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FullTerms&Conditionsofaccessandusecanbefoundathttps://www.
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com/action/journalInformationjournalCode=kaup20AutophagyISSN:1554-8627(Print)1554-8635(Online)Journalhomepage:https://www.
tandfonline.
com/loi/kaup20CriticalroleofCAV1/caveolin-1incellstressresponsesinhumanbreastcancercellsviamodulationoflysosomalfunctionandautophagyYinShi,Shi-HaoTan,ShukieNg,JingZhou,Na-DiYang,Gi-BangKoo,Kerrie-AnnMcMahon,RobertGParton,MichelleMHill,MiguelAdelPozo,You-SunKim&Han-MingShenTocitethisarticle:YinShi,Shi-HaoTan,ShukieNg,JingZhou,Na-DiYang,Gi-BangKoo,Kerrie-AnnMcMahon,RobertGParton,MichelleMHill,MiguelAdelPozo,You-SunKim&Han-MingShen(2015)CriticalroleofCAV1/caveolin-1incellstressresponsesinhumanbreastcancercellsviamodulationoflysosomalfunctionandautophagy,Autophagy,11:5,769-784,DOI:10.
1080/15548627.
2015.
1034411Tolinktothisarticle:https://doi.
org/10.
1080/15548627.
2015.
1034411ViewsupplementarymaterialAcceptedauthorversionpostedonline:06May2015.
Publishedonline:28May2015.
SubmityourarticletothisjournalArticleviews:2613ViewCrossmarkdataCitingarticles:32ViewcitingarticlesCriticalroleofCAV1/caveolin-1incellstressresponsesinhumanbreastcancercellsviamodulationoflysosomalfunctionandautophagyYinShi,1Shi-HaoTan,1,2ShukieNg,1JingZhou,1Na-DiYang,1Gi-BangKoo,3,4Kerrie-AnnMcMahon,5RobertGParton,5MichelleMHill,6MiguelAdelPozo,7You-SunKim,3,4andHan-MingShen,1,2,8,*1DepartmentofPhysiology;YongLooLinSchoolofMedicine;NationalUniversityofSingapore;Singapore;2NUSGraduateSchoolforIntegrativeSciencesandEngineering;NationalUniversityofSingapore;Singapore;3DepartmentofBiochemistry;AjouUniversitySchoolofMedicine;AjouUniversity;Suwon,Korea;4DepartmentofBiomedicalSciences;GraduateSchool;AjouUniversity;Suwon,Korea;5InstituteforMolecularBioscience;TheUniversityofQueensland;Brisbane,Australia;6DiamantinaInstitute;TheUniversityofQueensland;Brisbane,Australia;7IntegrinSignalingLaboratory;VascularBiologyandInammationDepartment;CentroNacionaldeInvestigacionesCardiovasculares;MelchorFernandezAlmagro;Madrid,Spain;8SawSweeHockSchoolofPublicHealth;NationalUniversityofSingapore;SingaporeKeywords:autophagy,breastcancer,caveolin1,lipidrafts,lysosomeAbbreviations:Baf,balomycinA1;CAV1,caveolin1;CHO,water-solublecholesterol;CQ,choloroquine;CTxB,choleratoxinsub-unitB;CTSL,cathepsinL;DRF,detergent-resistantfraction;DSF,detergent-solublefraction;EGF,epidermalgrowthfactor;KO,knockout;LAMP1,lysosomal-associatedmembraneprotein1;MAP1LC3/LC3,microtubule-associatedprotein1lightchain3;MBCD,methyl-b-cyclodextrin;MEF,mouseembryonicbroblasts;MTOR,mechanistictargetofrapamycin;PBS,phosphate-bufferedsaline;PI,propidiumiodide;PLA,proximityligationassay;PTRF,polymeraseIandtranscriptreleasefactor;tfLC3B,mRFP-GFPtandemuorescent-taggedLC3B;TFRC,transferrinreceptor;TSC,tuberoussclerosiscomplex;ATP6V0D1,ATPaseHCtransportinglysosomal38kDa,V0subunitd1;WT,wildtype.
CAV1(caveolin1,caveolaeprotein,22kDa)iswellknownasaprincipalscaffoldingproteinofcaveolae,aspecializedplasmamembranestructure.
Relatively,thecaveolae-independentfunctionofCAV1islessstudied.
Autophagyisaprocessknowntoinvolvevariousmembranestructures,includingautophagosomes,lysosomes,andautolysosomesfordegradationofintracellularproteinsandorganelles.
Currently,thefunctionofCAV1inautophagyremainslargelyelusive.
Inthisstudy,wedemonstrateforthersttimethatCAV1deciencypromotesbothbasalandinducibleautophagy.
Interestingly,thepromotingeffectwasfoundmainlyinthelatestageofautophagyviaenhancinglysosomalfunctionandautophagosome-lysosomefusion.
Notably,theregulatoryfunctionofCAV1inlysosomeandautophagywasfoundtobecaveolae-independent,andactsthroughlipidrafts.
Furthermore,theelevatedautophagylevelinducedbyCAV1deciencyservesasacellsurvivalmechanismunderstarvation.
Importantly,downregulationofCAV1andenhancedautophagylevelwereobservedinhumanbreastcancercellsandtissues.
Takentogether,ourdatarevealanovelfunctionofCAV1andlipidraftsinbreastcancerdevelopmentviamodulationoflysosomalfunctionandautophagy.
IntroductionCAV1istheprincipalscaffoldingproteinofcaveolae,whichisoneofthemajortypesoflipidraftsthatarepresentattheplasmamembrane.
1Lipidraftsareliquid-orderedmicrodomainsenrichedwithacharacteristicstructuralcomposition(sphingoli-pids,cholesterol,andsaturatedphospholipids)inplasmamem-braneandintracellularmembranesofvariousorganelles,includingGolgi,endoplasmicreticulum,mitochondria,andtheendosome/lysosome.
2Thecaveolae(ask-shapedstructures)andtheplanarlipidrafts(alsoknownasnoncaveolarrafts,referredtoaslipidraftshereafter)arethe2maintypesoflipidrafts.
3Atpresent,thecriticalroleofCAV1incaveolaeiswellstudied,whilethereisaccumulatingevidencesuggestingtheimportanceofCAV1inlipidrafts.
Forinstance,CAV1playsanimportantroleincholesteroltrafckingandhomeostasis,akeycomponentinlipidrafts.
4,5Inaddition,comparedtothelimiteddistributionontheplasmamembraneofcaveolae,6CAV1hasamoreexten-sivemembranedistributionincludingmitochondriaandendo-plasmicreticulum,aswellasthelateendosome/lysosome,indicatingtheexistenceofcaveolae-independentfunctionofCAV1intheintracellularmembranesystem.
7-9Emergingevidencedemonstratesthetumor-suppressionfunctionsofCAV1inseveraltypesofcancer,especiallyin*Correspondenceto:Han-MingShen;Email:han-ming_shen@nuhs.
edu.
sgSubmitted:07/09/2014;Revised:03/13/2015;Accepted:03/19/2015http://dx.
doi.
org/10.
1080/15548627.
2015.
1034411www.
tandfonline.
com769AutophagyAutophagy11:5,769--784;May2015;2015Taylor&FrancisGroup,LLCBASICRESEARCHPAPERbreastcancer.
10Forinstance,expressionofoncogenes(suchasABL1/v-ablandHRAS/H-ras)leadstoasignicantdecreaseinCAV1proteinandmRNAlevels.
11LossofCAV1inducescellulartransformationthroughtheactivationoftheMAPK1/ERK2/p41-MAPK3/ERK1/p44cascadeandsubse-quentdevelopmentofanunusualgrowthpatternwithincreasedphospho-MAPK1/3independentofEGF(epidermalgrowthfactor).
12,13Consistently,overexpressionofCAV1issufcienttodecreasecellproliferationandtumorsizeinvivo.
11Inbreastcancer,alargepercentageofpatientsaredecientinCAV1expression.
10SeveralhumanbreastcancercelllinesdisplayadecreasedCAV1expressionlevelcomparedtobenignmammaryepithelialcells.
14Moreover,about35%ofbreastcancercasescontainmutantCAV1.
15Forexample,adominantnegativemutantCAV1P132LhasbeenidentiedinER-positivepatientswithwell-differentiatedbreastcan-cer.
16AlthoughthestudiesdiscussedaboveindicateatumorsuppressiveroleforCAV1,thereisalsoconictingevidenceshowinganoppositeroleofCAV1.
Forinstance,CAV1expressioninbreasttumorstromaincreasestumorinvasionandmetastasisviabiomechanicalremodeling.
17Therefore,theexactbiologicalfunctionofCAV1inbreastcancerdevel-opmentandthemolecularmechanismsremaintobefurtherinvestigated.
