J. Biol. Chem.-2011-Imanishi-29249-60

Angiopoietin-2,GrowthLungandSurvivalanAngiogenicofBreastCancerRegulator,PromotesInitialLymphomathrough2(Bcl-2)theIntegrin-linkedMetastasestothePathway*Kinase(ILK)-AKT-BCellReceivedforpublication,February28,2011,andinrevisedform,June5,2011Published,JBCPapersinPress,June16,2011,DOI10.1074/jbc.M111.2356

YorihisaImanishi‡§¶,BoHu‡ʈ1,GutianXiao‡**,XuebiaoYao‡‡§§,andShi-YuanCheng‡§2Fromthe‡CancerInstitute,§DepartmentofPathology,ʈDepartmentofMedicine,and**DepartmentofMicrobiology&MolecularGenetics,UniversityofPittsburghSchoolofMedicine,Pittsburgh,Pennsylvania15213,the¶DepartmentofOtorhinolaryngology,HeadandNeckSurgery,KeioUniversity,SchoolofMedicine,Shinjuku,Tokyo160-8582,Japan,the‡‡AnhuiLaboratoryofCellularDynamics§§andChemicalBiology,HefeiNationalLaboratoryforPhysicalSciencesatNanoscale,Hefei230027,China,andtheDepartmentofPhysiology,MorehouseSchoolofMedicine,Atlanta,Georgia30310

Theearlyonsetsofbreastcancermetastasisinvolvecellreten-Breastcancersarethemostcommontumorsinhumansthattion,survival,andresistanttoapoptosisandsubsequentgrowthinflictwomenwithϳ210,000newcasesin2010withintheattargetvascularbedsandtissuesindistantorgans.Weprevi-UnitedStateswithpoorprognosis.Themajorcauseofbreastouslyreportedthatangiopoietin-2(Ang2),anangiogenicregu-cancerdeathisnotduetolocaltumorsbutdistanttumorlatorstimulatesMCF-7breasttumormetastasisfromtheirmetastasesthatareoftenundiscoveredduringinitialdiagnosisorthotopicsitestodistantorgansthroughthe␣5␤1integrin/andareresistanttoconventionaltherapies(1).Breastcancerintegrin-linkedkinase(ILK)/Aktpathway.Here,byusinganmetastasisisamultistageprocessduringwhichtumorcellsexperimentaltumormetastasismodelandinvitrostudies,wespreadfromtheprimarytumorsitestodistantorgans.Acqui-furtherdissecttheunderlyingmechanismbywhichAng2pro-sitionofametastaticphenotypebybreastcancercellsaltersmotestheinitialgrowthandsurvivalofMCF-7breastcancermultiplesignalpathwaysthatinducecellmotility,invasionintometastasisinthelungofanimals.WeshowthatAng2increasesperipheraltissuesandentranceintothecirculation,promotecellsurvivalandsuppressescellapoptosisthroughILK-inducedcellsurvivalinthenewmicroenvironmentandsubsequentphosphorylationofAkt1,Akt2,andup-regulationofBcl-2ingrowthofsecondarytumorsintheirtargetorgans(2,3).Studiesbreastcancercells.InhibitionofILK,Akt1,andAkt2,andtheirofgenediscoveryhaveidentifiedthedeterminantsofgenesig-effectorBcl-2diminishesAng2-stimulatedbreastcancercellnaturesandcriticalpathwaysthatinduceandpromotebreastsurvivalandAng2-attenuatedapoptosisinvitro,andinitialsur-cancermetastasistodistantorgansites(4).However,thevivalandgrowthofbreastcancermetastasisinthelungofani-mals.Additionally,siRNAknockdownofendogenousAng2inmolecularmechanismsunderlyingthebreastcancermetastaticthreehumanmetastaticbreastcancercelllinesalsoinhibitsprocess,especiallyduringinitialonsetoftumormetastasisatphosphorylationofAkt,expressionofBcl-2,andtumorcellsur-distantorgansremainslargelyunknown(5).

vival,migration,andincreasescellapoptosis.SinceincreasedAngiopoietins(Ang)3areTie2receptorligandsthatplayexpressionofAng2correlateswithelevatedpotentialofhumanimportantrolesinvasculardevelopment,vesselremodeling,breastcancermetastasisinclinic,ourdataunderscoretheandangiogenesis(6).Ang2wasinitiallyidentifiedasanantag-importancethatup-regulatedAng2notonlystimulatesbreastonistforAng1activationofTie2thatmodulatesvesselstabilitycancergrowthandmetastasisatlatestagesoftheprocess,butis(7).However,accumulatingevidencedemonstratedalinkofalsocriticalattheinitiatingstagesofmetastasesonset,therebyincreasedexpressionofAng2withinvasiveandmetastaticphe-suggestingAng2asapromisingtherapeutictargetfortreatingnotypesofvarioustypesofhumancancersincludingbreastpatientswithmetastaticbreastcancer.

cancers(8,9).EctopicexpressionofAng2byvarioustypesofcancerxenograftspromotedtumorangiogenesis,growth,inva-sion,andmetastasisinanimals(8–10).Moreover,preclinicalinvestigationsdemonstratedthatselectiveinhibitionoftumor-*Thisworkwassupported,inwholeorinpart,byGrantCA130966fromthe

derivedAng2byspecificreagentssuppressestumorangiogen-NationalInstitutesofHealthandthePennsylvaniaDept.ofHealthandInnovativeResearchScholarAwardsoftheHillmanFoundation(toS.Y.C.esisandgrowthbyinducingvesselnormalizationandprevent-andB.H.);NationalInstitutesofHealthCA116616,AmericanCancerSoci-ingVEGF-stimulatedneovascularizationinanimals(11,12).etyRSG-06-066-01-MGO(toG.X.),andNationalInstitutesofHealthGrantSignificantly,combinationinhibitionofAng2andVEGForCA1323;ChineseAcademyofSciencesKSCX-YW-R65andChinese973Projects2007CB914503and2010CB912103(toX.Y.).VEGFR-2furtherreducestumorangiogenesisandgrowth,1Towhomcorrespondencemaybeaddressed.Tel.:412-623-7791;Fax:412-accompaniedwithdecreasedtumorcellproliferationand623-4840;E-mail:hub@upmc.edu.2increasedapoptosis(13–16).Therefore,targetedinhibitionof

Towhomcorrespondencemaybeaddressed:CancerInstitute&Depart-mentofPathology,ResearchPavilionatHillmanCancerCenter,Suite2.26,5117CentreAve.,Pittsburgh,PA15213-1863.Tel.:412-623-3261;Fax:412-623-4840;E-mail:chengs@upmc.edu.

3Theabbreviationsusedare:Ang,angiopoietin;ILK,integrin-linkedkinase.

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Ang2andotherangiogenicsignalingsuchastheVEGF/VEGFRpathwaypresentsattractivetherapeuticoptionsfortreatingpatientswithbreastcancers(6,9).

WehavepreviouslyreportedthatectopicexpressionofAng2byhumanbreastcancerMCF-7cellsthatshowlowpotentialoftumorgrowthandmetastasis,significantlypromotecancermetastasesofprimarybreasttumorsfromorthotopicsitestoseveraldistantorgansofmice(10).However,ourstudyfocusedontheimpactsoftheAng2-␣5␤␤1integrin-integrin-linkedkinase(ILK)/Akt/GSK-3signalingonthefinaloutcomesofenhancedbreastcancermetastasis.Becausefirststepsofbreastcancermetastasisarecomprisedinitialtumorcellsurvivalandgrowthinthedistantorganssuchasinthelung(2,3),inthisreport,usinganexperimentaltumormetastasismousemodelandinvitrostudies,weaimedtogainbetterunderstandingofAng2stimulationofbreastcancerlungmetastasisintheinitialonsets.WeshowthatectopicexpressionofAng2byMCF-7cellsincreasedtumorcellsurvivalandgrowthinthelungatthefirst24hupto8dayspost-injectionoftumorcellsthroughtheAng2/ILK/Akt/Bcl-2-mediatedpathway.InhibitionofILK,Akt1/2,andBcl-2bysiRNAssignificantlyabrogatedAng2-stimulatedbreastcancercellsurvivalunderstressconditionsinvitroandinthelungofmice,therebysuppressingbreastcancermetastasistothelungofanimals.

