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ARTICLEReceived18Apr2013|Accepted19Sep2013|Published23Oct2013Rb1familymutationissufcientforsarcomainitiationYongqingLiu1,2,3,EsterSanchez-Tillo4,XiaoqinLu2,BrianClem1,SuchetaTelang1,AlfredB.
Jenson1,MiriamCuatrecasas5,JasonChesney1,AntonioPostigo1,4,6&DouglasC.
Dean1,2,3ItisthoughtthatgenomicinstabilityprecipitatedbyRb1pathwaylossrapidlytriggersaddi-tionalcancergenemutations,accountingforrapidtumouronsetfollowingRb1mutation.
However,recentwhole-genomesequencingofretinoblastomasdemonstratedlittlegenomicinstability,butinsteadsuggestedrapidepigeneticactivationofcancergenes.
TheseresultsraisethepossibilitythatlossoftheRb1pathway,whichisahallmarkofcancers,mightbesufcientforcancerinitiation.
Yet,mutationoftheRb1familyorinactivationoftheRb1pathwayinprimarycellshasproveninsufcientfortumourinitiation.
HerewedemonstratethattraditionalnudemouseassaysimposeanarticialanoikisandproliferationbarrierthatpreventsRb1familymutantbroblastsfrominitiatingtumours.
Bycircumventingthisbarrier,weshowthatprimarybroblastswithonlyanRb1familymutationefcientlyformsarcomasinnudemice,andaRas-ZEB1-Aktpathwaythencausestransitionofthesetumourstoaninvasivephenotype.
DOI:10.
1038/ncomms36501MolecularTargetsProgram,JamesBrownCancerCenter,UniversityofLouisvilleHealthSciencesCenter,529SouthJacksonStreet,Louisville,Kentucky40202,USA.
2DepartmentofOphthalmologyandVisualSciences,UniversityofLouisvilleHealthSciencesCenter,301EastMuhammadAliBoulevard,Louisville,Kentucky40202,USA.
3BirthDefectsCenter,UniversityofLouisvilleHealthSciencesCenter,301EastMuhammadAliBoulevard,Louisville,Kentucky40202,USA.
4GroupofTranscriptionalRegulationofGeneExpression,CIBERehd,IDIBAPS,Casanova143,08036BarcelonaSpain.
5DepartmentofPathology,HospitalClinic,Villarroel170,08036Barcelona,Spain.
6ICREA,LluisCompanys23,08010Barcelona,Spain.
CorrespondenceandrequestsformaterialsshouldbeaddressedtoA.
P.
(email:idib412@clinic.
ub.
es)ortoD.
C.
D.
(email:dcdean01@louisville.
edu).
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Althoughadultcancersarelinkedtoaseriesofmutationsthatarethoughttobeacquiredoveraprotractedperiod,pediatrictumourssuchasretinoblastomafrequentlyoccurinanarrowdevelopmentalwindowandinvolvealimitednumberofmutations.
Indeed,mutationofthesecondRb1alleleiscloselylinkedtoonsetofretinoblastoma,initiallyleadingtoatwo-hithypothesiswheremutationofbothRb1alleleswasthoughttobesufcientforretinoblastoma1.
Althoughconsistentadditionalmutationshaveyettobeidentiedinretinoblastoma,mutationoftheRb1pathwayhasbeenlinkedtogenomicinstability,anditisthoughtthatsuchinstabilityprecipitatedbytheRb1pathwaylosstriggersadditionalcancergenemutations2.
TherapidappearanceofthesemutationsasaresultofgenomicinstabilityisthenthoughttoberesponsibleforthecloselinkagebetweenmutationofthesecondRb1alleleandonsetofretinoblastoma.
However,recentwhole-genomesequencingofretinoblastomasdemonstratedremarkablylittlegenomicinstability,andinsteadsuggestedthatRb1mutationleadstorapidepigeneticderepressionofcancergenes3.
Alongtheselines,theRb1family,consistingofRb1,p107andp130,interactswithchromatin-modifyingenzymestorepresstranscription4.
SuchresultsraisetheinterestingpossibilitythatlossoftheRb1pathway,whichisahallmarkofcancers,mightbetheonlymutationrequiredforcancerinitiation.
However,eventhoughmutationoftheRb1familyinprimarycellssuchasmouseembryonicbroblasts(MEFs)leadstounrestrictedproliferation,suchmutationorinactivationoftheRb1pathwayhasneverbeenshowntobesufcientfortumourformationinxenograftexperiments5,6.
Instead,tumourinitiationfromRb1familymutantMEFsrequiresmutantRas6.
Insolidtumours,unrestrictedproliferationleadstolossofnormalcell–extracellularmatrixcontacts,whichcantriggeranchorage-dependentcelldeath(anoikis)7.
Anoikisisthoughttobeahurdlefortumourinitiation,andanchorage-independentsurvival(resistancetoanoikis)insuspensioncultureorinsoftagariscloselylinkedtotheRaspathwayandtheabilityofmutantcellstoformtumoursinnudemice.
GrowthfactorsignallingthroughRasisblockedwhencellslosematrixcontact8.
Suchsignallingactivatesphosphoinositide-3kinase(PI3K)andinturnAkt9,10,whichblockscelldeathbyphosphorylatingandinactivatingproapoptoticfactorssuchasFoxo3a,whosetranslocationtothenucleusissufcienttoinitiateanoikis11.
Formationofcell–cellcontactscansubstituteforlossofnormalcell–matrixcontactstorestoregrowthfactorsignalling,andsuchcell–cellcontactsinthecontextofaprimarytumourarethoughttoaffordcancercellresistancetoanoikis,butthisprotectionislostascellsabandontheprimarytumourtoinvadesurroundingtissues8,9,12.
HereweexaminedtheeffectsofmutantRasandcell–cellcontactsonanoikisandtumourformationwithRb1familymutantMEFs.
Wendthatifcell–cellcontactsaremaintained,cellsareprotectedfromanoikisandtheRb1familymutationissufcientfortumourinitiationinnudemice.
However,cellsthatabandontheprimarytumourdie,preventinginvasion.
MutantRasallowsforthesurvivalofcellsthatabandontheprimarytumour,therebyfacilitatingtumourinvasion.
ResultsRasorcell–cellcontactsblockanoikisandcauseAkt-S473.
WefoundthattheRb1familymutantMEFs(tripleknockout(TKO)DAPITrypanblue100a806040%Viablecells20011SS12311SSSS12233Ras-TKOtrypsinRas-TKO-trypsinTKONEDTKOtrypsinTKO-NEDTKO-trypsinTrypsinMEFtrypsinTKOTKORas-TKOTKOTKOSSTKOSSM123160kDa60kDa12333SS123MAkt473AktNEDNED3TrypsinNEDDAPIDAPIDAPIDAPIDAPIDAPIFoxo3aFoxo3aFoxo3aFoxo3aFoxo3aBrdUDAPIBrdUDAPIBrdUFoxo3aedfhgRas-TKO(day3)TKONED(day3)TrypsinTrypsinTKO(day2)bcFigure1|MutantRasorcell–cellcontactspreventanoikisandcauseconstitutiveAkt-S473andcytoplasmicretentionofFoxo3ainsuspensionculture.
(a)Cellswereplacedinsuspensioncultureandviabilitywasassessedbytrypanblueexclusion.
Numbersindicatedaysinsuspensionculture.
'NED'indicatesthatsinglecellsweregeneratednon-enzymaticallyinsteadofbytrypsinization,andthesecellsquicklyformedcell–cellcontactsinsuspension.
Trypanbluesinglecellsandtrypanblueassociatedcellsareshown.
'SS'indicatescellsintheabsenceofserum.
DAPI,4',6-diamidino-2-phenylindole.
(b)Cellsatday2insuspensioncultureweretreatedfor4hwithbromodeoxyuridine(BrdU)andthenimmunostainedasdescribed32.
