5.1. MAD2andchromosomalinstability
UpregulationofendogenousFAT10expressionbyTNF-␣aswell asoverexpressionofFAT10hasdirectlybeenlinkedto chromoso-malinstabilityinseveralreports(Gaoetal.,2015;Renetal.,2006, 2011;Thengetal.,2014).Itwasshownthatabout60%ofHCT116 andHepG2cellswhichhadbeentreatedforabouttenpassageswith IFN-␥andTNF-␣displayedabnormallyhighchromosomenumbers inakaryotypeanalysisandinhibition ofFAT10expressionwith silibinin,apotentinhibitoroftheNF-Bsignalingpathway,was abletoabolishthiseffect(Gaoetal.,2015).FAT10wasshownto becell-cycleregulatedwithapeakofexpressioninS-phasebut low expressionlevelsduring G2/Mphase (Lim etal.,2006; Liu etal.,2014)andaproteomicsbasedstudyprovidedevidencefor aroleofFAT10inmitoticregulation(Merbletal.,2013).Already 17yearsagoMAD2(Mitoticarrestdeficient2),amitoticspindle checkpointprotein,wasidentifiedasanon-covalentinteraction partnerofFAT10inayeasttwohybridscreenusingahumanBcell cDNAlibrary(Liuetal.,1999)andthisinteractionwasshownto prevailduringmitosis(Renetal.,2006).MAD2bindstothe kine-tochoresofsisterchromatidsinthemetaphaseofthecellcycle.
Thereit ensures thatbeforetheonset of anaphaseall
chromo-Fig.2.InteractionofFAT10withMAD2inducesaprematureonsetofanaphase,resultinginaneuploidy.
(A)Undernormalconditions,theonsetofanaphaseandtheseparationofthesisterchromatidsisinhibiteduntilallchromosomesareproperlyattachedviatheirkinetochores tothemitoticspindleapparatus.ThismechanismissurveilledbyaproteincomplexcontainingMAD2.(B)UpregulationofFAT10allowsthenon-covalentinteractionof FAT10withfreeMAD2andinturnreducestheamountofMAD2atthekinetochores.Asaresult,anaphasestartstooearlyandresultsininappropriateseparationofsister chromatidsandaneuploidy.Adetaileddescriptionisgiveninthetext.
somesbecomeproperlyattachedtothemitoticspindlealongthe metaphaseplate(Fig.2A).MAD2achievesthisbyinteractingwith andinhibitingCdc20,aco-activatorandsubstraterecruitment fac-toroftheanaphase-promotingcomplex/cyclosome(APC/C),which isamulti-subunitubiquitinE3ligase.Therebytheubiquitylation ofsecurinandcyclinisprevented.Securinbindsandinhibitsa pro-teasecalledsecurasewhichcleavescohesin,aproteinthatkeeps thetwo sister chromatidstogether (Lara-Gonzalezet al., 2012;
MusacchioandSalmon,2007).InFAT10overexpressingHCT116 cellsaswellasinTNF-␣-treatedHCT116cells,thelocalizationof MAD2atthekinetochoreswasmuchreducedintheprometaphase ofthecellcycle,indicatingthatFAT10interfereswiththebindingof MAD2tokinetochores(Fig.2B)(Renetal.,2006,2011).Moreover, afterreleasefromG1/Sarrest,FAT10overexpressingcellsshowed a delayedentryintomitosis. However,thesecells alsoshowed anunalteredreentryintoG1phaseascomparedtoparentalcells whichpointsatanabbreviatedmitoticphaseoranabbreviated mitoticarrestuponspindledamage.Whencomparedwithcontrol cells,ahighernumberofFAT10overexpressingcellsfailedtoarrest inmitosisupontreatmentwithnocadozoleorevenescapedthe mitoticarrest.Concurrentwiththesefindings,FAT10 overexpres-sioninducedtheformationofmultinucleatedcellswithabnormal nuclearmorphology.A constitutive overexpression of FAT10as wellasconstitutivetreatmentwithTNF-␣resultedinanincreased chromosomenumberascomparedtotheparentalHCT116cellline (Renetal.,2006,2011).However,whenthefiveputative interac-tionsitesofMAD2withintheN-terminalubiquitin-likedomainof FAT10weremutated(H11D,R13Q,H75D,T77DandK79Q), bind-ingofMAD2toFAT10wasgreatlydiminished.Ascomparedto cellsoverexpressingwildtypeFAT10,thechromosomenumbersof cellsexpressingthemutantFAT10returnedtonormalasfound inwildtypeHCT116cells.Thisfurthersupportsthenotionthat FAT10overexpressioncauseschromosomalinstabilityby interact-ingwithMAD2.Moreover,ascomparedtotheparentalcells,FAT10 overexpressingcellsshowedlong,incompletelycondensed chro-mosomesandthiswasalsorevertedbacktonormallycondensed, short“ribbon-like”chromosomes incellsexpressingthemutant