Macroautophagy(referredtoasautophagyhereafter)isanevolutionarilywell-conservedself-eatingprocessineukaryoticcellsthatresultsindegradationoflong-livedproteinsandorganellesviathelysosomalpathway,whichservesasapow-erfulboosterofmetabolichomeostasis.
18Onekeyfeatureofautophagyisthatitinvolvesvariousintracellularmembranestructures,includingautophagosomes,lysosomes,andautoly-sosomes.
Atpresent,severalstudieshaveimplicatedCAV1andlipidraftsintheregulationofautophagy.
Forinstance,CAV1deciencyinducesautophagyinadipocytesviasup-pressionofinsulinandlipolyticresponses.
19LossofCAV1promotesautophagyunderhypoxiaandoxidativestressinadipocytesandbroblasts.
20Thesestudiesindicateasuppres-siveroleforCAV1inautophagy.
Furthermore,therearesev-eralcluesindicatingthepotentialroleoflipidraftsinautophagy.
Forinstance,lipidraftspromotetheAKT-MTOR(mechanistictargetofrapamycin)pathway,21,22akeynegativeregulatorofautophagy.
23Incontrast,thereiscon-ictingevidenceindicatingthatsomecomponentsoflipidrafts,suchasceramidesandGD3ganglioside,playapositiveroleinautophagyregulation.
24-26Inthisstudy,weaimedtoevaluatetheinvolvementofCAV1inautophagy.
OurdataclearlydemonstratethatdeciencyofCAV1promotesautophagyandlysosomalfunctionviathedis-ruptionoflipidrafts,independentofcaveolae.
Moreover,theele-vatedautophagylevelinducedbyCAV1deciencyservesasacellsurvivalmechanismundernutritionalstress.
Importantly,down-regulationofCAV1andenhancedautophagylevelwereobservedinhumanbreastcancercelllinesandcanceroustissues.
Thus,ourdatarevealanovelfunctionofCAV1incellstressresponsesandpossiblybreastcancerdevelopmentviamodulationoflysosomalfunctionandautophagy.
ResultsCAV1deciencypromotesautophagyviadisruptionoflipidraftsWersttestedtheeffectofCAV1deciencyonautophagybyusingCav1wild-type(WT)andknockout(KO)mouseembry-onicbroblasts(MEFs)asreportedpreviously.
27TheabsenceoftheCAV1proteinwasconrmedinwesternblots(Fig.
1A).
WeassessedthelipidraftlevelusingAlexaFluor594–conjugatedcholeratoxinsubunitB(CTxB)staininginbothCav1C/Candcav1/celllines.
28AsshowninFigure1A,cav1/cellsshowedmarkedlyreducedCTxBsignalinbothintracellularandplasmamembranesincomparisontotheCav1C/CMEFs,indi-catingalowerleveloflipidraftsincav1/cells.
Theseresultswereconrmedbylabelingthecholesteroldirectlyusinglipin(Fig.
1B).
Next,wecomparedtheautophagylevelsbyevaluatingthewell-establishedautophagymarkerMAP1LC3B-II/LC3B-IIinnormalandaminoacidstarvationconditions.
Wefoundthatinbothconditions,LC3B-IIlevelswerehigherincav1/cells(Fig.
1C).
Moreover,tomeasuretheautophagicux,weinhib-itedtheautophagosomaldegradationbyadditionofthelyso-somalinhibitorchloroquine(CQ),andobservedanincreaseinLC3B-IIproteinlevelsincav1/cells,excludingthepossibilitythattheincreasedLC3B-IIwascausedbyablockageofautopha-gosomaldegradation(Fig.
1C).
ThesedatasuggestthatCAV1deciencypromotesbothbasalandinducibleautophagylevels.
SinceCAV1hasbeenestablishedasacriticalscaffoldingpro-teinincaveolae,weattemptedtotestwhethertheroleofCAV1inautophagyiscaveolae-dependent.
Todothis,weutilizedcellsdecientinPTRF(polymeraseIandtranscriptreleasefactor),anotherkeycomponentforcaveolaeformation.
1DeciencyofPTRFblockscaveolaeformationandpromotesCAV1degrada-tion.
29Consistently,wefoundlowerCAV1levelsinptrf/cells,andtheCQincreasedCAV1levelviablockageoflysosomaldeg-radation(Fig.
S1A).
Interestingly,wefoundthatbothCTxBandlipinstainingdidnotdiffersignicantlybetweenPtrfC/Candptrf/MEFs(Fig.
S1BandS1C).
Theautophagylevelwasalsocomparableinthese2celllinesinbasalandaminoacidstarvationconditions(Fig.
S1A).
Moreover,disruptionoflipidraftsbymethyl-b-cyclodextrin(MBCD)hadasimilareffectonbothPtrfC/Candptrf/cells(Fig.
S1A).
Takentogether,thesedataindicatethepossibilitythattheimplicationofCAV1inautoph-agyismainlyvianoncaveolarlipidrafts,butnotPTRF-relatedcaveolae.
TofurtherexaminewhetherCAV1isimplicatedinautophagyvialipidrafts,wetestedtheeffectofMBCDonautophagy.
MBCDdepletescholesterolfromtheplasmamembraneanddis-ruptslipidrafts.
30ThedisruptiveeffectsofMBCDonlipidraftswereconrmedbyusing3differentapproaches:(i)reductionofAlexaFluor594–CTxBstaining,(ii)reductionoflipinstainingand(iii)lossofCAV1intheisolatedraftfractionandtheaccu-mulationinthenonraftfraction.
31MBCDtreatmentsigni-cantlydecreasedtheintensityofbothCTxBstaining(Fig.
1D)andlipinstaining(Fig.
1B),andcausedCAV1andtheotherlipidraftproteinFLOT1(otillin1)toredistributefromthedetergent-resistantlipidraftfraction(DRF,representingthelipid770Volume11Issue5Autophagyraftcomponents)tothedetergent-solublefraction(DSF,repre-sentingthenonlipidraftcomponents,usingTFRC[transferrinreceptor]asthemarker)(Fig.
1EandS2),indicatingthatMBCDisabletodisruptlipidraftseffectively.
Asexpected,MBCDenhancedboththebasalandaminoacidstarvation-inducedautophagylevel,basedontheobservationsofFigure1.
Forgurelegend,seepage764.
www.
tandfonline.
com771AutophagyGFP-LC3Bpunctaformation(Fig.
1F)andincreasedLC3B-IIlevel(Fig.
1G)inbothnormalandaminoacidstarvationcondi-tions,withorwithoutthepresenceofCQ.
TheseobservationsdemonstratethatdisruptionoflipidraftsbyMBCDincreasesautophagicux.
Moreover,toexcludethepossibilitythatMBCD-mediatedautophagyobservedabovewascausedbymechanismsindepen-dentoflipidrafts,weusedcholesterol(CHO)replenishmenttorestorethelipidrafts.
AsshowninFigure1DandE,CHOreplenishmentrecoveredthedecreasedintensityofCTxBstain-ingandtheredistributionofCAV1causedbyMBCD.
Notably,CHOloadingdecreasesLC3B-IIux,asnoticedbythereduc-tionintheCQ-inducedaccumulationofLC3B-II(Fig.
1H).
TofurtherconrmthenotionthattheregulatoryroleofCAV1inautophagyisassociatedwithcholesterol,weperformedMBCDtreatmentincav1-KOcells.
AsshowninFigure1I,comparedtoCav1WTcells,MBCDtreatmentwaslesseffectiveininducingautophagyincav1-KOcells,suggestingthattheregulatoryroleofCAV1inautophagyisassociatedwithcholesterolandlipidrafts.
Inaddition,lipidraftsareimplicatedintheAKT-MTORsig-nalingpathway,whichisakeynegativeregulatorofautoph-agy.
21,22WenexttestedtheinvolvementofMTORC1inautophagyinducedbylipidraftdisruption.
AsshowninFigureS3A,MBCDdisruptiondidnotshowasignicantinhib-itoryeffectonMTORdownstreamtargetproteinRPS6/S6(Ser235/Ser236).
ConsistentlynodifferencewasfoundinthephosphorylationlevelofbetweenCAV1WTandKOMEFs(Fig.
S3B).
Moreover,wedisruptedlipidraftsbyMBCDtreat-mentintsc2(tuberoussclerosis2)-KOMEFs.
TSC2isanessen-tialcomponentintheformationofafunctionalheterodimertosuppressMTORC1activity,andlossofTSC2leadstothecon-stitutiveMTORactivationandimpairedautophagy.
32TheeffectsofMBCDdisruptionontheautophagicuxweresimilarinbothTsc2C/Candtsc2/MEFs(Fig.
S3A),suggestingthatMTORC1isunlikelytoplayaroleinautophagy-mediatedbyCAV1deciencyandlipidraftdisruption.
CAV1deciencypromoteslysosomalfunctionLysosomesarethekeyorganellesresponsibleforautophagydegradation.
