheappearanceofapoptoticfeaturesin THC-treatedcells(Figure5D).Inaddition,selectiveknockdown ofATG1(Figure5D)aswellasofAMBRA1orATG5(SupplementalFigure8)preventedTHC-inducedcaspase-3activation.Moreover,unliketheirwild-typecounterparts,Atg5-deficientimmortalizedMEFsdidnotundergophosphatidylserinetranslocation totheouterleafletoftheplasmamembrane(Figure5E),loss

ofmitochondrialmembranepotential(Figure5F),orincreased productionofreactiveoxygenspecies(SupplementalFigure9)in responsetocannabinoidtreatment.Thesefindingsindicatethat activationoftheautophagy-mediatedcelldeathpathwayoccurs upstreamofapoptosisincannabinoidantitumoralaction. Activation of autophagy is necessary for cannabinoid antitumoral action in vivo.Todeterminetheinvivorelevanceofourfindings,wefirst investigatedwhetherTHCpromotestheactivationoftheabovedescribedautophagy-mediatedcelldeathpathwayinU87MGcell derivedtumorxenografts,inwhichwehaverecentlyshownthat cannabinoidtreatmentreducestumorgrowth(specifically,THC administrationfor14daysdecreasedtumorgrowthby50%;ref. 13).AnalysisofthesetumorsrevealedthatcannabinoidadministrationincreasesTRB3expressionanddecreasesS6phosphorylation(Figure6A).Likewise,formationofLC3dotsaswellasincrease inLC3-IIandactivecaspase-3immunostainingwereobservedin THC-treated,butnotvehicle-treated,tumors(Figure6B). Tofurtherinvestigatewhetheractivationofthep8pathway mediatescannabinoidantitumoralaction,wealsoanalyzedtumors derivedfromp8+/+andp8/RasV12/E1A-transformedMEFs(inthis case,THCadministrationfor8daysdecreasedby45%thegrowth ofp8+/+tumorsbuthadnosignificanteffectonp8/tumors;ref. 13).THCtreatmentincreasedTRB3expression,decreasedS6phosphorylation,andincreasedautophagyaswellasTUNELandactive caspase-3immunostaininginp8+/+butnotp8/tumors(Figure6C andSupplementalFigure10).Moreover,THCtreatmentenhanced thenumberofcellswithLC3dotsandTUNEL-positivenucleiin p8+/+butnotinp8/tumors(Figure6C). Inordertoverifytheimportanceofautophagyforcannabinoid antitumoralaction,wenextgeneratedtumorswithAtg5+/+and Atg5/RasV12/T-largeantigentransformedMEFs.THCadministrationreducedbymorethan80%thegrowthoftumorsderived fromwild-typecellsbuthadnosignificanteffectonthosetumors generatedbyautophagy-deficientcells(Figure7A).Furthermore, cannabinoidadministrationincreasedautophagy,TUNEL(Figure7B),andactivecaspase-3immunostaining(SupplementalFigure11)inAtg5+/+butnotAtg5/tumors.Likewise,cannabinoid administrationincreasedthenumberofcellswithLC3dotsand TUNEL-positivenucleiinAtg5+/+butnotAtg5/tumors(Figure 7B).Takentogether,thesefindingsdemonstratethatactivationof theautophagy-mediatedcelldeathpathwayisindispensablefor cannabinoidantitumoralaction. Finally,weanalyzedthetumorsof2patientsenrolledinaclinical trialaimedatinvestigatingtheeffectofTHConrecurrentglioblastomamultiforme.ThepatientsweresubjectedtointracranialTHC administration,andbiopsiesweretakenbeforeandafterthetreatment(11).Inthe2patients,cannabinoidinoculationincreased TRB3immunostaininganddecreasedS6phosphorylation(Figure 8A).Interestingly,thenumberofcellswithautophagicphenotype (Figure8B)aswellaswithactivecaspase-3immunostaining(Figure8C)wasincreasedinthetumorsamplesobtainedafterTHC treatment.Althoughthesestudieswereonlyconductedinspecimensfrom2patients,theyareinlinewiththepreclinicalevidence shownaboveandsuggestthatcannabinoidadministrationmight alsotriggerautophagy-mediatedcelldeathinhumantumors. Discussion Inthisstudyweshowthatcannabinoids,anewfamilyofpotential antitumoralagents,induceautophagyofcancercellsandthatthis processmediatesthecelldeathpromotingactivityofthesecom1365

