NF-kb信号通路

notshown).Bycontrast,BrdUincorporationintoCT/I␬B␣(AA)tumorswasreducedafterLPSadministration.Conversely,theextentofapoptosiswasdramaticallyincreasedafterLPS-treat-mentofCT/I␬B␣(AA)tumors,whereasverylittleapoptosiswasseenafterLPStreatmentofCTorCT/vectortumors(Figure2Banddatanotshown).ThebasallevelsofcellproliferationandapoptosisinPBS-treatedtumorsofallthreecelltypeswerequitesimilar.Thus,inhibitionofNF-␬BactivityincancercellsconvertstheLPS-inducedproliferativeresponsetoanapoptoticresponse.WealsoexaminedthesurvivaltimesofmiceharboringCT/vectorandCT/I␬B␣(AA)metastaticlungtumorsthatwerechal-lengedwitheitherLPSorPBSaloneusingKaplan-Meiersurvivalanalysis.WefoundthatLPSadministrationshortenedthelifespanofCT/vectortumor-bearingmice,butprolongedthesur-vivaltimeofCT/I␬B␣(AA)tumor-bearingmice(Figure2C).TherewerenodifferencesinthesurvivalratesofmicebearingeitherCT/vectororCT/I␬B␣(AA)tumorsthatreceivedPBSalone.Theseresultsareconsistentwiththoseobtainedbymeasuringlungnodulenumbers,totallungweight,orlunghistology.LPSinducesNF-␬B-dependentgenesintumorcellsToexaminehowNF-␬BactivationmediatesLPS-inducedtumorgrowth,weanalyzedexpressionofNF-␬Btargetgenesknowntobeinvolvedincellproliferationandapoptosis(Karinetal.,2002;KarinandLin,2002).Tumorswereestablishedasabove,and24hrafteradministrationofLPSorPBS,themiceweresacrificed.Tumortissuewasmicrodissected,lysed,andana-lyzedbyimmunoblottingandzymography.ExpressionoftheantiapoptoticproteinsBcl-XL,cIAP1,andcIAP2(Figure3A)aswellasMMP9activity(Figure3B),werestronglyinducedinresponsetoLPStreatmentinCTandCT/vectortumors,butnotinCT/I␬B␣(AA)-generatedtumors.Thefailuretoinduceanti-apoptoticproteinsmayexplaintheapoptoticresponsetoLPSseenafterinhibitionofNF-␬B.ExpressionofMMP9,ontheotherhand,islikelytoaffecttumorinvasivenessandgrowth.WealsofoundthatexpressionofPCNA,anSphasemarker,correlatedwiththeNF-␬BactivationstateofthecellsbeinghighinCTandCT/vectortumorsfromLPStreatedmiceandlowinCT/I␬B␣(AA)tumors(Figure3A).TheseresultscorrelatewiththoseobtainedbytheanalysisofBrdUincorporation(seeFigure2A).TNF␣mediatesLPS-inducedtumorgrowthandNF-␬BactivationActivationofNF-␬BbyLPSrequiresToll-likereceptor4(TLR4),acellsurfacereceptorthatismostlyexpressedonmyeloidcells(Poltoraketal.,1998).WeexaminedwhetherLPSactivatedNF-␬BinCT26cellsthroughadirectmechanismorviaamyeloidcellintermediate.AlthoughdirectincubationwithLPSactivatedNF-␬BinCT26cellsinvitro,ratherhighdosesofLPSwererequired(Figure4A).ThelowsensitivitytoLPScouldbeduetolowTLR4mRNAexpressioninCT26cellsrelativetomacro-phages(Figure4B).TodetermineifTLR4inhostcellsisrequiredforNF-␬BactivationinCT26tumors,weinoculatedwild-type(WT)miceaswellasTlr4Lps-dmutantmice,whichexpressasignalingdefectiveformofTLR4(Poltoraketal.,1998),withCT26cells