FAT10deficientinMAD2binding.Inlinewiththisobservation,the inhibitionoftheFAT10andMAD2interactionalsorevertedthe capabilityofFAT10overexpressingcellstoescapethe nocadozole-inducedmitoticarrestbacktolevels,seenwithwildtypeHCT116 cells(Thengetal.,2014).Takentogether,thesedatacorroboratethe hypothesisthatthenon-covalentinteractionofFAT10andMAD2 is critical for thedevelopment of aneuploidyin cancer cells. It wasshownthat FAT10interacts withfree butnot withspindle boundMAD2asanimmunoprecipitationagainstFAT10ledtoa co-immunoprecipitationofMAD2only,andnotofadditionalproteins ofthespindlecomplexsuchasMAD1andBub1(Thengetal.,2014).
TheobservedlowlevelofendogenousFAT10expressioninG2/M phase(Limetal.,2006)suggeststhatcellskeeptheFAT10level lowduringthemitoticphaseinordertopreventmitotic dysfunc-tionswiththeriskofsubsequentcancerdevelopment.Consistently, HCT116cellsoverexpressingtheMAD2interaction-deficientFAT10 mutantretainedsignificantlylowerproliferationratesandshowed anincreasedsusceptibilitytoapoptosisascomparedtocells over-expressingwildtypeFAT10.Moreover,cellsexpressingtheMAD2 binding-deficientFAT10mutant failedtogrowin soft agarand losttheircapabilitytogrowinananchorageindependent man-ner.Moreimportantly,thisfindingcouldbesubstantiatedinvivo byinjectingHCT116cellsexpressingFAT10wildtypeortheMAD2 binding-deficientFAT10mutant intonudemice.Tumorsgrown fromHCT116cellsexpressingMAD2binding-deficientFAT10were significantlysmallerthanthoseexpressingwildtypeFAT10(Theng etal.,2014).Insummary,allthesedatafromwellcharacterizedcell linemodelsofhepatocellularcarcinomamakeaconvincingcase, thatthepro-malignantcapacitiesofFAT10relyextensivelyonits interactionwiththespindlecheckpointproteinMAD2,thereby pre-ventingtheproperfunctionofMAD2inmitoticcheckpointcontrol (Fig.2).
5.2. p53
Thetranscriptionfactorandtumorsuppressorp53playsa cen-tralroleinthemaintenanceofgenomicstabilityandasaninhibitor
Fig.3. FAT10negativelyregulatesp53transcriptionalactivity.
Undernormalconditions,theconcentrationofp53iskeptatalowlevelandthisismaintainedbyatightregulationofp53ubiquitylationanddeubiquitylationbyspecific E3ligasesanddeubiquitylationenzymes(DUBs).DNAdamageleadstoarapidupregulationofp53,itsactivationbyphosphorylationanditstrans-localizationintothe nucleuswhereitactseitherasanactivatororasaninhibitoroftranscriptionleadingtoDNArepairorinductionofapoptosis.HighFAT10concentrations,mediatedby pro-inflammatorycytokinesleadtoaninhibitionofthep53transcriptionalactivityandresultinDNAdamageandresistancetoapoptosis.Adetaileddescriptionisgiven inthetext.Arrowsmarkedwitha“+”illustrateactivatingpathways,arrowsmarkedwitha“−”illustratenegativelyregulatedpathways.Aredarrowwithtwoarrowheads representstheinteractionofp53andFAT10.
ofinflammation.Morethan50%ofallhumancancersdisplay dele-tionsormutationsinthep53gene.Undernormalconditions,the concentrationofp53iskeptatalowlevelandthisismaintained byatightregulationofp53ubiquitylationanddeubiquitylation.