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Figure 5
Autophagy 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-negative 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 6
THC 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 condition. 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-3positive cells relative to the total number of nuclei in each section 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 TUNELpositive 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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Figure 7
Autophagy 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). Photographs 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 TUNELpositive 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.

pounds.Severalobservationsstronglysupportthisidea:(a)THC inducedautophagyandcelldeathindifferenttypesofcancercells butnotinnontransformedastrocytes,whichareresistanttocannabinoidkillingaction,(b)pharmacologicalorgeneticinhibition ofautophagypreventedTHC-inducedcelldeath,(c)autophagydeficienttumorswereresistanttoTHCgrowth-inhibitingaction, and(d)THCadministrationactivatedtheautophagiccelldeath pathwayin3differentmodelsoftumorxenograftsaswellasin2 humantumorsamples. Dependingonthecellularcontextandthestrengthandduration ofthetriggeringstimulus,autophagyisinvolvedinthepromotion orinhibitionofcancercellsurvival(4,5,24,25).However,themolecularbasesofthisdualroleofautophagyincancerremainunknown.
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Datapresentedheredemonstratethatinductionofautophagyby cannabinoidsleadstocancercelldeathandidentifythesignaling routeresponsiblefortheactivationofthiscellularprocess.Thus, ourfindingssuggestthatTHCviaactivationoftheCB1receptorandstimulationofceramidesynthesisdenovoactivatesan earlyERstressresponsethatleadstoincreasedphosphorylationof eIF2onSer51.ExperimentsperformedwitheIF2S51Amutant cellshaveshownthatphosphorylationofthisresidue,whichis knowntoattenuategeneralproteintranslationwhileenhancingthe expressionofseveralgenesrelatedwiththeERstressresponse(17), isrequiredfortheupregulationofthestressproteinp8anditsER stressrelateddownstreamtargetsATF4,CHOP,andTRB3aswell asfortheinductionofautophagybycannabinoids.Furthermore,

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Figure 8
THC administration promotes autophagy in glioblastomas of 2 patients. Analysis of different parameters in 2 patients with glioblastoma multiforme 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 diaminobenzidine 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.

wedemonstratethattheupregulationofp8andTRB3,whichhas beenpreviouslyimplicatedincannabinoid-evokedcelldeath(13),is acrucialeventinthetriggeringofautophagy.CeramideaccumulationhasbeenproposedtoinduceERstress(26,27)andautoph

agy(28),andeIF2phosphorylationhasbeenimplicatedinthe inductionofautophagyinresponsetodifferentsituations(2931). However,themolecularmechanismsresponsiblefortheseactions havenotbeenclarified.Findingspresentedherenowsuggestthat