.Tumor-bearingmicewereadministeredLPSasde-scribedabove,andNF-␬Bactivationintumorswasanalyzed2hrlater.WhereasLPSinducedNF-␬BactivationintumorsgrowninWTmice,itfailedtodosointumorsgrowninTlr4lps-dFigure3.LPSinducesNF-␬B-dependentexpressionofantiapoptoticandprometastaticgenesintumorcellsA:ExpressionofNF-␬B-dependentgenesintumorcells.TumorsgeneratedbyinjectionoftheindicatedcelllineswereexposedtoPBSorLPSinvivoandisolated24hrlater.Tumorcelllysateswereanalyzedbyimmunoblottingforexpressionoftheindicatedproteins.B:InductionofMMPactivityintumorcells.Tumorcelllysatesgeneratedasabovewereanalyzedbyzymographyongelscopolymerizedwithgelatinorcasein/plasminogentodetectMMPactivity.Thisfigureshowsagelatin-containinggel.ThelocationsoftheindicatedMMPswereidentifiedbynegativestaining.mice(Figure4C).Thus,NF-␬BactivationintumorsrequiresfunctionalTLR4inoneofthehostcelltypes,possiblyamacro-phagewhoseinfiltrationintotumorswasenhancedafterLPSchallenge(SupplementalFigureS2athttp://www.cancercell.org/cgi/content/full/6/3/297/DC1).ThesefindingssuggestedthataLPS-inducedinflammatorymediatorproducedbyhostcellsisresponsibleforNF-␬Bactiva-tionintumors.Amajorproinflammatorycytokinewhoseproduc-tionisstronglyinducedbyLPSisTNF␣(TraceyandCerami,1994).Indeed,LPSadministrationtotumor-bearingWTmiceresultedinrapidandrobustinductionofcirculatingTNF␣(Figure4D).Administrationofananti-TNF␣antibody5minafterLPSchallengeneutralizedmostofthecirculatingTNF␣(Figure4E).NF-␬BactivationbyLPSwassimilarinitskineticstoTNF␣releaseandwasinhibitedbytheneutralizinganti-TNF␣antibody(Figure4F).Similarresultswereobtainedbystainingtumortissuewithanti-RelA(p65)antibody(SupplementalFigureS3).LPSadministrationinducedthenuclearappearanceofphos-300CANCERCELL:SEPTEMBER2004Figure5.TNF␣mediatesLPS-inducedtumorgrowthbutnotLPS-inducedtumorregressionAandB:TNF␣mediatesLPS-inducedtumorgrowth.MicereconstitutedwithWT,Tnfr1Ϫ/Ϫ,orTnf␣Ϫ/ϪbonemarrowwereinoculatedwithCT26tumorcellsandafter9dayswereadministeredPBSorLPS.Sevendayslater,thelungswereremovedandweighed(A),andthetumornodulesonthelungsur-faceswerecounted(B).(*,pϽ0.05;**,pϽ0.01).CandD:TNF␣isnotrequiredforLPS-inducedtumorregression.Micereconsti-tutedwithWTorTnf␣Ϫ/ϪbonemarrowwereinoculatedwithCT/I␬B␣AAtumorcellsandafter9dayswereadministeredPBSorLPS.Sevendayslater,thelungswereremovedandweighed(C),andtumornodulesonthelungsurfaceswerecounted(D)andstatisticallyanalyzed.