33Recentlywehavedemonstratedupregulationoflysosomalfunctioninthecourseofautophagyinducedbystarva-tionandMTORinhibition.
23Inthisstudy,weexaminedwhetherCAV1deciencyaffectslysosomalfunction,measuredbythefollowingassays:(i)lysosomalpHevaluationbyLyso-TrackerandLysoSensor,(ii)lysosomalproteolysisbyDQ-BSA,and(iii)detectionofcathepsinenzymeactivitycoupledwithowcytometryandconfocalmicroscopy.
First,therewasasignif-icantincreaseinLysoTrackerGreen,LysoSensorandDQ-BSAsignalincav1/MEFswhencomparingtoWTcells(Fig.
2Aand,S4AandB),indicatingenhancedacidicationoflysosomesandproteolysisactivity,respectively.
ThelysosomalpHwasalsosignicantlydecreasedincav1/cells(Fig.
S4C).
Second,asig-nicantincreaseinlysosomalCTSL(cathepsinL)enzymeactiv-ityincav1/cellswasdetectedbyowcytometry(Fig.
2B)andconfocalmicroscopy(Fig.
S4D).
Tofurtherunderstandtheroleoflipidraftsinregulationoflysosomalfunction,wealsoassessedtheeffectofMBCDandcholesterolreplenishmentonlysosomalfunction.
Consistently,ahigherlevelofLysoTrackerGreenstaining,LysoSensorstaining,DQ-BSAintensity,andCTSLenzymeactivitywerefoundincellstreatedwithMBCD(Fig.
2C,D,S4AandB).
MBCDtreatmentalsoledtoasignicantdecreaseofthelysosomalpH(Fig.
S4C).
Notably,thesamecholesterolreplenishmentwhicheffectivelyrecoveredlipidrafts(Figs.
1DandE)reversedthepro-motingeffectofMBCDonlysosomalfunction(Figs.
2CandD).
Toconrmwhethertheincreaseofthesemarkersusedforlysosomalfunctionwasduetoincreasedlysosomalmembranepermeabilityorevenincreasedlysosomenumbers,weexam-inedthedistributionofCTSLandtheLAMP1(lysosomal-associatedmembraneprotein1)level.
Inthisstudy,mostoftheCTSLmagicredstainingcolocalizedwithLysoTrackerGreeninbothCav1C/Candcav1/celllinesbeforeandafterraftdisruptionbyMBCD(Fig.
S4DandE),indi-catingthatincreasedCTSLbylipidraftdisruptionwasinthelysosomebutnotduetoitsleakageintothecytosol.
Moreover,inourdata,theLAMP1proteinlevelwasnotaffectedbyCAV1deciencyorMBCDtreatment(Fig.
S5AandB),thusexcludingthepossibilitythattheobservedchangesoflysosomalfunctionsareduetoincreasedlysosomenumbers.
Figure1(Seepreviouspage).
CAV1deciencypromotesautophagyviadisruptionoflipidrafts.
(A)LipidraftmarkerCTxB(Red)staininginCav1C/Candcav1/MEFs.
Cellidentitywasshownbywesternblots.
(B)Cholesterolindicatorlipin(blue)stainingofbothCAV1deciencyandMBCDpretreatment(5mM,1h).
Scalebar:10mm.
(C)CAV1deciencyincreasesbothbasalandaminoacidstarvation-inducedautophagy(AA-,2h).
Autophagicuxwasdetectedbythetreatmentswithorwithoutchloroquinediphosphate(CQ,20mM)for2h.
QuanticationoftheLC3B-II/ACTBratiosispresented(*P<0.
05).
(D)CholesterolreplenishmentrescuestheloweringeffectofMBCDonCTxBstaining.
MEFswerepretreatedwithorwithoutMBCD(5mM)for1h,thenincubatedinthepresenceorabsenceofcholesterol(CHO,30mg/ml)andthenstainedwithCTxB.
(E)CAV1redistributionindetergent-resistantfractions(DRF,proteinsrichinmembranerafts)anddetergentsolublefractions(DSF)afterthesametreatmentsasindicatedin(D).
Celllysatesweresep-aratedandimmunoblottedwiththeindicatedmarkers.
(F)MBCDincreasestheGFP-LC3Bpunctaformationinbothnormalandstarvationcondition.
MEFswithstableexpressionofGFP-LC3BwerepretreatedwithorwithoutMBCD(5mM)for1h,thenincubatedinbothnormalDMEMandAA-mediumwithorwithoutCQ(20mM)for2h.
TheGFP-LC3BpunctationandlipidraftmarkerCTxBwereobservedunderaconfocalmicroscope(600).
Scalebar:10mm.
(G)MBCDincreasesautophagicuxinbothnormalandaminoacidstarvation-inducedautophagy.
Cellswereexposedtothesametreatmentsindicatedinpanel.
QuanticationoftheLC3B-II/ACTBratiosispresented(*P<0.
05).
(F)thencelllysateswerecollectedandsubjectedtowesternblotanalysisfortheindicatedmarkers.
QuanticationoftheLC3B-II/ACTBispresented.
(H)CholesterolreplenishmentovercomestheeffectofMBCDdisrup-tiononautophagy.
MEFswerepretreatedwithorwithoutMBCD(5mM)for1h,thenincubatedinthepresenceorabsenceofCHO(30mg/ml)andCQ(20mM).
(I)MBCDdisruptiondoesnotfurtherenhanceautophagyuxincav1/MEFs.
Cav1C/Candcav1/MEFswerepretreatedwithorwithoutMBCD(5mM)for1h,thenincubatedinthepresenceorabsenceofCQ(20mM).
772Volume11Issue5AutophagyCAV1deciencypromotesV-ATPaseassemblyInordertounderstandthemolecularmechanismsunderlyinglysosomalactivationmediatedbyCAV1deciency,weexaminedchangesinlysosomalV-ATPaseassembly.
V-ATPaseisknownasthecrucialproteincomplexthatregulatestheacidicationpro-cessofthelysosomallumenandlysosomaldegradation.
34TheV-ATPase,aproton-pumpingmembraneproteincomplex,iscontrolledbytheassemblyof2domains(thecytosolicV1domainandtheintegralmembraneV0domain).
34SincetherearestudiesshowingthatlipidraftscontainV-ATPasesubunits,35wethusdeterminedwhetherCAV1deciencyregulatestheV-ATPaseassemblyvialipidraftdisruption.
First,morethan50%oflysosomes(labeledbyLysoTrackerGreen)werecolocal-izedwithlipidrafts(visualizedbyCTxB)(Fig.
3A),suggestingtheexistenceoflipidraftsonlysosomes.
Suchandingwasfur-thersupportedbytheevidentcolocalizationbetweenLAMP1andCAV1(Fig.
S6).
Second,isolatedlysosomemembranescon-tainedasignicantamountofCAV1(Fig.
3B),suggestingthepossibleexistenceofCAV1-dependentlipidraftsonthelysosomemembrane.
Andnally,weusedtheproximityligationassay(PLA)toassessthecolocalizationoftheV0andV1subunits.
Indeed,ahigherlevelofsignalwasdetectedinCAV1-decientFigure2.
CAV1deciencypromoteslysosomalfunction.
(A)cav1/MEFshasahigherDQTMRedBSA(red)andLysoTrackerGreenDND-26(green)stainingintensitywhencomparingtoCav1C/Ccells.
Cav1C/Candcav1/MEFswerepreloadedwithDQTMRedBSA(red)andcostainedwithLysoTrackerGreenDND-26(green).
Thenexaminedbyconfocalmicroscopy(600).
Scalebar:10mm.
ThemeanorescenceintensitywereanalyzedbyImageJ,means§SDwerepresented.
(B)CTSLenzymeactivityisincreasedincav1/MEFs.
Cav1C/Candcav1/MEFswereloadedwithMagicRedTMCTSLreagentfor15min.
Fluorescenceintensityof10,000cellspersamplewasdeterminedbyowcytometryusingtheBDFACScytometer.
Threeinde-pendentexperimentswereanalyzed,means§SDwerepresented.
(C).
MBCDincreasestheDQTMRedBSAandLysoTrackerGreenDND-26staininginten-sity,whilecholesterolreplenishmentovercomestheseeffects.
MEFswerecostainedwithDQTMRedBSAandLysoTrackerGreenDND-26,thenpretreatedwithMBCD(5mM)for1h,andthentreatedwithorwithoutCHO(30mg/ml)andexaminedbyconfocalmicroscopy(600).
Scalebar:10mm.
(D)MBCDincreasestheCTSLenzymeactivity,whilecholesterolreplenishmentovercomesthiseffect.
MEFswerepretreatedwithMBCD(5mM)for1h.
ThenCTSLenzymeactivitiesweremeasuredbyFACScytometer.