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upregulationofthep8-TRB3pathwayconstitutesamechanismby whichdenovosynthesizedceramideandeIF2phosphorylation promoteautophagy,thusidentifyingwhatwebelieveisanovelconnectionbetweenERstressandautophagy. Ourdataalsodemonstratethattheautophagy-promotingactivityofthep8-regulatedpathwayisbasedonitsabilitytoinhibitthe Akt/mTORC1axis.RegulationofmTORC1largelyreliesonthe activityoftheprosurvivalkinaseAkt,whoseinhibitionleadsto mTORC1inactivationand,inturn,toautophagy(20).OurfindingsrevealthatTHCupregulatesTRB3,promotingitsinteraction withAktandleadingtodecreasedphosphorylationofthiskinase aswellasofitsdirectsubstratesTSC2andPRAS40,whichtriggersmTORC1inhibitionandinductionofautophagy.TRB3has beenpreviouslyshowntoinhibitAkt(21,22),althoughtheprecise contributionofthispseudo-kinasetotheregulationofAktactivityindifferentcellularcontextsisunclear(32).HerewedemonstratethatTRB3inhibitionoftheAkt/mTORC1axisisessential forcannabinoid-inducedautophagyofcancercells.Moreover,we showthatthispathwayisessentialforcannabinoidantitumoral action.Thus,THCadministrationleadstoTRB3upregulation, mTORC1inhibition,inductionofautophagy,andreductionof tumorgrowthindifferentmodelsoftumorxenografts,butnotin p8-deficienttumorsthataredefectiveintheupregulationofthe p8/TRB3pathway.Furthermore,activationofthispathwaywas alsoevidentin2gliomapatientsthathadbeentreatedwithTHC. TheseresultsthusuncoveraroleforTRB3thatmaybeofgreat importanceintheregulationofcancercelldeath. Autophagyhasbeenproposedtoprotectfromapoptosis,act asanapoptosis-alternativepathwaytoinducecelldeath,oract togetherwithapoptosisasacombinedmechanismforcelldeath (6,33).However,verylittleisknownabouttheroleoftheinterplay betweenthese2cellularprocessesinthecontroloftumorgrowth inresponsetoanticanceragents.Ourresultsnowclearlydemonstratethatinductionofautophagyisinvolvedinthemechanism bywhichcannabinoidspromotetheactivationofthemitochondrialpro-apoptoticpathway.Thus,neithertumorsinwhichthe p8-regulatedpathwayhasbeenablated(andinwhich,therefore, THCtreatmentdoesnotinduceautophagy)nortumorsintrinsicallydeficientinautophagyundergoapoptosisinresponseto THC,andsotheyareresistanttoTHCantitumoralaction.These findingsrevealthatautophagyisrequiredfortheactivationof apoptosisinresponsetocannabinoidtreatmentinvivo. ItisworthnotingthattheconcentrationsofTHCusedinthis studyareinthesamerangeasthoseadministeredintracranially tothepatientsinwhichweobservedactivationoftheautophagymediatedcelldeathpathway(11)andcouldbethusconsidered clinicallyrelevant.Ofinterest,intraperitonealadministrationof THCtoU87MGtumorxenograftsproducesasimilardecreasein tumorgrowth(thatoccursinconcertwithincreasedautophagy andapoptosis)tothatobservedwhenthecannabinoidisadministeredperitumorally(ourunpublishedobservations).Considering thatnosignsoftoxicitywereobservedintheclinicaltrialpatients (11)orintumor-bearinganimalstreatedintracranially,peritumorally,orintraperitoneallywithTHC(refs.34and35anddatanot shown),andthatnooverttoxiceffectshavebeenreportedinother clinicaltrialsofcannabinoiduseincancerpatientsforvarious applications(e.g.,inhibitionofnausea,vomiting,andpain)and usingdifferentroutesofadministration(e.g.,oral,oro-mucosal) (9,36),ourfindingssupportthatsafe,therapeuticallyefficacious dosesofTHCmaybereachedincancerpatients.
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Insummary,inthisstudyweidentifywhatwebelieveisanew routethatlinkstheERstressresponsetotheactivationofautophagyandpromotestheapoptoticdeathoftumorcells(Figure7C). Theidentificationofthispathwaywillhelptounderstandthe moleculareventsthatleadtoactivationofautophagy-mediated celldeathbyanticancerdrugsandmaycontributetothedesignof newtherapeuticstrategiesforinhibitingtumorgrowth. Methods

