The ubiquitin-like modifier FAT10 in cancer development
Annette Aichem
a, Marcus Groettrup
a,b,∗aBiotechnologyInstituteThurgauattheUniversityofKonstanz,CH-8280Kreuzlingen,Switzerland
bDivisionofImmunology,DepartmentofBiology,UniversityofKonstanz,D-78457Konstanz,Germany
a b s t r a c t
Duringthelastyearsithasemergedthattheubiquitin-likemodifierFAT10isdirectlyinvolvedincancer development.FAT10expressionishighlyup-regulatedbypro-inflammatorycytokinesIFN-␥andTNF-␣ inallcelltypesandtissuesanditwasalsofoundtobeup-regulatedinmanycancertypessuchasglioma, colorectal,liverorgastriccancer.Whilepro-inflammatorycytokineswithinthetumormicroenvironment probablycontributetoFAT10overexpression,anincreasingbodyofevidencearguesthatpro-malignant capacitiesofFAT10itselflargelyunderlieitsbroadandintenseoverexpressionintumortissues.FAT10 therebyregulatespathwaysinvolvedincancerdevelopmentsuchastheNF-B-orWnt-signaling.More- over,FAT10directlyinteractswithandinfluencesdownstreamtargetssuchasMAD2,p53or-catenin, leadingtoenhancedsurvival,proliferation,invasionandmetastasisformationofcancercellsbutalsoof non-malignantcells.InthisreviewwewillprovideanoverviewoftheregulationofFAT10expressionas wellasitsfunctionincarcinogenesis.
Contents
1. Introduction...452
2. Theubiquitin-likemodifierFAT10,aproteinoftheimmunesystem...452
2.1. ThestructureofFAT10andhowittargetsforproteasomaldegradation...452
2.2. FAT10inadaptiveandinnateimmunity...452
2.3. WhicharethetargetsofFAT10andhowisitconjugatedtoitssubstrates...453
3. RegulationofFAT10expressionunderinflammatoryconditionsandduringcancerdevelopment...454
3.1. FAT10expressionlevelsindifferentcancertypes...454
3.2. ThetransformingcapacityofFAT10...454
4. WhatarethemechanismsbehindFAT10overexpression?...455
5. FunctionalconsequencesofFAT10up-regulationininflammationandcancer ... 455
5.1. MAD2andchromosomalinstability...455
5.2. p53...456
5.3. -catenin...458
5.4. Smad2...459
6. Summaryandfutureprospects...459
Conflictofinterest ... 459
Acknowledgements...459
References...459
Abbreviations:AIPL1,aryl-hydrocarbonreceptorlike-1;APC/C,anaphase-promotingcomplex/cyclosome;DUB,deubiquitylatingenzyme;EMT,epithelial-mesenchymal transition;FAT10,HLA-Fadjacenttranscript10;GSK,glycogensynthasekinase;HCC,hepatocellularcarcinoma;HLA,humanleukocyteantigen;HOX,homeobox;IFN, interferon;IL,interleukin;MAD2,mitoticarrestdeficient2;mTEC,medullarythymicepithelialcells;NF-B,nuclearfactorkappa-B;NUB1L,NEDD8-ultimatebuster1long;
STAT,signaltransducerandactivatoroftranscription;SUMO,small-ubiquitin-likemodifier;TGF,transforminggrowthfactor;TNF,tumornecrosisfactor;UBD,UbiquitinD;
ULM,ubiquitin-likemodifier;UPS,ubiquitinproteasomesystem;USE1,UBA6-specificE2enzyme1.
∗Correspondingauthorat:DivisionofImmunology,DepartmentofBiology,UniversityofKonstanz,Universitaetsstr.10,D-78457Konstanz,Germany.
E-mailaddress:marcus.groettrup@uni-konstanz.de(M.Groettrup).
Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-347923
Erschienen in: The International Journal of Biochemistry & Cell Biology ; 79 (2016). - S. 451-461 https://dx.doi.org/10.1016/j.biocel.2016.07.001
1. Introduction
Acausallinkbetweenchronicinflammationorinfectionand tumorigenesisisnowadaysagenerallyacceptedprincipleleading tothedevelopmentofvariouscancertypes.Theunderlyingmech- anismisinpartbasedontheupregulationandsecretionofseveral proinflammatorycytokinessuchastumornecrosisfactor(TNF)␣, interferon(IFN)␥orinterleukin-6(IL-6)bystromalcells aswell asbyinfiltratingimmunecells inthetumormicroenvironment, orby cytokines,secreted bytumor cells themselves(Zamarron andChen,2011).Suchinflammatoryconditionsaregeneratedto promote healing and regeneration of the affected tissue. How- ever,thepersistingexpressionofthesecytokinescanalsoprovide anicheforcancerdevelopmentbypromotingDNAdamageand abnormal tissue healing. This is based on the fact that pro- inflammatory cytokines cause the activation of transcription factorssuchasnuclearfactorkappa-B(NF-B)andsignaltrans- ducerandactivatoroftranscription(STAT)3,whichplayacentral roleasactivatorsofanti-apoptoticgeneexpressionandcellprolif- eration.Furthermore,thesecytokinespromoteoptimalconditions for tumorcell proliferation,invasion and metastasisformation, apoptosisresistanceaswellasangiogenesis,i.e.processeswhich areallprerequisitesforcancerdevelopment(Grivennikovetal., 2010;Karin,2006).