Threeindependentexperimentswereanalyzed,means§SDwerepresented(*P<0.
05,**P<0.
01).
www.
tandfonline.
com773AutophagycellsandafterMBCDtreatment(Fig.
3C),indicatingenhancedV1translocationtolysosomesandV-ATPaseassemblyincellsafterlipidraftdisruption.
Inaddition,wefoundthatthecolocali-zationbetweenCAV1andATP6V0D1(ATPase,HCtransport-ing,lysosomal38kDa,V0subunitd1)wasdecreasedafterlipidraftdisruptionbyMBCD(Fig.
S7).
Interestingly,theaminoacid-freestarvation,whichpromoteslysosomalfunction,23alsodecreasedthecolocalizationbetweenCAV1andATP6V0D1(Fig.
S7).
TheseobservationssupportthenotionthattheCAV1-dependentlipidraftslocalizeatlysosomemembranesanddisrup-tionoftheselipidraftsincreasestheV-ATPaseV0andV1assembly.
Disruptionoflipidraftspromotesautophagosome-lysosomefusionItisknownthatactivationoflysosomalfunctioninthecourseofautophagyisalsodependentonautophagosome-lysosomefusion.
23Here,weassessedtheeffectoflipidraftsonthisfusionprocessbyusingtheL929cellswithstabletrans-fectionoftfLC3B(mRFP-GFPtandemuorescent-taggedLC3B)asatool.
First,inthesecells,theRFPismoreresistantthanGFPtotheacidiclyso-someenvironment.
Therefore,RFP-positive/GFP-negativepunctacanbeusedtoevaluatethefusionofautopha-gosomesandlysosomes.
36WeobservedincreasedRFPpunctaafterMBCDpretreatmentandCav1knockdown,indicatingapositiveeffectofraftdis-ruptiononautophagosome-lysosomefusion(Figs.
4A,BandS8).
Second,asshowninFigure4CandD,incellstreatedwithbalomycinA1(Baf)for15h,allofthepunctaweredoublepositiveforGFPandRFP,indicatingacompleteblockageofautophagosome-lysosomefusion.
RemovalofBaffromthecellculturemediumwouldelimi-natetheblockageandresumetheauto-phagosome-lysosomefusionprocess.
37AfterBafwasremoved,thenonfusedautophagosomes(bothGFPandRFPpositive)wouldproceedwithlysosomefusiontoformautolysosomes,whicharedetectablewithRFPduetothequenchingofGFPintheacidicautoly-sosome.
36Therefore,bycomparingtheratioofGFP/RFPsignalsafterBafremovalweareabletoevaluatetheautophagosome-lysosomefusionpro-cess.
Incomparisontocontrolcells,MBCDtreatmentmarkedlyreducedtherelativeratioofGFP/RFP,indicat-ingahigherrateofautophagosome-lysosomefusionandautolysosomeformationinMBCD-treatedcells(Figs.
4CandD).
Notably,theeffectofMBCDwasevenmoreevidentthanthoseobservedinthepositivecontrolwithaminoacidstarvation(Figs.
4CandD).
AutophagyinducedbyCAV1deciencypromotescellsurvivalunderstarvationOneimportantphysiologicalroleofautophagyistoprolongcellsurvivalunderstarvationbyrecyclingcytoplasmiccompo-nentstoprovidetheessentialnutrientsneeded.
38-40Here,weexaminedthecellviabilitybymorphologicalchangesandpropi-diumiodide(PtdIns)exclusionassaytotestifCAV1deciency-inducedautophagywouldhaveanyimpactonthecelldeathinducedbyaminoacidstarvation.
Asexpected,weobservedthatcav1/MEFsweremoreresistanttoaminoacidstarvationthanWTMEFs(Figs.
5AandB).
Figure3.
CAV1deciencypromotesV-ATPaseassembly.
(A)LipidraftmarkerCTxB(red)andlyso-somemarkerLysoTrackerGreenDND-26(green)arecolocalized.
MEFswerecostainedwithCTxB(red)andLysoTrackerGreenDND-26(green),thenobservedbyconfocalmicroscopy(600).
Scalebar:10mm.
(B)CAV1isaccumulatedinlysosomefractions.
LysosomefractionswerecollectedasdescribedinMaterialsandMethods.
LAMP1wasusedasamarkerforthelysosomefraction,andMT-CYBwasusedasamarkerformitochondria.
Lysosomefractionsandtotalcelllysateswereblottedforindicatedproteins.
(C)V-ATPaseassemblyisincreasedinCAV1-decientcellsorafterMBCDtreat-ment.
Cav1C/CMEFs,whichweretreatedwithorwithoutMBCD(5mM)for1h,andcav1/MEFswerexedandfollowedbyproximityligationassay(PLA).
ThenucleiwerecounterstainedwithDAPI.
RepresentativePLAimageswereselectedandshown.
Scalebar:10mm.
774Volume11Issue5AutophagyNext,toconrmwhetherthissurvivaladvantageinCAV1-decientcellsisautophagydependent,wecomparedtheeffectofMBCDtreatment,whichmimicsthelipidraftdisruptioneffectofCAV1deciency,onthecellviabilityinbothAtg5C/Candatg5/MEFsafterstarvation.
First,wefoundthattreatmentwithMBCDenhancedautophagicuxlevelonlyinAtg5C/CMEFs(Fig.
5C).
Second,MBCDalonedidnotaffectcellviabilityinnormalgrowthmediuminbothAtg5C/Candatg5/cells,whilepretreatmentwithMBCDofferedsignicantprotectionagainststarvation-medi-atedcelldeathinAtg5C/Ccells,butnotinatg5/cells(Figs.
5DandE).
Asexpected,theatg5/MEFsweremuchmoresusceptibletocelldeathunderstarvation(Figs.
5DandE).
Theseresultsthusindicatethattheprotec-tiveeffectbylipidraftdisruptionagainststarvation-inducedcelldeathisautophagyorAtg5-dependent.
Altogether,weprovideevidencetodemonstratethatthehigherlevelofautophagyinducedbyCAV1deciencyvialipidraftdisruptionpromotescellsurvivalunderstarvationconditions.
ReducedCAV1expressionlevelinhumanbreastcancercellsandre-expressionofCAV1inMCF7cellsrecoverslipidraftsandsuppressesautophagyandlysosomefunctionDatafromanearlierpartofourstudydemonstratethatCAV1deciencypromotesautophagy.
SinceautophagyiswidelyFigure4.
Disruptionoflipidraftspromotesautophagosome-lysosomefusion.
(A)MBCDpretreatmentincreasestheRFPsignalinthetfLC3B-L929cells.
ThetfLC3B-L929cellswerepretreatedwithorwithoutMBCD(5mM)for1h,thenincubatedinbothnormalDMEMandaminoacid-freemediumfor2h.
Cellswereexaminedusingaconfocalmicroscope(600).
Scalebar:10mm.
(B)Statisticalanalysisoftheguresinpanel(A)bytheaverageRFPuores-cencepercell.
Valueswererepresentedasmeans§SDof3experiments(*P<0.
05).
(C)MBCDpretreatmentincreasesautophagosome-lysososmefusion.
ThetfLC3B-L929cellsweretreatedwithbalomycinA1(Baf,50nM)for15h.
Thenmediumwaschangedtotheindicatedtreatmentsandcellswereobservedunderconfocalmicroscopyateachtime(3,6,and9h).
Representativedatafrom9-htreatmentwerepresented.
Scalebar:10mm.
(D)Sta-tisticalanalysisofGFP/RFPratio.
Therateofautophagosomematurationwasmeasuredbythegreen/reduorescenceratio(RelativeRatioofGFP/RFP)ofindicatedtreatmentsateachtime(3,6,and9h)afterBafremoval(0).
TheratioofGFP/RFPwasnormalizedtothevalueatT0.
Valueswererepresentedasmeans§SDof3experiments.
www.
tandfonline.
com775Autophagyconsideredtobeatumor-suppressivemechanism,38,39,41wehypothesizedthatCAV1expressionisassociatedwithcancerdevelopmentviamodulationofautophagy.
WerstmeasuredtheCAV1proteinlevelinasetofdifferentbreastcancercelllinesandanontumorigenicMCF10A.
Amongthem,ZR75–1,SK-BR3andMCF7hadlowlevelsofCAV1expression,whiletheCAV1proteinlevelsinMDA-MB-231,MDA-MB-436werefoundtobehigher,similartoMCF10Acells(Fig.
6A).
SuchresultsaregenerallyconsistentwiththeearlierreportswithregardtoCAV1expressioninbreastcancercells.
14Wenextchose2breastcancercelllinesMDA-MB-231andMCF7toelucidatethefunctionofCAV1inautophagyandcellsurvival.
ThemRNAlevelofCAV1wasfoundtobeconsistentwiththepro-teinlevelinthese2celllines(Fig.
