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REVIEWOpenAccess RacandRhoGTPasesincancercellmotility control MatteoParri 1 ,PaolaChiarugi 2* Abstract RhoGTPasesrepresentafamilyofsmallGTP-bindingproteinsinvolvedincellcytoskeletonorganization,migration, transcription,andproliferation.Acommonthemeoftheseprocessesisadynamicreorganizationofactincytoske- letonwhichhasnowemergedasamajorswitchcontrolmainlycarriedoutbyRhoandRacGTPasesubfamilies, playinganacknowledgedroleinadaptationofcellmotilitytothemicroenvironment.Cellsexhibitthreedistinct whichmaintaintheadherensjunctionsandmovebyphotolyticdegradationofmatrixbarriers.Singlecell mesenchymal-typemovementischaracterizedbyanelongatedcellularshapeandagainrequiresextracellularpro- teolysisandintegrinengagement.InadditionitdependsonRac1-mediatedcellpolarizationandlamellipodiafor- mation.Conversely,inamoeboidmovementcellshavearoundedmorphology,themovementisindependent fromproteasesbutrequireshighRhoGTPasetodriveelevatedlevelsofactomyosincontractility.Thesetwomodes ofcellmovementareinterconvertibleandseveralmovingcells,includingtumorcells,showanhighdegreeof plasticityinmotilitystylesshifting adhoc betweenmesenchymaloramoeboidmovements.Thisreviewwillfocus ontheroleofRacandRhosmallGTPasesincellmotilityandinthecomplexrelationshipdrivingthereciprocal controlbetweenRacandRhograntingfortheopportunisticmotilebehaviourofaggressivecancercells.Inaddi- tionweanalysetheroleoftheseGTPasesincancerprogressionandmetastaticdissemination. Review RhoandRacGTPases RhoproteinsbelongtotheRassuperfamily.Theyare small(21-25kDa)moleculesthatsharestructuralhomo- logyandbecomeactivatedonlywhenboundtoGTP. Thebest-characterizedmoleculesareRho,whichcon- RacandCdc42,whichregulatemembraneruffling,and filopodiumformation,respectively.Astructuralfeature thatdistinguishestheRhoproteinsfromothersmall GTPasesistheso-calledR hoinsertdomainlocated betweena b strandandan a helixwithinthesmall GTPasedomain[1-3].TypicallyRhoproteinsare190- 250residueslongandconsistonlyoftheGTPasedomain andshortterminalC-terminalextensions.Withintheir GTPasedomains,theyshareapproximately30%amino acididentitywiththeRasproteinsand40-95%identity withinthefamily.Allmemberscontainthesequence motifscharacteristicofallGTP-bindingproteins,bindto GDPandGTPwithhighaffinity.Inaddition,themajor- ityofmembersundergoC-terminalpost-translational modificationbyisoprenoidlipids.Togetherwithother C-terminalmodificationsors equences,isoprenoidaddi- tionfacilitatestheirsubcellularlocationandassociation withspecificmembranesororganelles.Theselipidmodi- ficationsaremainlypalmitoylationorprenylations,being post-translationmodifications[4]. RhoGTPasesworkassensitivemolecularswitches existingeitherinaninactive,GDP-boundformoran activeGTP-boundform.TheyareendowedwithGTP hydrolyticactivity,mainlyinvolvedincytoskeletonrear- rangementsandcellmotility,butalsoinvolvedincell proliferation,transformati onanddifferentiation[2]. Amongothermembers,wewillfocusourattentionon theRacandRhosubfamilies,astheyarethemaineffec- torsofcellmotility. TheexchangeofGDPtoGTPandthustheactivation ofRhoGTPasesiscatalyzedbyguaninenucleotide *Correspondence:paola.chiarugi@unifi.it 2 DepartmentofBiochemicalSciences,UniversityofFlorence,TuscanyTumor Instituteand “ CenterforResearch,TransferandHighEducationDenoTHE ” , 50134Florence,Italy Fulllistofauthorinformationisavailableattheendofthearticle ParriandChiarugi CellCommunicationandSignaling 2010, 8 :23 http://www.biosignaling.com/content/8/1/23 ©2010ParriandChiarugi;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited. exchangefactors(GEFs),whichactdownstreamofnumerousgrowthfactorreceptors,integrins,cytokinereceptors,andcadherins.RhoGTPasesarekeyintegrat-ingmoleculesfromdifferentextracellularsignals,astheycanbeactivatedbydifferentGEFs.Inturn,GTP-boundactiveGTPasescaninteractwithaplethoraofdifferenteffectorswhichmediatethedifferentcellularfunctionsofthisfamilyofproteins.RhoGTPaseeffec-torsarealargegroupofproteinsandincludeactinnucleationpromotingmolecules,adaptors,aswellaskinases.TwofactorsconcurtodeterminespecificRhoGTPasefunction:tissuespecificityofGTPaseeffectorsanddistinctintracellularlocalizationsofcloselyrelatedRhoGTPases,duetodifferentlipidmodifications[1].TheGEFfamilyisreallylarge,consistingofover70pro-teinsmainlybelongingtotheDblortheDockfamilies[5,6].LipidmodificationofRhoandRacGTPasesarealsostrategicforsubcellularcompartmentalization,allowinginteractionwithmembrane-localisedGEFsuponmaskingofisoprenoidsbyGDI.ThehydrolysisofGTPandcontactwithGAPsallowsanewassociationoftheGTPaseswithGDIandreturntothecytosol[7].Inaddition,RhoGTPasescanalsoberegulatedbyphos-phorylation.RhoAhasbeenreportedtobephosphory-latedbyproteinkinaseAandG(PKAandPKG)atserineatposition188,withoutanymodificationofitsinteractionwithGEFs,butincreasingitsinteractionwithGDIandleadingtoextractionofRhoAfromplas-mamembrane[8].InactivationofRhoGTPasesisduetoanintrinsicGTPaseactivity,whichhydrolysesGTPtoGDP.How-ever,thisactivityisveryweakandneedstobeup-regu-latedbyGTPaseactivatingenzymes(GAPs).Ofnote,Rnd1-3[9]andRhoH[10,11]arenotregulatedviaGAPs,duetotheirinabilitytohydrolyseGTP,andarethereforeregulatedthroughgeneexpressionandproteindegradation.AnadditionalnegativecontrolisachievedthroughRhoguaninenucleotidedissociationinhibitors(GDIs).TheybindRhoGTPasesandpreventtheiracti-vationbymeansofblockinginteractionoftheGTP-boundformwitheffectors,sequesteringGDP-boundRhoproteinsinthecytoplasmawayfromtheGDP-GTPcycle,aswellasbychangingmembranecompartmenttoGTPases[12].BesidetheGFfamily,theGAPgroupisalsohuge:moreorless100membershavebeenfoundinthehumangenome,buttheirregulationareevenlessclearthanthoseoftheGEFs.Indeed,externaltotheirGEForGAPdomains,theseproteinsstronglydivergeinstructureandsecondaryfunctions[6