Cell culture and viability.Corticalastrocyteswerepreparedfrom24-hour-old miceaspreviouslydescribed(13).Primaryculturesofbraintumorcells werepreparedandculturedasdescribedintheSupplementalMethods. U87MG,T98G,U373MG,andMiaPaCa2cells,p8+/+andp8/RasV12/E1A MEFs,Atg5+/+andAtg5/T-largeantigenMEFs(providedbyNoboru Mizushima,TokyoMedicalandDentalUniversity,Tokyo,Japan),Bax/ Bakwild-typeandBax/BakDKOT-largeantigenMEFs(providedbyLuca Scorrano,DulbeccoTelethonInstitute,Milan,Italy,andPatriziaAgostinis,CatholicUniversityofLeuven,Leuven,Belgium),eIF2S51SWTand eIF2S51AT-largeantigenMEFs(providedbyRichardKaufman,UniversityofMichigan,AnnArbor,Michigan,USA,andCesardeHaroand JuanJ.Berlanga,CentrodeBiologaMolecularSeveroOchoa,Autonoma University,Madrid,Spain),Tsc2+/+andTsc2/p53/MEFs,emptyvector (pBABE)andpBABE-myr-AktMEFs,andAtg5+/+andAtg5/RasV12/T-large antigenMEFswereculturedinDMEMcontaining10%FBSandtransferredtomediumcontaining0.5%FBS(exceptRasV12/E1A-transformed MEFs,whichweretransferredtomediumcontaining2%FBS)18hbefore performingthedifferenttreatments.p8+/+andp8/RasV12/E1AMEFsas wellasAtg5+/+andAtg5/RasV12/T-largeantigenMEFscorrespondtoa polyclonalmixofatleast20differentselectedclones.Unlessotherwise indicated,THCwasusedatafinalconcentrationof5M.Cellviabilitywas determinedbytheMTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide]test(Sigma-Aldrich). Flow cytometry.Briefly,cells(approximately5105cellsperassay)were trypsinized,dividedin2tubes,washed,andcollectedbycentrifugation at1,500gfor5min.Onealiquotwasincubatedfor10minat37Cwith AnnexinVFITC(BDBiosciences).Propidiumiodide(1g/ml)wasadded justbeforecytofluorometricanalysis.Theotheraliquotwassimultaneouslylabeledwith3,3-dihexyloxacarbocyanineiodide(DiOC6[3],40nM; Invitrogen)andhydroethidium(5M;Invitrogen)for10minutesat 37C,followedbycytofluorometricanalysis.Cells(10,000)wererecorded ineachanalysis.FluorescenceintensitywasanalyzedinanEPICSXLflow cytometer(BeckmanCoulter). Western blot.Westernblotanalysiswasperformedfollowingstandard procedures.AlistoftheantibodiesusedcanbefoundinSupplementalMethods.DensitometricanalysiswasperformedwithQuantityOne software(Bio-Rad). Transfections. U87MG cells (75% confluent) were transfected with siRNAduplexesusingtheDharmaFECT1Transfectionreagent(Dharmacon).Cellsweretrypsinizedandseeded24haftertransfection,ata densityof5,000cells/cm2.Transfectionefficiencywasgreaterthan70% asmonitoredwithacontrolfluorescent(red)siRNA(siGLORISC-Free siRNA;Dharmacon).Inimmunofluorescenceexperiments,controland selectivesiRNAswereusedina1:5ratio,andcellswithredspotswere scoredastransfected. Infections with adenoviral vectors.U87MGcells(75%confluent)weretransducedfor1hwithsupernatantsobtainedfromHEK293cellsinfectedwith adenoviralvectorscarryingEGFP(providedbyJavierG.Castro,Hospital InfantilUniversitarioNioJess,Madrid,Spain),ratHA-taggedTRB3 (donatedbyPatrickIynedjian,UniversityofGeneva,Geneva,Switzerland) (32),orhumanEGFP-LC3(providedbyAvivaTolkovskyandChristoph