6B).
WethencomparedCTxBandlipinstainingintensityasdirectevidenceforlipidraftlevels.
ConsistentwithCAV1expressiondifferences,MDA-MB-231cellswithhighCAV1expressionhadarelativelyhigherCTxBandlipinlevelwhencomparingtoMCF7cells(Figs.
6CandD).
ThesedatathussuggestthatCAV1expressionlevelisassociatedwithlipidraftsinbreastcancercelllines.
InordertoevaluatetheroleofCAV1inautophagyregulation,weestablishedastablecelllineinwhichtheMCF7cellswerereconstitutedwithCAV1.
Asexpected,thelipidraftmarkersCTxBandlipinstainingwererecoveredinMCF7cellswithCAV1expression(Figs.
6EandF).
Moreover,expressionofCAV1ledtoreducedLysoTrackerRedstaining(Fig.
6G).
Moreimportantly,theautophagymarkerLC3B-IIlevelwasdecreasedinMCF7cellswithexogenousexpressionofCAV1withorwithoutCQtreatment(Fig.
6H),consistentwiththeearliernotionthatCAV1negativelyregulatesautophagy.
Finally,theexpressionofFigure5.
AutophagyinducedbyCAV1deciencypromotescellsurvivalunderstarvation.
(AandB)cav1/MEFsaremoreresistanttoaminoacidstar-vationcomparedtoCav1C/Ccells.
Cav1C/Candcav1/MEFsweretreatedwithaminoacid-freemediumfor48h.
Thenthecellswereobservedunderphase-contrastmicroscopy(A)Scalebar:200mm.
Thepercentageofdeadcellswasmeasuredwiththepropidiumiodide(PtdIns)uptakeassaycoupledwithowcytometry(B)Valueswererepresentedasmeans§SDof3experiments(*P<0.
05).
(C)MBCDpretreatmentinducesautophagicuxinAtg5C/Ccells,andautophagyisdecientinatg5/cells.
WTandatg5/MEFswerepretreatedwithorwithoutMBCD(5mM)for1h,thenincubatedinthepresenceorabsenceofCQ(20mM).
(DandE)MBCDprotectsagainstcelldeathinATG5WTcellsafteraminoacidstarvation(AA-).
Atg5C/Candatg5/MEFswerepretreatedwithMBCD(5mM)for1h,followedbyaminoacidstarvation.
Thenthecellswereobservedunderphase-contrastmicros-copy.
Scalebar:200mm.
ThepercentageofdeadcellswasmeasuredwiththePtdInsuptakeassaycoupledwithowcytometry.
776Volume11Issue5AutophagyFigure6.
ReducedCAV1expressionlevelinhumanbreastcancercellsandre-expressionofCAV1inMCF7cellsrecoverslipidraftsandsuppressesautophagyandlyso-somefunction.
(A)TheCAV1levelindifferentbreastcancercelllines.
ThelysatesofMCF-10Aand5breastcancercelllineswereimmunoblottedwiththeindicatedmarkers.
(B)mRNAlevelofCAV1inMCF7andMDA-MB-231cells.
Quantitativereal-timeRT-PCRanalysisofmRNAlevelsofCAV1intheMDA-MB-231andMCF7cells.
TheamountofCAV1expressioninMDA-MB-231cellswassetto100%(CandD).
LipidraftmarkerinMDA-MB-231andMCF7cells.
MDA-MB-231andMCF7cellswerestainedbyraftmarkerCTxB(C)andlipin(D)andobservedbyconfocalmicroscopy(X600).
(EandF)Re-expressionofCAV1inMCF7recoverslipidraftlevels.
MCF7cellswithsta-bleexpressionofEGFPandEGFP-CAV1werestainedbytheraftmarkerCTxB(E)andlipin(F).
(G)Re-expressionofCAV1decreaseslysosomefunction.
MCF7cellswithsta-bleexpressionofEGFPandEGFP-CAV1wereincubatedwithLysoTrackerRedandassessedbyconfocalmicroscopy(600).
Toavoidsignalsaturation,thepowerofthegreenlaserwasreduced.
(H)Re-expressionofCAV1decreasestheautophagylevelinMCF7cells.
MCF7cellswithstableexpressionofEGFPandEGFP-CAV1weretreatedwithCQ(20mM)for2h,thenthecelllysateswereblottedfortheindicatedmarkers.
(I)Re-expressionofCAV1decreasescellsurvivalafteraminoacidstarvation.
EGFPandEGFP-CAV1MCF7cellsweretreatedwithorwithoutwortmannin(Wort,50nM)underbothnormalandaminoacidsstarvationconditionsafter72h,thenstainedwithPI.
ThePtdInsstainingwasobservedthroughuorescencemicroscopy.
Scalebar:200mm.
(J)ThepercentageofPI-positivecellswerecountedandanalyzed.
Threeindependentexperimentswereanalyzed,Means§SDarepresented(**P<0.
01).
www.
tandfonline.
com777AutophagyCAV1sensitizedtheMCF7cellstocelldeathinducedbyaminoacidstarvation(Figs.
6IandJ),indicatingthatdeciencyofCAV1inMCF7cellspromotescellsurvivalunderstarvationcon-ditions.
Inaddition,theinhibitionofautophagybywortmannin(Wort)almosttotallydiminishedtheprosurvivaladvantagesinCAV1-decientcells(Figs.
6IandJ).
Theseresultsfurthercon-rmthattheprotectiveeffectofCAV1deciencyagainststarva-tion-inducedcelldeathisautophagy-dependent.
Figure7.
DownregulationofCAV1withenhancedautophagyinhumanbreastcancertissues.
(A)LevelsofCAV1andautophagymarkersinbreasttumorandadjacentnormaltissues.
Thelysatesof7pairsoftumorsandtheiradjacentnormaltissuesampleswereimmunoblottedwiththeindicatedmarkers.
(B)mRNAlevelofCAV1inbreasttumorandtheiradjacentnormaltissuesamples.
Quantitativereal-timeRT-PCRanalysisofmRNAlevelsofCAV1inthe7pairsoftumorandtheiradjacentnormaltissuesamples.
TheamountofCAV1expressioninthenormaltissueoftherstpairwassetto100%.
(C)Immu-nostainingofCAV1inparafn-embeddedsectionsfrombreasttumorandtheiradjacentnormalsamples.
Sampleswereimmunostainedwithanti-CAV1,thencounterstainedwithhematoxylin(400X).
ThetissuemorphologywasconrmedbyH&Estaining(200X).
(D)Thestatisticalanalysisof7pairsofbreasttumorandadjacentnormalsamples.
TheintensitiesofCAV1proteinswerequantiedusingtheMoticImagesAdvancedsoftware,followedbysta-tisticalanalysis.
Means§SDarepresented(**P<0.
01).
(E)ModeloftheregulatoryroleoflipidraftsandCAV1inautophagyandthecellularstressresponse.
LossofCAV1andtheassociateddisruptionoflipidraftspromoteautophagybypromotinglysosomefunctionandpossiblyincreasingofauto-phagosomeformation.
Inducibleautophagyservesasacellsurvivalmechanismunderstressconditionssuchasstarvation.
778Volume11Issue5AutophagyDownregulationofCAV1withenhancedautophagyinhumanbreastcancertissuesInordertounderstandthepathologicalimplicationofCAV1inhumanbreastcancer,weanalyzedandcomparedtheCAV1mRNAandCAV1proteinlevelsin7pairsofbreastcancerandtheiradjacentnormaltissues.
AsshowninFigure7A,wefoundmarkeddownregulationforCAV1proteinlevelsinthecanceroustissues.
Similarly,themRNAlevelofCAV1wassignicantlylowerorundetectableinthecanceroustissues(Fig.
7B).
Consistently,inallofthesamples,weobservedalowerlevelofCAV1inbreasttumortissues(onerepresentativepairwasshowninFig.
7CandthestatisticalanalysisofthesemiquanticationoftheCAV1levelin7pairsoftissuesampleswereshowninFig.
7D).
SinceCAV1isassociatedwithautophagyasshownearlier,wefur-theranalyzedandcomparedtheautophagicmarkersinthosehumanbreastcancerandadjacenttissuesamples.
AsshowninFig-ure7A,amongthe7pairstested,LC3B-IIlevelwasincreasedincanceroustissuesin4pairsofthesamples(#11,#20,#19and#86),withcorrespondingreductionoftheleveloftheautophagysubstrateSQSTM1/p62inallofthe7samples.
Takentogether,thesedatasuggestthatdownregulationofCAV1isassociatedwithupregulatedautophagyinhumanbreastcancertissues.
DiscussionInthisstudy,wereportanovelfunctionofCAV1inmodula-tionoflysosomalfunctionandautophagy.
Thisregulatoryfunc-tionofCAV1isbelievedtoactvialipidraftsindependentofcaveolae.