NotethatTKOMEFsthatfailedtoformcell–cellcontactsdidnotincorporateBrdU.
(c)WesternblottingsshowingexpressionoftotalAktandAkt-S473.
'M'indicatesmonolayerculture.
(d)PhaseimageofRas-TKOcellsinsuspension.
(e)ImmunostainingshowingcytoplasmicretentionofFoxo3ainRas-TKOMEFsindafter3daysinsuspension.
(f)PhaseimageoftrypsinizedTKOMEFsaftertwodaysinsuspension.
(g)ImmunostainingshowingtranslocationofFoxo3atothenucleusincellsfromf.
(h)ImmunostainingshowingcytoplasmicretentionofFoxo3AinTKOMEFswithcell–cellcontactsafter3daysinsuspension.
Scalebars,20mM.
Errorbarsrepresents.
d.
ofthreereplicates.
Allresultsarerepresentativeofatleastthreeindependentexperiments.
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MEFs)showedinitialresistancetoanoikiswhentheyweretrypsinizedtosinglecellsandplacedinsuspensionculture,buttheyfailedtoproliferateandlostviabilityafter2daysinsus-pension(Fig.
1a,b).
Bycontrast,expressionofmutantRasintheTKOMEFs(Ras-TKOMEFs)ledtoproliferationandlong-termsurvivalinsuspension(Fig.
1a,b),consistentwiththetumorigenicphenotypeofRas-TKOMEFsinnudemice6.
Ascell–cellcontactsrestoregrowthfactorsignallingandresistancetoanoikistobroblastsinsuspension8,12,weaskedwhethersuchcell–cellcontactsmightblockanoikisofTKOMEFsinsuspension.
WefoundthatifTKOMEFswerepipettedornon-enzymaticallydissociated(NED)tosinglecells,asopposedtotrypsinization,thecellsrapidlyestablishedcell–cellcontactsinsuspension(Fig.
1a).
Moreover,similartoRas-TKOMEFswheregrowthfactorsig-nallingisshortcircuitedbymutantRas,NEDTKOcellsthatestablishedcell–cellcontactswereresistanttoanoikis(Fig.
1a).
Cellsthatfailedtoestablishsuchcontactsdidnotproliferate(Fig.
1b).
Consistentwiththeimportanceofgrowthfactorsig-nallingforviabilityofTKOMEFs,removalofserumfromtheculturemedialedtoarapidlossofviabilityofNEDcellsinsuspension,butitdidnotcauselossofviabilityoftrypsinizedsingleRas-TKOMEFs,wherethegrowthfactorsignallingpath-wayisshortcircuited(Fig.
1a).
Theseresultsdemonstratethatcell–cellcontactsbypassarequirementforRasmutationforproliferationandresistancetoanoikisinsuspension,butNEDTKOMEFsthatestablishsuchcell–cellcontactsarestilldepen-dentonserumgrowthfactorsignallingforproliferationandviabilityinsuspension.
AktcanbeactivatedbyphosphorylationofS473(Akt-S473)andT308(Akt-T308),andthesephosphorylatedAktformshavedistinctfunctions11,13.
Akt-S473caninhibitanoikisbyphosphorylationandinactivationofproapoptoticfactorssuchasFoxo3a,whosenucleartranslocationissufcienttoinitiateanoikis11.
WefoundpreviouslythatAkt-S473accumulatesinTKOMEFswithmutationoftheRb1family,andthatAkt-S473levelsarefurtherincreasedinRas-TKOMEFs14.
Akt-S473levelsdiminishedasTKOMEFslostviabilityinsuspension,butAkt-S473wasmaintainedinRas-TKOMEFsinsuspension(Fig.
1c).
Accordingly,Foxo3awasretainedinthecytoplasmofRas-TKOMEFsinsuspension(Fig.
1d,e),whereasitaccumulatedinthenucleusofTKOMEFsinsuspension(Fig.
1f,g).
AswithRas-TKOMEFs,Akt-S473wasmaintainedinNEDTKOMEFsthatestablishedcell–cellcontactsinsuspensionandFoxo3aremainedcytoplasmic(Fig.
1h),butAkt-S473rapidlydiminishedwhenserumgrowthfactorswereremoved(Fig.
1c).
TheseresultslinkresistancetoanoikistoretentionofAkt-S473andcytoplasmicFoxo3ainsuspension,andtheydemonstratethatserumgrowthfactorsarerequiredforresistanceofNEDTKOMEFstoanoikis.
GrowthfactorsignallingorRasmaintainsZEB1expression.
Incarcinomas,inductionofthetranscriptionfactorZEB1causesepithelial-to-mesenchymaltransition(EMT)thattriggersamigratorybroblasticphenotypelinkedtoinvasion15.
However,ZEB1expressionhasalsobeenlinkedtoresistancetoapoptosis,andlungandpancreaticcancercellswithaRasmutationsomehowbecomeaddictedtotheirelevatedlevelofZEB1(refs16,17).
ZEB1isrepressedbytheRb1family,andthusitisinducedinTKOMEFs18.
WefoundthatZEB1expressiondiminishedinTKOMEFsinsuspension,buttheexpressionwasmaintainedandevenincreasedinNEDTKOMEFsthatmaintainedcell–cellcontactsinsuspension(Fig.
2a,b).
However,aswithviability,ZEB1expressiondiminishedwhenserumgrowthfactorswereremovedfromNEDTKOMEFs(Fig.
2a,b).
Bycontrast,ZEB1expressionwasnotdiminishedwhenRas-TKOMEFswereplacedinsuspension,nordiditdiminishwhenserumgrowthfactorswereremoved(Fig.
2a,b).
Next,weexaminedtheexpressionofZEB1inmono-layerculture.
ZEB1expressionwasdependentonseruminbothwild-typeandTKOMEFs,butnotinRas-TKOMEFs(Fig.
3a).
Together,theseresultsdemonstratethatalthoughZEB1isdere-pressedinTKOMEFswithlossoftheRb1family,itsexpressionisstilldependentonserumgrowthfactors.
Thelinkbetweenintegrinsandgrowthfactorreceptorsiswellknown8,19–24.
Clusteringofintegrinsoncelladhesionconcentratesgrowthfactorreceptorstositesrequiredforefcientsignalling.
Integrinligationisrequiredforintegrin-associatedfocaladhesionkinase(FAK)activationinresponsetogrowthfactors,andalackofgrowthfactorclusteringandFAKactivationinsuspensioncausesablockingrowthfactorsignalling.
SuchalossofgrowthfactorsignallinginsuspensionthenleadstolossofZEB1expression.
Bycontrast,whenmutantRasispresent,FAKandgrowthfactorreceptorsareconstitutivelyactivatedinanintegrin-independentfashion,allowingforadhesion-independentgrowthfactorsignalling(andevengrowthfactor-independentgrowthfactorsignalling).
AsgrowthfactorsignallingisshortcircuitedwithRasmutation,Ras-TKOMEFsarenotdependentonintegrinligationorgrowthfactorsforthemaintenanceofZEB1expression,providingforcontinuedZEB1expressioninsuspension.
Similartoadhesiontomatrix,cell–cellcontactshavealsobeenshowntomaintaingrowthfactorsignalling12,23,andthisallowsthemaintenanceofZEB1inNEDTKOMEFsinsuspension.
ButincontrasttoRas-TKOMEFs,NEDTKOMEFsarestilldependentonserumgrowthfactorstomaintainZEB1.
ZEB125a2015Relativeabundance10501MEFTKORas-TKO112223TKOtrypsinRas-TKOtrypsinRas-TKOTKONEDTKONEDNEDTrypsinTrypsinZeb1180kDa42kDaMonolayerActb33331SSSSSSbFigure2|RetentionofZEB1expressioninTKOMEFinsuspensionrequireseithercell–cellcontactsandserumgrowthfactorsignalling,ormutantRas.