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Goemans,UniversityofCambridge,Cambridge,UnitedKingdom).Infectionefficiencywasgreaterthan80%asdeterminedbyEGFPfluorescence. RNA interference.Double-strandedRNAduplexeswerepurchasedfrom Dharmacon.AlistofsequencescanbefoundintheSupplementalMethods. RT-PCR analysis. RNAwasisolatedusingTrizolReagent(Invitrogen). cDNA was obtained with Transcriptor Reverse transcriptase (Roche AppliedScience).Primersandamplificationconditionscanbefoundin theSupplementalMethods. Real-time quantitative PCR.cDNAwasobtainedusingTranscriptor(Roche AppliedScience).Real-timequantitativePCRassayswereperformedusing theFastStartUniversalProbeMastermixwithRox(RocheAppliedScience),andprobeswereobtainedfromtheUniversalProbeLibrarySet (RocheAppliedScience).PrimersequencescanbefoundintheSupplementalMethods.Amplificationswererunina7900HT-FastReal-Time PCRSystem(AppliedBiosystems).Eachvaluewasadjustedbyusing18S RNAlevelsasareference. Immunoprecipitation.U87MGcellswerelysedinHEPESlysisbuffer(see SupplementalMethodsforbuffercomposition).Lysate(14mg)waspreclearedbyincubatingwith520lofproteinGSepharoseconjugated topre-immuneIgG.Thelysateextractswerethenincubatedwith520 lofproteinGSepharoseconjugatedto520goftheanti-TRB3antibodyorpre-immuneIgG.TRB3antibody(aminoterminalend,ab50516; Abcam)wascovalentlyconjugatedtoproteinGSepharoseusingdimethyl pimelimidate.Immunoprecipitationswerecarriedoutfor1hat4C onarotatorywheel.Theimmunoprecipitateswerewashed4timeswith HEPESlysisbuffer,followedby2washeswithHEPESkinasebuffer.The immunoprecipitateswereresuspendedin30lofsamplebuffer(notcontaining2-mercaptoethanol)andfilteredthrougha0.22-mSpin-Xfilter, and2-mercaptoethanolwasaddedtoaconcentrationof1%(vol/vol).Samplesweresubjectedtoelectrophoresisandimmunoblotanalysis. Ceramide levels. Ceramidelevelsweredeterminedaspreviouslydescribed(37). Confocal laser scanning microscopy. Standard protocols for immunofluorescencemicroscopywereused(seeSupplementalMethodsforthe antibodiesused).ToquantifythepercentageofcellswithLC3orPDIdots, atleast200cellsperconditionwerecountedinrandomlyselectedfields. Inallcases,onlythosecellswith4ormoreprominentdotsofeitherLC3 orPDIwerescoredpositively. In vivo treatments.TumorsderivedfromU87MGcellsandp8+/+andp8/ MEFs were induced and treated as previously described (13). Tumors derivedfromAtg5+/+orAtg5/RasV12/T-largeantigenMEFs(seeSupplementalMethodsfortheprocedureusedtogeneratethesecells)wereinduced innudemicebysubcutaneousinjectionof107cellsinPBSsupplemented with0.1%glucose.Tumorswereallowedtogrowuntilanaveragevolumeof 200250mm3,andanimalswereassignedrandomlytothedifferentgroups. Atthispoint,vehicleorTHC(15mg/kg/d)in100lofPBSsupplemented with5mg/mlBSAwasadministereddailyinasingleperitumoralinjection. Tumorsweremeasuredwithanexternalcaliper,andvolumewascalculated as(4/3)(width/2)2(length/2).Allproceduresinvolvinganimalswere performedwiththeapprovaloftheComplutenseUniversityAnimalExperimentationCommitteeaccordingtoSpanishofficialregulations. Human tumor samples.Tumorbiopsieswereobtainedfrom2recurrent glioblastomamultiformepatientswhohadbeentreatedwithTHC.The characteristicsofthepatientsandtheclinicalstudyhavebeendescribed indetailelsewhere(11).Briefly,THCdissolvedin30mlofphysiological salinesolutionplus0.5%(wt/vol)humanserumalbuminwasadministered intratumorallytothepatients.Patient1receivedatotalof1.46mgofTHC for30days,whilepatient2receivedatotalof1.29mgofTHCfor26days (itwasestimatedthatdosesof610MTHCwerereachedatthesiteof administration;ref.11).Sampleswerefixedinformalin,embeddedinparaffin,andusedforimmunomicroscopy.