Thereversiblepost-translationalmodificationofproteinswith asingleubiquitinorwithubiquitinchainsisanimportantmech- anismtoregulatethefunction,activity,cellulardistributionbut alsothestabilityofproteinsbymediatingtheirproteolysisbythe 26Sproteasome.The ubiquitinproteasomesystem(UPS)is not onlyanimportantregulatorofproteindegradationbutalsoreg- ulates cancer-relevantprocesses such ascell cycle progression, proliferation,DNAdamageresponse,angiogenesisandapoptosis (Ciechanover,1998;HaglundandDikic,2005).Moreover,inhibitors oftheproteasomearealreadyinuseorunderfurtherinvestiga- tionforthedevelopmentofcancertherapies(ChenandDou,2010;
Tuet al.,2012).Ubiquitylation aswellaspoly-ubiquitylationis achievedbyanenzymaticcascadewherefirstubiquitinisadeny- latedatitsC-terminaldiglycineresiduebyoneofitstwoubiquitin E1activatingenzymesUBE1(Ciechanoveretal.,1981)orUBA6(Jin etal.,2007;Pelzeretal.,2007)andthentransferredontotheactive site-cysteineoftherespectiveE1enzymetoformathioesterbond.
Inasecondstep,ubiquitinistransferredtotheactivesitecysteineof acognateubiquitinconjugatingenzyme(E2)byatransthiolation reaction.Finally,differentclassesofubiquitinligases(E3s)catal- ysetheisopeptidelinkageofubiquitintothe-amino-groupofan internallysineresidueofasubstrateprotein(Finley,2009;Kerscher etal.,2006).Ubiquitinitselfcontains7lysineresidues(atpositions 6,11,27,29, 33,48and 63)whichareusedtobuildupchains ofdifferentlinkagetypesconveyingdifferentoutcomesforsuch modifiedproteins.Modificationofaproteinwithasingleubiqui- tinisnotenoughtoguideproteinstoproteasomaldegradationbut requirestheattachmentofaubiquitinchainofatleastfourubiqui- tinmolecules,linkedthroughoneoftheabovementionedinternal lysineresidues.Alldifferentlinkagetypes,withtheexceptionof K63,leadtothedegradationofthemodifiedproteinbythe26S proteasome(Xuetal.,2009),abarrelshapedmulti-enzymecom- plex,consistingofa20Scoreparticleandoneortwo19Sregulatory particles(Finley,2009).Beforedegradation,ubiquitinisremoved fromtheproteinsbysocalleddeubiquitylatingenzymes(DUBs) andrecycled(Reyes-Turcuetal.,2009;Wolberger,2014).
Besidesubiquitin,severalubiquitin-likemodifiers(ULMs)have beenidentified.TheULMsfamilyincludesHumanLeukocyteAnti- gen(HLA)-F adjacenttranscript10(FAT10),small-ubiquitin-like modifier(SUMO)-1/2/3,ISG15,NEDD8,ATG8,ATG12,UFM-1,and URM-1.TheULMsshareasimilarthree-dimensionalstructure,the ubiquitinor-graspfold,aswellastheC-terminaldiglycinemotif
foractivationandcovalentattachmenttosubstrateproteinsbyan E1–E3enzymaticcascade(Hochstrasser,2009).OfalltheseULMs, FAT10istheonlymodifierwhichactsasanautonomoustransfer- ablesignalfordegradationbythe26Sproteasomeanditwasshown thatthisprocesscanoccurindependentlyofubiquitin(Hippetal., 2005;Schmidtkeetal.,2009).FAT10isaproteinoftheimmunesys- temanditisstronglyup-regulatedbypro-inflammatorycytokines (Fan et al.,1996; Raasietal., 1999)and during thematuration of antigen presenting cells (Buerger et al., 2015;Ebstein et al., 2009;Lukasiaketal.,2008).Moreover,numerousrecentpublica- tionspointtoadirectinvolvementofFAT10incancerdevelopment.
Theserecentinsightswillbepresentedinthisreviewalongwith thecurrentstateofknowledgeontheregulationandfunctionof FAT10expressionininflammationandtumorigenesis(Fig.1).
2. Theubiquitin-likemodifierFAT10,aproteinofthe immunesystem
2.1. ThestructureofFAT10andhowittargetsforproteasomal degradation
TheFat10gene(originallydesignatedUbiquitinD(Ubd))was identifiedbygenomicsequencingofthehumanmajorhistocom- patibilitycomplex(MHC)byShermanWeissmanandcolleaguesin 1996(Fanetal.,1996).Thestructuralmodelofthe165aminoacid long,18kDaproteinFAT10aswellasapreviouslypublishedNMR structureoftheN-terminalubiquitin-likedomainofFAT10predicts twoubiquitin-likedomains,bothwithatypical-graspfold.TheN- andC-terminalubiquitin-likedomainsare29%and36%identicalto ubiquitin,respectively,arrangedinatandemarrayandseparated byashortlinker(Fanetal.,1996;Groettrupetal.,2008;Thengetal., 2014).Incontrasttoubiquitinandotherubiquitin-likemodifiers whichareexpressedasinactiveprecursorsandwhichneedtobe activatedbyproteolyticprocessingattheirC-terminusbyspecific proteases(Kerscheretal.,2006),FAT10isalreadysynthesizedasa matureproteinwithafreediglycinemotifatitsC-terminusandcan directlybeactivatedandconjugatedtosubstrateproteins(Raasi etal.,2001).AcentralfunctionofFAT10modificationistotarget proteinsfordegradationbytheproteasome(Hippetal.,2005;Raasi etal.,2001;Schmidtkeetal.,2014).Thisprocesswasshown to beindependentofubiquitylationbuttobedependentonanon- covalentinteraction partner of FAT10, namelyNEDD8-ultimate buster1long(NUB1L)(Hippetal.,2005;Schmidtkeetal.,2009, 2006).NUB1L,alikewiseinterferon-inducibleprotein(Kitoetal., 2001),actsasasolublereceptorandbringsFAT10andFAT10ylated proteinstotheproteasomewhereitbindstothe19Sregulatory subunitsRPN10 (S5a) andRPN1 (S2).It wasshown thatFAT10 itselfcouldalsodirectlybindtotheVWAdomainofRPN10inthe absenceofNUB1LbutthatthedockingofNUB1Ltotheproteasome wasnecessaryfortheaccelerateddegradationofFAT10(Ranietal., 2012;Schmidtkeetal.,2009,2006).Incontrasttoubiquitin,FAT10 hasaveryshorthalflifeofabout1handseemstobedegraded bytheproteasomealongwithitssubstrates(Aichemetal.,2014;
Hippetal.,2005).SinceNUB1Lnegativelyinfluencesthestability ofFAT10,theFAT10degradationratemightbedifferentindiffer- entcelllinesortissuesexpressingdifferentamountsofNUB1L,as e.g.inHEK293cells, whichexpressonlylowamountsofNUB1L andwhereFAT10ismorestable(Aichemetal.,2014).Inlinewith this,noFAT10-deconjugatingenzymes,whichmightcontributeto FAT10recycling,havebeenidentifiedtodate.