(a)Real-timePCR.
Numbersindicatedaysinsuspensionand'SS'indicatesserum-starvedcellsasinFig.
1.
(b)WesternblottingsshowingthelevelofZEB1.
NEDandtrypsinizedcellswereinsuspensionfor2days.
TheTKOMEFblotisexposedlongersothatthelevelofZEB1intrypsinizedcellscanbeseen(notethelevelofActb).
Errorbarsrepresents.
d.
ofthreereplicates.
Allresultsarerepresentativeofatleastthreeindependentexperiments.
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WethenusedlentiviralshorthairpinRNA(shRNA)toknockdownZEB1inTKO(TKOZEB1shRNA)andRas-TKO(Ras-TKOZEB1shRNA)MEFstoalevelsimilartothatinparentwild-typeMEFs(Fig.
3a).
TKOZEB1shRNAMEFsshowedsimilarproliferationinmonolayerculturebuttheylostviabilitymorerapidlythanTKOMEFsassinglecellsinsuspension,andNEDcellsalsounderwentanoikisinsuspension(Fig.
3b).
Ras-TKOZEB1shRNAMEFslostviabilityeveninmonolayercultureandcouldnotbepassaged(Fig.
3c–e).
TheseresultsdemonstratethatinductionofZEB1seeninTKOMEFsiscriticalforresistancetoanoikis,andthatelevatedZEB1isalsocriticalforviabilityofRas-TKOMEFs.
ZEB1isrequiredforAkt-S473phosphorylation.
WewonderedwhetherinductionofZEB1mightbelinkedtoconstitutiveAkt-S473seeninTKOandRas-TKOMEFs.
Indeed,wefoundthatconstitutiveAkt-S473waslostwithZEB1knockdowninbothTKOandRas-TKOMEFs(Fig.
3f–h).
Althoughshort-circuitingRasmutationsleadtoconstitutiveactivationofsignallingpath-ways,includingErkandAkt,thesepathwaysarenormallyinducedonlytransientlybyserumgrowthfactorsinwild-typecells9,10.
Accordingly,whenwild-typeMEFswereserumstarvedandthenretreatedwithserumgrowthfactors,bothAkt-S473andAkt-T308wereinducedwithin30min,andtheirlevelthendiminishedafter2h(Fig.
3i).
TodeterminewhetherZEB1mightbeimportantforthisgrowthfactor-inducedphosphorylationofAktinwild-typeMEFs,weassessedtheeffectofZEB1mutationonthisserum-inducedAktphosphorylation.
Akt-S473wasdiminishedinagenedosage-dependentmannerinZEB1(andZEB1(MEFs,butAkt-T308wasunaffectedbyZEB1mutation(Fig.
3i).
Surprisingly,theseresultsdemonstratethatabasallevelofZEB1isnecessaryforserumgrowthfactor-inducedAkt-S473inwild-typeMEFs.
However,inductionofZEB1whentheRb1pathwayislostandRasismutatedsomehowmediatesconstitutiveAkt-S473.
BecauseofthisdependenceofAkt-S473onZEB1,itisinterestingtonotethatAktcanregulatetheexpressionofthemiR-200familyinamannerthatdependsonthebalancebetweenAkt1andAkt2(ref.
25).
ZEB1andmiR-200compriseamutualrepressionloop15,raisingthepossibilitythatabalanceofAktisoformexpressionmayalsofeedbacktoregulatethelevelofZEB1.
TKOandRas-TKOMEFsareaddictedtoAkt.
DependenceofTKOandRas-TKOMEFsonZEB1,andinturntherequirementMonolayerTKORas-TKOZEB1shRNARas-TKODAPIAkt-473DAPIGFPAkt-473ZEB130a543210Trypsin100806040%Viablecells20011112TKOTKOMEFTKOZEB1shRNATKOZEB1shRNA3123NED60kDa60kDa30min1h2h4h–Zeb1(+/+)Zeb1(+/–)Zeb1(–/–)60kDa60kDa60kDa60kDa60kDa60kDa60kDa60kDaAkt473Akt-473Akt-473Akt-473Akt-308Akt-308AktAktAktAktTKOTKOZEB1shRNAMEFMEFSSTKOTKOSSRas-TKORas-TKOSSRasTKOZEB1shRNATKOZEB1shRNARelativeabundancebhicdefgFigure3|ZEB1isrequiredforserumgrowthfactor-inducedAkt-S473inwild-typeMEFsanditmediatesconstitutiveAkt-S473inTKOandRas-TKOMEFs.
(a)Real-timePCRshowingthatZEB1expressionisinducedinTKOcomparedwithwild-typeMEFs,andthatitisfurtherinducedinRas-TKOMEFs.
ZEB1expressiondiminishedinwild-typeandTKOMEFsserumstarved(SS)overnight,butserumstarvationhadnoeffectinRas-TKOMEFs.
LentiviralknockdownofZEB1inTKOandRas-TKOMEFstoalevelsimilartothatinwild-typeMEFsisshown.
(b)KnockdownofZEB1restoresanoikisinNEDTKOMEFs.
Numbersindicatedaysinsuspension.
(c–e)PhaseimagesshowingthatknockdownofZEB1inRas-TKOMEFstoalevelsimilartothatinwild-typeMEFscausedrapidlossofviability.
FollowingshRNAlentiviralinfections,mostcellsdiedandthosethatremainedbecamelargeandat(resemblingsenescentcells),andfailedtoproliferate.
(f,g)ImmunostainingofcellsindandeforAkt-S473.
GreenuorescentproteinisexpressedfromthelentiviralshRNAvector.
(h)KnockdownofZEB1inTKOMEFsleadstolossofconstitutiveAkt-S473.
AwesternblottingshowingtotalAktandAkt-S473.
(i)Wild-typeorZEB1mutantMEFs35wereserumstarvedovernightandthenretreatedwith20%serumfortheindicatedtime.
WesternblottingsshowingtotalAkt,Akt-S473andAkt-308.
Errorbarsrepresents.
d.
ofthreereplicates.
Scalebars,20mM.
Allresultsarerepresentativeofatleastthreeindependentexperiments.
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forZEB1forconstitutiveAkt-S473inthesecells,raisedthepossibilitythatelevatedZEB1isimportantforviability,becausethesecellshavebecomeaddictedtoconstitutivelyactiveAkt.
Totestthispossibility,weusedsmallinterferingRNAtoknockdownthethreeAktisoformsinwild-type,TKOandRas-TKOMEFs.
KnockdownofallthreeAktisoformsledtorapidlossofRas-TKOMEFviabilityinamonolayerculture(Fig.
3a,b).
Moreover,lossofviabilitywasalsoevidentwithknockdownofeitherAkt1orAkt2,buttheeffectwasmostpronouncedwithlossofAkt1(Fig.
4a,b).
Likewise,knockdownofAktinTKOMEFsalsoledtolossofviability.
Bycontrast,knockdownofAktisoformsinMEFsdidnotreduceviabilityinthemonolayerculture(Fig.
4a,b).
TheseresultssuggestthatTKOandRas-TKOMEFshavebecomeaddictedtoconstitutiveAkt-S473,andtheco-addictiontoele-vatedZEB1reectsarequirementforelevatedZEB1tosomehowcauseconstitutiveAkt-S473.
However,howhaveTKOandRas-TKOMEFsbecomeaddictedtoAktPreviously,wedemonstratedthatmutationoftheRb1familyinTKOMEFsledtoelevatedAkt-S473,butinterestinglyitalsoledtolossofErkactivation14.
Moreover,introductionofRasintothesecellsdidnotrestoreErkactivation.
WeprovidedevidencethatconstitutiveAkt-S473wasfeedingbacktoinhibitRaf,thusresultinginlossofErkactivation.