MATERIALSANDMETHODS

CellLinesandReagents—HumanMCF-7,MDA-MB-231,MDA-MB-468,andSK-BR-3breastcancercellswereobtainedfromATCC,andclone1834cellsderivedfromMDA-MB-231cellswerefromDr.J.MassagueatMemorialSloan-KetteringCancerCenter,NewYork.Thesecellswereculturedasprevi-ouslydescribed(10,17,18).Thefollowingantibodieswereusedforthisstudy:goatanti-Ang2antibody(AF623,R&DSys-tems,Minneapolis,MN);mouseanti-phospho-Akt(Ser-473;587F11),rabbitanti-Akt(11E7),anti-phospho-Akt(Ser-473,193H12),anti-phospho-Akt(Thr-308;244F9),anti-phospho-Bad(Ser-136,D25H8),andanti-Bax(D2E11)antibodies(CellSignaling,Danvers,MA);rabbitanti-ILK,anti-Akt1,andanti-Akt3antibodies(UpstateBiotechnologies,LakePlacid,NY);mouseanti-Akt2(F-7),rabbitanti-Bad(C-7),anti-Bcl-2(N-19),andanti-␤-actin(I-19)antibodies(SantaCruzBiotechnology,SantaCruz,CA);peroxidase-conjugatedanti-rabbit,anti-mouse,andanti-goatantibodies(Dako,Carpinteria,CA).Otherchem-icalsandreagentswerefromThermoFisherScientific(Hano-verPark,IL),Bio-Rad,SigmaChemicals,orInvitrogen(Carls-bad,CA).MCF-7celllinesthatstablyexpressAng2andGFP,expression,andpurificationofAng2andimmunoblot(IB)andimmunoprecipitation(IP)aredescribedindetailinourprevi-ousreports(10,19).

XenograftAssays—Allanimalworkwasperformedinaccor-dancewithNIHguidelinesundertheprotocolapprovedbytheInstitutionalAnimalCareandUseCommitteeatUniversityofPittsburgh.Inanexperimentalmetastasismodel,1ϫ106ofMCF-7controlGFP,Ang2#1orAng2#52cells(referredastoAng2#1andAng2#52hereafter)wereseparatelyinjectedintothetailveinsof8-week-oldovariectomizedfemalenudemice(Taconic,Germantown,NY)supplementedwith17␤-estradiol(E2)pellets(60-dayslowrelease;InnovationResearchofAmer-

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ica,Sarasota,FL).Micewereeuthanized12–14weekspost-injectionorwhenpathologicalsymptomsdeveloped.Thelung,liver,spleen,kidney,ovary,bone,lymphnodes,andbrainwereremoved,processed,andsectionedaspreviouslydescribed(10).MetastasisintheorganswasfirstdeterminedbydirectepifluorescentexaminationofGFP-positivecellsusinganOlympusSZX12stereomicroscopebeforeembeddinginOCT,andanOlympusBX51microscopeaftercryo-sectioning,andthenfollowedbyH&Estainingusingtheirsistersections.ImageswerethencapturedwithSPOTdigitalcameras(Diag-nosticInstrument)equippedonthesemicroscopes.

InVivoCellSurvivalAssays—Toevaluatecellsurvival,2ϫ106ofcontrol/GFPorAng2#1/GFPMCF-7cellsweresepa-ratelyinjectedintothetailveinsofnudemicesupplementedwith21-dayrelease17-␤-E2pellets.ThecellstransfectedwithindicatedsiRNAswereinjectedintothetailveinsofanimals72haftertransfection.Ateachindicatedtimepointafterinjec-tion,4micepereachgroupwereeuthanizedandthelungswereharvested.Imagesof10randomframesperlungwerecapturedunderanepifluorescentstereomicroscope(OlympusSXZ12)atϫGFP-positive108magnificationcellswithinandanalyzedtheareabyusingcountingtheImagethenumbersProPlusofsoftware(Version4.1;MediaCybernetics).Thepercentageofmetastaticcellsurvivalwasdeterminedbynormalizingthemeannumbersofcellsateachindicatedtimepointwiththoseat4hforeachgroup.DifferenceofthepercentcellsurvivalateachtimepointwasstatisticallyanalyzedusingMann-WhitneyUtest.

InVivoTUNELAssays—Toexamineapoptotictumorcellsinthelungofmicedescribedabove,8␮mcryostatsectionsofthelungswereanalyzedusingtheinsituCellDetectionKit,TMRred(RocheAppliedScience,Indianapolis,IN).Theassaywasperformedaccordingtothemanufacturer’sinstructions,fol-lowedbycounterstainingwithHochest33258.Imagesof10randomframespersectionwerecapturedatϫ200magnifica-tionandanalyzedtocalculatetheapoptoticindex(AI)oftumorcellasaratioofapoptotictumorcellnumbertototalGFP-positivetumorcellnumber.DifferenceofAIoftumorcellsateachtimepointwasstatisticallyanalyzedusingattest.

InVitroCellSurvivalAssays—MCF-7controlGFP,Ang2#1,andAng2#52cellsweregrowninmediacontaining10%FBSuntil80%confluence,followedbyinaserum-free/phenolred-freemediumfor48h.Then,thecellsweretrypsinized,counted,separatelyseededat4ϫ105cells/wellin6-wellplates.ControlGFPorAng2#1cellstransfectedwithindicatedsiRNAswerepreparedsimilarly72haftertransfectionwithoutpreserum-starvation.Thecellswereincubatedwithserum-free/phenolred-freemedia,orwith10%charcoal-treatedFBS/phenolred-freemediacontaining100␮Mcobaltchloride(CoCl2,Sigma)tomimichypoxiacondition(20)inthepresenceorabsenceof1.0or5.0␮MofHA14–1(aninhibitorforBcl-2)forvarioustimeperiodsasindicated.Thenumbersofsurvivalcellsateachtimepointwereanalyzedbytrypanbluedyeexclusion,counted,andnormalizedtothenumberofthecellseededonday0.

InVitroMigrationAssays—Invitromigrationandinvasionassayswereperformedaspreviouslydescribed(10).Briefly,var-iousserum-starvedbreastcancercellsthatweretransientlytransfectedwithasiRNApoolforAng2orcontrolsiRNAswere

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Downloaded from www.jbc.org by guest, on February 28, 2013separatelysuspendedat1ϫ106cells/mlinserum-freeDMEMcontaining0.5%BSA.50␮lofeachcellsuspensionwasseededintoupperwellsoftheBoydenchambers.Thecellswerethenallowedtomigratethroughthe12␮mporesizemembranesprecoatedwithgelatinfor12hat37°C.Afterthemembranewasfixedandstained,non-migratingandnon-invadingcellswereremoved.Thenumberofmigratingcellsandinvadingcellswasquantifiedunderamicroscope(10).

CellDeathDetectionELISA—Cytoplasmiccellextractswerepreparedandnormalizedonthebasisoftotalcellnumberofbothaliveanddyingcells.Cellapoptosiswasmeasuredintrip-licatebyquantificationofcytoplasmichistone-boundDNAfragmentsusingacelldeathdetectionELISAkit(RocheAppliedScience)accordingtothemanufacturer’sinstructions.Theresultswereshownasrelativeapoptosisbynormalizingeachabsorbancevaluetothatofeachcontrol.