2.2. FAT10inadaptiveandinnateimmunity
TheFAT10geneisencodedintheMHCclassIlocusandthehigh- estFAT10mRNAexpressionismeasuredinorgansoftheimmune
Fig.1. SchemeshowingtheregulationofFAT10expressionaswellasdownstreamtargetswhichareinfluencedbyFAT10overexpressionintumortissues.
ThebroadandintenseoverexpressionofFAT10intumortissues,inducedbypro-inflammatorycytokinessuchasIFN-␥,TNF-␣andIL-6withinthetumormicroenvironment, contributestothemalignantcapacitiesofFAT10itself.FAT10regulatespathwaysinvolvedincancerdevelopmentsuchasNF-B-,Akt-,orWnt-signalingandalsodirectly interactswithandinfluencesdownstreamtargetssuchasp53,-catenin,SMAD2,andMAD2,leadingtoenhancedsurvival,proliferation,invasionandmetastasisformation ofcancercellsbutalsoofnon-malignantcells.Arrowsmarkedwitha“+”illustrateanactivatingpathway,arrowsmarkedwitha“−”illustratenegativelyregulatedpathways.
systemsuchasthymus,lymphnodes,fetalliverandspleen(Canaan etal.,2006;Leeetal.,2003;Liuetal.,1999;Lukasiaketal.,2008).
ThehighexpressionofFAT10inthethymuscouldbeallocated tomedullarythymic epithelialcells (mTECs)and it wasshown thatFAT10modifiesthymicT-cellselection,probablydue toan alteredpeptidepresentationonMHCclassImolecules(Buerger etal.,2015).AdditionaldatapointtoaroleofFAT10inMHCclassI antigenpresentation.Ebsteinandcolleagues(Ebsteinetal.,2012) showedthatFAT10couldenhancetheMHCclassIpresentation ofthehumancytomegalovirus(HCMV)-derivedantigenpp65in HeLacellsbyabouttwo-foldascomparedtountaggedpp65and Schlieheand colleagues(Schlieheet al.,2012)showedthatthe degradationrateaswellasthepresentationofspecificlymphocyte choriomeningitisvirus(LCMV)epitopesonMHCclassImolecules wasenhancedwhenFAT10wasfusedtotheN-terminusofthe LCMVnucleoprotein.ApartfromitsexpressioninmTECsFAT10is alsoconstitutivelyexpressedinmatureBcellsanddendriticcells (Batesetal.,1997)anditsexpressionishighlyandsynergistically inducibleonmRNAandproteinlevelinnumeroustissuesandcell typesbythepro-inflammatorycytokinesIFN␥andTNF␣(Aichem etal.,2010;Raasietal.,1999).Arecentpublicationshowed,that ahighinductionofFAT10expressionisalsoachievedbyasyner- gistictreatmentofHepG2cellswithTNF-␣incombinationwith IL-6(Choietal.,2014).UpregulationofFAT10expressioninduced caspase-dependentapoptosisinHeLacells,renaltubularepithe- lialcellsaswellasmousefibroblasts(Liuetal.,1999;Raasietal., 2001;Rossetal.,2006).However,theroleofFAT10inapoptosis iscontradictorybecauseotherpublicationsshowedapro-survival roleofFAT10(Canaanetal.,2006).LymphocytesofFAT10knock outmicewereshowntobemoresusceptibletospontaneouscell death than lymphocytes from wildtype mice and human and mousecardiacmyocyteswereprotectedfromapoptosisinduced bymyocardialinfarctionassociatedhypoxia/reoxygenationstress throughup-regulationofFAT10(Pengetal.,2013).
NexttotheroleofFAT10inadaptiveimmuneresponsesthere arerecentpublicationswhichassignafunctionininnateimmu- nitytoFAT10.FAT10wasshowntoplayaroleintheintracellular defense against bacteria (Spinnenhirn et al., 2014). Intracellu- larSalmonellaTyphimuriumwhich weretargetedforxenophagy
bybeingdecoratedwithubiquitin,theautophagyreceptorsp62 and NDP52as wellas withLC3weresimultaneouslydecorated withFAT10.Spinnenhirnandcolleagues(Spinnenhirnetal.,2014) showedthatthepresenceof FAT10resultedina prolongedlife span of S. Typhimurium-infected mice,pointing to a protective roleofFAT10.ItisassumedthatFAT10playsadditionalrolesin innateimmunitysinceanup-regulationofFAT10expressionwas described in HIV-infected renal tubular epithelial cells (RTECs) (Rossetal.,2006;Snyderetal.,2009)andduringKaposisarcoma- associatedherpesvirusinfection(Hongetal.,2004).Moreover,the groupofJ.-Y.YooshowedthatFAT10interferedwithRIG-I(retinoic acidinduciblegene1)mediatedantiviralsignalingnecessaryfor theproductionoftypeIinterferons.Bynon-covalentlyinteracting withRIG-I,FAT10sequesteredRIG-Iintoinsolubleaggregatesand thisresultedinadiminishedanti-viralimmuneresponse(Nguyen etal.,2016).