WesuggestthatitistheswitchfromErksignallinginwild-typeMEFstoAktsignallinginTKOandRas-TKOMEFsforproliferation/survivalthatiscausingthesecellstobecomedependentonAkt.
ZEB1regulatesmTorexpressiontocontrolAkt-S473.
Akt-T308iscatalysedbyPdk1,butAkt-S473iscatalysedbymammaliantargetofrapamycin(mTor)inthecontextofmTorcomplex2(mTorc2)11,13.
WefoundthatmTorexpressionwaslowinMEFs,itwasupregulatedinTKOMEFsandfurtherinducedinRas-TKOMEFs(Fig.
5a;ref.
14).
Furthermore,mTordiminishedalongwithAkt-S473inTKOMEFsinsuspensionculture,andmTorexpressionwasmaintainedalongwithAkt-S473andresistancetoanoikisinNEDTKOMEFsandinRas-TKOMEFsinsuspension(Figs1band5a).
ThisinductionofmTorthenparalleledAkt-S473andresistancetoanoikis,raisingthepossibilitythatmTorexpressionmightberatelimitingfortheactivityofmTorc2.
WefoundthatoverexpressionofmTorinMEFs(MEF-mTor)wassufcientforconstitutiveAkt-S473andatleastpartialresistancetoanoikis(Fig.
5b,c).
Moreover,knockdownofmTorinTKOMEFs(TKOmTorshRNAMEFs)toalevelsimilartothatinwild-typeMEFs(Fig.
5a,b)diminishedconstitutiveAkt-S473anditrestoredanoikiswithin24hinsuspensionculture(Fig.
5b,c).
TheseTKOmTorshRNAMEFsshowedsimilarsizeandmorphologytoTKOMEFs,andtheirproliferationandviabilityinmonolayerculturewasalsosimilar,implyingthatthislevelofmTorknock-downhadnotcausedinactivationofmTorc1,whichiscriticalforproteinsynthesis.
However,mTorknockdowninRas-TKOMEFs(alsotoalevelsimilartothatinwild-typeMEFs)ledtoarapidlossofviability,aswithknockdownofZEB1orAkt.
Althoughover-expressionofmTorledtoconstitutiveAkt-S473inMEF-mTorcellsinmonolayerculture,wefoundthatremovalofserumgrowthfactorsfromthemediastilldiminishedAkt-S473(Fig.
5b).
Con-sistently,wealsofoundthatAkt-S473wasdiminishedinMEF-mTorcellsinsuspensionculturewheregrowthfactorsignallingisblocked(Fig.
5b).
Takentogether,ourresultsdemonstratethatinductionofmTorisrequiredtoreachathresholdforconstitutiveAkt-S473,butmTorc2isstilldependentonserumgrowthfactorsorRasmutationforPI3K-mediatedactivation.
Interestingly,PI3KactivationofmTorc2involvesassociationofmTorc2withtheribosome,whichmightservetolinkmTorc2tocellmetabolism26.
AsmTorc1isinvolvedinribosomebiogenesis,thisndingmayalsoservetoindirectlylinkmTorc2activitytomTorc1.
Dependingonpromotercontext,ZEB1canbindthetranscriptionalco-activatorp300andserveasatranscriptionalactivator—p300competeswiththecorepressorCtBPforbindingtoZEB1,therebyeliminatingdefaultrepressionbyZEB1(ref.
27).
WefoundthatmTorwasinducedalongwithZEB1inTKOandRas-TKOMEFs(Fig.
5d),leadingustohypothesizethatZEB1wasresponsibleforinductionofmTor.
Indeed,knockdownofZEB1inTKOorRas-TKOMEFsdiminishedmTorexpression(Fig.
5d),demonstratingthatmTorispositivelyregulatedbyZEB1.
ConsistentwithadirecteffectofZEB1onmTorexpression,wefoundmultipleconsensusE-boxbindingsitesforZEB1inthemTorpromoter,andZEB1boundtothemTorpromoterinvivoinchromatinimmunoprecipitation(ChIP)assays(Fig.
5e).
WeproposethatZEB1isimportantformaintainingathresholdlevelofmTorinwild-typeMEFsrequiredforthegenerationofAkt-S473inresponsetoserumgrowthfactors.
Moreover,inductionofmTorresultingfromoverexpressionofZEB1inTKOandRas-TKOMEFsleadstoconstitutiveAkt-S473.
TKOMEFsareabletomaintainthisconstitutiveAkt-S473insuspensiononlyifcellsformcell–cellcontactstoallowforgrowthfactorsignallingandinductionofZEB1.
However,Ras-TKOMEFsareabletomaintainconstitutiveAkt-S473assinglecellsinsuspension,becausemutantRasshortcircuitsthisgrowthfactorsignalling,therebymaintainingZEB1andthusmTor.
Rb1familymutationissufcientforsarcomainitiation.
Next,wewonderedhowtheeffectsonanoikisthatwedescribedaboveinsuspensionculturemightrelatetotumourformation.
Beyond706050%Trypanblue+cells40302010MEFsRas-TKOTKOActbAkt3Akt2Akt1siAktsiAktsiAktsiAktMEFsTKOTKO-Ras60kDa60kDa60kDa42kDaCCCCCsiAkt1siAkt2siAkt3siAktns0siControlsiControlsiControlsiAktsiAktsiAktsiAkt1siAkt2siAkt3abFigure4|TKOandRas-TKOMEFsbecomeaddictedtoAkt.
(a)WesternblottingsshowingtheeffectsofsmallinterferingRNA(siRNA)onthelevelofAktisoforms.
'C'indicatesascrambledcontrolsiRNA.
siAktindicatesapansiRNAthattargetsallthreeisoforms.
'ns'indicatesanonspecicband.
(b)EffectofAktisoformknockdownonviabilityinmonolayerculture.
Cellswereanalysed3daysaftertransfectionforviabilitybytrypanblueexclusion.
Errorbarsrepresents.
d.
ofthreereplicates.
Allresultsarerepresentativeofatleastthreeindependentexperiments.
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theirroleincarcinomas(forexample,pancreaticandlungcarcinomas),mutationsintheRaspathwayarealsoimportantinprogressionofsarcoma28.
WhenRas-TKOMEFsweretrypsinizedtosinglecellsandinjectedintonudemice,thecellsweretumorigenic(Table1).
Tumourswereallhistologicallysimilarspindle-cellsarcomasandwerehighlyinvasiveintothesurroundingsofttissues(Fig.
6).
Bycontrast,TKOMEFswerenottumorigenic(Table1),demonstratingdependenceonRasfortumourinitiationundertheseconditions.
MutationofRb1andp53leadstosarcoma29,30.
AlthoughRb1mutantMEFsarearrestedbyp53,activationofp53inTKOMEFsdoesnotleadtogrowtharrest5,6,demonstratingthatp53functionisdependentontheRb1family.
Accordingly,mutationofRb1andp107issufcientforsarcomainvivo31.
Yet,TKOMEFswerenottumorigenicinnudemice(Table1).
Onthebasisofourndingsinsuspensionculture,wereasonedthattrypsinizationofTKOMEFstosinglecellsuspensionsforinjectionintonudemicemightblockproliferationandinduceanoikis,therebypreventingtumourinitiation.
Therefore,insteadoftrypsinization,NEDTKOMEFswereinjectedintonudemicetoallowthepotentialforcell–cellcontactstoestablish.
Remarkably,NEDTKOMEFsformedtumours,andthetumoursizewassimilartothoseformedwithRas-TKOMEFs(Table1).