KnockdownofILK,Ang2,Akt1,Akt2,andBcl-2—ILKsiRNA(5Ј-AAGGACACAUUCUGGAAGGGG-3Ј)(21)andsiRNApoolsforAng2andBcl-2werefromDharmacon,Lafayette,CO.The25-nucleotideStealthTMsiRNAforAkt1(5Ј-ACGUCG-GAGACUGACACCAGGUAUU-3Ј),Akt2(5Ј-GGCACGGGC-UAAAGUGACCAUGAAU-3Ј)(22)andStealthTMRNAineg-ativecontrolmediumGCduplexwerefromInvitrogen,SanDiego,CA.MCF-7controlGFPandAng2#1cells,MDA-MB-231,clone#1834,MDA-MB-468andSK-BR-3wereseparatelytransfectedwithsiRNAsforcontrol(C),ILK(I),Akt1(A1),Akt2(A2),Akt1ϩAkt2(A1&2),Ang2(A),andBcl-2(B)usingLipo-fectamine2000(Invitrogen)accordingtothemanufacturer’sprotocol.EfficaciesofspecificknockdownofILK,Akt1,Akt2,Ang2,andBcl-2inthetransfectedcellswereconfirmedbyIBanalyses.

RESULTS

ExpressionofExogenousAng2EnablesMCF-7BreastCancerCellstoMetastasizetoSeveralDistantOrgansofAnimals—Inourpreviousstudy,wedescribedasignificantcorrelationbetweenup-regulationofAng2andmetastaticpotential,tumorgrade,andlymph-vascularinvasionduringhumanbreastcan-cerprogression.ByoverexpressionofexogenousAng2atlevelscomparablewiththatdetectedinclinicalhumanbreastcancerspecimensinpoorlymetastaticMCF-7breastcancercells,wedemonstratedthatAng2stimulatesbreastcancermetastasisfromorthotopicsites(mammaryfatpads)tovariousorgansincludingthelungthroughthe␣5␤1integrin-ILKpathway(10).Becausetheearlystepsoftumormetastasisdecidethefateofmetastaticcancercellsafterarrivalatdistantorgans,inthisstudy,weaimedtodissectthemechanismsbywhichAng2pro-motesinitialgrowthandsurvivalofmetastaticMCF-7cellsinthelungofanimalsusinganexperimentalmetastaticxenograftmodel.WefirstsoughttodeterminewhetherAng2stimulationofMCF-7breastcancermetastasisisinvolvedintheinitialonsetoftumormetastasis.WeintravenouslyadministeredMCF-7Ang2/GFP-expressingandcontrol/GFP-expressingcellsintothetailveinsofnudemice.Twelveto14weekspost-injection,themicewereeuthanizedandvariousorgansofthemicewereexamined.AssummarizedinFig.1A,expressionofAng2byMCF-7cells(Ang2#1andAng2#52)stimulatedbreastcancermetastasestothelung,liver,spleen,andovarywitha

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preferencetothelungofmice.Incontrast,injectionofGFP-expressing(MCF-7)controlcellsdidnotcauseanyobservablemetastatictumorgrowthintheseorgans.AsillustratedinFig.1B,themetastatictumorsweregrosslyvisibleonthesurfacesoflung(Fig.1B,panelseandi).MicrometastaticAng2#1andAng2#52tumorcellscouldbefounddirectlyasgreenfociingrosstissuesofthelung(Fig.1B,panelsfandj).Cryosectionsofthelungtissuesunderepifluorescence(panelsd,h,andl)andH&Estainingofidenticalsections(panelc,g,andk)furthervalidatedourobservation.Incontrast,nometastatictumorfociweredetectedintheorgansincludingthelungofmicethatreceivedMCF-7controlGFPcells(Fig.1,AandB).Inanagree-mentwithourpreviousreport(10),thefrequencyoftumormetastasistovariousorgansinthesemicecorrelateswiththeexpressionlevelsofAng2inMCF-7cells.Ang2#1cellsexpressAng2athighlevelsandAng2#52cellsexpressAng2atmediumlevelswhileAng2wasundetectableinMCF-7controlGFPcells(10).MicereceivedAng2#1cellsdisplayedhigherincidence(91%)oftumormetastasisthanthatinmicereceivedAng2#52cells(75%).

Ang2EnhancesInitialGrowthofMetastaticMCF-7TumorsintheLungbyStimulationofCellSurvivalandInhibitionofCellApoptosis—TodeterminetheeffectsofexpressionofAng2byMCF-7cellsoninitialonsetoftheexperimentaltumormetas-tasis,atotalof24miceintwogroupswith4miceateachtimepointthatseparatelyreceivedAng2#1orGFPcellswereeutha-nizedat4,12,24,48h,5and8dayspost-injections.VariousGFP-expressingcellsinthelungofeachmiceateachtimepointwereevaluated(Fig.2A)andstatisticallyquantifiedaspercent-agecellsofeachgroupandeachtimepointcomparedwiththatat4hpost-injections(Fig.2B).AsshowninFig.2A,withinthefirst4h,Ang2#1andcontrolGFP(MCF-7)cellswereefficientlyretainedinthelungsofmice(Fig.2A;4h).However,thenum-berofthecellsinthelungofmiceinbothgroupswasrapidlydecreasedat12hpost-injections.Althoughthecellnumbersineachgroupwerefurtherdeclinedat24hpost-injections,markeddifferencesinnumberoftumorcellsretainedinthelungofmicebetweentwogroupswereobservedat24and48hofpost-injections.Comparedwiththatat4hinbothgroups,at24hpost-injection,28.3Ϯ3.3%ofAng2#1cellsversus11.0Ϯ3.7%ofGFPcellsandat48h,3.2Ϯ1.2%ofAng2#1cellsversus0.9Ϯ0.5%ofGFPcellswereretainedinthelung,respectively.Significantly,at5and8dayspost-injection,theretained/sur-vivedAng2#1cellsstartedtogrowforminggreenfociorcolo-nies,indicatinganinitialgrowthoftumormicro-metastasisinthelung.Incontrast,minimalnumbersofMCF-7controlGFPcellswerefoundassinglecellsinthelunginbothtimepoints(Fig.2,AandB)andeventuallyfailedtosurviveandgrowtometastatictumors(Fig.1).Inthisexperimentaltumormetas-tasismodel,between12and48hpost-injection,wecouldnotdeterminewhethertherapidlydecreasedGFP-expressingcellsinbothgroupswasduetotheflushbythecirculatingbloodortumorcellswerenotabletosurvive.Toassessthecellsurvivalofbreasttumorcellsretainedinthelung,weperformedinvivoTUNELassayat12and24hpost-injections.AsshowninFig.2,CandD,althoughnodifferenceinapoptoticindexofcontrolGFP(MCF-7,10.8Ϯ3.6%)versusAng2#1(8.4Ϯ1.8%)cellswasfoundinthelungsat12hpost-injection,asignificantlyhigher