2.3. WhicharethetargetsofFAT10andhowisitconjugatedtoits substrates
UponIFN-␥/TNF-␣mediatedinductionofFAT10inthehuman embryonic kidney cell line HEK293, the FAT10 mRNA was detectable already after 4–5h, however, FAT10 protein was prominentlydetected inwestern blotsonly 24haftercytokine stimulation withthe highlysensitive monoclonalantibody 4F1 (Aichemetal.,2012,2010).Nevertheless,uponimmunoprecipita- tionofFAT10anditsmodifiedsubstrateswithmAb4F1,combined withasubsequentwesternblotanalysiswithapolyclonalFAT10 specificantibody,bulkFAT10conjugatesofalargerangeofsizesare detectable,similartoubiquitin,ISG15orSUMOconjugates.Amass spectrometryanalysisidentified 571putativeFAT10 interaction partners,ofwhich176proteinswereclassifiedasputativeconju- gationsubstratesandtheremaining395proteinsasnon-covalent interactionpartners.Interestingly,theseproteinsdidnotbelong todistinctcellularpathwaysbutcoveredalotofdifferentcellu- larfunctions,showingthatFAT10mightplayaroleinnumerous cellbiologicalprocesses(Aichemetal.,2012;Lengetal.,2014).Of importanceisthatFAT10apparentlyisnotonlyfunctionalwhen covalentlyconjugatedtoitssubstrateproteins,suchastheubiqui-
tinactivatingenzymeUBE1(Bialasetal.,2015;Ranietal.,2012), butalsowheninteractingnon-covalentlywithinteractionpartners suchasHDAC6(Kalverametal.,2008),aryl-hydrocarbonreceptor like-1(AIPL1),animportantproteinwithintheretina(Bettetal., 2012),ortheautophagosomalreceptorp62/SQSTM1whichinter- actsboth,covalentlyandnon-covalentlywithFAT10(Aichemetal., 2012).ActivationandconjugationofFAT10toitssubstrateproteins isperformedmostprobablybyathreestepenzymaticcascade,as describedforubiquitin.TheFAT10E1activatingenzymeUBA6acts asabi-specificE1enzyme,becauseitisabletoactivateboth,FAT10 andubiquitin(Chiuetal.,2007;Pelzeretal.,2007).Indoingso, UBA6isabletodistinguishbetweenthetwomodifiersandbinds FAT10withahigheraffinitythanubiquitin,althoughtheadeny- lationand transthiolationreaction isslowerfor FAT10than for ubiquitin(Gavinetal.,2012).TheactivatedFAT10isthentrans- ferredontotheactivesitecysteineofthelikewisebi-specificE2 conjugatingenzymeUBA6-specificE2enzyme1(USE1,alsocalled UBE2Z)whichdirectlyundergoesauto-FAT10ylationincis,mainly onLys323(Aichemetal.,2014,2012;Guetal.,2007;Jinetal.,2007).
Aportionoftheauto-FAT10ylatedUSE1issubsequentlydegraded bytheproteasome,albeit theremainingUSE1-FAT10conjugate remainsactivewithrespecttoFAT10andubiquitintransfer.There- fore,thismechanismmightrepresentanegativefeedbackloopto limittheFAT10conjugationpathway(Aichemetal.,2014).Sofarno FAT10-specificE3ligaseshavebeenidentified,butsinceallULMs whichgetcovalentlyconjugatedtosubstratesneedsuchligasesto befinallytransferredontotheirsubstrates,itisassumedthatsuch E3ligasesmustalsoexistforFAT10.
3. RegulationofFAT10expressionunderinflammatory conditionsandduringcancerdevelopment
3.1. FAT10expressionlevelsindifferentcancertypes
FAT10expressionishighlyup-regulatedinmanydifferentcan- certypessuchascoloncancer, hepatocellularcarcinoma(HCC), gastrointestinalandpancreaticcarcinomas,gynecologicalcarcino- mas,andglioma(Leeetal.,2003;Lukasiaketal.,2008;Qingetal., 2011;Sunetal.,2014;Yuanetal.,2012).InliverneoplasiaFAT10 isevendescribedasanepigeneticmarker(Frenchetal.,2010a,b;
Olivaetal., 2008).Moreover,inseveral cancertypesa statisti- callysignificantassociationbetweenahighFAT10expressionlevel andtheprogressionandseverityofthediseaseincludingahigher propensityformetastasisformationandpoorprognosis,hasbeen reported.Theseincludetriple-negativebreastcancer(Hanetal., 2015),glioma(Daietal.,2016;Yuanetal.,2012),pancreaticductal adenocarcinoma(Sunetal.,2014),HCC(Liuetal.,2014),orgastric cancer(Jietal.,2009).Furthermore,coloncancerpatientstreated withapostoperativechemotherapyandbearingtumorswithhigh FAT10expressiondisplayedahigherrecurrenceratethanthose withFAT10-negativetumorsandFAT10expressionwasassociated withasignificantlyshortersurvivaltime(Zhaoetal.,2015).