AssummarizedinFigure7E,CAV1deciencyandlipidraftdisruptionpromotelysosomalfunction,increaseautophagicuxandpromotecellsurvivalunderstarvation.
Moreover,inbreastcancercellsandtissues,downregulatedCAV1wasassociatedwithanenhancedautophagylevel.
ThusourdatarevealanovelfunctionofCAV1incellstressresponsesandpossiblycancerdevelopmentviamodulationoflysosomalfunctionandautophagy.
Caveolaeandlipidraftsarestructurallyandfunctionallydis-tinct,whileCAV1hasbeenwellestablishedtoplayacriticalroleinthestructureandfunctionofcaveolae.
6Atpresent,thereare2linesofevidencedemonstratingthatCAV1isalsoinvolvedinlipidrafts:(i)CAV1playsanimportantroleintheregulationofcholesterolhomeostasisinvariousmembranestructures,4,6and(ii)distributionofCAV1incellularorganellessuchasmitochondria,endoplasmicreticulum,andthelateendosome/lysosome,hasalsobeenwellreported.
7-9Inthisstudy,weprovideconvincingevi-dencedemonstratingthatthefunctionofCAV1inautophagyismostprobablylipidraft-dependentbutcaveolae-independent.
First,depletionofanothercriticalcaveolaeregulatorPTRFdidnotaffectautophagy(Fig.
S1).
Second,depletionofcholesterolbyMBCDtreatmenthadasimilareffectasCav1deciencyonautophagy,andmoreover,MBCDwasunabletofurtherpromoteautophagyinCav1-KOcells(Fig.
1I).
Weacknowledgethattreat-mentwithMBCDwouldhaveadifferentimpactonlipidraftsthanCAV1deciency,thefactthatMBCDisineffectiveinCav1-KOcellstopromoteautophagy,indicatingtheproautophagyfunctionofMBCDandCAV1deciencyaremostlikelylipidraft-related.
Third,therewaspartiallycolocalizationoflipidraftswithlysosomalmarkers,andalargequantityofCAV1accumula-tioninthelysosomalfraction(Figs.
3A,B).
BasedonthefactthatcaveolaeprimarilylocalizeattheplasmamembraneandPTRFsta-bilizesCAV1proteinattheplasmamembrane,29itisthusbelievedthatCAV1regulateslysosomalfunctionandautophagyinacaveolae-independentandlipidraft-dependentmanner.
Therefore,datafromthisstudydemonstrateanovelfunctionofCAV1inregulationoflysosomalfunctionsandautophagy.
OurresultsaregenerallyconsistentwithseveralearlierstudiesinwhichCAV1andlipidraftsareabletonegativelyregulateautophagy,viamultiplemechanisms.
First,inadipocytes,Cav1deciencysuppressesinsulinandlipolyticresponses,whichwillleadtoautophagyinduction.
19Second,lossofCAV1promotesautophagyviaenhancedoxidativestressandNFKB1/NF-kBactivation.
20,42Third,arecentstudyprovidesdirectevidencetoshowacompetitiveinteractionofCAV1andtheATG12–ATG5system,whichsuppressestheformationandfunctionoftheATG12–ATG5complexinlungepithelialcells.
43Incontrast,somecomponentsoflipidraftsarereportedtopromoteautoph-agy.
Forinstance,aparadigmaticcomponentoflipidrafts,GD3ganglioside,playsapositiveroleinautophagosomeformation.
25Inaddition,anothercomponentoflipidrafts,ceramides,hasalsobeenimplicatedinautophagyinductionviablockageofAKTactivation,upregulationofBECN1orsuppressionofnutrienttransporterexpression.
24,26,44Notably,thesestudiesdonotpro-videthedirectevidencetoshowwhetherthefunctionofthoselipidsinautophagyregulationislipidraftsdependentandwhetherCAV1isinvolvedintheregulationoftheselipids.
Itremainstobefurtherdeterminedwhetherthoseindividualcom-ponentsoflipidraftsactthroughlipidraftsperseintheregula-tionofautophagyandwhethertheCAV1-dependentlipidraftsareaffectedbythechangesoftheselipids.
Inourattempttounderstandthemolecularmechanismsunder-liningtheregulatoryroleofCAV1inautophagy,wefocusedourattentiononlysosomes.
Therolesoflipidraftsindifferentcellularorganelleshavebeenreportedpreviously.
Forinstance,thelipidraftadaptorCDKN2C/p18controlsendosomedynamicsbyanchoringtheMAP2K1/7-MAPK1/3(formerlyalsoknownasMEK–ERK)pathwaytolateendosomes.
45Lipidraftsalsoplayanimportantroleinthetrans-Golginetworkformembranetrafck-ing.
46Furthermore,severallysosomemembraneproteinsassociatewithlipidrafts,suchasV-ATPase,chloridechannelsCLCN6-CLCN7,andLAMP2A,suggestingapossibleroleoflipidraftsinregulationoflysosomalfunctions.
35,47However,theinvolvementofCAV1inlysosomeregulationisunknown.
Here,weprovidedclearevidencetoshowtheinvolvementofCAV1andlipidraftsintheregulationoflysosomalfunction.
DisruptionoflipidraftsbyCAV1deciencyandMBCDtreatmentenhanceslysosomefunc-tionviapromotionofV-ATPaseassembly(Figs.
2and3).
IthasbeenreportedthatCAV1andlipidraftslocalizetolateendosomeandlysosomemembranes.
9CAV1movestoandfromthelysosomemembraneduringintracellularcholesteroltrafckingbasedontheevidenceofincreasedlysosomalCAV1accumulationafterpertur-bationoflysosomepHandblockageofcholesteroltrafcking.
9Therefore,lysosomesmightbecriticalfortheregulationofCAV1www.
tandfonline.
com779Autophagyandcholesteroltrafcking.
However,thefunctionsofCAV1onlysosomesarenotwellunderstood.
Insomelysosomalstoragedis-eases,ahigherleveloflipidraftsisfoundonlateendosomesorlysosomes.
48,49Arecentstudyalsodemonstratesthattheabnormalaccumulationof'cholesterol-enrichedregions'leadstolysosomaldisease.
37Suchobservationsthussuggestthepossibilitythatlipidraftsmayactasanegativeregulatoroflysosomalfunction.
Datafromourstudyareindeedsupportiveofsuchahypothesis.
ItisthusbelievedthatmodulationofCAV1andlipidraftsregulateslysosomefunction.
Interestingly,thedegradationofCAV1requiresacidiclysosomalorlateendosomes.
49Here,weshowedthattheCAV1deciencywillpromotelysosomalfunction,indicatingapositivefeedbackloopbetweenCAV1andlysosomalfunction.
InordertounderstandthemolecularmechanismsunderlyingtheregulatoryrolesofCAV1andlipidraftsonlysosomalfunc-tion,weexaminedthechangesofV-ATPaseassembly.
V-ATPaseisthemaindeterminantinlysosomeacidicationandfunctions.
34TheassemblyofV1andV0domainsiscriticalforitsenzymaticactivity34andtheV-ATPasecomplexlocalizestolipidrafts.
35WefoundthatdeciencyofCAV1anddisruptionoflipidraftsbyMBCDincreasedtherateofV-ATPaseassembly(Fig.
3),suggest-ingthatCAV1andlipidraftsmayhindertheassemblyprocessoftheV-ATPasecomplex.
Inaddition,wehaveprovidedevidenceshowingthatbothlipidraftsandaminoacidstarvationleadtothedecreasebetweenCAV1andaV-ATPaseV0subunit(Fig.
S7),whichprovidesapossibleexplanationforthepromotedV-ATPaseassembly.
ThemembranebondedV0subunitlocalizedinlipidraftsandmightinteractwithCAV1directly.
TheCAV1andlipidraftsmightpreventtheV0andV1subunit-assemblyprocess.
Thus,disruptionoftheselipidraftswillreleasetheV0subunitstobeassembledwiththeV1subunits.
However,thishypothesisstillneedstobefurtherconrmedandassessedastoexactlyhowthelipidraftsregulatetheV-ATPaseassembly.
Autophagyhasbeenwellestablishedasacellsurvivalmecha-nismunderstressconditionssuchasstarvation.
50Consistently,inthisstudywefoundthatautophagymediatedbyCAV1de-ciencyvialipidraftdisruptionprotectsagainstcelldeathinducedbystarvation(Figs.
5,6I,andJ).
Theprosurvivalfunctionofautophagyisimplicatedincancerdevelopment.
Autophagyfunc-tionsasatumorsuppressorduringtheinitiationstageofcancerprogression;onthecontrary,onceatumorisformed,autophagyinthetumorcellswillprovideasurvivaladvantagetoresistcelldeathinducedbycancertherapeuticagents.