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FIGURE1.ExpressionofexogenousAng2enablesMCF-7breastcancercellstometastasizetoseveralorgansofmice.A,summaryoftheincidenceoforganmetastasesinmicethatseparatelyreceivedMCF-7Ang2-expressing(Ang2#1orAng2#52)orGFP-expressing(MCF-7)cells.Fisher’sexacttestshowedsignificantlyhigherincidenceofmetastasisinmicereceivedAng2#1cells(*,pϽ0.0001)orAng2#52(*,pϭ0.007)ascomparedwithmicereceivedMCF-7/GFPcells.Dataarecombinedfromtwoindependentexperimentswith3–11micepergroup.B,representativeimagesofthelungsofmiceinAanalyzedbydifferentmethods.MCF-7breastcancermetastasesinthelungofmicewereexamined12–14weeksaftertail-veininjectionbygrossobservation(panelsa,e,andi)andepi-fluorescentobservationofGFP(panelsb,f,andj).Cryo-sectionswerefurtherexaminedbyepi-fluorescence(panelsd,h,andl)followedbyH&Estaining(panelsc,g,andk).MicethatreceivedAng2#1orAng2#52cellsshowedlungmetastases(panelsetol)withahighincidence.Grosslyvisiblemetastases(bluearrowheads)inthelung(panelseandi)wereidentifiedasgreenfoci(panelsfandj,whitearrowheads)andcellsexpressingGFPinthenuclei(panelshandl,redarrowheads),andfurtherconfirmedbyH&Estaining(panelsgandk,redarrowheads).Scalebars:panelsbtod,ftoh,andjtol,120␮m.DataarerepresentativefromtwoindependentexperimentsshowninAwithsimilarresults.

numberofapoptoticcellsofcontrolGFPcells(35.4Ϯ5.2%)hypoxia-mimeticagent(20)incellculture,100␮MofCoCl2thanthatofAng2#1cells(11.7Ϯ3.0%)wasobserved24hafter(23)causedarapidlydecreaseinsurvivalofcontrolGFPcellsthecellsenteredcirculation,suggestingthatincreasedcell(MCF-7,from45.9Ϯ3.1%atday2to9.4Ϯ1.8%atday8)deathcontributedtotherapidlossofMCF-7cellsinthelungswhereasAng2expressionsignificantlypromotedcellsurvivalofmiceandoverexpressionofAng2protectsMCF-7cellsfrom(94.1Ϯ3.1%forAng2#1cellsand90.9Ϯ1.3%forAng2#52cellsapoptosisinthelung.Takentogether,theseresultsshowthatatday2;38.1Ϯ2.7%forAng2#1and32.8Ϯ1.3%forAng2#52expressionofAng2byMCF-7cellsaugmentscellsurvival,sup-cellsatday8,respectively).Furthermore,Ang2alsosuppressedpressescellapoptosisinthelungofmiceontheinitiatingstageCoCl2-inducedcellapoptosisatday4.Ang2#1cellshad35.7Ϯofonsetofmetastases,whichcontributestoinitiationoftumor1.7%andAng2#52cellshad55.0Ϯ2.2%apoptoticindexcom-growthofbreastcancermicrometastasesinmultipledistantparedwiththatofMCF-7controlcells(Fig.3D).However,weorgans(Fig.1A).

didnotobservesignificantdifferencesofgrowthrateoftheseAng2AugmentsCellSurvivalandSuppressesApoptosisoftwogroupsofMCF-7Ang2-expressingcellsundercompleteMCF-7CellsundertheStressConditions—Next,wetestedmediacontaining10%FBS.Nonetheless,theseresultssuggestwhetherAng2promotesbreastcancercellsurvivalunderthethatAng2augmentscellsurvivalandattenuatescellapoptosisconditionsofgrowthstress.AsshowninFig.3A,underserum-ofbreastcancercellsunderthestressconditionsinvitro.

starvationconditions,comparedwithcellsatday0post-treat-Ang2ActivatesAkt1andAkt2andInducesExpressionofment,atdays4and6,Ang2#1cellsdisplayedasurvivalrateofBcl-2inMCF-7Cells—BecauseAktisphosphorylatedbyILK,76.7Ϯ5.7%and49.8Ϯ4.5%,Ang2#52cellsshowed67.2ϮthemTORC2complexandDNA-dependentproteinkinase4.5%and46.5Ϯ6.3%whereascontrolGFP(MCF-7)cellsonly(DNA-PK)atSer-473(p-AktSer473)andbyPDK1atThr-308hadasurvivalrateof47.5Ϯ6.9%and26.5Ϯ3.1%,respectively.(p-AktThr308)(24,25)andAng2inducesp-AktSer473throughSimilarly,atday4,comparedwithcontrolGFPcells,Ang2#1the␣5␤1integrin-ILKsignaling(10),weexaminedwhethercellsshowedapoptoticindexof69.9Ϯ3.3%andAng2#52cellsAng2activatesAkttopromotetumorcellsurvivalandinhibithad78.9Ϯ1.9%,respectively(Fig.3B).AsshowninFig.3C,cellapoptosisunderserum-starvationcondition.Asshowninwhenvariouscellsexposedtocobaltchloride(CoCl2),a

Fig.4A,inabsenceofserum,expressionofAng2byMCF-7cells

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FIGURE2.Ang2enhancesbreastcancercellsurvival,suppressesapoptosis,andpromotestheinitialgrowthofbreastcancermicrometastasesinthelung.A,ateachindicatedtimepointpost-injection,4micepereachgroupwereeuthanizedandthelungswereharvestedandexamined.Imagesof10randomframesperlungwerecapturedunderanepifluorescentstereomicroscope(OlympusSXZ12).Representativeimagesofthelungateachtimepointareshown.Scalebars:100␮m.B,numbersofGFP-expressingcellsofeachgroupwithintheareaof10randomframesperlungwereanalyzedusingtheImageProPlussoftware.Thepercentmetastaticcellsurvivalwasdeterminedbynormalizingthemeannumbersofthecellsat12horlaterwiththoseat4hforeachgroup.DifferenceofthepercentcellsurvivalateachtimepointwasstatisticallyanalyzedusingMann-WhitneyUtest:*,pϽ0.05anderrorbars:ϮS.D.C,representativeimagesofTUNELassaysofMCF-7tumorcellsinthelung24haftertail-veininjection.Greencolor,viableGFP-expressingtumorcellnuclei;redcolor,totalapoptoticcellnuclei,andbluecolor,cellnuclei.Yellowcolor,mergedofgreen-andred-stainedapoptotictumorcellnuclei(arrowhead);Scalebars:50␮m.D,apoptoticindexoftumorcellsinthelungs12and24haftertail-veininjectionwasstatisticallyanalyzedusingttest:*,pϽ0.001anderrorbars:meanϮS.D.DatainA–Darerepresentativeofthreeindependentexperimentswithsimilarresults.

stimulatedp-AktSer473(Ser-473)butnotp-AktThr308(Thr-308)orphosphorylationofBadandproteinexpressionofBaxinatboth24and48hpost-treatmentcomparedwiththatofMCF-7cells(Fig.4E).

MCF-7controlcells.WhenMCF-7controlcellswereexposedAng2PromotesCellSurvivalthroughILK-Akt1/2Signaling—topolylysine(poly-L,anon-stimulativesubstrateforinteg-Next,weexaminedeffectsofknockdownofILKonp-AktSer473,rins),recombinantAng2proteinsorfibronectin(FN)pre-expressionofBcl-2andcellsurvivalandapoptosisofAng2-coatedincellcultureplates,Ang2,butnotpoly-LorFNexpressingandcontrolMCF-7cells.AsshowninFig.5A,deple-inducedp-AktSer473butnotp-AktThr308(Fig.4B).Ang2tionofILKexpressionbyasiRNApoolmarkedlysuppressedinductionofp-AktSer473,butnotp-AktThr308validatesourAng2-inducedp-AktSer473butnotp-AktThr308,andexpressionpreviousobservationthatAng2inducesp-AktSer473throughofBcl-2,indicatingthatAng2inducesp-AktSer473andBcl-2the␣5␤1integrin-ILKsignaling(10).