3.2. ThetransformingcapacityofFAT10
Lukasiaket al.(2008) showedthat thehighupregulation of FAT10expressionincancersofliverandcolonstronglycorrelated withtheexpression of the proteasomesubunit LMP2which is inducedintheseorgansbypro-inflammatorycytokinesIFN-␥and TNF-␣inthetumormicroenvironment.Byperformingacolonyfor- mationassaywithFAT10transfected NIH3T3cells,noevidence forcolonyformation andtransformation capacityofFAT10was observed,arguingagainstafunctionofFAT10asaproto-oncogene
inthisexperimentalmodel.Thereforeitwasquestionedwhether FAT10 hastransforming capacity and whether sucha property couldexplainitsoverexpressioninthesecancers.However,FAT10 overexpressionintheimmortalizedlivercelllineNeHepLxHTcould confermalignantpropertiestotheseotherwisenon-tumorigenic cells.NeHepLxHTcells,overexpressingclinicallyrelevantamounts ofFAT10,showedadose-dependenteffectofFAT10onprolifera- tion,anchorageindependentgrowth–ahallmarkoftransformed malignantcells–andresistancetoapoptosis(Gaoetal.,2014).In addition,ascompared tocontrolcells, FAT10expressingNeHe- pLxHTcellssignificantlyincreasedtheirinvasivecapacity across matrigelaswellastheirmigratorycapacityinascratch-wound- healingassay,againinaFAT10dose-dependentmanner.Thesame resultswereobtainedwhenusingasecondcelllinestablytrans- fectedwithaFAT10expressionplasmid,namelythetumorigenic colorectalcancercelllineHCT116.FAT10expressingNeHepLxHT cellsinducedtumorformation,againinadose-dependentmanner, wheninoculatedintonudemiceascomparedtomice,whichhad beentreatedwithemptyvector-transfected,FAT10-negativecon- trolcells.Furthermore,FAT10overexpressioninHCT116cellseven greatlyenhancedtumorgrowthascomparedtoFAT10-negative HCT116cells,stronglyarguingforadirectinvolvementofFAT10in tumorformation(Gaoetal.,2014).ThereforeitseemsthatFAT10 cannotonlyconfermalignantpropertiestonon-tumorigeniccells butthatitcanevenaugmentthemalignancyassociatedtraitsofa cancercellline.
AdditionaldataalsosuggestedFAT10tobeaproto-oncogene:
knockdownofFAT10intheFAT10-positivehepatocellularcarci- nomacelllineHep3Bspecificallyinhibitedcellproliferationand colony formation under in vitroconditions, most probably due toablockageoftheentryintotheS-phaseofthecellcycleand byinducingapoptosis,asmeasuredbyAnnexinVstaining.Under invivoconditionsusingnudemicewhichhadreceivedasubcuta- neousinjectionofHep3BcellsitwasfurthershownthattheHep3B xenograftgrowthwasmuchreducedwhenthemiceweretreated withanintratumoralinjectionofAd-siRNA/FAT10twoweeksafter injectionoftheHep3Bcells.TheknockdownofFAT10evenpro- longedthelifespanofthesemiceascomparedtomice,whichhad beentreatedwithacontrolvirusorPBS(Chenetal.,2014).
ThenotionthatFAT10isdirectlyinvolvedinthedevelopment oftumorsisatleastconsistentwithrecentinvestigationsofFAT10 geneknockoutmice.Thesemiceareviableandfertileandexhibit onlyminimalphenotypicdifferencestotheirwildtypecounter- partssuchase.gahighersensitivitytowardsendotoxinchallenge.
Inaddition,asalreadymentioned,lymphocytesfromthesemice aremorepronetospontaneousapoptoticcelldeath(Canaanetal., 2006).Noteworthy,whenexaminingolderFAT10-deficientmice, itcouldbeobservedthatthesemiceshoweddelayedagingand anappox.20%increaseinlifespan.Thisphenotypewasaccompa- niedwithasignificantreductioninadiposetissueandanincreased metabolisminmuscletissue.InmusclesofFAT10deficientmice, decreasedlevelsofpro-inflammatorycytokinesandenhancedlev- elsofanti-inflammatorycytokinessuchasIL-10weremeasured, therebycreatinganinflammation-suppressivemilieuinskeletal muscleandbloodwhichmayaccountfortheabsenceoftumorsin thesemice(Canaanetal.,2014).
Taken all these findings together, several publications sup- port thenotion that FAT10itself is a proto-oncogene and that itsupregulationduringinflammatoryprocessesisinvolvedinthe developmentofdifferentcancertypes.Thisconceptionposesfur- therquestions:(1)HowistheexpressionofFAT10regulatedduring inflammationandcancer?(2)Whicharethedownstreamtargetsof FAT10?and(3)HowdoesFAT10enhancethemalignantproperties oftheseproteins.
4. WhatarethemechanismsbehindFAT10overexpression?
What are the molecular mechanisms how FAT10 exerts its malignanteffects?Onepotentialmechanismcouldbebasedona recentlydescribedinvolvementofFAT10inNF-Bsignaling(Fig.1).