Immunomicroscopy of tumor samples. Samples from tumor xenografts weredissected,Tissue-Tek(Sakura)embedded,frozen,and,beforethe stainingprocedureswereperformed,fixedinacetonefor10minatroom temperature.Samplesfromhumantumorsweresubjectedtodeparaffinization,rehydration,andantigenretrievalbeforethestainingprocedureswereperformed.Standardprotocolsforimmunofluorescenceor immunohistochemistrymicroscopywereused(seeSupplementalMethods).NucleiwerecounterstainedwithTOTO-3iodide(U87MGandhuman tumorsamples;Invitrogen)orHoechst33342(MEFtumors;Invitrogen). FluorescenceimageswereacquiredusingMetamorph-Offline6.2software (UniversalImaging)andZeissAxioplan2Microscope. TUNEL.Tumorsampleswerefixed,blocked,andpermeabilized,and TUNELwasperformedaspreviouslydescribed(13). Electron microscopy.Ultrastructuralanalysisofvehicle-andTHC-treated cellswasassessedbyconventionalembeddingintheepoxy-resinEML-812 (TaabLaboratories).Ultrathin(20-to30-nm-thick)sectionsofthesampleswereobtainedusingaLeica-Reichert-Jungultramicrotomeandthen stainedwithsaturateduranylacetateleadcitratebystandardprocedures. UltrathinsectionswereanalyzedinaJEOL1200-EXIItransmissionelectronmicroscopeoperatingat100kV. Statistics.StatisticalanalysiswasperformedbyANOVAwithapost-hoc analysisusingtheStudent-Neuman-Keulstest.DifferenceswereconsideredsignificantwhenthePvaluewaslessthan0.05.

Acknowledgments ThisworkwassupportedbygrantsfromtheSpanishMinistry ofEducationandScience(MEC)(HF2005/0021,toG.Velasco; SAF2006/00918, to M. Guzmn; and BFU2006-00508, to P. Boya),Santander-ComplutensePR34/07-15856,toG.Velasco), Comunidad de Madrid (S-SAL/0261/2006, to M. Guzmn), andLaLiguecontreleCancerandCanceropolePACA(toJ.L. Iovanna). M. Salazar was the recipient of a fellowship from theMEC.A.Carracedowastherecipientoffellowshipsfrom GobiernoVasco,theFederationofEuropeanBiochemicalSocieties,andtheEuropeanMolecularBiologyOrganization.M. LorenteandP.BoyahaveaJuandelaCiervaandaRamny CajalcontractfromtheMEC,respectively.S.Hernndez-Tiedra hasatechniciancontractfromtheSpanishMinistryofEducationandtheFondoSocialEuropeo.TheauthorsthankDario Alessi(UniversityofDundee,Dundee,UnitedKingdom)for donatinganti-PRAS40antibodiesandfortechnicalsupportfor immunoprecipitationexperiments;GemmaFabris,Josefina Casas,andEvaDalmau(InstitutodeInvestigacionesQumicas yAmbientales,Barcelona,Spain)foranalyzingceramidesamples;JosLizcano,JosBayascas,MaraM.Caffarel,andPatrizia Agostinisfortheirexperimentalsuggestions;andothermembersofourlaboratoryfortheircontinualsupport. Received for publication November 3, 2008, and accepted in revisedformFebruary11,2009. Addresscorrespondenceto:GuillermoVelasco,DepartmentofBiochemistryandMolecularBiologyI,SchoolofBiology,Complutense University,c/JosAntonioNovaiss/n,28040Madrid,Spain.Phone: 34-913944668;Fax:34-913944672;E-mail:gvd@bbm1.ucm.es. Arkaitz Carracedo and Ainara Egias present address is: CancerGeneticsProgram,BethIsraelDeaconessCancerCenterand DepartmentofMedicine,BethIsraelDeaconessMedicalCenter, HarvardMedicalSchool,Boston,Massachusetts,USA.
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