throughILK.Asexpected,knockdownofILKdecreasedAng2-BecauseAktisoformsdisplayeddistinctrolesinpromotingstimulatedcellsurvival(Fig.5B,cellsurvival,comparetheinitiationandmetastaticphasesofbreasttumorprogres-ILK.siRNA(I)of102.5Ϯ6.6%withcontrol.siRNA(C)ofsion(26),wedeterminedAng2stimulationofAktisoformsin143.6Ϯ3.3forAng2#1,Iof103.1Ϯ5.7%withCof132.1Ϯ7.8%MCF-7cells.AsshowninFig.4C,whileexpressionofAng2-forAng2#52)andincreasedcellapoptosis(Fig.5C,cellapopto-inducedp-AktSer473inMCF-7cells,expressionsofAkt1andsis,compareIof93.5Ϯ6.9%withCof62.1Ϯ8.4forAng2#1Akt2butnotAkt3weredetectedinMCF-7cellsandexogenousandIof95.6Ϯ7.6%withCof72.3Ϯ6.7%forAng2#52).

expressionofAng2didnotchangetheexpressionlevelsofAkt1TodeterminewhetherAkt1orAkt2orbothmediatesAng2andAkt2inMCF-7cells.Furthermore,expressionofAng2stimulation,weusespecificsiRNAstoknockdownofAkt1andinducedp-AktSer473ofAkt1andAkt2butnotAkt3inMCF-7Akt2,individuallyortogetherinAng2#1cells.AsshowninFig.cells(Fig.4D).Additionally,weexaminedtheimpactofAng25B,separateinhibitionofAkt1(A1)orAkt2(A2)attenuatedonthreedeathmoleculesdownstreamofAkt,Bcl-2,Bcl-2-Ang2-inducedproteinexpressionofBcl-2whereassimultane-associatedagonistofcelldeath(Bad),andBcl-2-associatedXousdepletionofbothAkt1andAkt2(A1&2)bysiRNAsshowedprotein(Bax)(25).WefoundthatAng2markedlyinducedprofoundsuppressionofAng2-inducedexpressionofBcl-2expressionofBcl-2buthadnoeffectsonproteinexpression

(Fig.5D).Tosimilarextents,separateknockdownofAkt1(A1)

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FIGURE3.Ang2augmentscellsurvivalandsuppressescellapoptosisofMCF-7cellsinvitroundertheconditionsofgrowthstress.MCF-7controlGFPcells,Ang2#1andAng2#52cellswereincubatedwithserum-free/phenolred-freemediumasserum-starvationmodel(A)and(B)orwith10%charcoal-treatedFBS/phenolred-freemediumcontaining100␮Mofcobaltchloride(CoCl2)asahypoxiamodel(CandD).InAandC,thenumbersofsurvivalcellsateachtimepointwereanalyzedbytrypanbluedyeexclusion,counted,andnormalizedtothecellnumberofeachgroupseededonday0.InBandD,apoptosisinthecellsculturedunderserum-starvation(B)orahypoxiacondition(D)for4dayswereanalyzedusingacelldeathdetectionELISAkit.DatawereshownasrelativeapoptosisbynormalizingeachabsorbancevaluetothatofMCF-7controlcells.Alldatawerestatisticallyanalyzedusingttest.*,pϽ0.001anderrorbars:meanϮS.D.ResultsinA–Darerepresentativeofthreeindependentexperimentswithsimilarresults.

orAkt2(A2)decreasedAng2-stimulatedcellsurvivalorAkt1andAkt2weresimultaneouslyknockeddown,Ang2-pro-inducedcellapoptosisatmoderatelevelswhereasdoublemotedcellsurvivalwasdiminishedunderbothstresscondi-depletionofAkt1&Akt2(A1&2)furtherinhibitedcellsurvivaltions(Fig.6,BandC,righthalvesofthebargraphs).Further-orenhancedcellapoptosis(Fig.5E,cellsurvival,compareA1&2more,asshowninFig.6,DandE,at24hpost-injection,miceof34.6%Ϯ6.1withA1of66.4Ϯ7.4%orA2of81.3Ϯ5.1%;Fig.thatreceivedAng2#1cellstransfectedwithcontrolsiRNAshad5F,cellapoptosis,compareA1&2of199.8Ϯ6.6%withA1of33.5Ϯ11.1%cellsretainedinthelungcomparedwiththatat4h154.8Ϯ8.9%orA2of123.4Ϯ7.4%).Takentogether,thesewhereasmiceinjectedwithAng2#1cellsinwhichendogenousresultssuggestthatbothAkt1andAkt2contributetoAng2Akt1andAkt2wereknockeddownbysiRNAsonlyhad3.8Ϯmodulationofbreastcancercellsurvivalandapoptosisand0.8%ofcellsleftinthelung.After5days,micereceivedAng2#1simultaneousinhibitionofAkt1andAkt2bysiRNAknock-cellsthatweretransfectedwithcontrolsiRNAsshowed4.3ϮdowndisplaymarkedinhibitionofAng2stimulationofbreast1.4%cellsstayedinthelungassmalltumorcellclusterswhereascancercells.

micereceivedAng2#1cellsinwhichAkt1andAkt2wereKnockdownofAkt1andAkt2DiminishesAng2-stimulatedknockeddownonlyhad0.3Ϯ0.3%cellleftinthelung(Fig.6E).CellSurvivalandProliferationofMCF-7CellsinVitroandinTakentogether,thesedatafurthersupportourobservationthattheLungofAnimals—Afterward,wedeterminedtheeffectsofAng2stimulationofbreastcancercellsurvivalandmetastaticknockdownofAkt1ϩAkt2(siRNAAkt1&2)onAng2-enhancedMCF-7tumorgrowthinthelung(Fig.1)ismediatedbythecellsurvivalofMCF-7cellsinvitroandinthelungofmice.Asintegrin/ILK/Aktsignalpathway.

showninFig.6A,comparedwithcontrolsiRNA,knockdownofInhibitionofBcl-2SuppressesAng2-stimulatedCellSurvivalAkt1ϩAkt2withtheircorrespondingsiRNApools(Akt1&2)andProliferationofMCF-7CellsinVitroandintheLungofeffectivelyinhibitedAng2-inducedp-AktSer473andexpressionAnimals—Next,wedeterminedwhetherBcl-2,adownstreamofBcl-2inAng2#1cells.AsshowninFig.6,BandC,whentheseeffectorofAktsignalingmediatestheAng2-stimulatedMCF-7siRNAtransfectedcellswereusedtoassesstheimpactoncellbreastcancercellsurvivalandinitialgrowthoftumormetasta-survival,after6daycellcultureinabsenceofserum,comparedsisinthelung.VariousMCF-7cellswereexposedto0,1.0,andwithcellsatday0inculture,Ang2#1cellsdisplayedanincrease5.0␮MofHA14–1,apharmacologicalinhibitorforBcl-2thatincellsurvivalrateof43.5Ϯ3.6%whereasMCF-7controlcellsdidnotsignificantlyaffectMCF-7cellviability(27)underonlyhadasurvivalrateof20.8Ϯ1.8%ofcells.Toasimilarserumstarvationconditionfor48h.AsshowninFig.7,AandB,extent,whenvariouscellsweretreatedwithCoCl2,Ang2#1comparedwithMCF-7controlcells,expressionofAng2cellsdisplayedasurvivalrateof63.6Ϯ3.0%whereasMCF-7enhancedAng2#1cellsurvival(a40Ϯ4.3%increase)andinhib-controlcellshadasurvivalrateof22.3Ϯ2.1%.However,whenitedcellapoptosis(a30.7Ϯ3.2%decrease)underserumstar-29254JOURNALOFBIOLOGICALCHEMISTRY