Twomaincellulareventsregulatedbyp53arecell cyclearrest uponDNAdamageandinductionofapoptosis.DNAdamageleads toarapidup-regulationofp53,itsactivationbyphosphorylation anditstrans-localizationintothenucleuswhereitactseitheras anactivatororasaninhibitoroftranscription.Down-stream tar-getsof p53aregenesinvolved intheregulationof DNArepair, apoptosisandcellcyclecontrol.Uponup-regulationofp53,the cellcycleisarrestedatthetransitionofG1/SuntilDNArepairis performedandthecellre-entersthecellcycle.However,ifthe DNAdamageistoostrong,apoptosiswillbeinduced.Mutationsin p53whichinhibititsactivityastranscriptionfactoraretherefore linkedtocancerdevelopment(Gudkovetal.,2011;Levine,1997).
p53wasrecentlyidentified as aFAT10 interactingproteinin a massspectrometryscreensearchingforFAT10interactionpartners (Aichemetal.,2012).Additionaldatashowedthatp53gets cova-lentlymodifiedbyoverexpressedFAT10inHEK293cells,andthat overexpressionofwildtypebutnotofmutantFAT10,inwhichthe diglycinemotifattheC-terminuswasmutated,increasedthe tran-scriptionalactivityofa luciferasereporterconstruct,containing bindingsidesforp53(Lietal.,2011).However,under inflamma-toryconditionsthep53transcriptionalactivitytowardsluciferase reporter constructs containingeitheran artificialp53-regulated promoteroranaturaltargetpromoterofp53(p21WAF1/CIP1)was decreased upon FAT10induction byIL-6 and/orTNF-␣. Consis-tently,overexpressionofFAT10indifferentcellslinesalsoreduced thetranscriptionalactivity ofp53 withoutaffecting theoverall
p53proteinamount.Insupportofthisfinding,silencingofFAT10 induced theopposite effectand ledtoan increase inthe tran-scriptionalactivityofp53andtoanincreaseinapoptosis(Choi etal.,2014).Thesystemseemstobetightlyregulatedbya neg-ativefeedbackmechanismbecauseitwasshown,thatp53itself negativelyregulatestheexpressionofFat10mRNAbybindingto aninhibitorysitewithintheFat10promoter.Itwasshownthat thetranscriptionalactivityof-galactosidasereporterconstructs underthecontroloftheFat10promoterwassignificantlyhigherin thep53negativecelllineHep3Bascomparedtothep53expressing celllinesHepG2andKB3-1.Overexpressionofp53inHep3B like-wisedecreasedFat10reporteractivityaswellasendogenousFAT10 expression.Conversely,whenp53wasdownregulatedbyspecific siRNAinHep3Bcellsoverexpressingp53,Fat10reporteractivity increased.ThesameincreaseinendogenousFAT10mRNA expres-sionwasobtainedwhenendogenousp53wasdownregulatedby specificsiRNAinHEK293cells,clearlyunderliningthatp53 nega-tivelyregulatestheFat10promoter.Thesedataimplythatmutant p53,asfoundinmanytumors,wouldelevateFAT10expressionand alongwithitFAT10‘spro-malignantcapacities(Zhangetal.,2006).
Insupportofthishypothesis,themRNAandproteinexpression levelsofFAT10positivelycorrelatedwiththemRNAandprotein expressionlevelsofmutantp53ingastriccancertissueandpatients withhighFAT10expressionintheirtumorsshowedatendency towardsunfavorableprognosisregardingtheoverallsurvivalrate (Jietal.,2009).
Taken allthese datatogether,one canconstruct amodel of howtheexpressionlevelsofp53andFAT10mustbetightly regu-latedtopreventcellsfrombecomingmalignant(Fig.3).Innormal cellsexpressingfunctionalp53,FAT10expressioniskeptatalow
Fig.4.FAT10regulates-cateninstabilitybyinfluencingtheWntandAktsignaltransductionpathways.
TheWntsignalingpathwayplaysacriticalroleinpromotingHCCcarcinogenesisandmetastasis.Itleadstotheactivationofthetranscriptionfactor-cateninbypreventing itsphosphorylationbyGSK3andthusitssubsequentubiquitylationandproteasomaldegradation.FAT10contributestothemalignantcapacitiesof-cateninbydirectly interactingwithitandinhibitingitsubiquitin-dependentdegradationbytheproteasome.Inasecondline,upregulationofFAT10leadstoanincreaseofphosphorylatedand thusactivatedAkt,whichinturnphosphorylatesandinactivatesGSK3,whichagainresultsinstabilizationof-catenin.Adetaileddescriptionisgiveninthetext.Anarrow markedwitha“+”illustratesanactivatingpathway,anarrowmarkedwitha“−”illustratesanegativelyregulatedpathway.Redarrowwithtwoarrowheadsrepresentsthe interactionof-cateninandFAT10.
levelduetothenegativeregulationoftheFAT10promoterbyp53.