(*,pϽ0.05;**,pϽ0.01).Figure4.TNF␣mediatesLPS-inducedNF-␬BactivationintumorsA:ActivationofNF-␬Binculturedcells.CT26cellsweretreatedwithdifferentdosesofLPS(in␮g/ml)orTNF␣(10ng/ml).NF-␬BandNF1DNAbindingactivitiesweredeterminedbyEMSA.B:TLRmRNAexpression.RNAwasisolatedfromtheindicatedtumorcellsorbonemarrow-derivedmacrophagesandanalyzedbysemiquantitativeRT-PCRforexpressionofTLR2andTLR4mRNAs.C:LPSisanindirectactivatorofNF-␬Bintumors.Tumor-bearingmice(WTandTlr4lps-d)wereadministeredLPSasdescribedabove.Tumorswerecollected2hrlater,andNF-␬BandNF1DNAbindingactivitieswereexaminedbyEMSA.D:LPSinducesreleaseofcirculatingTNF␣.CT26tumor-bearingmicewereinjectedwithLPS.andserumsampleswerecollectedattheindicatedtimepoints.TNF␣concentrationwasdeterminedbyELISA.E:NeutralizationofcirculatingTNF␣.Tumor-bearingmicewereinjectedwithLPS,and5minlaterwereinjectedwith30␮lofanti-TNF␣antibody(AF-410-NA,Pharmingen)orPBS.Serumwascollectedafter2hr,andTNF␣levelwasdeterminedbyELISA.F:ActivationofNF-␬BisTNF␣-dependent.Tumor-bearingmicewereadminis-teredLPSwithoutorwithanti-TNF␣antibodyasdescribedabove.Tumorswerecollectedattheindicatedtimepoints,andNF-␬BDNAbindingactivitywasexaminedbyEMSA.thelungswereremovedandweighedandtumornodulesonthelungsurfaceswerecounted.ThelungtumorburdensinchimericmicereconstitutedwitheitherWTorTnfr1Ϫ/ϪbonemarrowweresignificantlyincreasedafterLPSchallenge(Fig-ures5Aand5B).However,noLPS-inducedincreaseintumorburdenwasevidentinlungsfrommicereconstitutedwithTnf␣Ϫ/Ϫbonemarrow.Similarresultswereobtainedbymeasur-ingNF-␬BDNAbindingactivityintumortissues:whileLPScausedNF-␬BactivationintumorsgrowninWTorTnfr1Ϫ/Ϫchimericmice,nosuchinductionwasseeninTnf␣Ϫ/Ϫchimericmice(datanotshown).Thus,TNF␣productionbyhosthemato-poieticcellsisrequiredforactivationofNF-␬Bincancercellsandstimulationoftumorgrowth.TRAILmediatesLPS-inducedregressionofNF-␬B-deficienttumorsInadditiontopreventionofLPS-inducedtumorgrowth,inhibi-tionofNF-␬BactivityinCT26cellsconvertedthegrowthre-sponsetoadeathresponse.WethereforesearchedforthemediatorthroughwhichLPSinducestheapoptoticresponse.WefirstsuspectedthatTNF␣maybethismediator,asitwasshownthathighdosesofTNF␣cankilltumorcellswhenNF-␬Bactivityisinhibited(Wangetal.,1999).WethereforegeneratedeitherWTorTnf␣Ϫ/ϪchimericmiceandinoculatedthemwithCT/I␬B␣(AA)tumorcells.Curiously,LPSadministrationdecreasedtumorburdentoasimilarextentinbothWTandTnf␣Ϫ/ϪradiationphorylatedRelA.ThisresponsewasblockedbytheI␬B␣super-repressor.WeconcludethatTNF␣isthemainmediatorthroughwhichLPSactivatesNF-␬Bintumors.WeexaminedwhetherablationofTNF␣productionorsig-nalingpreventsLPS-inducedNF-␬Bactivationandtumorgrowth.BonemarrowfromWT,type1TNFreceptor-deficient(Tnfr1Ϫ/Ϫ),orTNF␣-deficient(Tnf␣Ϫ/Ϫ)micewasindividuallytransplantedintolethallyirradiatedBALB/cmicetogeneratechimericmice,whichwereinoculatedsixweekslaterwithCT26tumorcells.LPSorPBSwereadministered,and7dayslaterCANCERCELL:SEPTEMBER2004301Figure6.TRAILmediatesLPS-inducedtumorre-gressionA:LPS-inducedexpressionofproapoptoticmole-cules.MicebearingCT/vectororCT/I␬B␣(AA)tu-morswereadministeredLPSorPBS.After12hr,themiceweresacrificed,andtumorswerecare-fullymicrodissectedusingneedlestoavoidbloodcontaminationfromoneofthelungs,whereastheotherlungwasleftasis.RNAwasisolatedandgeneexpressionwasanalyzedbyRNaseprotectionassay.B:LPS-inducedTRAILproteinexpression.MicebearingCT/vectororCT/I␬B␣(AA)tumorswereadministeredLPSorPBS.After14hrthemiceweresacrificed,andtumorsweremicrodis-sectedfromoneofthelungs,whereastheotherlungwasleftasis.ProteinwasisolatedandTRAILexpressionwasexaminedbyimmunoblotting.C:TRAILmediatesLPS-inducedtumorregression.MicebearingCT/I␬B␣(AA)tumorswereadminis-teredPBSorLPS.Thirtyminutesbeforeand3daysaftertheLPSchallenge,micewerei.p.injectedwitheitheranti-TRAILmonoclonalantibodyoracontrolIgG2a(300␮geach).SevendaysafterLPSinjection,miceweresacrificedandtheirlungsremoved,weighed,andphotographed,andtumornodulesonthelungsurfacewerecountedandstatisticallyanalyzed.(*,pϽ0.05;**,pϽ0.01).D:TRAILblockadepreventsLPS-inducedcelldeath.ApoptoticcellswereidentifiedbyTUNELstaining(green).chimeras(Figures5Cand5D).Thus,TNF␣isnotacriticalmedia-torofLPS-inducedregressionofNF-␬B-deficienttumors.TosearchforotherdeathmediatorsinducedbyLPS,weexaminedexpressionofTNFfamilymembers,theirreceptors,andeffectormoleculesinmicrodissectedtumorcellsandtumor-bearinglungs.WeobservedthatbothTRAILandFASmRNAswerestronglyinducedintumor-bearinglungtissue,butnotinmicrodissectedtumorcellsafterLPSchallenge(Figure6A).WealsoobservedweakinductionofthemRNAsforFASLanddownstreameffectorsofapoptosissuchasRIP1andcaspase8.AsFASisnotexpressedbyCT26tumors(Chenetal.,1998),itisratherunlikelythatFASListhemediatorofLPS-inducedtumorregression.AmorelikelycandidatewasTRAIL,whosereceptorDR5(AshkenaziandDixit,1998)wasexpressedbymicrodissectedtumorcells(seeFigure7D).ConsistentwiththemRNAexpressiondata,thelevelsofTRAILproteinwerehighlyelevatedfollowingLPSadministrationintumor-bearinglungtissues,butnotinmicrodissectedtumorcells(Figure6B).ToexaminetheroleofTRAIL,micebearingCT/I␬B␣(AA)tumorswerei.p.injectedwithaneutralizinganti-TRAILantibody(Takedaetal.,2001)oracontrolIgG2a30minbeforeand3daysafterLPSchallenge.Aftersevendays,themiceweresacrificedandtheirlungswereremoved,microscopicallyexam-ined,andweighed,andtumornodulesonthelungsurfacewerecounted.LPSchallengeofmicetreatedwithcontrolIgG2aresultedinamarkedlyreducedtumorburden,andinjectionofanti-TRAILantibodycompletelyblockedthiseffect(Figure6CandSupplementalFigureS4athttp://www.cancercell.org/cgi/content/full/6/3/297/DC1).ATUNELassaydemonstratedthattheanti-TRAILantibodyentirelyablatedtheapoptoticresponseofNF-␬B-deficienttumorstoLPSstimulation(Figure6D).TheseresultsstronglysuggestthatTRAIListhemaineffectorthatmediatesLPS-induceddeathandregressionofNF-␬B-deficienttumors.ExpressionofTRAILisinducedbytypeIandtypeIIinterfer-302CANCERCELL:SEPTEMBER2004