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FIGURE4.Ang2stimulatesphosphorylationofAkt1andAkt2atSer-473andinducesexpressionofBcl-2inMCF-7cells.IB(AtoC,E)orIP/IB(D)analysesusingwholecelllysatesofMCF-7Ang2-expressingcellsandcontrolGFPcellswiththeindicatedantibodies.A,phosphorylationofAktatSer-473(S473)butnotThr-308(T308)wasenhancedinAng2-expressingcellscomparedwithMCF-7controlGFPcellsundertheserum-starvationconditions.B,phosphorylationofAktatSer-473(S473)butnotThr-308(T308)inMCF-7cellswasinducedbyseedingMCF-7cellsontorecombinantAng2-coatedcellcultureplates,butnotontothepoly-lysine(poly-L)-orfibronectin(FN)-coatedplates.C,IBanalyses.ExpressionofAkt1andAkt2,butnotAkt3wasdetectedinMCF-7Ang2-expressingandcontrolcells.ExpressionofexogenousAng2inducesphosphorylationofAktatSer-473(S473)inMCF-7cells.D,IB/IPanalyses.ExpressionofexogenousAng2inducesphosphorylationatSer-473(S473)ofAkt1andAkt2,butnotAkt3inMCF-7cells.E,IBanalyses.ExpressionofAng2enhancedexpressionofBcl-2,butnotBaxorphosphorylationofBadinMCF-7Ang2-expressingcellscomparedwithGFPcontrolcells.InAandCtoE,wholecelllysatesofphosphataseandtensinhomolog(PTEN)-mutatedhumangliomaU87MGcellswereusedaspositivecontrolsfortheexpressionandactivation(phosphorylationatSer-473)ofAkt,Akt1,Akt2,andAkt3.TotalproteinsofAktand␤-actinwereusedasloadingcontrolsforallexperimentsexceptD.TheexperimentsofA–Ewereperformedthreeindependenttimeswithsimilarresults.

FIGURE5.Ang2enhancescellsurvivalandinhibitscellapoptosisofMCF-7cellsthroughtheILK-Akt1/2signaling.AandD,IBanalyses:knockdownofILK(I)(A)andAkt1(AbutnotatThr-3081)andAkt2(A(T308)andinhibited2)separatelyortogether(Akt1ϩAkt2,AtheAng2-inducedexpression1&2)bysiRNAs(D)blockedtheAng2-stimulatedphosphorylationofAktatSer-473(S473)ofBcl-2inMCF-7cells.TotalproteinsofAktand␤-actinwereusedasloadingcontrols.BandE,cellsurvivalandCandF,cellapoptosisinthecellscultureunderserum-starvation.Ang2#1cellswereseparatelytransfectedsiRNApoolsforcontrol(C),siRNApoolsforILK(I),Akt1(A1),Akt2(A2),orAkt1ϩAkt2(A1&2).48-hlater,thecellswereincubatedwithserum-free/phenolred-freemedium(serumstarvation)for4days.Thenumbersofsurvivalcellswereanalyzedbytrypanbluedyeexclusion,countedandnormalizedtothecellnumberofeachgroupseededonday0(BandE).CellapoptosiswasanalyzedusingacelldeathdetectionELISAkit(CandF).DataofB,C,E,andFwerestatisticallyanalyzedusingttest.*,pϽ0.001anderrorbars:meanϮS.D.TheresultsofA–Farerepresentativefromthreeindependentexperimentswithsimilarresults.

vationconditionfor2days(alsoseeFig.3,AandB).WhileBcl-2trationssignificantlyabrogatedAng2-enhancedcellsurvivalsinhibitorHA14–1hadnoimpactoncellsurvivalorapoptosis(35.7Ϯ3.9%and39.7Ϯ5.1%decreasescomparedwithsolvent-ofMCF-7controlcells,HA14–1atboth1.0and5␮Mconcen-treatedAng2#1controlcells,Fig.7A)anddiminishedthe

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FIGURE6.KnockdownofAkt1andAkt2diminishesAng2-stimulatedcellsurvivalofMCF-7cellsinvitroandinthelungofmice.A,IBanalyses:knockdownofAkt1ϩAkt2(A1&2)blockedtheAng2-stimulatedphosphorylationofAktatSer-473(S473)andAng2-inducedexpressionofBcl-2proteinsinAng2#1cells.C,controlsiRNAs.TotalproteinsofAktand␤-actinwereusedasloadingcontrols.BandCinvitrocellsurvivalassays.KnockdownofAkt1&Akt2bysiRNAs(siRNA.Akt1ϩ2)abrogatedtheAng2-enhancedcellsurvivalinvitrounderserum-starvationcondition(B)orafterthecellswereexposedtoCoClpϽ0.001byttest.Errorbars:meanϮS.D.D,knockdownofAkt1ϩAkt2(siRNA.Akt1ϩ2)inMCF-7Ang2#1cellsattenuatedtheAng2-enhancedcellsurvival2(C).*,inthelungofmice(4micepergroup)in24hand5daysafterthetail-veininjections.Representativeimagesofthelungofvariousmiceateachtimepointareshown.Scalebars:100␮m.E,percentageofsurvivedbreastcancercellsinthelungofmiceofeachgroupinDwasanalyzedbynormalizingthemeannumbersofthecellsin24hor5daysafterinjectionswiththoseofeachgroupat4hpost-injections.DifferenceofthepercentageofcellsurvivalateachtimepointwasstatisticallyanalyzedusingMann-WhitneyUtest.DatainB,C,andE,*,pϽ0.05anderrorbars:meanϮS.D.TheresultsofA–Garerepresentativefromtwoorthreeindependentexperimentswithsimilarresults.

Ang2-attenuatedapoptosis(25.3Ϯ4.3%and32.5Ϯ3.3%retainedinthelung.Fivedaysaftertheinjection,micereceivedincreasescomparedwithsolvent-treatedAng2#1controlcells,Ang2#1/controlsiRNAhad3.5Ϯ0.8%cellsremainedintheFig.7B).TofurtherexaminetheroleofBcl-2inAng2-Akt-lungassmalltumorclusters.Incontrast,Ang2#1/Bcl-2siRNAstimulatedbreastcancercellsurvivalandmetastasis,wecellsonlyhad0.1Ϯ0.2%cellsleftinthelung.Takentogether,knockeddownBcl-2usingasiRNApoolinMCF-7/Ang2#1andthesedatademonstratethattheup-regulatedBcl-2mediatescontrolGFPcells(Fig.7C).Asexpected,depletionofAng2-up-Ang2-promotedMCF-7breastcancermetastasisthroughregulatedBcl-2markedlyattenuatedAng2-promotedcellsur-enhancingcellsurvivalandattenuatingcellapoptosisinthevival(102.3Ϯ6.6%inBcl-2siRNA-transfectedcellsversuslung.

135.0Ϯ3.3%incontrolsiRNAcells,Fig.7D)andreversedDepletionofEndogenousAng2bysiRNAsinBreastCancerAng2-suppressedcellapoptosis(94.9Ϯ5.4%inBcl-2siRNACellsInhibitsp-AktSer473,ProteinExpressionofBcl-2,CellSur-transfectedcellsversus.5Ϯ8.7%incontrolsiRNAcells,Fig.vival,CellMigration,andIncreasesCellApoptosis—Increased7E).Incontrast,becauseminimallevelsofBcl-2proteinswereexpressionofAng2hasbeendemonstratedtocorrelatewithdetectedinMCF-7GFPcells,knockdownofBcl-2hadminimalinvasiveandmetastaticphenotypesofvarioustypesofhumanimpactsoncellsurvivalandapoptosisofcontrolGFPcells(Fig.cancersincludingbreastcancers(8,9).Apreviousstudy7D,leftbars).WhenthesesiRNAknockdowncellswereshowedthatendogenousAng2proteinsaredetectedathighinjectedintothetailveinsofmice,asshowninFig.7,FandG,atlevelsinhumanbreastcancerMDA-MB-468andSK-BR-3cells24hpost-injection,micereceivedAng2#1cellstransfectedwhereasMCF-7andMDA-MB-231cellsexpressAng2atlowwithcontrolsiRNAhad34.7Ϯ9.0%cellsinthelungcomparedlevels(17).Therefore,wereasonedthatinhibitionofendoge-withthatat4hpost-inoculationwhereasmiceinjectedwithnousAng2inMDA-MB-468andSK-BR-3cellswouldsuppressBcl-2knockdownAng2#1cellsonlyhad4.3Ϯ3.5%cellstheAkt-Bcl-2signaling,cellsurvival,andmigration,and