Furthermore,thetumourswereallspindle-cellsarcomassimilartothoseformedbyRas-TKOMEFs210kDaabc60kDa60kDaMEFMEFMEFMEF-mTorMEF-mTorMEF-mTorMEF-mTorMEF-mTorRas-TKOTKOmTorshRNATKOTKOTKOmTorshRNATKOmTorshRNA42kDaMonolayerMonolayerMonolayerMEFMEF-mTorTKOTKOmTorshRNAMonolayerTrypsinSSAkt473AktMEF10d86420mTorRelativeabundancee100mTorPromoter80604020197bp0InputZEB1H3IgGRelativeabundanceActbTKOTKOZEB1shRNARas-TKOZEB1shRNARas-TKO3TrypsinTrypsin10080%Viablecells6040200111133TKOTKOmTorshRNA2132TrypsinNEDNEDmTor12312TKOTKO3123Ras-TKOActbFigure5|ZEB1regulatesmTorexpressiontocontrolAkt-S473.
(a)WesternblottingsshowingtheexpressionofmTor.
Numbersindicatedaysinsuspensionculture.
Theleft-handtwolaneshavevetimesmoreproteinaddedsothattherelativelevelofmTorcanbecomparedinwild-typeandTKOmTorshRNAMEFs.
MEF-mTorindicatesMEFswheremTorisoverexpressed.
(b)WesternblottingsshowingthatthelevelofmTorcorrelateswiththatofAkt-S473.
However,Akt-S473isstilldependentonserumgrowthfactorsinMEF-mTorcells(SS),anditislostwhensingletrypsinizedMEF-mTorcellsareculturedinsuspensionfor3days(3).
(c)KnockdownofmTorinNEDTKOMEFstoalevelsimilartothatinwild-typeMEFsrestoresanoikis.
Viabilityismeasuredbytrypanblueexclusion.
Numbersindicatedaysinsuspension.
(d)Real-timePCRanalysisshowingthatmTormRNAisdependentonZEB1.
SimilartoZEB1,mTorisinducedinTKOMEFsanditisfurtherinducedinRas-TKOMEFs,andknockdownofZEB1diminishesmTorexpression.
(e)ChIPassayshowingZEB1andhistoneH3(positivecontrol)bindingtothemTorpromoterinvivoinTKOMEFs.
IgGindicatespre-immunerabbitantiserumasacontrolforZEB1antiserum.
InputindicatestotalgenomicDNAusedforimmunoprecipitationintheassays.
Real-timePCRwasusedtoquantifyabundance,andagelofPCRproductsisshownbelow.
Errorbarsrepresents.
d.
ofthreereplicates.
Allresultsarerepresentativeofatleastthreeindependentexperiments.
Table1|MutationoftheRb1familyinMEFsissufcientforsarcomainitiationinnudemiceandRasmutationisrequiredforinvasionofthesetumours.
CelltypeTumourformationTumourmassTumourtypeRas-TKO(trypsin)6/6882212Spindle-cellsarcoma(invasive)Ras-TKO(NED)6/669175Spindle-cellsarcoma(invasive)TKO(trypsin)0/6——TKO(NED)6/6582107Spindle-cellsarcoma(non-invasive)TKOZEB1shRNA(trypsin)0/6——TKOZEB1shRNA(NED)0/6——TKOmTorshRNA(trypsin)0/6——TKOmTorshRNA(NED)0/6——MEF,mouseembryonicbroblast;mTor,mammaliantargetofrapamycin;NED,non-enzymaticallydissociated;shRNA,shorthairpinRNA;TKO,tripleknockout.
Cells(50,000)weretrypsinizedorsubjectedtoNEDbeforeinjectionintonudemice.
Tumourswerecollectedafter31days.
Averagetumourmassingramsisshownalongwiths.
d.
SeeFig.
6forrepresentativetumourhistology.
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(Fig.
6).
AsimilarinjectionofNEDTKOZEB1shRNAMEFsdidnotleadtotumourformation(Table1),demonstratingthatinductionofZEB1intheTKOMEFsisrequiredfortumourinitiation.
Likewise,NEDTKOmTorshRNAMEFsdidnotformtumours(Table1).
Takentogether,ourresultsprovideevidencethatRb1familymutationinMEFsissufcientforsarcomaformationinnudemicewhencell–cellcontactsaremaintained.
Rasmutationisrequiredfortumourinvasion.
AlthoughthetumoursformedwithRas-TKOMEFsandNEDTKOMEFsweresimilarinsizeandhistology,Ras-TKOMEFtumourswerehighlyinvasiveintothesurroundingsofttissues,whereasthoseformedwithNEDTKOMEFswereallnon-invasive(Table1andFig.
6).
TheseresultsprovideevidencethatmutantRasintheTKOMEFsisrequiredforthetumourcellstotransitiontoaninvasivephenotype.
Next,weimmunostainedTKONEDandRas-TKOtumoursfortheproliferationmarkerKi67andAkt-S473.
BothtumoursshowedKi67andAkt-S473cellsthroughoutthetumour,buttherewasaslightlyhigheroverallproliferationrateintheRastumours(Fig.
7a,b).
AsTKONEDtumourswerenon-invasive,wewonderedwhethercellsattheedgeofthetumourmightshowdiminishedKi67andAkt-S473astheydetachedfromthetumour,causingcelldeathandtherebypreventinginvasion.
Asanticipated,wefoundthatinvadingRas-TKOcancercellsexpressedbothAkt-S473andKi67,butTKONEDtumoursalsoexpressedAkt-S473andKi67atthetumouredge(Fig.
7a).
Oneinterpretationisthatsimilartothatinsuspensionculture,cellsthatleavethetumourdierapidlyandthustheyarenotdetected.
Nevertheless,theTKOtumourslackinvadingcells.
DiscussionWeproposethattrypsinizingandsuspendingcellsinsuspensionbeforeinjectionplacesanarticialanoikisandproliferativebarrierontumorigenesi1sinnudemicebydisruptingthepotentialforcell–cellcontacts,which,wedemonstrate,arecriticalfortumourinitiationwithsomecells.
DifferencesinsarcomainitiationandinvasioninourexperimentswithTKOandRas-TKOMEFsaremirroredbypropertiesofthetwocellsinAkt-473DAPI353025%Ki67+cells20151050TKONEDRas-TKOTKONEDAkt-473Akt-473Ki67Ki67DAPIDAPIDAPIMMMMMMMMMMMMMMMMMMAdiposeDAPIKi67DAPIAdiposeAdiposeRas-TKOabFigure7|ProliferationandAkt-S473inTKONEDandRas-TKOtumours.
(a)ImmunostainingforKi67andAkt-S473inTKONEDandRas-TKOtumoursinFig.
6.
Arrowsindicatethetumourborderand'M'indicateshostmuscletissue.
(b)ThepercentageofKi67cellswascountedinfourdifferentregionsofthreedifferenttumours.
Scalebars,20mM.
Errorbarsrepresents.
d.
ofthreereplicates.
Ras-TKOMEFsTKOMEFsNEDTKOMEFsNEDFigure6|MutantRasisrequiredforinvasionofNEDTKOMEFsarcomasinnudemice.
Tumour-hostbordersareshownaboveandhigherpowerviewsofthetumourinteriorisshownbelow.
NotetheinvasionofRas-TKOMEFsarcomasintohostskeletalmuscleandadiposetissue,andthelackofcorrespondinginvasionofNEDTKOMEFsarcomasintothehosttissues.
TwodifferentsectionsofNEDTKOMEFtumoursareshowntoillustratethelackininvasionintohostskeletalmuscleandadiposetissue.
TheborderofNEDTKOMEFtumoursisshownbyyellowarrows.
Scalebars,20mM.
TumourhistologyisrepresentativeofresultsinTable1.
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suspensionculture(Fig.
8a–c).
AlthoughTKOMEFssurvivewhentheyformcell–cellcontactsinsuspension,whichweputforthasanalogoustoaprimarytumour,cellsthatdonotformthesecontactsundergorapidanoikis.