,13].TheRac-relatedsubfamilyincludesRac1(anditssplicevariantRac1b),Rac2andRac3[4].SeevaralRac-relatedproteins,sharingmorethan80%identity,theystimulatetheformationoflamellipodiaandmembraneruffles,presumablythroughinteractionwiththeWAVEcomplex[14].ThesplicevariantRac1bcontainsanadditionalC-terminal19-residueinsertandisconstitu-tivelyactiveduetoanincreasedintrinsicguaninenucleotideexchangerate,decreasedintrinsicGTPaseactivity,itsinabilitytointeractwithRhoGDIandenhancedassociationwiththeplasmamembrane[15,16].Inaddition,Rac1canalsoberegulatedbyphos-phorylationbyAktonSer71,therebyleadingtoinhibitthebindingofGTPbutnotRac1GTPaseactivity[17].Rac1isubiquitouslyexpressed,whereasRac2isexpressedonlyinhematopoieticcells,whereitseemstohavespecializedfunctions[18].Rac2inactivationhasbeencorrelatedwithseveralneutrophilic,phagocyticandlymphocyticdefects[19].Indeed,Rac2ismainlyresponsibleforactivationofNADPHoxidaseandconse-quentgenerationofreactiveoxygenspecies(ROS)inhematopoieticcells[20].FinallyRac3,highlyexpressedinbrainandupregulateduponserumstimulationoffibroblasts[21],isstronglylocalizedtothemembraneswhereitappearstobehyperactive[22].Animalshave3Rhoisoforms,RhoA,RhoB,andRhoC,sharing85%aminoacidsequenceidentity[1,6].Despitetheirsimilarity,bothmodulators(GEFsandGAPs)anddownstreameffectorsshowfavouredinteractionwithsingleRhoisoforms,andthethreeproteinsplaydiffer-entialrolesincells.RhoAandRhoCplaykeyrolesintheregulationofactomyosincontractilityandincelllocomotion,whileRhoB,primarilylocatedinendo-somes,hasbeenshowntoregulateintracellulartraffick-ingandcellsurvival[23].Mostly,thefunctionaldifferencesareaconsequenceofdivergenceintheirC-terminal15aminoacids,wherethehighestlevelofdifferenceisfound.MolecularmechanismofcellmigrationCellmigrationintridimensionalextracellularmatrix(ECM)isamultistepprocessinvolvingchangesinthecytoskeleton,cell-substrateadhesionsandtheextracellu-larmatrixcomponents.Cellmigrationisgenerallyinitiatedinresponsetoextracellularstimuli,whichcanbediffusiblefactors,signalsonneighboringcells,and/orsignalsfromtheextracellularmatrix.TheideathatRhofamilyGTPasescouldregulatecellmigrationderivesfromobservationsthattheymediatetheformationofspecificactincontainingstructures[24,25].Furthermore,Rhoproteinsregulateseveralotherprocessesrelevanttocellmigration,includingcell-substrateadhesion,cell-celladhesion,proteinsecretion,vesicletraffickingandCellmigrationinthree-dimensionalECMcanbesche-matizedintofiveseparatesteps[26](figure1):1.lamellipodiumextensionattheleadingedge2.formationofnewfocaladhesionscomplexesParriandChiarugiCellCommunicationandSignalinghttp://www.biosignaling.com/content/8/1/23Page2of14 3.secretionofsurfaceproteasetoECMcontactsand focalizedproteolysis 4.cellbodycontractionbyactomyosincomplexes 5.taildetachment Lamellipodiumextensionattheleadingedge involves actinpolymerization,anditisknownthatlamellipodia consistofbranchedorunbrancedfilamentnetworks formedthroughtheactin-nucleatingactivityofthe actin-relatedproteins2/3(Arp2/3)proteincomplex [27,28].Racstimulatesnewactinpolymerization,acting onArp2/3complex,whichbindstoafamilyofproteins callednucleatingpromotingfactors(fordetaileddescrip- tionofactinnucleationfactorsreferto[29,30])and initiatestheformationofn ewactinfilamentsonthe sidesofexistingfilamentstoformabranchingactinnet- work[27].TheArp2/3complexisactivatedbyRac throughitstargetinsulinreceptortyrosinekinasesub- stratep53(IRSp53)[31].RacinteractswithIRSp53, whichinturninteractsthroughanSrc-homologous domain3(SH3)domainwithamemberoftheWASP family,whichthenbindstoandactivatestheArp2/3 complex.Racisrequiredforlamellipodiumextension inducedbygrowthfactors,cytokinesandextracellular matrixcomponents[32].Racactivationbybothtyrosine kinasesandG-protein-coupledreceptorsisdependent onphosphoinositol3-kinase(PI3K)activity,andinhibi- torsofPI3KblockRacactivation[33].During lamellipodiaextensionphosphoinositolphosphates (PIPs)alsobindandactivateGEFsthatregulatethe activityofRacthatbindtheArp2/3complex[34]. Anumberofmyosins,themainmotorproteinineukar- yoticnon-musclecells,havebeenimplicatedincellmigra- tion[35].Myosinlightchain(MLC)phosphorylationis enhancedinthelamellipodi alregionofcells[36],which suggestsaroleformyosinsinlamellipodiumextension. Raccanaffectthephosphorylationofbothmyosinheavy chain(MHC)[37]andMLCviaactivationofitsdown- streamkinasep21activated-kinase(PAK)[38]. Formationofnewfocaladhesionscomplexes isloca- lizedinthelamellipodiaofmostmigratingcells.Upon theattachmentoftheextendinglamellipodiumtothe extracellularmatrix,integrinscomeintocontactwith ECMligandsandclusterinthecellmembraneinteract- ingwiththefocaladhesionkinase(FAK), a -actininand talin.Alltheseproteinscanbindadaptorproteins throughSH2,SH3orprolinerichdomainstorecruit actinbindingproteins(vinculin,paxillinand a -actinin) aswellasregulatorymoleculesPI3Ktofocalcomplexes [39,40].Racisrequiredforfocalcomplexassembly[41] andcelladhesiontotheextracellularmatrixitselfacti- vatesRac[42]. SecretionofsurfaceproteasetoECMcontactsand focalizedproteolysis iscrucialforcellstomigrateina three-dimensionalmatrixand,evenonatwo-dimen- sionalmatrix,proteaseproductioncanbeimportantfor Figure1 Cellmigrationin3Dmatrix .Seetextfordetailedexplanationofmotilitysteps. ParriandChiarugi CellCommunicationandSignaling 2010, 8 :23 http://www.biosignaling.com/content/8/1/23 Page3of14 