IncaseofDNAdamage,cellcyclearrestandDNArepair is per-formedtoletthecellre-enterthecellcycle,or,iftheDNAdamage istoosevere,apoptosisisinduced.Chronicinflammatory condi-tionswhichinducehighFAT10expressionleadtoatranscriptional downregulationofp53-dependentgeneexpressionandpromote theinteractionofFAT10withMAD2.Thisleadstoatooearlyonset ofanaphase, chromosomal instabilityand protectionfrom apo-ptosis.Thepresence ofmutant, non-functional p53might even enhancethiseffectandfurthersupportthetransforming capaci-tiesofFAT10.ThemaintenanceofthebalancebetweenFAT10and p53expressionisthereforeveryimportanttoprotectcellsfrom gettingmalignant.
5.3. ˇ-catenin
Thewntsignalingpathwayleadingtotheactivationofthe tran-scriptionfactor-cateninplaysacrucialroleinthedevelopmentof cancer.-catenin,aslongasitisnotphosphorylated,can translo-cateinto the nucleus where it forms heterodimers with other transcriptionfactorssuchasT-cellfactorandlymphoid enhancer-bindingfactor1,leading tothetranscriptionof genes,involved intheregulationofproliferation,differentiationandcellsurvival (Fig.4).Uponphosphorylationof-cateninbytheserine/threonine glycogensynthasekinase3(GSK3)andbycaseinkinaseI, -cateningetsubiquitylatedbytheE3ligase-TRCPanddegradedby theproteasome(Amitetal.,2002;Polakis,2000,2012).Mutations in-cateninwhichpreventitsphosphorylationandsubsequent ubiquitylation,andwhichresultinaconstitutiveactive transcrip-tion factor are found withsignificant frequency in HCC and it wasshown that-cateninplays akeyrole inthemetastasisof humanhepatocellularcarcinoma(deLaCosteetal.,1998;Laietal., 2011).Recently,itwasreportedthattheup-regulationofFAT10 expressionpromotedtheinvasion andmetastasisof hepatocel-lularcarcinomacellsbybindingnon-covalentlyto-cateninand preventingitsubiquitylationandsubsequentproteasomal degra-dation(Fig.4).Asaresult,theexpressionofHomeoboxB9(HOXB9),
amember oftheclassIhomeobox(HOX)genesand atargetof
-catenin/TCF4(Hatzisetal.,2008),wasfoundtobehighly up-regulatedin HCCandtheHOXB9mRNAandproteinexpression levelscorrelatedwiththemRNAandproteinexpressionlevelsof FAT10.HOX genesarenormally involved inembryogenesis but a role in cancerdevelopment has been proposed(Abate-Shen, 2002).HOXB9regulatestheexpressionofgenesinvolvedinthe regulationof cellmigration, invasionand metastasisformation.
FAT10andHOXB9overexpressioncorrelatedwithHCCtumorsize, tumormicrosatelliteformationandvascularinvasion,pointingto an involvement of both proteinsin HCC aggressiveness. Taken together,thesedatashowthatFAT10up-regulationstabilizesthe
-catenin/TCF4 signaling pathway by preventing its ubiquitin-dependentproteasomaldegradation,andthisinturninducesan increaseinHOXB9expression,leadingtoHCCinvasionand metas-tasisgeneration(Yuanetal.,2014).
Concurrentwiththesefindings,FAT10wasfoundtoinfluence theAkt/GSK3signalingpathway(Fig.4).The serine/threonine kinaseAkt(orPKB)isacentralnodeincellsignalingdownstream ofcytokines,growthfactorsandotherstimuli.Aktsignalingplays aroleinregulatingcellsurvival,differentiation,proliferationand angiogenesis,pointing toa roleoftheAktsignalingpathwayin carcinogenesis(Manningand Cantley, 2007).One ofthe down-streamtargetsofAktisGSK3,whichisinactivatedwhengetting phosphorylatedbyAkt,andsignaltransductionviaAkt/GSK3was showntopromoteTNF-␣-mediatedepithelial-mesenchymal tran-sition(EMT)(Wangetal.,2013).FAT10overexpressingHCCcell linesHepG2andSMMC-7721wereshowntoundergoEMTbecause theirmorphologychangedtowardsspindle-likefibroblasticcells, theexpressionoftheepithelialmarkerE-cadherinwasdecreased, andtheexpressionofthemesenchymalmarkerN-cadherinand vimentinwereincreased.Inaddition,theamountofsnail,an E-cadherin transcriptional repressor, was elevated in these cells.