NF-Bis animportanttranscription factorfor theregulationof genesinvolvedininflammation,proliferation,cell-cyclecontrol, apoptosis,andtheimmuneresponseandplaysthereforeacriti- calroleincarcinogenesis(HoeselandSchmid,2013).Ontheone handFAT10mayactasaregulatorofNF-Bsignalingandonthe otherhandFAT10expressionitselfisregulatedbyNF-B.Asalready pointedout, FAT10mRNAand proteinexpressionis highlyand synergisticallyinducibleinallcelltypesbythepro-inflammatory cytokinesIFN-␥andTNF-␣(Aichemetal.,2010;Raasietal.,1999;
Renetal.,2011)orbyacombinationofTNF-␣andIL-6asshown inHepG2cells(Choietal.,2014).InHCT116cellsitwasshown, thatTNF-␣signalingviatheTNF-␣receptor1 (TNFR1)induced FAT10expression,areceptormainlyfoundontumorandstromal cellswhereitmediatestheactivationofNF-B(Balkwill,2009;Ren etal.,2011).TheanalysisoftheFAT10promotersequencerevealed sevenpotentialp65/NF-Bbindingsitesaswellasthreebinding sitesfortranscriptionfactorsofthefamilyofsignaltransducerand activatoroftranscription(STAT)3(Canaanetal.,2006;Choietal., 2014;Gaoetal.,2015).NF-BandSTAT3cooperativelyregulate anumberoftargetgenesanditwasshownthatbindingofone ofthesetranscriptionfactorstotherespectivepromotersequence facilitatesrecruitmentoftheother,andviceversa.STAT3activa- tionismediatedmainlybyIL-6andthisleadstotheexpression ofgenesinvolvedincellproliferation,angiogenesisandsurvival.
STAT3isconstitutivelyactiveinmanycancertypessuchasbreast andcoloncanceranditsexpressionlevelpositivelycorrelateswith theaggressivenessof thetumor (Bosch-Barrera andMenendez, 2015;HoeselandSchmid,2013;WakeandWatson,2015).Choi etal.(2014)haveshownthatSTAT3andp65/NF-Bactsynergisti- callytoactivateFAT10expressionuponIL-6andTNF-␣induction.
FurthersupportfortheregulationofFAT10expressionbyNF-B signalingisderivedfromexperimentswithasubstancecalledsilib- inin,aflavonoidisolatedfromseedextractsoftheherbmilkthistle (Silybummarianum).Silibininexhibitsseveralpotentanti-cancer propertiessuchasactingasanantioxidant,havingimmunomodu- latoryandanti-proliferativepropertiesanditwasshowntoinhibit multiplecancersignalingpathwaysasforinstanceNF-Bsignal- ing(Bosch-BarreraandMenendez,2015;Singhetal.,2004;Tyagi etal.,2012).CarolineLeeandcolleaguesfoundthatupontreatment ofIFN-␥/TNF-␣-stimulatedHCT116cellswithincreasingamounts of silibinin, the FAT10 mRNA expression was diminished in a dose-dependentmannerandthiswasduetoasilibinin-mediated preventionofthenucleartranslocationofp65mostprobablydue toaninhibitionofthedegradationoftheNF-BinhibitorIB␣(Gao etal.,2015).TumorformationbyTNF-␣-inducedandthusFAT10 expressingHCT116cellsinnudemicewasdiminishedwhenthe miceweretreatedatthesametimewithsilibininwhichcorrelated withreducedFAT10expressioninthetumors.Thedependencyof endogenousFAT10expressiononNF-Bsignalingwasfurthersup- portedbydatashowingthattheinductionofFAT10expressionby TNF-␣wasgreatlydiminishedwhenp65wasdownregulatedby specificsiRNAsinHCT116andHepG2cells(Gaoetal.,2015;Ren etal.,2011).
TheinterplaybetweenFAT10and NF-Bmaynotbea one- waystreetsinceinapreviousstudyusingrenaltubularepithelial cells, FAT10was reportedtoactivate NF-B signaling,pointing to a potential positive feedback loop. Consistently, the TNF-
␣-induced IB␣ degradation, as well as the expression of the NF-B-dependentgenesCXCL2andMCP-1wasimpairedinFAT10- deficient cells (Gong et al., 2010).Arguing against a RTEC cell type-confined effectofFAT10 onNF-Bsignaling,a microarray
basedtranscriptomeanalysisofFAT10overexpressingNeHepLxHT andHCT116cellsrevealedinbothcelllinesthattheNF-Btran- scriptional activity was significantly enhanced as compared to controlcells, notexpressingFAT10.Thiswasaccompaniedbya highernuclearlocalizationofNF-B/p65,whilethetotalcellular amountofp65remainedstable.Atthesametime,theamountof theNF-BinhibitorIB␣wasdiminishedinFAT10overexpressing NeHepLxHTcells.Asimilarincreaseinnuclearp65wasobserved inHCT116cells,overexpressingFAT10.Theincreaseinnuclearp65 couldbereversedbyknockingdownFAT10withFAT10-specific siRNAs(Gaoetal.,2014).Takentogether,itwasshownthatthe increasedFAT10expressionindirectlyaugmentedNF-Bactivity byalleviatingtheinhibitionofNF-B.NF-Bisatranscriptionfac- torwithmultipletargetgenes,sothequestionarises,whicharethe genesdownstreamofFAT10andNF-B?Themicroarrayexpression analysisofFAT10overexpressingNeHepLxHTandHCT116cellsby Gao etal.(2014)revealedthat thechemokinereceptorsCXCR4 and CXCR7 were highly up-regulated in FAT10 overexpressing NeHepLxHTcells,consistentwithotherdatashowingthatNF-B isa transcriptionfactorforboth genes(KatohandKatoh,2010;
Tarnowskietal.,2010).CXCR4and−7arebothdescribedtopro- moteinvasionandtumorgrowthinseveralcancertypes(Gaoetal., 2010;Heinrichetal.,2012;Liangetal.,2005;Zhengetal.,2010).In accordancewiththesedata,ansiRNA-mediatedknockdownofp65 oroverexpression ofIB␣inFAT10overexpressingNeHepLxHT cellssignificantlyattenuatedcellinvasionandmigrationinvitro, precisely assilencingofCXCR4 andCXCR7 did.Taken together, these datapoint towards a mechanism where up-regulationof FAT10leadstoactivationofNF-Bsignalingwhichinturnactivates theexpressionofCXCR4and−7,resultinginenhancedmigration andinvasionofFAT10overexpressingcells(Gaoetal.,2014).