migration[43].TherearesomeindicationsthatRhoGTPasescouldplayaroleinregulatingthesecretionand/oractivationofsecretedproteases.Forexample,Racisrequiredforshearstress-inducedmatrixmetallo-proteinase9(MMP9)expressioninchondrocytes[44],andactivatedRaccaninduceexpressionoftheMMP1infibroblasts[45].ConstitutiveexpressionofactivatedRacinducesactivationofJunN-terminalkinase(JNK),whichphosphorylatesandactivatesthetranscriptionfactorJun.Junisacomponentoftheactivatorprotein1(AP-1)transcriptionfactorcomplexandregulatestran-scriptionofmanygenes,includingMMPgenes[46].Furthermore,inHT1080cells,Rac1mediatesMMP2activationandmembranetypematrixmetalloproteinase(MT1-MMP)expression/processingduringtheencoun-terbetweeninvadingtumorcellsandtypeIcollagen-richstroma,therebyfacilitatingcollagenolysisandcellinvasion[47].Cellbodycontractionisdependentonactomyosincontractility.Thecontractionofactinfilamentsispro-videdbymyosinII.Stress-fiberassemblyandcontrac-tion,whicharecontrolledbymyosinII,arepredominantlyinducedbythesmallG-proteinRhoanditsimportantdownstreameffector,theRho-associatedserine/threoninekinase(ROCK).RhoactsviaROCKstoaffectMLCphosphorylation,bothbyinhibitingMLCphosphataseandbyphosphorylatingMLC[48].ItislikelythatROCKsandMLCKactinconcerttoregulatedifferentaspectsofcellcontractility,becauseROCKappearstoberequiredforMLCphosphorylationasso-ciatedwithactinfilamentsinthecellbody,whereasMLCKisrequiredatthecellperiphery[49].Thisallowsthecelltoseparatelycontrolcorticalactindynamicsfromcontractionsininnerregions.Taildetachmentoccurswhencell-substratelinkagesispreferentiallydisruptinthebackofthecell,whereastheleadingedgeremainsattachedtotheECMandfurtherelongates[50].Atthetrailingedge,focalcomplexdis-sassemblyoccursthroughseveralmechanismsdepen-dentonthetypeofcellandstrengthofadhesiontotheextracellularmatrix[51].Inslowlymovingcellstaildetachmentappearstodependontheactionofthepro-teasecalpain,whichcleavesfocalcomplexcomponentsliketalinandcytoplasmictailof1and3integrinsattherearofcells[52].AreductioninRhoactivitycouldinhibittaildetachment,throughdecreasedactomyosincontractility[53].DiversityoftumorinvasionmechanismsAcombinationofinvivoimagingand3Dinvitromod-elshaveshownthatcellscouldmoveusingdifferentmotilitystyles.Indeedcellscanmoveasindividualcellsorinsolidmulticellularcomponent.Single-cellmigra-tionincludesmesenchymalandamoeboidmigrationstrategies,whereascollectivemigrationisreferredtomulticellularstrands,sheets,clusterandcohorts.Differ-encesinextracellularproteaseactivities,integrin-mediatedcell-matrixadhesion,cadherin-mediatedcell-celladhesion,cellpolarityandcytoskeletalarrangementdefinethetypeofcellmigrationandinvasion.MesenchymalmotilityMesenchymalmotilityischaracterisedbyanelongated,fibroblast-like,cellmorphologywithestablishedcell-polarityandisdependent,uponproteolysis,tothedegradationoftheECM[54,55].Inthiskindofmotility,cellspeedisrelativelyslow(0.1-1m/min).Uponsev-eralstimuli,phosphatidylinositol(3,4,5)-triphosphate(PtdIns(3,4,5))isgeneratedattheleadingedgeofthecellandleadstocellpolarizationthroughactivationofthesmallGTPaseRac1,whichinturnorganizesactinpolymerizationandlamellipodiumformation[56,57].Activationofcelldivisioncontrolprotein42homolog(Cdc42)andtherecruitmentofadaptorproteinscanalsopromoteactinpolymerisation.ThedirectionalityofcellmovementismaintainedbyCdc42,whichcoordi-natesactinpolymerisationatthefrontofthecellwithmicrotubuleattachmentandalignment[57,58].Together,theseeventsleadtotheformationofanactin-richprotrusion.Aftertheextensionoftheprotru-sion,smallintegrin-dependentfocalcomplexesareformedthatattachthenewprotrusiontotheECM.SomefocalcomplexesthendevelopintolargefocaladhesionsthatenableactomyosincontractileforcetobetransmittedtotheECM[57].TheroleofRhoAanditseffectorsROCKinmesenchymalmotilityiscomplex;theiractivityneedstobereducedtoextendprotrusionsatthefrontofthecell[59],buttheypromotetheretrac-tionofthelaggingtail[57].Asaresult,theoveralleffectofinhibitingtheseproteinsinmesenchymalcellsisoftenminimal[60].Mesenchymalcellsareabletomovethroughamatrix-filledspacebyusingproteases,suchasMMPsandurokinase-typeplasminogenactivator(uPA),thatdegradeECMproteinsandcreatesthepath[61,62].AmoeboidmotilityAmoeboidmovementofcellsislikelytousesimilarmechanismsofthemigrationofleukocytesandliumdiscoideum[63].ThismovementisverysimilartotheroundedRho-andROCK-dependentformofmotilitythathasbeendescribedinA375m2melanomaandLS174Tcoloncarcinoma[60];Withtheadventofmulti-photonmicroscopy,intravitalimagingofmammaliansys-temshasgreatlyimprovedandhasopenedupnewwaystoexplorechemotaxis,cell-cellinteractionsandthemetastaticcascadewithintheinvivomicroenvironment.HighresolutionintravitalimaginghavedemonstratedthatsomecarcinomacellsmoveatveryhighspeedwithParriandChiarugiCellCommunicationandSignalinghttp://www.biosignaling.com/content/8/1/23Page4of14 anamoeboidmorphology(upto4m/min)invivo[54,64].Thismotilitystyleislargelyindependentfromcell-ECMcontactandfromproteolyticdegradationofECMfromMTorsolubleMMPs.Amoeboidmovingcellsshowroundedmorphologyandgreatlyexploitasapropulsoryforcetheacto-myosincytoskeletoncontracti-lity,withoutRac-drivencellpolarization,butrequiringRhoactivation.CorticalactincontractiondrivenbyRho-ROCKsignallingthroughmyosinactivationmightpromotetherapidremodellingofthecellcortexcharac-teristicofamoeboidmovement[54,60,65].Furthermore,cell-ECMattachmentsofamoeboidmovingcellsarenotorganizedinlargefocaladhesionsbutareverydiffuseandmuchweakercell-ECMattach-mentsarerequired,becauseamoeboidmovementcan-notbeblockedbyinhibitionofintegrinfunction[55,66].Notably,proteasesarenotrequiredforthisstyle,becausecellsareabletosqueezethroughgapsintheECMinsteadofdegradingit[66].Thesedifferencespathgeneratingbyproteolysisformesenchymalmovingcellsorinpathfindingbysqueezingforamoeboidcellscouldexplainthedifferentspeedsofthetwostyles.CollectivemotilityAthirdformofmotilityiscollectivecellmotility.Col-lectivelymigratingcellsmaintaintheircell-celljunctionsandmigrateinsheets,strands,tubesandcluster,eitherstillinconnectionwiththeiroriginatingtissueorasseparated,independentlymigratingcluster.Incancer,collectivecellmigrationandinvasionisfoundindistinctcancertypes,includinghighandintermediatedifferen-tiatedtypesoflobularbreastcancer,epithelialprostatecancer,largecelllungcancer,melanoma,rhabdomyosar-coma,andmostprominentlyinsquamouscellcarci-noma.High-resolutionmultimodalmicroscopyhasshownthattheguidingcellsuse1-integrin-mediatedfocaladhesionsandlocalexpressionofMT1-MMPattheirleadingedgestocleavecollagenfibersandorienttheminawaythatgeneratestube-likemicrotracksintowhichthecollectivemassmigrationoffollowercellscanoccur[67,68].Mechanistically,thisissimilartoacollec-tiveformofmesenchymalmotility,withthecellsatthefrontproducingMMPsandgeneratingapathforthefollowingcells[68].Incontrasttosinglecellmovement,whichrequiresthelossofadherensjunctions,themain-tenanceofadherensjunctionsisimportantforthisformofmovement[67].Themechanicsofthisformofmoti-lityarepoorlyunderstoodbecauseofthedifficultiesofmodellingitinvitro.However,theregulatorypathwaysunderlyingcollectivecellmigrationhavejustbeguntobeelucidatedanditsclinicalmanifestations,prognosticvalue,andactualcontributiontometastasisremaintobeassessedPlasticityoftumor-cellmigrationCellsfromseveralorigins,andamongthemcancercellsareparticularlytalented,areabletoengageadhocgenetic/ontogeneticprogrammesenablingthemtoadapttoenvironmentalchanges.Thisabilityofcells,com-monlyreferredascellplasticity,isoftenrelatedtodif-ferentstrategiestomovein3Dtissues[65,69].Lossofepithelial-likecellmorphologytoadoptamotilephenotypehasbeentermedepithelial-mesenchy-maltransition(EMT).Thisisaprofoundchangeincellphenotypethatcausesimmotileepithelialcellstoacquiretraitssuchasmotility,invasiveness,andresis-tancetoapoptosisortheabilitytoadapttoenvironmen-talchangesandcontinuetoinvadesuccessfully.Thesefeaturesaredrivenbyextracellularsignals,mostofwhicharestillunknown,whichinturninduceexpres-sionofaseriesoftranscriptionfactorsguidingtheachievementofthenewplastic,adjustablephenotype.IntheEMTprocess,thecellslosetheirepithelialcharacteristics,includingtheirpolarityandspecializedcell-cellcontacts,andacquireamesenchymalmigratorybehaviour,allowingthemtomoveawayfromtheirorigi-nalsitetowardsremotelocations[70,71].EMTillus-tratesthedifferentiationplasticityduringdevelopment,butiscommonlyexploitedbycancercellstoinvadeandmetastatize[69,71,72].Mesenchymalmotilityischarac-terisedbyelongatedandpolarizedcellmorphology;itdependsuponECMproteolysisofthemovingcellswhich,throughproductionofMMPs,generatesapathAlthoughseveralsolublefactorsthatpromotethispro-cesshavebeenidentified,thepathophysiologiccontextinwhichtheyactremainunclear.InflammationisakeyconspiratorintheemergenceofEMTinadults,althoughitisabsentduringembryonicdevelopment,suggestingtheexistenceofmultiplestimulielicitingEMTandpossiblemultipledifferentsubtypesofEMT.RecentlyKalluriandWeinbergproposedaclassificationofEMT:type1EMTservesforembryonicdevelopment,type2fortissuerepairandtype3formetastaticspread-ingofcancer[71].Whiletype1EMTisindependentfrominflammationandinjuries,bothtype2and3sharetheirdependencefrominflammationandarecharacter-izedfromtheirenduranceuntiltheprovokingspurisremoved.Ofnote,exogenousadditionofMMP-3,MMP-2orMMP-9facilitateEMTlikelythroughclea-vageofE-cadherin[73,74].Finally,type3EMTisfacili-tatedbygenomicandepigeneticalterationsacquiredbycancercells,andsomeofthesealterationshavebeenreportedalsointumor-associatedstroma.TheEMTtranscriptionalprogrammehasbeenassociatedwithactivationofseveralkeytranscriptionsfactors,includingSnail-1andSnail-2(Slug),Twist,ZEB-1-2,etc.Thelargenumberoftranscriptionfactorswhichcanbeengagedtoelicitthesamephenotype,i.e.ParriandChiarugiCellCommunicationandSignalinghttp://www.biosignaling.com/content/8/1/23Page5of14 thetransitionfromanepithelial-liketoamesenchymal-likecellbehaviour,isnotnecessaryindicativeofredun-dancy.Indeed,differentstimuliabletoelicitEMTappeartoactondifferenttranscriptionfactors.Tumormicroenvironmentalcuesasinflammation[75]orstro-malfibroblasts(Chiarugi,P.,unpublisheddata)driveaSnail-1-dependentEMT,whileintratumoralhypoxiaeli-citsaTwist-mediatedEMT[76-78].Inaddition,miR-NAsareabletoregulateEMTactingmainlyonZEB-1andZEB-2[79,80].Thetranscriptionalprogrammeleadstoregulationofaseriesofproteins:decreaseofE-cad-herinfordisruptionofadherensjunctions,increaseinN-cadherinandMetproto-oncogenetodrivemotility,aswellasincreaseinMMPsanduPA/uPARproteolyticsystemstodegrade3Dbarriers[71,72].Inresponsetoparticularenvironmentalcues,cancercellscandenovoacquireanamoeboid-likemotility,thusundergoingtowhathasbeentermedmesenchymaltoamoeboidtransition(MAT).Thelatterisaprimitiveformofcellmigrationthatallowscellstoglidethrough,ratherthandegrade,ECMbarriersthroughweakenedcell-ECMattachments.Converselytomesenchymalmotility,cellsmovingthroughanamoeboidmodeshowindependencefromproteolyticsystemstodegrade3Dbarrierandthemovementofcellsdependsontheirabil-itytosqueezebetweengapsofECMinsteadfromtheabilitytodegradeit[55,65].MATcanbeinducedincellsbybothenvironmentalorepigeneticcues.InfibrosarcomaandmelanomacellstheinhibitionofintegrinorMMPfunction,leadstoswitchfrommesenchymaltoanamoeboid-likemigra-tionprogram,therebyrescuingmotilitybyalternativemechanismsandsustainingthedisseminationofsinglecancercells[55,66,81].Indeedfibrosarcomaandmela-nomacells,inthepresenceofacocktailofabroadspectrumproteaseinhibitors,converttheirmotilitystylefromproteolytictoamoeboid,thusundergoingMAT.Inkeepingwiththedifferentdependenceofmesenchymaloramoeboidmotilitiesfromintegrinengagement,treat-mentofsarcomacellswithintegrinantagonistselicitsaclearMAT[55,66,81,82].Besideenvironmentalregulation,MATcanalsobeinducedbyepigeneticexpressionofregulatingfactors.First,prostatecarcinomacellsmovethroughandEphA2-mediatedamoeboidmotility[83,84].Second,aggressivemelanomacellsareabletoshiftadhocbetweenmesenchymalandamoeboidmotility:inresponsetopro-inflammatorycytokinestheyundergoEMT,whileafterre-expressionofembryonicEphA2receptor,