FurtherinvestigationsshowedthatFAT10mediatedthiseffectvia theAkt/GSK3pathway.PhosphorylatedandtherebyactivatedAkt wasfoundtobehighly elevatedinFAT10overexpressing cells,
accompaniedbyanincreaseintheamountofphosphorylatedand thusinactivatedGSK3andastabilizationof-catenin(Liuetal., 2014).Anincreasein-catenininHCCwasalreadyreported ear-lierandit hadbeenshownthat thiscontributestodegradation of E-cadherin and inductionof EMT(Nejak-Bowen and Monga, 2011).In summary, FAT10seemsto elicitits stabilizing effects onto-cateninleadingtocellmigration,invasionandmetastasis ofHCCintwoways(Fig.4).Inafirstline,FAT10directlyinteracts with-cateninwhich prevents itsubiquitylation and proteaso-maldegradationleading toincreased-catenin levelsand thus toincreasedexpressionof downstream targetssuchasHOXB9.
Inasecondline,overexpressionofFAT10elevatestheamountof activeAkt,whichinturnphosphorylatesGSK3,renderingit inac-tivewhichinturnstabilizes-catenin.TheAkt-GSK3signaling pathwayisthereforeanotherintriguingmechanismofhowFAT10 exertsitscarcinogeniceffectsinlivercarcinoma.
5.4. Smad2
Still another downstream target of FAT10 wasidentified by investigating the contribution of FAT10 upregulation onto the developmentofglioma,aneuroepithelialtumorarisingfromglia cellsandaccountingfornearlyhalfofallintracranialtumors(Filbin andSuva,2016).Gliomasareclassifiedintofourgrades(I–IV)with increasingseverity.FAT10isprominentlyexpressedin gliomas, withthehighestmRNAandproteinexpressioningradeIVgliomas.
TheincreaseinFAT10expressionpositivelycorrelateswith pro-gressionofthediseaseandFAT10-positivepatientshavethelowest survivalrate(Daietal.,2016;Yuanetal.,2012).Gliomacelllines stably overexpressingFAT10 displayedenhanced tumorigenesis invitrobecausetheydevelopedsignificantlymorespheresand pos-sessedincreasingmigrationandinvasioncapacity.Wheninjected intonudemice,thesecellsdevelopedlargertumorsascompared tomock-transfectedcells,implicatingthatFAT10isalsopromoting gliomacarcinogenesis.FAT10seemstoexertitseffecton glioma-genesisviathetransforminggrowthfactorbeta(TGF1)signaling pathway.WhereasinnormalcellsTGFinhibitsproliferation,it turnsouttobeanoncogenicfactorinhighgradegliomabecause itinfluencesgliomaproliferation,survival,migration,invasionand theescapefromhostimmunity.DimericTGFbindingtooneofthe TGFtypeIandIIreceptordimersinducesconformationalchanges andactivateskinasedomainsofthereceptors.Thisleadstothe phosphorylationandactivationofSmad2andSmad3transcription factors,whichinteractwithSmad4andaccumulateinthenucleus wheretheyregulatethetranscriptionofTGFexpressionitselfbut alsoofgenese.g.involvedincellcycleregulation(Kaminskaetal., 2013).OverexpressionofFAT10inthegliomacelllineU251ledto significantlyincreasedlevelsofphosphorylatedSmad2(p-Smad2) andofothercancerstem-cellmarkerssuchasCD133,OCT4,nestin, andBmi-1.Inturn,knockdownofFAT10ortheapplicationofan inhibitoroftheTGFsignalingpathway(LY364947)reversedthis effect.Althoughtheexactmechanism ofthis functionofFAT10 needsfurtherclarification,itappearsthataberrantFAT10might influencetheprogressionofgliomabyenhancingthelevelof p-Smad2(Daietal.,2016).