51Basedonthisunderstanding,suppressionofautophagyusingchemicalinhibi-torsbecomesanovelapproachincancertherapy,includingcom-binationaltherapywithautophagyinhibitorsandestablishedtherapeuticagents.
38CAV1possessesatumorsuppressionfunc-tioninseveraltypesofcancer,especiallyinbreastcancer,10basedonobservationsthatCAV1isdownregulatedorexhibitslossoffunctioninmutants,inbreastcancercells.
10,14,52Moreover,inbreastcancer,lipidraftsandCAV1arecrucialforsomekeyprocesses,includingapoptosis,cellfateregulation,growthfactorreceptorsignaling,andseveralimportantsignalingpath-ways.
53–55Forinstance,theoncogenicproteinsEGFR,ERBB2/HER2,andIGFLareassociatedwithlipidrafts.
55-57Targetinglipidraftsmayhaveananticancereffectbypromotingtheinternalizationanddegradationofthoseoncogenicproteins.
58Inthisstudy,wealsofounddownregulatedCAV1levelsinbothbreastcancercelllinesandtissuesamples(Figs.
6A,7A,andD).
OurdataalsoindicatethatahigherautophagylevelcorrelatedwithlowerCAV1expressioninthebreastcancertissues.
There-fore,breastcancercellsuseautophagyasaprosurvivalmecha-nismtoresistcelldeath,especiallythosecancercellswithlowerlevelsofCAV1andimpairedlipidraftfunction.
Theresultspre-sentedinthisstudyalsosuggestapossibleexplanationforthepoorclinicaloutcomesofthosebreastcancerpatientswithCAV1deciency,asreportedpreviously.
10,52,59Atpresent,thereisnoreportedbiomarkerintheselectionofcancerpatientsforautophagy-targetedchemotherapies.
Thus,ourstudyindicatesthatthosebreastcancerpatientswithCAV1deciencymightbemoresuitableforautophagy-inhibitortherapies.
Insummary,ourdatarevealanovelfunctionofCAV1incellstressresponsesandbreastcancerdevelopmentviaregulationoflysosomalfunctionsandautophagy,aprocessdependentonlipidraftsbutindependentofcaveolae.
UnderstandingsuchanovelfunctionofCAV1providesaclueforsuppressingautophagyasapotentialtherapeuticapproach,especiallyforbreastcancerpatientswithdecientCAV1expression.
MaterialsandMethodsReagentsandantibodiesThechemicalsusedinourexperimentswere:methyl-b-cyclo-dextrin(MBCD;Sigma,C4555),cholesterol-watersoluble(Sigma,C4951),chloroquinediphosphate(CQ,Sigma,C6628),balomycinA1(Baf,Sigma,B1793),rapamycin(Sigma,R8781),choleratoxinsubunitBconjugatedwithAlexaFluor594(CTxB,Invitrogen,C34777),LysoTrackerGreenDND-26(Invitrogen,L7526),LysoTrackerRedDND-99(Invitro-gen,L7528),MagicRedTMCTSLreagent(ImmunochemistryTechnologies,LLC,942),DQTMRedBSA(dyequenched;MolecularProbes,D12051).
Theantibodiesusedinourexperi-mentswere:anti-MAP1LC3B/LC3B(Sigma,L7543),anti-SQSTM1/p62(Abnova,H00008878–2C11),anti-ACTB/b-actin(Sigma,A5441),p-RPS6(S235/236)(CellSignalingTechnology,2211),anti-TFRC/transferrinreceptor(Invitrogen,136800),anti-CAV1/caveolin-1(BDPharmingen,610059),anti-TSC2(CellSignalingTechnology,3990s),mouseanti-ATP6V1B2/V-ATPaseB2(SantaCruzBiotechnology,sc-166122),goatanti-ATP6V0D1/V-ATPaseD1(SantaCruzBio-technology,sc-69105),anti-PTRF/Cavin-1(Merck,ABT131).
CellcultureMEFs,MCF7,andL929cellsweregrowninDulbecco'smodiedEagle'smedium(DMEM;Sigma,D7777)with10%fetalbovineserum(Hyclone,SH30071.
03)and1%penicillin-streptomycin(Pan-Biotech,P06–07100)(denedasnormalmediuminthisstudy)ina5%CO2incubatorat37C.
TheCav1C/Candcav1/MEFsweredescribedpreviously.
27MCF-7cells(1106cells)weretransfectedwith5mgpEGFPDNAand5mgpEGFP-CAV1DNAusingLipofectamine2000(Life780Volume11Issue5AutophagyTechnologies,11668)accordingtothemanufacturer'sinstruc-tions.
G418(SigmaAldrich,A1720)wasusedasaselectiondrugat500mg/mlfor3wktoestablishstablelines.
PTRFWTandKOMEFshavebeenpreviouslydescribed.
60TheAtg5C/Candatg5/andAtg5Tet-offinducibleMEFs(m5–7)withstablyexpressingGFP-LC3BwereallkindlyprovidedbyDr.
N.
Mizushima(TokyoMedicalandDentalUniversity).
TheTsc2C/Candtsc2/MEFswereagenerousgiftfromDr.
D.
Kwiatkowski(HarvardMedicalSchool).
ThetfLC3BstablytransfectedL929cellshavebeenreportedpreviously.
61PlasmidsThemRFP-GFPtandemuorescence-taggedLC3Bconstruct(tfLC3B)wasakindgiftprovidedbyDr.
T.
Yoshimori(OsakaUniversity).
36WesternblottingAfterthedesignatedtreatments,LaemmliSDSbufferwereusedtolysethecollectedsamples(62.
5mMTris,pH6.
8,25%glyc-erol,2%SDS[Vivantis,PB0640],phosphataseinhibitor[ThermoScientic,78428]andproteinaseinhibitorcocktail[Roche,11697498001]).
AnequalamountofproteinofeachsamplewasresolvedbySDS-PAGE,andthentheSDS-PAGEgelsweretrans-ferredontoPVDFmembrane(Bio-Rad,162–0177).
Membraneswereblockedfor30minwithblockingbuffer(Thermo,37538)andfollowedbyincubationswiththeindicatedprimaryandsec-ondaryantibodies.
Thenthemembraneswerevisualizedwiththeenhancedchemiluminescencemethod(ThermoScientic,34076)bytheImageQuantLAS500(GE).
Quantitativereal-timeRT-PCRTheCAV1primerset(CAV1:forward,CGTAGACTCG-GAGGGACATCandreverse,ACTTGCTTCTCGCT-CAGCTC)wereusedforquantitativeRT-PCR.
SsoFastEvaGreen(Bio-Rad,1725201AP)wasusedinathermalcycler(modelC1000TM,Bio-Rad).
AllsampleswerenormalizedtoGAPDH(glyceraldehyde-3-phosphatedehydrogenase)expressionlevels.
MeasurementofintralysosomalfunctionusingLysoTrackerTheintralysosomalfunctionwasestimatedbyLyso-Tracker.
62,63Cellswereculturedoncoverglassslidechamber,andthenfollowedwiththedesignatedtreatments.
After30minincubationwith50nMLysoTrackerGreenDND-26orLysoTrackerRedDND-99,theuorescenceintensitywasobservedunderaconfocalmicroscope(OlympusFluoviewFV1000,Shinjuku-ku,Tokyo,Japan)andrepresentativecellsfrom3eldsofviewwereselectedandphotographed.
Alltheimagesweresinglefocalplanes.
CathepsinactivityassayTheCTSLenzymaticactivitywasmeasuredbyMagicRedTMreagent(ImmunoChemistrytechnologies,942)withthemethoddescribedearlierwithslightmodications.
64Cellswereculturedin24-wellplatesandtreatedwithindicatedtreatments.
ThencellswerefurtherloadedwithMagicRedTMCTSLreagentfor15min.
Theuorescenceintensitiesof10,000cellspersamplewerequantiedbyaBDFACScytometer(BDBiosciences).
CellfractionationIsolationofdetergent-resistantlipidraftfractionfromthedeter-gent-solublefractionwasperformedbasedonapreviouslycon-ductedstudywithminormodications.
31Briey,afterindicatedtreatments,cellswerewashedwithice-coldphosphate-bufferedsaline(PBS[Vivantis,PB0344];137mMNaCl,2.
7mMKCland10mMPhosphateBuffer,pH7.
4)andcollectedbycentrifugationat400gat4Cfor3min.
ThencellswerehomogenizedwithTNEbuffer(25mMTrisHCl,pH7.
4,150mMNaCl,3mMEDTA,andproteaseinhibitorcocktail)with1%TritonX-100(Sigma,X100)bypassagethrougha27-gaugeneedlefor20timesonice.
Cellswerecollected,lysedandkeptonicefor30min.
After-wards,sampleswerespundownat16,000gat4Cfor20min,andthesupernatantfractionswerecollectedastheDSF.