experienceanewkindofmotilityprogramundergoingMAT[81].Inaddition,fibrosarcomacellshavebeenreportedtoundergoMATduringforcedacti-vationofstathmin,aknownmicrotubulecytoskeletonregulator,orduringinhibitionoftheE3-ubiquitinligaseforRhoASmurf1[85,86].Finally,lossofp53orp27tumorsuppressorspromotesRhoA/ROCK-dependentcellmigrationandinvasionin3Dmatricesforhumanmelanomacells,suggestingthatMATisassociatedwithworseprognosiscancers[87-89].NeverthelessseveralinterestingdataindicatethatMATisanefficientplasticityprogrammeforcellmoti-lity,theidentificationofthemolecularplayersregulatingMATisstillatitsinfancy.Inanycase,asthedifferentreportedexamplesofMATsharesomekeyfeatures,ascellbodyconstrictionandindependencefromproteases,wespeculatethatMAT,aswellasEMT,shouldbedri-venbyatranscriptionalresponse.OnethefirsteventcouldbetherepressionofEMT,i.e.antran-scriptionalprogramme,andofitstranscriptionalexecu-torsSnailandTwist,butitislikelythatMATswitchesonitsowntranscriptionfactors.SimilarlywithrespecttoEMT,thetransitionfromcollectiveinvasiontoamoeboidmovementreliesinweakeningcell-cellandcell-ECMinteractions,i.e.dis-ruptionofE-cadherinmediatedadherensjunctionandintegrin-linkedfocalcomplexes[68,90].Melanomacellshavebeenindicatedtomoveincohortsofmulticellularclustersbutthecontextualinhibitionof1integrinsabolishedthesecollectivemovement,therebyinducingdetachmentofindividualsinglemovingcellsusingamoeboidstyletoinvade,i.eacollectivetoamoeboidtransition(CAT)[91].TodateitisunknownthatCATconvertscollectivemigrationtotheamoeboidonedirectlyorviaanintermediatemesenchymalmigrationstep[55].ReciprocalcontrolofRacandRhosmallGTPasesAmutualantagonismbetweentheRacandRhoGTPaseshasbeenobservedinseveralcellularsettings,raisingthesignificantquestionofitsintegratedincellbehaviour.InA375M2melanomacells,displayingapre-dominantlyamoeboidphenotypewithaminorityofcellsmigratinginamesenchymalfashion,Sanz-Morenoiden-tifiedDOCK3asaGEFspecificforRac1,NEDD9asanadaptorproteinofthep130CasfamilybindingDOCK3,andWAVE2asaproteinthatpromotesactinnucleationdownstreamofRac[92].InthiscellmodelthereisareciprocalinhibitoryrelationshipbetweenRacandRhosignalingcascadesestablishingaregulatoryswitchbetweenthemesenchymalandamoeboidphenotypes.MesenchymalmelanomamorphologyandinvasivenessstyleiscontrolledbyaRac1activationpathway,mediatedbyadaptorproteinNEDD9andDOCK3,actingasaRac1-GEF.Cellelongationandactinpoly-merizationdownstreamtoRac1ismediatedbytheactin-nucleationproteinWAVE2.WAVE2isalsoresponsiblefordownregulationofactomyosincontracti-lity,cytoplasmblebbingandamoeboidmotility.OntheParriandChiarugiCellCommunicationandSignalinghttp://www.biosignaling.com/content/8/1/23Page6of14 contraryinamoeboidmovingcells,Rhoactivationsti-mulatesaROCK-mediatedactomyosinandcellbodycontractility.Inparallel,amoeboidsignallingleadstodownregulationofmesenchymalmovement,mainlythroughinhibitionofRac1byactivatingtheRac-GAPARHGAP22,therebycompletingthecircuitryofRac1-RhoAantagonism.BesideARHGAP22andDOCK3/NEDD9signaling,otherpathwaysleadingtoshiftofRac/RhobalanceinfavorofthelastinduceMATaswell.TheseincludetheinterferencewithRab5-mediatedendocytosisandrecy-clingofRactocellprotrusions[93]andtheinhibitionofE3ubiquitinligaseSmurf1,whichleadstoRhodegra-dationdirectlyattheleadingedgeandtherebygrantsfordominanceofRacatthefrontofpolarizedcells[86].Studiesonspecificextracellularsignalsactinginfavourofmesenchymaloramoeboidmovementsarestillattheirinfancy.Clearly,extracellularfactorsactondifferentinvasivestylesthroughRacandRhomodula-tionandtheroleoftheirGEFsandGAPsismandatory.PresumablybothRac1andRhoactivationareultimatelycontrolledbyGFsandintegrinactivity,therebysuggest-ingtheexistenceofadditionalmechanismsbywhichRaccaninhibittheRho-mediatedamoeboidphenotype.ActivatedRacinresponsetointegrinengagementhasbeenshowntostimulatetheactivityofp190RhoGAP(whichdown-regulatestheactivityofRhoisoforms)bypromotingitsphosphorylation.Indeed,theoxidativecascadeinvolvingRac1,reactiveoxygenspecies(ROS)andap190RhoGAPphosphatasehasbeencorrelatedwiththeantagonisticcrosstalkbetweenRac1andtheRhoA[94].ExtracellularactivationofRac1leadstoenhancementofROSproductionandthisleadsinturntoredoxinhibitionofLow-Mwproteintyrosinephos-phatase(LMW-PTP),finallyenhancingthephosphoryla-tionofitssubstratep190RhoGAP[94,95].Theredoxcircuitryengagedbymesenchymalstimuliisclosedbecausephosphorylatedp190RhoGAPdownregulatesRhoAandsuppressesamoeboidactivity.Inaspecularfashion,activationoftherepulsiveEphA2receptorinprostatecarcinomacellsisaccompaniedbyreducedRac1activityandattenuatedgenerationofROS,whichleadstoLMW-PTPactivation,p190RhoGAPdepho-sphorylationandtoanincreaseofRhosignaling[83,96].EphA2receptoractivationbyitscognateligandephrinA1isapowerfulsignaltoactivateRhoAanditsoverexpressionandactivationcausesachievementofamoeboidinvasivestylesfrombothprostatecarcinomaandmelanomacells[81,83,84,96].Inaddition,p120-cateninsupportsRac-Rhocrosstalkbycontrollingthecorticallocalizationofp190RhoGAPandtherebyallow-ingRhoinhibitionthroughactivationofRac[97,98].Indeed,inp120-deficientcells,p190RhoGAPwasacti-vatedviaitsredoxpathwaysbyaconstitutivelyactiveRacmutantbutwasunabletoinhibitRho[97].ThiscoordinatedandopposedactivityofRac1andRhoAiscrucialtocellulardynamics,theformerpromotingmembraneprotrusion,cellpolarityandspreading,thesecondcytoskeletoncontractilityandtailretractionCol-lectively,theaboveevidenceindicatethattheintricatecross-talkbetweenRhofamilyGTPasethatunderliesdynamiccellresponsesisinlargepartredoxregulated(figure2).Moreover,Radiskyetal.reportedthatactivationofEMTinbreastcarcinomacellsisassociatedwithexpres-sionofthealternativelysplicedRac1isoform,theconsti-tutivelyactiveRac1b,andtherebytoROSgeneration.AlthoughtheidentificationofthedirectredoxproteinsensorsdrivingEMTisstilllacking,theseoxidanthavebeenprovedtobegenotoxic,therebycontributingtobothcarcinogenesisandtumorinvasiveness[74].OurpreliminaryobservationsinthiscontextindicatethatincancercellstheROSgenerateduponEMTcommitmentalsoretainsignallingroles,enhancingexpressionoftranscriptionfactorscorrelatedwithmesenchymalorinflammatoryprogramsasSnail-1,Twist,hypoxiaindu-ciblefactor-1ornuclearfactor-B(Chiarugi,P.,unpub-lishedresults).InthecontextofEMTthedownregulationofRhoproteins,althoughhighlyfeasi-ble,remainstobedescribedinitsmoleculardetails.AberrantregulationofRacandRhoproteinsincancerAsaconsequenceofthelargenumberofkeyfunctionsassignedtoRhoproteins,likeproliferation,apoptosis/survival,cellpolarity,celladhesionandplasticityofcellmigration[99,100],itisnotsurprisingthattheyplayimportantrolesintumorbiology[101].Aclearconnec-tioncanbeestablishedbetweenRhoproteinsoverex-pressionandalargevarietyofhumantumors[102,103].RhoGTPaseshavebeenreportedtocontributetomoststepsofcancerinitiationandprogressionincludingtheacquisitionofunlimitedproliferationpotential,survivalandevasionfromapoptosis,angiogenesis,tissueinvasionandtheestablishmentofmetastases(figure3).SomeRhoGTPasesstimulatecellcycleprogressionandregu-lategenetranscription,andthiscouldinpartexplaintheirpro-oncogenicproperties,forexampleinpromot-ingRas-inducedtransformation[104].SomeRhoGTPasesarethoughttobeabletoregulatethereleaseofpro-angiogenicfactorstopromoteneovascularisation[105].TheabilityofRhoGTPasefamilymemberstoregulatelooseningofepithelialcell-cellcontacts,MMPsexpressionandtheplasticityofcellmigration(EMT,MAT)[103]pointstoacentralroleincancercellinva-sionandmetastasis(figure3).Primarytumorsgenerallyariseasaconsequenceofmultiplemutationsandepigeneticchangesaffectingkeygenesthatultimatelyaffectproliferationandsurvival.ParriandChiarugiCellCommunicationandSignalinghttp://www.biosignaling.com/content/8/1/23Page7of14 Unexpectedly,todate,nomutationshavebeenfound inRhoproteins.OnlyonememberoftheRhofamily ofsmallGTPases(RhoH)hasbeenreportedtobe geneticallyalteredinnon-Hodgkin ’ slymphomasand multiplemyeloma.SincemutationsinRhoproteins havenotbeenfound,deregulationofRhoGTPasesig- nallingcouldoccuratthelevelofexpressionoractiva- tionofRhoGTPases,accom plishedbythelevelof expressionoractivationoftheirregulatorsordown- streameffectors.RhoAandRhoCexpressionand/or activityisfrequentlyincreasedinhumantumors, whereasRhoBisoftendownregulated[106].Increased RhoAexpressionwasdescribedinvarioushuman tumorsincludingliver[107],skin[108]andcoloncan- cer[109].Inliver,increasedRhoAexpressioncorre- latedwithincreasedRhoAac tivity,poorprognosisand recurrence[107].ElevatedRhoAlevelsalsocorre- spondedtoprogressionofovarian[110],bladder[111], gastric[112]esophagealsquamouscell[113],andtesti- cularcancer[114](table1).RhoAhasbeenimplicated invirtuallyallstagesofcancerprogression:forexam- ple,invitro,constitutive lyactiveRhoAcanstimulate transformation[115].Innormalepithelia,RhoAcontri- butestothegenerationofepithelialpolarityandjunc- tionassemblyandfunction[116]butalsoaffects epithelialdisruptionduringtumorprogression.RhoA activitycanbeinhibiteddow nstreamofcadherinslead- ingtoamoremotilephenot ype[97].DifferentGEFs andGAPsinfluencehowRhoproteinscanactindif- ferentcontextseitherpromot ingepithelialorganization andpolarityorepithelialEMT,asseeninstudiesof RhoAGEFs/GAPsinDrosophilamodels[117]. A3Dinvitroinvasionmodelusingco-culturesof SSC12carcinomacellswithstromalfibroblasts,has shownhowthisfibroblastgeneratesthetractionforce andremodelsthematrixthroughMMPs.DifferentRho GTPaseswerefoundtoberequiredintheleadingfibro- blastandthefollowingcarcinomacellswithaRhoA regulationofMLCintheformerandmainlyCdc42and myotonicdystrophykinase-relatedCdc42-bindingkinase (MRCK)functioninthelatter[118].Incontrastto RhoA,RhoChasnoapparenttransformingactivity. RhoCwasidentifiedinascreenforgenesupregulatedin melanomametastases[119],andhassubsequentlybeen proposedasamarkerforpoorprognosisincancersof differentorigins[120].RhoCisupregulatedinmany cancersincludingbreastcancer[121]andsquamouscell carcinoma(SCC)ofskin[122](table1).Increased expressionofRhoCcorrela teswithprogressionand poorprognosisofductaladenocarcinomaofpancreas [123],hepatocellularcancer[124],breastcancer[109], ovariancancer[110],bladdercancer[111],gastric Figure2 ReciprocalregulationbetweenRhoandRacduringmesenchymaloramoeboidmotilitystyles .ROSactasabalanceforRac-1/ RhoAantagonism.IndeedRac-1,whichdrivesorientedmesenchymalmotility,leadingedgeprotrusionandlamellipodiaformation,isakey molecularplayerofregulatedintracellularROSsources.Rhoactivationisresponsibleforamoeboidmotility,anon-orientedmovementwhich enablesthecelltosqueezebetweengapsofECMinsteadofproteolyticallydegradeit.Hence,uponRacactivationoxidation/inactivationofthe LMW-PTPwhichnormallyactivatetheRhoregulatorp190Rho-GTPase,leadstoRhoAdown-regulation.Conversely,lowROSintracellularcontent leadtoRhoAactivation,throughLMWPTPactivationandp190RhoGAPdephosphorylation/inactivation.ActivatedRhoAisabletoinhibitRac-1 throughtheARHGAP2(alsonamedchimerin-2),whileRac1activatesWAVE2whichinturninhibitsRhoA. ParriandChiarugi CellCommunicationandSignaling 2010, 8 :23 http://www.biosignaling.com/content/8/1/23 Page8of14 cancer[125],esophagealSCC[113],headandneckSCC [120],prostatecancer[126],andnon-smallcelllung carcinoma(NSCLC)[127].IncontrasttoRhoC,expres- sionofRhoAandRhoBdidnotcorrelatewithpoor prognosisinpancreaticcancer[123].IncreasedRhoC expressionhasbeenclaimedasthepossiblecausefor theinductionininvasionandmetastasistriggeredby theoverexpressionofthemicroRNA-10binbreastcan- cer[128].RhoCexpressionisincreasedduringEMTin acoloncancermodelandcontributestoEMT-induced Table1AberrantregulationofRhoproteinsincancer Rho proteins Mechanismof deregulation Tumortype RhoA Highproteinlevels, highsignallingactivity