Wesuggestthatthesenon-associatedcellsarerepresentativeofpotentiallyinvasivecellsthatshedfromtheprimarytumour.
InthecaseofRas-TKOMEFs,RasinductionofZEB1,upregulationofmTorandresultingconstitutiveAkt-S473allowsthesecellstosurviveandinvadesurroundingtissues.
Incontrast,dependenceofTKOMEFsoncell–cellcontactswithintheprimarytumourtomaintainZEB1,mTorandAkt-S473leadstoanoikisofcellswhentheyareshedfromthetumour,therebyblockinginvasion.
Thereiscontroversyregardingtheoriginofcancerstemcells/cancer-initiatingcellsintumours.
However,wehavedemon-stratedpreviouslythatcellswithcancerstemcellpropertiescanbeinducedfromthepopulationofcancercells,andthisinductionrequiredZEB1(ref.
32).
Recently,Weinbergandcolleagues33publishedsimilarndings,demonstratingthatZEB1modulatesareversibletransitionofexistingbreastcancercellstocancerstemcells.
Thereismountingevidencethatearlyinvasive/metastaticcellsinpancreaticandbreastcancerhaveinducedZEB1andshowpropertiesofcancerstemcells33,34.
Remarkably,inRas-inducedpancreaticcancer,invasive/metastaticZEB1cellswithcancerstemcellpropertieswerealreadyevidentatthestageofprecancerousprostaticintraepithelialneoplasia(PIN)lesionbeforeafranktumourhaddeveloped34.
Together,thesestudiesprovideevidencethatinatleastsomecancers,cancerstemcellscanarisefromtheexistingcancercellpopulationthroughinductionofZEB1,andtheylinkthesecancerstemcellstoearlyinvasion/metastasis.
MethodsCellculture.
Rb1familymutantMEFsfromfourseparateembryoswereobtainedfromSageetal.
6andusedwithsimilarresults.
CellswereculturedinDMEMwith10%heat-inactivatedfetalbovineserum14.
Ras-TKOMEFswerecreatedbyinfectionwithhumanH-rasV12mutantretrovirus(agiftfromDrRMitchell,UniversityofLouisville,Louisville,KY,USA).
RasactivitywasassessedusingtheEZ-DetectRasActivationKit(PierceBiotechnology,Rockford,IL)andglutathioneS-transferasefusionproteincontainingtheRas-bindingdomainofRaf14.
Tumourformationinnudemice.
MousestudieshavebeenapprovedbytheUniversityInstitutionalAnimalCareandUseCommittee.
Cellsweretrypsinized,pipettedwithaP200pipetman,ortreatedwithcelldissociationbufferobtainedfromInvitrogen(Carlsbad,CA),togeneratesinglecellsinsuspension.
Cellsinsuspensionwereinjectedsubcutaneouslyintothehindlimbofnudemice31.
Tumourswerexedin10%bufferedformalin,embeddedinparafnandsectionedforhaematoxylinandeosinstainingorimmunostaining.
Real-timePCRandChIPassays.
RNAwasextractedusingTRIzol(Invitrogen).
ComplementaryDNAwassynthesizedusingtheInvitrogenRTKitaccordingtothemanufacturer'sprotocol.
SYBRGreenreal-timequantitativePCRwasper-formedusingtheMx3000PReal-TimePCRSystem(Stratagene,CedarCreek,TX)accordingtothemanufacturer'sinstructions.
PCRprimersareshowninTable2.
Threeindependentsamples,eachintriplicate,wereanalysedforeachreal-timePCRcondition,andproductswereanalysedforsizebyagarosegel.
ChIPassayswerebasedontheUpstateBiotechnologyprotocol(http://www.
millipore.
com/userguides/tech1/789mrn)usingformaldehydetocross-linkgenomicDNA.
Poly-clonalantiserumforZEB1(ref.
35),histoneH3andhistoneH4(SantaCruzBiotechnology,Inc.
,SantaCruz,CA)wereusedforimmunoprecipitation.
Equalamountsofanti-IgGorpre-immuneserumwereusedasnegativecontrols,whereasTKOMEFtrypsinacbTKOMEFNEDRas-TKOMEFtrypsinTumourNon-invasivePrimarytumoursTumourInvasionProliferationAnoikisZEB1mTorAkt-473ProliferationAnoikisZEB1mTorAkt-473ProliferationAnoikisZEB1mTorAkt-473ProliferationAnoikisZEB1mTorAkt-473ProliferationAnoikisZEB1mTorAkt-473ProliferationAnoikisZEB1mTorAkt-473NotumourFigure8|Trypsinizingcellsinsuspensionbeforeinjectionintonudemiceplacesanarticialproliferationandanoikisbarrierontumourinitiation.
(a)WhenTKOMEFsaretrypsinizedandplacedinsuspension,thesinglecellsarenolongergrowthfactorresponsive,leadingtodownregulationofZEB1and,inturn,lossofAkt-S473.
Thesecellsstopproliferationandundergoanoikis,preventingthemfromformingtumourswheninjectedintonudemice.
(b)Maintainingcell–cellcontactsinTKOMEFsinsuspension(TKOMEFNED)allowsgrowthfactorsignallingtomaintainZEB1andthusconstitutiveAkt-S473,andthecellscontinuetoproliferateandareresistanttoanoikis.
Thesecellsformsarcomaswheninjectedintonudemice,andasinsuspensioncultureweproposethatcell–cellcontactsbetweenTKOMEFswithintheprimarytumourallowgrowthfactorsignallingtomaintainelevatedZEB1andconstitutiveAkt-S473.
However,aswithsinglecellsinsuspensionculture,TKOMEFsthatabandontheprimarytumour(redcircles)losegrowthfactorsignalling,leadingtolossofZEB1andinturnAkt-S473.
Thesecellsthenstopproliferationandundergoanoikis,preventingtheirinvasion.
(c)MutantRasshortcircuitsgrowthfactorsignalling,leadingtoretentionofZEB1andthusconstitutiveAkt-S473whenRas-TKOMEFsaretrypsinizedandplacedinsuspensionculture.
Thesecellscontinueproliferationandareresistanttoanoikis.
Theyformtumoursintumourmiceand,similartothatinsuspensionculture,cellsthatdissociatefromtheprimarytumour(greencircles)maintainZEB1andAkt-S473;thus,theycontinueproliferatingandareresistanttoanoikis,allowingthemtoinvadesurroundingtissues.
Theseresultsplaceanoikisasacriticalhurdlefortransitiontotumourinvasion,asopposedtotumourinitiation.
WesuggestthatRasmutationactsatthisstepoftumourinvasionbyblockinganoikisofcellsthatabandontheprimarytumour.
Moreover,thiseffectofRasismediatedbyitsabilitytomaintainZEB1expressionundertheseconditions,whichinturncausesconstitutiveAkt-S473.
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histoneH4servedasapositivecontrol.
ChIPPCRreactionsweresimilartothosedescribedaboveforreal-timePCRusingprimersets(Table2)toamplifypromotersequencesofmTor.
Geneknockdowns.
TogeneratealentivirusexpressingZEB1ormTORshRNA14,32,aprimercontainingaZeb1shRNAsequence(50-CTGTCTAGACAAAAAAAGACAACGTGAAAGACAATCTCTTGAATTGTCAAACACGTTGTCTTGGGGATCTGTGGTCTCATACA-30)oranmTORshRNAsequence(50-CTGTCTAGACAAAAGAGATGGAGGAGATCACCCTCTCTTGAAGGGTGATCTCCTCCATCTCGGGGATCTGTGGTCTCATACA-30)wasusedwithaT3primertoamplifya500-bpfragmentcontainingtheH1promoterusingthepSupervectorasatemplate.
TheresultingPCRproductwasdigestedwithSpeIandXbaI,andclonedbackintothelentiviralvectordigestedwithNheI.