Theinsol-ublepelletfractionswereresuspendedandlysedinLaemmliSDSbuffer,andwereregardedastheDRF.
Theisolationoflysosomeswasperformedaccordingtothemanufacturer'sprotocoloftheLysosomeEnrichmentKitforTissueandCulturedCells(ThermoScientic,89839).
Three150-mm2cellculturedisheswereusedforeachindicatedtreatment.
Theisolatedlysosomefractionswerethensubjectedtowesternblotanalysis.
VisualizationoflipidraftsbyCTxBstainingThelipidraftswerestainedwithCTxBconjugatedwithAlexaFluor594asreportedpreviouslywithminormodications.
28Briey,MEFswereseededoncoverglassslidechambers.
Aftertheincubationandindicatedtreatments,cellswereloadedwith1mg/mLCTxBfor15minoniceandthenwashedwithPBSandfollowedbya30minincubationat37C.
Theuorescenceintensitywasobservedunderaconfocalmicroscope(OlympusFluoviewFV1000)andrepresentativecellsfrom3eldsofviewwerephotographed.
Alltheimagesweresinglefocalplanes.
VisualizationoflipidraftsbylipinstainingForcholesterolstaining,thecellswerelabeledbyFilipinIII(Sigma,F4767)asreportedpreviously.
65Briey,cellswereseededandtreatedoncoverglassslidechambers.
Aftertreatments,cellswerexedwith4%paraformaldehydefor30minandquenchedin50mMNH4Cl3for10min.
Thencellswereblocked,permeabilizedandstainedinthesolutionwhichcon-taining0.
2%BSA(Sigma,A7906),0.
2%shskingelatin(Sigma,G7041)with50mmoflipinIIIfor20min.
After3washesinPBS,theuorescenceintensitywasdetectedunderaconfocalmicroscope(OlympusFluoviewFV1000).
Representa-tivecellsfrom3eldsofviewwereselectedandphotographed.
Alltheimagesweresinglefocalplanes.
ProteolysisactivityassayProteolysisactivitywasperformedbyDQTMRedBSAstaining,conductedwiththemethodsreportedpreviously.
66MEFswerecul-turedoncoverglassslidechambersandpreloadedwithDQ-BSA(10mg/ml)for1h.
After3washeswithPBS,themediumwaschangedtothedesignatedtreatments.
Confocalmicroscopy(OlympusFluoviewFV1000)wasusedtodetecttheuorescenceintensity.
Therepresentativecellsfrom3eldsofviewwereselectedandphotographed.
Alltheimagesweresinglefocalplanes.
www.
tandfonline.
com781AutophagyProximityligationassay(PLA)Cellswereculturedincoverglassslidechambersandtreatedasindicated.
Afterxationwith4%paraformaldehydeandpermea-bilizationby0.
1%saponin(Sigma,47036),thecellsweresub-jectedtoPLAbyDuolinkdetectionkit(OlinkBioscience,[PLAProbeAnti-GoatMINUS,92006;PLAProbeAnti-MousePLUS,92001;DetectionReagentRed,92008]).
67Briey,per-meabilizedcellswereblockedandincubatedwithmouseanti-ATP6V1B2andgoatanti-ATP6V0D1antibodiesovernightat4C.
ThenafterincubationwithsecondaryantibodiesconjugatedtouniqueDNAprobes(anti-mouseandanti-goatfor2primaryantibodies,providedbythekit),arollingcircleamplicationstepwasusedtosubjecttotheproximityligation(<40nm)andcircularizationoftheDNA.
Aftertheamplicationprocess,repli-cationsoftheDNAcirclewerelabeledbycomplementaryoligo-nucleotideprobesandthesignalswereobservedunderaconfocalmicroscope(OlympusFluoviewFV1000).
Representativecellsfrom3eldsofviewwereselectedandphotographed.
Alltheimagesweresinglefocalplanes.
CAV1immunohistochemistryTheparafn-embeddedtissuesectionsof7breastcancersam-plesandtheiradjacentnormaltissuesampleswerecollectedfromaTissueRepository(NUH,Singapore)fordeterminationofCAV1expression.
Sectionsaredewaxedandrehydratedusingstandarddewaxingprotocol.
Thenthesamplesweresubjectedtoheatantigenretrievalbyexposureto10mMcitricacidbuffer(pH6.
0for15minat105C).
Endogenousperoxidaseactivitywasblockedbya10minincubationwith3%H2O2.
Thenthesampleswereblockedby3%BSAandincubatedwiththe1:100dilutionoftheanti-CAV1primaryantibodyovernightatroomtemperature(RT)inahumidchamber.
EnvisionHRPanti-rab-bitpolymer(Dako,K401011)wasaddedfor30minatRT.
ColorisdevelopedbyincubationwithDAB(Dako,K401011)for5min,followedacounterstainwithMayerhaematoxylin(Sigma,MHS1).
Thenthesectionsweredehydratedthroughascendinggradedalcohols,clearedinxylene,mountedandvisu-alized.
Representativeregionswereselectedandphotographed.
Toquantifytheimmunohistochemistryresultofpositivestainingintensity,3observationsofeachsamplewereanalyzedbyanexperiencedpathologist.
MoticImagesAdvancedsoftware(ver-sion3.
2,MoticChinaGroup)wasusedtoanalyzetheproteinlevel.
Meanvalues§SDwerepresented.
Studentttestswereusedtoidentifythesignicanceofdifferences.
DetectionofcelldeathSeveralmethodswereusedtodetectcelldeathquantitativelyandqualitatively,whichare(i)morphologicalchangesunderphase-contrastmicroscopy,(ii)PtdInsexclusionassaycoupledwithowcytometry(5mg/ml).
(iii)PtdInsstainingobservedunderuorescencemicroscopy.
ForthePtdInsexclusionassay,trypsinizedcellswiththemediumineachwellwerecollected.
ThepelletswereresuspendedintoPBScontaining5mg/mlPtdIns.
Then10,000cellsfromeachsamplewereanalyzedbyusingaBDFACScytometer(BDBiosciences).
ForPtdInsstainingassay,cellsstainedwithPtdIns(5mg/ml).
Then,thecellswithPtdInsstain-ingwereobservedthroughuorescentmicroscope.
ThepercentageofPtdIns-positivecellswerecountedandanalyzedbyImageJ.
MeasurementoflysosomepHSeveralmethodswereusedtoevaluatelysosomepH,whichare(1)LysoSensorGreenDND189(1mM;LifeTechnologies,L7535)stainingobservedunderaconfocalmicroscope;(2)Lyso-SensorGreenDND189(1mM)stainingcoupledwithowcytometry;(3)lysosomepHquantication.
ForlysosomepHquantication,briey,cellswereseededin96-wellplatesandlabeledwith2mMLysoSensorYellow/BlueDND160(LifeTech-nologies,L7545)for45mininregularmedium,thenwashedinHEPESsolution(10mMHEPES,133.
5mMNaCl,2.
0mMCaCl2,4.
0mMKCl,1.
2mMMgSO4,1.
2mMNaH2PO4,11mMglucose,pH7.
4).
Theuorescenceemittedat440and535nminresponsetoexcitationat340and380nmwasmea-suredimmediatelybyamicroplatereaderat37C.
Theratiooflightemittedwith340and380nmexcitationwascalculatedforeachwell.
ThepHcalibrationcurvefortheuorescenceprobewasgeneratedbyusingthe2-(N-morpholino)ethanesulfonicacid(MES)calibrationbuffer(10mMmonensin[Sigma,M5273],10Mnigericin[Sigma,N7143],25mMMES[Sigma,M2933],5mMNaCl,115mMKCland1.
2mMMgSO4,pH3.
5to6.
0).
StatisticalanalysisAllimageandwesternblotdatapresentedabovearerepresen-tativesfrom3independentexperiments.
Thenumericdataarepresentedasmeans§SDfromatleast3independentexperi-mentsandanalyzedbytheStudentttest.
DisclosureofPotentialConictsofInterestNopotentialconictsofinterestweredisclosed.
AcknowledgmentsWethankNUH-NUSTissueRepository(TR)forprovidingthetissuesamples,andthepersonneloftheNUHSResearchCoreFacilityfortechnicalassistance.
WethankDr.
DavidJ.
KwiatkowskiforprovidingTsc2C/Candtsc2/MEFs.
WethankDr.
NMizushimaforprovidingtheatg5/MEFsandAtg5Tet-offinducibleMEFswithstableexpressionofGFP-LC3B.
FundingThisworkwassupportedbyresearchgrantsfromSingaporeNationalMedicalResearchCouncil(NMRC-1260/2010andCIRG/1346/2012).
TheworkinYSK'slabwassupportedinpartbytheNationalResearchFoundationofKorea(NRF,No.
2011–0030043).
SupplementalMaterialSupplementaldataforthisarticlecanbeaccessedonthepublisher'swebsite.
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