Liver[107],skin[108],colon[109],ovarian[110],bladder[111],gastric[112],esophagealsquamouscell(SCC) [113],testicular[114],breast[109] RhoB Overexpressionor downregulation Breast(overexpression)[109],lung(downregulation)[151] RhoC Highproteinlevels, highsignallingactivity Melanomametastases[119],breast[121],squamouscell(SCC)[122],pancreas[123],liver[124],ovarian[110], headandneck[120],prostate[126],non-smallcelllung(NSCLC)[127],gastriccancer[125] RhoH Rearrangementand mutations (5 ’ UTR) non-Hodgkin ’ slymphomasandmultiplemyeloma[152] Rac1 Highproteinlevels, highsignallingactivity Testicular,[114]gastric[112],breast[136],squamouscell(SCC)[137] Rac1B AlternativesplicingColon[148];Breast[136] Rac2 HighproteinlevelsHead[108]necksquamous-cellcarcinoma(SCC)[122] Rac3 Hyperactiveor overexpression Breast[22] Figure3 InvolvementofRhoproteinsatdifferentstagesoftumorprogression .A)Maintenanceofnormalepithelialcellpolarity.B)Benign tumors:onceatumorisinitiated,Rhoproteinscancontributetotumordevelopmentbystimulatingdedifferentiation,growthandlossofcell polarity.C)Locallyinvasivetumors:Rhoproteinscancontributetotumordevelopmentbyalteringcell-cellandcell-matrixadhesion,Rho proteinsallowtumorcellstobecomeinvasive.D)Metastasistodistantsite:RhoandROCKarerequiredfortumorcellstocrossendothelialcell layers.RhoCpromotesexpressionofangiogenicfactors,leadingtoanincreaseinvascularizationofthetumor. ParriandChiarugi CellCommunicationandSignaling 2010, 8 :23 http://www.biosignaling.com/content/8/1/23 Page9of14 migration,whereasRhoAlevelsgodown[129].ItisnotyetclearhowRhoCincreasesinvasionandmetastasisorwhyitseffectsdifferfromRhoA.SomereportsindicatethatRhoA,RhoCandtheirdownstreamtargetROCKareneededforcancercellextravasation[130].Interest-ingly,RhoCcaninducetheproductionofangiogenicfactorsinbreastcancer,andthiscouldhelppromoteentryintobloodvesselsandtherebymetastasisdissemi-nation[105].UnlikeRhoAandRhoC,RhoBisoftendownregulatedinhumantumorsanditsexpressioninverselycorrelateswithtumoraggressiveness.IthasbeenproposedthatRhoBcanworkasatumorsuppres-sorasitisactivatedinresponsetoseveralstressstimuliincludingDNAdamageorhypoxia,andithasbeenreportedtoinhibittumorgrowth,cellmigrationandinvasionandhaveproapoptoticfunctionsincells[131].RhoBknock-outmicedevelopnormallybuthaveenhancedcarcinogen-inducedskintumorformation,inagreementwitharoleofRhoBasatumorsuppressor[132].RhoBalsosuppressesinvasion:forexampleithasbeenpostulatedtoactdownstreamofproteinkinaseCintheregulationofcancercellinvasioninvitro[133]anditwasalsoreportedtoinhibitRas-inducedinvasionandmetastasis[134].TheexactmechanismwherebyRhoBsuppressestumorgrowthandinvasionisnotclear,althoughitsroleinendosomaltraffickingcouldbeimportant.RhoBregulatesthedeliveryofsignallingpro-teins,includinggrowthfactorreceptorsandthetyrosinekinaseSrc,tospecificintracellularcompartments[135],andthiscouldcertainlyinfluenceproliferationandTheRacsubfamilyofRhoGTPasesincludesRac1,Rac2,Rac3.Rac1isover-expressedinvarioustumorsandaccumulatingevidenceindicatethatRac1-depen-dentcellsignallingisimportantformalignanttransfor-mation[106].OverexpressionofRac1correlateswithprogressionoftesticular[114],gastric[112],andbreastcancer[136].Rac1isalsooverexpressedinoralSCC[137](table1).Rac1knock-outinmiceisembryoniclethal[138]butconditionalknock-outmicehavebeenstudiedextensively[139].InaconditionallungcancermousemodelRac1functionwasrequiredforK-Ras-dri-venproliferationandtumorigenicity[140].Similarly,micelackingtheRac-specificGEFTiam1areprotectedfromRas-inducedskincancer,developingfewertumors,althoughthetumorsthatdoformaremoreaggressive[141].TheseresultssuggestthatRacproteinsnormallystimulatetumorcellproliferationbutinhibittumordis-semination.Rac1couldcontributetocancercellprolif-erationviaregulationofthecellcycle:forexample,itstimulatesexpressionofcyclinD1,andinducescelltransformationinvitro[104].ActiveRaccanmediatethelossofadherensjunctioninsomesituations,pro-motingamoreinvasivephenotype[142].Rac1canalsocontributetocancercellinvasionbyregulatingthepro-ductionofMMPsandtheirnaturalinhibitors,thetis-sue-specificinhibitorsofMMP(TIMPs)[143].LikeRac1,Rac2andRac3areover-expressedinsometumors.Rac3ishyperactiveand/orderegulatedinbreastcancers[22].ThecontributionofdifferentRacisoformstomigrationislikelytodependonthecelltypeandtheirrelativeexpressionlevels.Rac2isrequiredforneu-trophilmigrationbutwhetheritactssimilarlyintumorsisnotknown[144].Incontrast,Rac1andRac2aredis-pensableforcellmigrationinmacrophages,althoughRac1isrequiredforinvasion[145].StudiesofRac3-nullmiceindicatethatRac3butnotRac1orRac2specifi-callycontributestothedevelopmentofBrc/Abl-inducedlymphomasinvivo[146].However,infibroblasts,Rac1butnotRac3suppressionbyRNAiaffectslamellipodiumformationalthoughcellinvasionisreducedinbothcases[147].Itisnotyetclearhowtheseresultscanbetranslatedtocancercellinvasioninvivo.ThesplicevariantofRac1,Rac1b,wasinitiallyidenti-fiedtobeup-regulatedincoloncancers[148].ItdoesnotbindRhoGDIandthusispresentpredominantlyintheGTP-boundstate.AlthoughRac1bisdefectiveinactivatingseveralRac1-regulatedsignalingpathways,insomecelltypesitstimulatescellsurvivalandcellcycleprogressionthroughnuclearfactor-kappaB,andislesssusceptibletoubiquitinationanddegradation,whichcouldexplainitsincreasedexpressionincancers[149].RhoGTPasesareinvolvedinallstagesduringcancerprogression(figure3).Althoughtheirinitialdiscoveryasregulatorsofcytoskeletondynamicsimpliedthattheyaremostlikelytocontributetocancercellmigrationandinvasion,itisnowclearthatthefunctionofRhoGTPasesisnotrestrictedtotheseeventsandthattheycanaffecttumorcellsthroughmodulationofgenetran-scription,celldivisionandsurvival,intracellulartrans-portofsignallingmoleculesormodifyingtheinteractionofcancercellswithsurroundingstromalcells.ThismakesthedetailedanalysisofhowRhoGTPasesworkincellsandcontributetotumorsverycomplexbutatthesametimepromisingforpotentialfuturetherapeuticalintervention.TheinvolvementofspecificGEFsorGAPsindefinedprocessesregulatedbyRhoGTPasesmakesthemparticularlysuitableastherapeutictargets[150].ConclusionsMetastasisisamultistageprocessneedingastrongadaptabilityofcellstothedifferentmicroenvironmentswithinprimarytumors,intheECM,inbloodorlym-phaticstreamsandfinallyinthemetastaticniche.Intra-vitalimagingofGFP-expressingcancercellsinsubcutaneoustumorsillustratedthisadaptability.InthecoreofthetumorneoplasticcellsmainlymovedusingParriandChiarugiCellCommunicationandSignalinghttp://www.biosignaling.com/content/8/1/23Page10of14 