Insummary,itemergesthatFAT10expressionuponTNF-␣/IFN-
␥ or TNF-␣/IL-6 stimulation is mediated mainly by NF-Band STAT3signaling.Thereby,a FAT10-dependent positivefeedback loopseemstoexistwhichenhancesNF-Bactivationandwhich maycontributetothemalignantpropertiesofFAT10.Thisdirectly leadstothenextquestion,namelywhichproteinsgettargetedby FAT10and whataretheconsequences oftheirinteractionwith FAT10withrespecttocancerdevelopment.
5. FunctionalconsequencesofFAT10up-regulationin inflammationandcancer
5.1. MAD2andchromosomalinstability
UpregulationofendogenousFAT10expressionbyTNF-␣aswell asoverexpressionofFAT10hasdirectlybeenlinkedtochromoso- 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).MAD2bindstothekine- tochoresofsisterchromatidsinthemetaphaseofthecellcycle.
Thereit ensures thatbeforetheonset of anaphaseallchromo-
Fig.2.InteractionofFAT10withMAD2inducesaprematureonsetofanaphase,resultinginaneuploidy.
(A)Undernormalconditions,theonsetofanaphaseandtheseparationofthesisterchromatidsisinhibiteduntilallchromosomesareproperlyattachedviatheirkinetochores tothemitoticspindleapparatus.ThismechanismissurveilledbyaproteincomplexcontainingMAD2.(B)UpregulationofFAT10allowsthenon-covalentinteractionof FAT10withfreeMAD2andinturnreducestheamountofMAD2atthekinetochores.Asaresult,anaphasestartstooearlyandresultsininappropriateseparationofsister chromatidsandaneuploidy.Adetaileddescriptionisgiveninthetext.
somesbecomeproperlyattachedtothemitoticspindlealongthe metaphaseplate(Fig.2A).MAD2achievesthisbyinteractingwith andinhibitingCdc20,aco-activatorandsubstraterecruitmentfac- toroftheanaphase-promotingcomplex/cyclosome(APC/C),which isamulti-subunitubiquitinE3ligase.Therebytheubiquitylation ofsecurinandcyclinisprevented.Securinbindsandinhibitsapro- 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,FAT10overexpres- sioninducedtheformationofmultinucleatedcellswithabnormal nuclearmorphology.A constitutive overexpression of FAT10as wellasconstitutivetreatmentwithTNF-␣resultedinanincreased chromosomenumberascomparedtotheparentalHCT116cellline (Renetal.,2006,2011).However,whenthefiveputativeinterac- tionsitesofMAD2withintheN-terminalubiquitin-likedomainof FAT10weremutated(H11D,R13Q,H75D,T77DandK79Q),bind- ingofMAD2toFAT10wasgreatlydiminished.Ascomparedto cellsoverexpressingwildtypeFAT10,thechromosomenumbersof cellsexpressingthemutantFAT10returnedtonormalasfound inwildtypeHCT116cells.Thisfurthersupportsthenotionthat FAT10overexpressioncauseschromosomalinstabilitybyinteract- ingwithMAD2.Moreover,ascomparedtotheparentalcells,FAT10 overexpressingcellsshowedlong,incompletelycondensedchro- mosomesandthiswasalsorevertedbacktonormallycondensed, short“ribbon-like”chromosomes incellsexpressingthemutant
FAT10deficientinMAD2binding.Inlinewiththisobservation,the inhibitionoftheFAT10andMAD2interactionalsorevertedthe capabilityofFAT10overexpressingcellstoescapethenocadozole- inducedmitoticarrestbacktolevels,seenwithwildtypeHCT116 cells(Thengetal.,2014).Takentogether,thesedatacorroboratethe hypothesisthatthenon-covalentinteractionofFAT10andMAD2 is critical for thedevelopment of aneuploidyin cancer cells. It wasshownthat FAT10interacts withfree butnot withspindle boundMAD2asanimmunoprecipitationagainstFAT10ledtoaco- immunoprecipitationofMAD2only,andnotofadditionalproteins ofthespindlecomplexsuchasMAD1andBub1(Thengetal.,2014).
TheobservedlowlevelofendogenousFAT10expressioninG2/M phase(Limetal.,2006)suggeststhatcellskeeptheFAT10level lowduringthemitoticphaseinordertopreventmitoticdysfunc- tionswiththeriskofsubsequentcancerdevelopment.Consistently, HCT116cellsoverexpressingtheMAD2interaction-deficientFAT10 mutantretainedsignificantlylowerproliferationratesandshowed anincreasedsusceptibilitytoapoptosisascomparedtocellsover- expressingwildtypeFAT10.Moreover,cellsexpressingtheMAD2 binding-deficientFAT10mutant failedtogrowin soft agarand losttheircapabilitytogrowinananchorageindependentman- ner.Moreimportantly,thisfindingcouldbesubstantiatedinvivo byinjectingHCT116cellsexpressingFAT10wildtypeortheMAD2 binding-deficientFAT10mutant intonudemice.Tumorsgrown fromHCT116cellsexpressingMAD2binding-deficientFAT10were significantlysmallerthanthoseexpressingwildtypeFAT10(Theng etal.,2014).Insummary,allthesedatafromwellcharacterizedcell linemodelsofhepatocellularcarcinomamakeaconvincingcase, thatthepro-malignantcapacitiesofFAT10relyextensivelyonits interactionwiththespindlecheckpointproteinMAD2,therebypre- ventingtheproperfunctionofMAD2inmitoticcheckpointcontrol (Fig.2).