WerstclonedtheshRNAsequenceintoacytomegalovirus–greenuorescentproteinlentiviralvec-tor,whereitsexpressionwasdrivenbythemouseU6promoter32.
Briey,theshRNAconstructwasgeneratedbysynthesizingan83-meroligonucleotidecontainingthefollowing:a19-nucleotidesensestrandanda19-nucleotideantisensestrand,separatedbyanine-nucleotideloop(50-TTCAAGAGA-30);astretchofveadeninesasatemplateforthePolIIIpromoterterminationsignal;21nucleotidescomplementarytothe30-endofthePolIIIU6promoter;anda50-endcontainingauniqueXbaIrestrictionsite.
Thelongoligonucleotidewasused,togetherwithasp6oligonucleotide(50-ATTTAGGTGACACTATAGAAT-30),toPCR-amplifyafragmentcontainingtheentireU6promoterplusshRNAsequences;theresultantproductwasdigestedwithXbaIandSpeIligatedintotheNheIofthelentivirusvector,andtheinsertwassequencedtoensurethatnoerrorsoccurredduringthePCRorcloningsteps.
Lentiviralparticleswereproducedbyafour-plasmidtransfectionsystem32.
Briey,293Tcellsweretransfectedwiththelentiviralvectorandpackagingplasmids,andthesupernatants,containingrecombinantpseudolentiviralparticles,werecollectedfromculturedishesonthesecondandthirddaysaftertransfection.
TKOMEFsandRas-TKOMEFsweretransducedwiththeselentiviralparticlesexpressingshRNAstargetingZEB1.
Atransductionefciencyofnear100%wasachievedbasedongreenuorescentprotein-positivecells.
BeyondtheseoriginalshRNAs,weusedveadditionalshRNAlentiviruseswithdifferentshRNAsequencesobtainedfromOpenBiosystems,forknockdownswithsimilareffects.
LentiviruswithascrambledshRNAsequence50-CAACAAGATGAAGAGCACCAATCTCTTGAATTGGTGCTCTTCATCTTGTTG-30wasusedasacontrol—thiscontrolsequencewasblastedagainstallmouseRNAsequencestoensurethatitdidnottargetamessengerRNA.
MouseAktisoformsmallinterferingRNAswerefromOriGene.
Immunostaining.
CellswerewashedwithPBS,xedwith4%formaldehydefor10minandthenwashedagainwithPBS.
Thecellswerethentreatedwithmethanolat20°Cfor10minandblockedwith4%goatserumatroomtemperaturefor1h.
Cellswereincubatedwithprimaryantibodyovernightat4°C.
Thefollowingprimaryantibodieswereused:rabbitanti-phospho-Akt(Ser473)polyclonalantibody(1:10;CellSignalingTechnology,Inc.
;Boston,MA),rabbitanti-Foxo3apolyclonalantibody(1:10;SantaCruzBiotechnology,Inc.
),rabbitanti-mTorpolyclonalantibody(H-266)(1:10;SantaCruzBiotechnology,Inc.
)andrabbitanti-Ki67polyclonalantibody(1:20;NovusBiologicals).
Thenextday,cellswerewashedwithPBSfollowedbyincubationwithsecondaryantibody(AlexaFluorgoatanti-rabbitIgG;Invitrogen)for1hatroomtemperature.
Imageswerecap-turedwithaZeissconfocalmicroscope.
Westernblotting.
CellextractswerepreparedbywashingwithcoldPBSandadding100mloflysisbuffer(PierceBiotechnology)withproteaseinhibitorcocktail(Sigma-Aldrich,SaintLouis,MO),followedbyhigh-speedcentrifugation.
ProteinconcentrationwasassayedbytheBradfordMethod(Bio-RadLaboratories,Richmond,CA).
Fortymicrogramsofproteinfromeachsamplewassubjectedtoelectrophoresisona4–20%gradientSDS–polyacrylamidegel(Bio-RadLabora-tories)andtransferredtonitrocellulosemembrane(GEHealthcareBiosciences,Piscataway,NJ).
Membraneswereblockedfor1hin5%powderedmilkand0.
1%Tween20,andthenincubatedovernightwithprimaryantibody,followedbya1-hincubationwithhorseradishperoxidase-conjugatedsecondaryantibody(PierceBiotechnology),anddetectionwasdonewithenhancedchemiluminescence(Per-kinElmer,Waltham,MA).
Thefollowingantibodieswereused:rabbitanti-phospho-Akt(Ser473)polyclonalantibody(1:1,000;CellSignalingTechnology,Inc.
),rabbitanti-phospho-Akt(Thr308)polyclonalantibody(1:1,000;SantaCruzBiotechnology,Inc.
),rabbitanti-Aktpolyclonalantibody(1:1,000;SantaCruzBiotechnology,Inc.
),rabbitanti-FRAPpolyclonalantibody(1:1,000;SantaCruzBiotechnology,Inc.
),rabbitanti-phospho-Raf-1(Ser259)polyclonalantibody(1:1,000;SantaCruzBiotechnology,Inc.
)andanti-ZEB1(1:1000;agiftfromDouglasSDarling)35.
References1.
Knudson,A.
G.
Mutationandcancer:statisticalstudyofretinoblastoma.
Proc.
NatlAcad.
Sci.
USA68,820–823(1971).
2.
Alderton,G.
K.
Chromosomeinstability:RBmoonlightsinmitosis.
Nat.
Rev.
Cancer10,536(2010).
3.
Zhang,J.
etal.
Anovelretinoblastomatherapyfromgenomicandepigeneticanalyses.
Nature481,329–334(2012).
4.
Harbour,J.
W.
&Dean,D.
C.
TheRb/E2Fpathway:expandingrolesandemergingparadigms.
GenesDev.
19,2393–2409(2000).
5.
Dannenberg,J.
H.
,vanRossum,L.
,Schuijff,L.
&teRiele,H.
AblationoftheretinoblastomagenefamilyderegulatesG1controlcausingimmortilizationandincreasedcellturnoverundergrowth-restrictingconditions.
GenesDev.
14,3051–3064(2000).
6.
Sage,J.
etal.
TargeteddisruptionofthethreeRb-relatedgenesleadstolossofG(1)controlandimmortilization.
GenesDev.
14,3037–3050(2000).
7.
Taddei,M.
,Giannoni,E.
,Fiaschi,T.
&Chiarugi,P.
Anoikis:anemerginghallmarkinhealthanddiseases.
J.
Pathol.
226,380–393(2012).
8.
Klein,E.
A.
,Yung,Y.
,Castagnino,P.
,Kothapalli,D.
&Assoian,R.
K.
Celladhesion,cellulartension,andcellcyclecontrol.
MethodsEnzymol.
426,155–176(2007).
9.
Mitin,N.
,Rossman,K.
L.
&Der,C.
J.
SignalinginterplayinRassuperfamilyfunction.
Curr.
Biol.
15,563–574(2005).
10.
Buday,L.
&Downward,J.
ManyfacesofRasactivation.
Biochem.
Biophys.
Acta1786,178–187(2008).
11.
Hay,N.
InterplaybetweenFoxo,Tor,andAkt.
Biochem.
Biophys.
Acta1813,1965–1970(2011).
12.
Horbinski,C.
,Mojesky,C.
&Kyprianou,N.
Livefreeordie:talesofhomeless(cells)incancer.
Am.
J.
Pathol.
177,1044–1052(2010).
13.
Oh,W.
J.
&Jacinto,E.
mTORcomplex2signalingandfunctions.
CellCycle10,2305–2316(2011).
14.
El-Naggar,S.
,Liu,Y.
&Dean,D.
C.
MutationoftheRb1pathwayleadstooverexpressionofmTor,constitutivephosphorylationofAktonserine473,resistancetoanoikisandablockinc-Rafactivation.