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FIGURE7.Bcl-2isadownstreameffectoroftheAng2-ILK-Akt1/2signalingthatmediatesAng2-enhancedcellsurvivalandAng2-attenuatedcellapoptosisofMCF-7cells.AandB,apharmacologicalinhibitorofBcl-2,HA14–1,significantlyinhibitedAng2-enhancedcellsurvival(A)andAng2-protectedcellapoptosis(B)underserum-starvationconditions.*,pϽ0.001byttestanderrorbars:meanϮS.D.C,IBanalyses:knockdownofBcl-2withacontrol(C)orsiRNApoolforBcl-2(B)incontrolGFPorAng2#1cellsabrogatedtheAng2-enhancedcellsurvival(D)andreversedAng2-attenuatedcellapoptosis(E)invitrounderserumstarvationcondition(A).*,pϽ0.001byttest.Errorbars:meanϮS.D.F,knockdownofBcl-2withasiRNApool(siRNA.Bcl-2)butnotthecontrolinAng2#1cellsattenuatedtheAng2-enhancedcellsurvivalinthelungofmice(4micepergroup)in24hand5daysafterthetail-veininjections.Representativeimagesofthelungofvariousmiceateachtimepointareshown.Scalebars:100␮m.G,percentageofsurvivedbreastcancercellsinthelungofmiceofeachgroupinFwasanalyzedbynormalizingthemeannumbersofthecellsin24hor5daysafterinjectionswiththoseofeachgroupat4hpost-injections.DifferenceofthepercentageofcellsurvivalateachtimepointwasstatisticallyanalyzedusingMann-WhitneyUtest.*,pϽ0.05anderrorbars:meanϮS.D.TheexperimentsofA–Garerepresentativefromtwoorthreeindependentexperimentswithsimilarresults.

increasecellapoptosis.Tothisend,weknockeddownendoge-obtainedinbreastcancerMDA-MB-468andSK-BR-3cellsnousAng2inthesetwobreastcancercelllinesusingasiRNA(Fig.8).Incontrast,knockdownofAng2inparentalMDA-MB-poolandcontrolsiRNAs.AsshowninFig.8A,depletionof231cellshadminimalimpactonthesecellularbehaviors(Fig.9,Ang2inhibitedp-AktSer473andexpressionofBcl-2proteininB–D),possiblybecauseoflowlevelsofendogenousAng2inMDA-MB-468andSK-BR-3cells.Asexpected,whencom-thesecells(Fig.9A).Together,inhibitionofendogenousAng2paredwithcontrols,inhibitionofendogenousAng2intheseinmetastaticbreastcancercellsimpairedtheactivatedAkt-twocelllinesattenuatedcellsurvival(Fig.8B),cellmigrationBcl-2signaling,cellsurvival,andmigration,andincreasecell(Fig.8C),andincreasedcellapoptosis(Fig.8D)underserum-apoptosis.

starvedconditions.

Finally,wevalidatedourobservationsinabreastcancerDISCUSSION

MDA-MB-231/#1834cellclone,aninvivo-derivedcellpopu-Inthisstudy,weprovidenovelmolecularinsightsintoAng2lationthatpreferentiallymetastasizetothelungandseveralstimulationofbreastcancermetastasistothelungofanimalsbyotherorganswheninjectedintothetailveinsofmice(18).Sig-focusingoninitialonsetofbreasttumormetastasis.Weshownificantly,geneprofileanalysisshowedthatexpressionlevelsofthatatearlystagesofMCF-7breastcancermetastasisintheAng2mRNAiselevatedϳ20-foldwhencomparedwiththeirlung,Ang2stimulatesbreastcancermetastasisthroughtheparentalMDA-MB-231cells(28).ConsistentwiththereportedILK-Akt1/2-Bcl-2signaling,increasingtumorcellsurvivalanddataofcDNAarrays,wedetectedincreasedlevelsofendoge-suppressingcellapoptosis,resultinginsubsequenttumornousAng2proteinsintheMDA-MB-231/#1834cellswhengrowthinthelungofanimals.InhibitionofILK,Akt1/2,andcomparedwiththatinparentalMDA-MB-231cells(Fig.9A).Bcl-2bysiRNAsorBcl-2byapharmacologicalinhibitorabro-WetransientlytransfectedparentalandMDA-MB-231/#1834gatesAng2stimulationoftumorcellsurvivalinvitroundercellswithsiRNAsforAng2orcontrol.AsshowninFig.9A,growthstressconditionsandAng2-promotedtumormetasta-whencomparedwithcontrolsiRNAs,depletionofendogenoussisinthelungofmice.Conversely,depletionofendogenousAng2inhibitedincreasedp-AktSer473andBcl-2expressionAng2bysiRNAsinthreemetastaticbreastcancercelllinescomparedwiththatinparentalcells(Fig.9A),attenuatedcellinhibitedtheAkt-Bcl-2signaling,cellsurvival,andmigration,survival(Fig.9B)andcellmigration(Fig.9C)butincreasedcellandincreasedcellapoptosis.Ourworkdemonstratesthatapoptosis(Fig.9D)invitro,consistentwiththedatathatwe

increasedexpressionofAng2enhancesbreastcancercell

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FIGURE8.KnockdownofendogenousAng2inhibitstheAkt-Bcl-2signal-inganddecreasescellsurvival,migration,andinhibitscellapoptosisofMDA-MB-468andSK-BR-3cellsinvitro.A,IBanalyses:knockdownofendogenousAng2Ser473byAng2.siRNAs(A)oracontrol(C)abrogatedAng2-in-duecedp-AktandproteinexpressionofBcl-2inMDA-MB-468andSK-BR-3cells.TotalproteinsofAktand␤-actinwereusedasloadingcontrols.Cellsurvival(B),cellmigration(C),andapoptosis(D)inthecellscultureunderserum-starvation.Ang2#1cellswereseparatelytransfectedsiRNApoolsforcontrol(C)orAng2(A).48-hlater,thecellswereincubatedwithserum-free/phenolred-freemedium(serum-starvation)for4days.Thenumbersofsur-vivalcellswereanalyzedbytrypanbluedyeexclusion,countedandnormal-izedtothecellnumberofeachgroupseededonday0(B).C,transfectedMDA-MB-468orSK-BR-3cellswereseededintoupperwellsofinaBoydenchamberswith1ϫ105cellperwell.Variouscellswereallowedtomigratethroughthe12␮mporesizemembranesprecoatedwithgelatinfor12hat37°C.Afterthemembranewasfixedandstained,non-migratingcellswereremoved.Thenumberofmigratingcellswasquantifiedunderamicroscopeaswepreviouslydescribed(10).Impactoncellmigrationwasexamined.D,cellapoptosiswasanalyzedusingacelldeathdetectionELISAkit.DataofB–Dwerestatisticallyanalyzedusingttest.*,pϽ0.001anderrorbars:meanϮS.D.TheresultsofA–Darerepresentativefromthreeindependentexperimentswithsimilarresults.

metastasisbypromotingtheinitialtumorcellsurvivalandgrowthintheirnewmicroenvironmentthroughstimulationoftheILK/Akt/Bcl-2signaling.

Breastcancermetastasisisaninefficientprocessandeachstepisequallyimportantfortumorcellstosuccessfullyestab-lishmetastatictumorsinthetargetingorgans(2).Anumberofstudiesshowedthatthepresenceoftumorcellsinthebloodcirculationdoesnotpredictwhethertumormetastasiswilloccurbecausemostofthetumorcellsthatenterthebloodstreamarerapidlyeliminated(2).Ourdatavalidatedtheseobservations.Atthefirst4hafterintravenousinjectionofMCF-7breastcancercellsintomice,alargenumberofGFP-expressingtumorcellswereretainedinthelung,perhapsbefore,duringorafterextravasationfromthevasculatures.At12and24hpost-injection,near90%ofGFPtumorcellsthatwereinitiallyretainedwereeliminatedinthelungofmicethatreceivedAng2-expressingorGFP-expressingcontrolcells.Incontrolgroups,by48h,lessthan2%ofGFPcontrolcellswereretainedandviable.At8days,lessthan0.5%GFPcontrolcellswereviable(Fig.2,AandB).Importantly,thesecellsfailedtosurvive,proliferateandgrowintomacrometastastictumors(Fig.1,AandB).Incontrast,at24hpost-injection,Ang2#1cellsdisplayedahigherretentionratethanGFPcontrolcells(Fig.29258JOURNALOFBIOLOGICALCHEMISTRY

FIGURE9.KnockdownofendogenousAng2inmetastaticMDA-MB-231/#1834cellsinhibitstheAkt-Bcl-2signaling,cellsurvival,andmigrationbutenhancescellapoptosisinvitro.A,IBanalyses:knockdownofendoge-nousAng2byAng2.siRNAs(A)oracontrol(C)attenuatedp-AktSer473andexpressionofBcl-2inclone1834cellsderivedfromMDA-MB-231cells.TotalproteinsofAktand␤-actinwereusedasloadingcontrols.Cellsurvival(B),cellmigration(C),andapoptosis(D)inthecellcultureunderserum-starvation.B,Ang2#1cellswereseparatelytransfectedsiRNApoolsforacontrolorAng2.48-hlater,variouscellswereincubatedwithserum-free/phenolred-freemedium(serumstarvation)for4days.Thenumbersofsurvivalcellswereanalyzedbytrypanbluedyeexclusion,counted,andnormalizedtothecellnumberofeachgroupseededonday0.C,impactoncellmigration.Trans-fectedMDA-MB-231/#1834cellswereseededintoupperwellsofBoydenchamberswith1ϫ105cellsperwell.Variouscellswereallowedtomigratethroughthe12␮mporesizemembranesprecoatedwithgelatinfor12hat37°C.Afterthemembranewasfixedandstained,non-migratingcellswereremoved.Thenumberofmigratingcellswasquantifiedunderamicroscopeaswepreviouslydescribed(10).D,cellapoptosiswasanalyzedusingacelldeathdetectionELISAkit.DataofB–Dwerestatisticallyanalyzedusingttest.*,pϽ0.001anderrorbars:meanϮS.D.TheresultsofA–Darerepresentativefromthreeindependentexperimentswithsimilarresults.

2B).Furthermore,from48hto8dayspost-injection,Ang2#1cellsnotonlystayedassmalltumorclustersbutalsostartedtogrow,accompaniedasignificantdecreaseincellapoptosis(Fig.2,A–D).ApreviousstudydemonstratedthattumorsgrowmuchslowerinAng2-deficientmicecomparedwiththatinwildtypeanimalsattheirearlystagegrowth,suggestingthatAng2isinvolvedintumorinitiation(29).Ourdatacorroboratewiththisreport.Byusinganexperimentalmetastasismodel,wedemonstratethatAng2actsoninitialstepsofbreastcancermetastasisthroughpromotingtumorcellretentioninthelungandincreasecellsurvival,therebystimulatingbreasttumormetastasistothelungofanimals.

OurmechanisticdatashowedthatAng2playsacriticalroleatearlystagesofbreastcancermetastasisinthelungthroughtheILK/Akt/Bcl-2pathway.Aktisakeymodulatorforvarietyofcellularfunctionsincludingcellproliferation,growth,pro-teinsynthesis,cellmetabolism,andsurvival(25).AlthoughallthreeAktisoformshavealterationsinclinicalbreasttumorspecimens(30),onlyAkt1andAkt2showedimpactsonbreastcancerdevelopmentandmetastasis(31).Intransgenicmam-maryglandtumormousemodels,activationofAkt1acceleratesv-erb-b2erythroblasticleukemiaviraloncogenehomolog2(ErbB2)/Neu-mediatedmammaryglandtumordevelopment

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Downloaded from www.jbc.org by guest, on February 28, 2013(32).Moreover,activationofAkt2markedlyenhancesmam-maryglandtumormetastasistothelung(26).Conversely,abla-tionofAkt1inhibits,whereasdeficiencyinAkt2acceleratestheprogressionandgrowthoftheErbB2/neu-mediatedmammarycarcinomainmice(33).Additionally,studiesusinginvitroandinvivomodelsfurthersupportthenotionthatAkt1andAkt2playdistinctrolesinmodulationofbreastcancerprogression,growth,invasion,andmetastasis(34–37).Ourresultsagreebutalsodifferfromtheseobservations.WedemonstratethatAkt1andAkt2,andoneoftheireffectors,Bcl-2arecriticalforAng2-stimulatedMCF-7breastcancermetastasisattheinitialonsetofmetastaticprocessinthelungofanimals.WeshowthatAkt1andAkt2butnotAkt3areexpressedinMCF-7cellsandAng2stimulationinducesproteinphosphorylationofAkt1andAkt2andexpressionofBcl-2.InhibitionofAkt1andAkt2,theirdirectupstreamactivatorILK(10)ordownstreamBcl-2(25)abrogatesAng2-stimulatedcellsurvivalunderstressconditionsinvitroandinthelungofanimals.Wealsoshowthatknock-downofeitherAkt1orAkt2aloneortogetherhaveinhibitoryimpactsonAng2-stimulatedcellsurvivalunderstresscondi-tionsinvitro.AlthoughthesedatadoesnotdifferentiatewhetherAkt1andAkt2playdistinctrolesintheearlyonsetofbreastcancermetastasisinthelung,ourobservationswarrantfurtherinvestigationtostudyhowAkt1andAkt2affectAng2-orotherstimuli-promotedpulmonarymetastasisofbreastcancers.Finally,wedetectedhighlevelsofendogenousAng2pro-teinsintwobreastcancercelllines,MDA-MB-468andSK-BR-3(17)andMDA-MB-231/#1844cellsthatarehighlymeta-statictothelung(18).KnockdownoftheendogenousAng2inthesecellsinhibitedstimulatedAkt/Bcl-2signaling,cellsur-vival,migrationandincreasedcellapoptosis,furtherdemon-stratingtheroleofAng2inpromotingbreastcancercellsur-vival,migrationandsuppressingcellapoptosisthroughtheAkt-Bcl-2signaling.

Insummary,thisstudyinvestigatesthemechanismsofAng2stimulationofbreastcancerlungmetastasisatearlystages.Byusinganexperimentaltumormetastasismodel,werevealthatthatAng2enhancesbreastcancercellsurvivalandsuppressescellapoptosisundergrowthstressconditionsthroughtheILK/Akt/Bcl-2-mediatedsignalpathway,therebypromotingbreastcancermetastasistothelungandseveralotherorgansofani-mals.WeshowthatILK,Akt1,andAkt2aswellasBcl-2arecriticalfortheenhancedbreastcancercellsurvivalandtumormetastasisinthelung.BecausehighlevelsofAng2expressioniscloselylinkedwithincreasedpotentialofangiogenesis,tumorgrowth,andmetastasisofbreastcancers,ourdatasuggestAng2asapromisingtherapeutictargetfortreatingpatientswithmet-astaticbreastcancers.

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