mesenchymalandelongatedstyle,whilecellsatthetumoredgeescapethetumorlimitandentertheECMusingarounded/amoeboidmotility[92].GeneticorpharmacologicaltreatmentofARHGAP22orROCKshiftonemotilitystyletoanother,therebyconfirmingthekeyroleofRacandRhoGTPasesinplasticityofcellmotility.Moreintriguingly,thecombinedtreatmentsaimedatblockingsimultaneouslybothmodesofmigra-tionstronglyinhibittheopportunisticbehaviourofcan-cercells,therebylimitingtheirinvasivepotential.Thesedataindicatethatthewinningstrategytocombatsuc-cessfulmetastaticdiffusionofaggressivecancercellsiseitheracombinatorytreatmenttargetingbothinvasivestyles,ortheidentificationofsinglemoleculartargetsdrivingtheabilityofcancercellstoadapttoenviron-mentalchanges,i.e.cellplasticityitself.Unfortunately,theidentificationofthemolecularmediatorsofplasticityincellmotilityisstillatitsinfancy,butitwillbesurelythenextchallengetoreallytargettheopportunisticmotilityofcancercells[69].AcknowledgementsThisworkwassupportedbytheAssociazioneItalianaRicercasulCancro(AIRC),byIstitutoToscanoTumori,andRegioneToscana.AuthordetailsExternautics-R&D,viaFiorentina1,Siena,SI,53100,Italy.DepartmentofBiochemicalSciences,UniversityofFlorence,TuscanyTumorInstituteandCenterforResearch,TransferandHighEducationDenoTHE,50134Florence,Italy.PCandMPorganized,wroteandeditedthemanuscripttogether.Bothauthorsredandapprovedthefinalmanuscript.CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.Received:26May2010Accepted:7September2010Published:7September20101.Etienne-MannevilleS,HallA:RhoGTPasesincellbiology.2.RidleyAJ:RhoGTPasesandactindynamicsinmembraneprotrusionsandvesicletrafficking.TrendsCellBiol3.ValenciaA,ChardinP,WittinghoferA,SanderC:Therasproteinfamily:evolutionarytreeandroleofconservedaminoacids.4.BoscoEE,MulloyJC,ZhengY:Rac1GTPase:aofalltrades.CellMolLifeSci5.CoteJF,VuoriK:GEFwhat?Dock180andrelatedproteinshelpRactopolarizecellsinnewways.TrendsCellBiol6.SchmidtA,HallA:GuaninenucleotideexchangefactorsforRhoGTPases:turningontheswitch.GenesDev7.PechlivanisM,KuhlmannJ:HydrophobicmodificationsofRasproteinsbyisoprenoidgroupsandfattyacidsMorethanjustmembraneanchoring.BiochimBiophysActa8.EllerbroekSM,WennerbergK,BurridgeK:SerinephosphorylationnegativelyregulatesRhoAinvivo.JBiolChem9.ChardinP:FunctionandregulationofRndproteins.NatRevMolCellBiol10.LiX,BuX,LuB,AvrahamH,FlavellRA,LimB:Thehematopoiesis-specificGTP-bindingproteinRhoHisGTPasedeficientandmodulatesactivitiesofotherRhoGTPasesbyaninhibitoryfunction.MolCellBiol11.Schmidt-MendeJ,GeeringB,YousefiS,SimonHU:LysosomaldegradationofRhoHproteinuponantigenreceptoractivationinTbutnotBcells.EurJImmunol12.DerMardirossianC,BokochGM:GDIs:centralregulatorymoleculesinRhoGTPaseactivation.TrendsCellBiol13.MoonSY,ZhengY:RhoGTPase-activatingproteinsincellregulation.TrendsCellBiol14.EdenS,RohatgiR,PodtelejnikovAV,MannM,KirschnerMW:MechanismofregulationofWAVE1-inducedactinnucleationbyRac1andNck.15.FiegenD,HaeuslerLC,BlumensteinL,HerbrandU,DvorskyR,VetterIR,AhmadianMR:AlternativesplicingofRac1generatesRac1b,aself-activatingGTPase.JBiolChem16.MatosP,CollardJG,JordanP:Tumor-relatedalternativelysplicedRac1bisnotregulatedbyRho-GDPdissociationinhibitorsandexhibitsselectivedownstreamsignaling.JBiolChem17.KwonT,KwonDY,ChunJ,KimJH,KangSS:AktproteinkinaseinhibitsRac1-GTPbindingthroughphosphorylationatserine71ofRac1.JBiol18.DidsburyJ,WeberRF,BokochGM,EvansT,SnydermanR:rac,anovelras-relatedfamilyofproteinsthatarebotulinumtoxinsubstrates.JBiol19.AmbrusoDR,KnallC,AbellAN,PanepintoJ,KurkchubascheA,ThurmanG,Gonzalez-AllerC,HiesterA,deBoerM,HarbeckRJ,OyerR,JohnsonGL,RoosD:HumanneutrophilimmunodeficiencysyndromeisassociatedwithaninhibitoryRac2mutation.ProcNatlAcadSciUSA20.WernerE:GTPasesandreactiveoxygenspecies:switchesforkillingandJCellSci21.HaatajaL,GroffenJ,HeisterkampN:CharacterizationofRAC3,anovelmemberoftheRhofamily.JBiolChem22.MiraJP,BenardV,GroffenJ,SandersLC,KnausUG:hyperactiveRac3controlsproliferationofbreastcancercellsbyap21-activatedkinase-dependentpathway.ProcNatlAcadSciUSA23.WheelerAP,RidleyAJ:WhythreeRhoproteins?RhoA,RhoB,RhoC,andcellmotility.ExpCellRes24.HallA:RhoGTPasesandtheactincytoskeleton.25.VanAL,DSouza-SchoreyC:RhoGTPasesandsignalingnetworks.26.LauffenburgerDA,HorwitzAF:Cellmigration:aphysicallyintegratedmolecularprocess.27.PollardTD,BlanchoinL,MullinsRD:Molecularmechanismscontrollingactinfilamentdynamicsinnonmusclecells.AnnuRevBiophysBiomol28.UrbanE,JacobS,NemethovaM,ReschGP,SmallJV:Electrontomographyrevealsunbranchednetworksofactinfilamentsinlamellipodia.NatCell29.CampelloneKG,WelchMD:Anucleatorarmsrace:cellularcontrolofactinassembly.NatRevMolCellBiol30.ChesaroneMA,GoodeBL:Actinnucleationandelongationfactors:mechanismsandinterplay.CurrOpinCellBiol31.MikiH,YamaguchiH,SuetsuguS,TakenawaT:IRSp53isanessentialintermediatebetweenRacandWAVEintheregulationofmembrane32.KnightB,LaukaitisC,AkhtarN,HotchinNA,EdlundM,HorwitzAR:Visualizingmusclecellmigrationinsitu.CurrBiol33.RoyalI,Lamarche-VaneN,LamorteL,KaibuchiK,ParkM:Activationofcdc42,rac,PAK,andrho-kinasein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