Mol.
Cell.
Biol.
29,5710–5717(2009).
15.
Brabletz.
,S.
&Brabletz,T.
TheZEB/miR-200feedbackloop--amotorofcellularplasticityindevelopmentandcancerEMBORep.
11,670–677(2010).
16.
Singh,A.
etal.
Ageneexpressionsignatureassociatedwith''K-Rasaddiction''revealsregulatorsofEMTandsurvival.
CancerCell15,489–500(2009).
17.
Smit,M.
A.
&Peeper,D.
S.
Zeb1isrequiredfortrkB-inducedepithelial-mesenchymaltransition,anoikisandmetastasis.
Oncogene30,3735–3744(2011).
18.
Liu,Y.
etal.
Thezincngertranscriptionfactor,ZFHX1A,islinkedtocellproliferationbyRb/E2F1.
Biochem.
J.
408,79–85(2007).
19.
Spassov,D.
S.
etal.
Trasklossenhancestumorigenicgrowthbyliberatingintegrinsignalingandgrowthfactorreceptorcross-talkinunanchoredcells.
CancerRes.
73,1168–1179(2013).
20.
Carloni,V.
etal.
Celladhesionregulatesplatelet-derivedgrowthfactor-inducedMAPkinaseandPI-3kinaseactivationinstellatecells.
Hepatology36,582–591(2002).
Table2|PCRprimersusedinthisstudy.
PrimernameSequenceTemperature(°C)Amplicon(bp)Primersforreal-timePCRMmZeb1LP50-TGGCAAGACAACGTGAAAGA-3060200MmZeb1RP50-AACTGGGAAAATGCATCTGG-3060MmmTORLP50-ACTGAGGAGGGAGAACAGCA-3057.
9153MmmTORRP50-TGGCTCCATCTGCTAGTGTG-3056.
8MmACTBLP50-GGCTGTATTCCCCTCCATCG-3057.
6154MmACTBRP50-CCAGTTGGTAACAATGCCATGT-3055.
9MmGAPDHLP50-AGGTCGGTGTGAACGGATTTG-3057.
6123MmGAPDHRP50-TGTAGACCATGTAGTTGAGGTCA-3055.
1PrimersforChIPassaysmTORPRMTLP50-ggatgttcctccccatcttc-3055197mTORPRMTRP50-actccaggccccagactc-3059.
2ChIP,chromatinimmunoprecipitation.
NATURECOMMUNICATIONS|DOI:10.
1038/ncomms3650ARTICLENATURECOMMUNICATIONS|4:2650|DOI:10.
1038/ncomms3650|www.
nature.
com/naturecommunications9&2013MacmillanPublishersLimited.
Allrightsreserved.
21.
Morozevich,G.
E.
etal.
Integrinalpha5beta1simultaneouslycontrolsEGFR-dependentproliferationandAkt-dependentpro-survivalsignalinginepidermoidcarcinomacells.
Aging4,368–374(2010).
22.
Zeng,Z.
Z.
etal.
Roleforfocaladhesionkinaseandphosphatidylinositol3'-kinaseinintegrinbronectinreceptor-mediated,matrixmetalloproteinase-1-dependentinvasionbymetastaticprostatecancercells.
CancerRes.
66,8091–8099(2006).
23.
Walker,J.
L.
,Fournier,A.
K.
&Assoian,R.
K.
Regulationofgrowthfactorsignalingandcellcycleprogressionbycelladhesionandadhesion-dependentchangesincellulartension.
CytokineGrowthFactorRev.
16,395–405(2005).
24.
Xia,H.
etal.
Focaladhesionkinaseisupstreamofphosphatidylinositol3'-kinase/Aktinregulatingbroblastsurvivalinresponsetocontractionoftype1collagenmatrixesviabeta1integrinviabilitysignalingpathway.
J.
Biol.
Chem.
279,33024–33034(2004).
25.
Illopoulos,D.
etal.
MicroRNAsdifferentiallyregulatedbyAktisoformscontrolEMTandstemcellrenewalincancercells.
Sci.
Signal.
2,ra62(2009).
26.
Zinzalla,V.
,Stracka,D.
,Oppliger,W.
&Hall,W.
W.
ActivationofmTORC2byassociationwiththeribosome.
Cell144,757–768(2011).
27.
Postigo,A.
A.
OpposingfunctionsofZEBproteinsintheregulationoftheTGFb/BMPsignalingpathway.
EMBOJ.
22,2443–2452(2003).
28.
Young,N.
P.
,Crowley,D.
&Jacks,T.
Uncouplingcancermutationsrevealscriticaltimingofp53lossinsarcomagenesis.
Cancer.
Res.
71,4040–4047(2011).
29.
Berman,S.
D.
etal.
MetastaticosteosarcomainducedbyinactivationofRbandp53intheosteoblastlineage.
Proc.
NatlAcad.
Sci.
USA105,11851–11856(2008).
30.
Walkley,C.
R.
etal.
Conditionalmouseosteosarcoma,dependentonp53lossandpotentiatedbylossofRb,mimicsthehumandisease.
GenesDev.
22,1662–1676(2008).
31.
Dannenberg,J.
H.
etal.
Tissue-specictumorsuppressoractivityofretinoblastomagenehomologsp107andp130.
GenesDev.
18,2952–2962(2004).
32.
Liu,Y.
etal.
MousebroblastslackingRB1functionformspheresandundergoreprogrammingtoacancerstemcellphenotype.
CellStemCell4,336–347(2010).
33.
Chaffer,C.
L.
etal.
PoisedchromatinattheZEB1promoterenablesbreastcancercellplasticityandenhancestumorigenicity.
Cell154,61–74(2013).
34.
Rhim,A.
D.
etal.
EMTanddisseminationprecedepancreatictumorformation.
Cell148,349–361(2012).
35.
Liu,Y.
,El-Naggar,S.
,Darling,D.
S.
,Higashi,Y.
&Dean,D.
C.
ZEB1linksepithelial-mesenchymaltransitionandcellularsenescence.
Development135,579–588(2008).
AcknowledgementsWethankT.
JacksforRb1familymutantandwild-typeMEFs,andGuirongLiuforhistologicalsections.
ThesestudiesweresupportedbygrantsfromtheNIHtoD.
C.
D.
(EY019113andEY018603);anInstitutionalDevelopmentAward(IDeA)fromtheNationalInstituteofGeneralMedicalSciences(P20GM103453)toY.
L.
;NIHCoreGrantEY015636andFOT,AVON-SAU,AECC,ACMCB-BRB-2013andMICINN-BFU2010-15163toA.
P.
AuthorcontributionsY.
L.
performedmostoftheexperiments,preparedguresandwrotethemanuscript.
E.
S.
-T.
performedexperimentsonZEB1expression.
X.
L.
performedimmunostaining.
B.
C.
andS.
T.
performedthenudemousetumourformationassays.
A.
B.
J.
andM.
C.
providedpathologyofthemousetumours.
J.
C.
andA.
P.
plannedexperimentsandcommentedonthemanuscript.
D.
C.
D.
plannedtheexperimentsandwrotethemanuscript.
AdditionalinformationCompetingnancialinterests:Theauthorsdeclarenocompetingnancialinterests.
Reprintsandpermissioninformationisavailableonlineathttp://npg.
nature.
com/reprintsandpermissions/Howtocitethisarticle:Liu,Y.
etal.
Rb1familymutationissufcientforsarcomainitiation.
Nat.
Commun.
4:2650doi:10.
1038/ncomms3650(2013).
ARTICLENATURECOMMUNICATIONS|DOI:10.
1038/ncomms365010NATURECOMMUNICATIONS|4:2650|DOI:10.
1038/ncomms3650|www.
nature.
com/naturecommunications&2013MacmillanPublishersLimited.
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