5.2. p53
Thetranscriptionfactorandtumorsuppressorp53playsacen- 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%ofallhumancancersdisplaydele- tionsormutationsinthep53gene.Undernormalconditions,the concentrationofp53iskeptatalowlevelandthisismaintained byatightregulationofp53ubiquitylationanddeubiquitylation.
Twomaincellulareventsregulatedbyp53arecell cyclearrest uponDNAdamageandinductionofapoptosis.DNAdamageleads toarapidup-regulationofp53,itsactivationbyphosphorylation anditstrans-localizationintothenucleuswhereitactseitheras anactivatororasaninhibitoroftranscription.Down-streamtar- 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).Additionaldatashowedthatp53getscova- lentlymodifiedbyoverexpressedFAT10inHEK293cells,andthat overexpressionofwildtypebutnotofmutantFAT10,inwhichthe diglycinemotifattheC-terminuswasmutated,increasedthetran- scriptionalactivityofa luciferasereporterconstruct,containing bindingsidesforp53(Lietal.,2011).However,underinflamma- 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).Thesystemseemstobetightlyregulatedbyaneg- ativefeedbackmechanismbecauseitwasshown,thatp53itself negativelyregulatestheexpressionofFat10mRNAbybindingto aninhibitorysitewithintheFat10promoter.Itwasshownthat thetranscriptionalactivityof-galactosidasereporterconstructs underthecontroloftheFat10promoterwassignificantlyhigherin thep53negativecelllineHep3Bascomparedtothep53expressing celllinesHepG2andKB3-1.Overexpressionofp53inHep3Blike- wisedecreasedFat10reporteractivityaswellasendogenousFAT10 expression.Conversely,whenp53wasdownregulatedbyspecific siRNAinHep3Bcellsoverexpressingp53,Fat10reporteractivity increased.ThesameincreaseinendogenousFAT10mRNAexpres- sionwasobtainedwhenendogenousp53wasdownregulatedby specificsiRNAinHEK293cells,clearlyunderliningthatp53nega- 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 howtheexpressionlevelsofp53andFAT10mustbetightlyregu- 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 isper- formedtoletthecellre-enterthecellcycle,or,iftheDNAdamage istoosevere,apoptosisisinduced.Chronicinflammatorycondi- tionswhichinducehighFAT10expressionleadtoatranscriptional downregulationofp53-dependentgeneexpressionandpromote theinteractionofFAT10withMAD2.Thisleadstoatooearlyonset ofanaphase, chromosomal instabilityand protectionfromapo- ptosis.Thepresence ofmutant, non-functional p53might even enhancethiseffectandfurthersupportthetransformingcapaci- tiesofFAT10.ThemaintenanceofthebalancebetweenFAT10and p53expressionisthereforeveryimportanttoprotectcellsfrom gettingmalignant.
5.3. ˇ-catenin
Thewntsignalingpathwayleadingtotheactivationofthetran- scriptionfactor-cateninplaysacrucialroleinthedevelopmentof cancer.-catenin,aslongasitisnotphosphorylated,cantranslo- cateinto the nucleus where it forms heterodimers with other transcriptionfactorssuchasT-cellfactorandlymphoidenhancer- 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,andwhichresultinaconstitutiveactivetranscrip- 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 preventingitsubiquitylationandsubsequentproteasomaldegra- dation(Fig.4).Asaresult,theexpressionofHomeoboxB9(HOXB9),
amember oftheclassIhomeobox(HOX)genesand atargetof
-catenin/TCF4(Hatzisetal.,2008),wasfoundtobehighlyup- 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,leadingtoHCCinvasionandmetas- 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 ofthedown- streamtargetsofAktisGSK3,whichisinactivatedwhengetting phosphorylatedbyAkt,andsignaltransductionviaAkt/GSK3was showntopromoteTNF-␣-mediatedepithelial-mesenchymaltran- sition(EMT)(Wangetal.,2013).FAT10overexpressingHCCcell linesHepG2andSMMC-7721wereshowntoundergoEMTbecause theirmorphologychangedtowardsspindle-likefibroblasticcells, theexpressionoftheepithelialmarkerE-cadherinwasdecreased, andtheexpressionofthemesenchymalmarkerN-cadherinand vimentinwereincreased.Inaddition,theamountofsnail,anE- cadherin transcriptional repressor, was elevated in these cells.
FurtherinvestigationsshowedthatFAT10mediatedthiseffectvia theAkt/GSK3pathway.PhosphorylatedandtherebyactivatedAkt wasfoundtobehighly elevatedinFAT10overexpressing cells,
accompaniedbyanincreaseintheamountofphosphorylatedand thusinactivatedGSK3andastabilizationof-catenin(Liuetal., 2014).Anincreasein-catenininHCCwasalreadyreportedear- 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 andproteaso- maldegradationleading toincreased-catenin levelsand thus toincreasedexpressionof downstream targetssuchasHOXB9.
Inasecondline,overexpressionofFAT10elevatestheamountof activeAkt,whichinturnphosphorylatesGSK3,renderingitinac- 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.
TheincreaseinFAT10expressionpositivelycorrelateswithpro- gressionofthediseaseandFAT10-positivepatientshavethelowest survivalrate(Daietal.,2016;Yuanetal.,2012).Gliomacelllines stably overexpressingFAT10 displayedenhanced tumorigenesis invitrobecausetheydevelopedsignificantlymorespheresandpos- sessedincreasingmigrationandinvasioncapacity.Wheninjected intonudemice,thesecellsdevelopedlargertumorsascompared tomock-transfectedcells,implicatingthatFAT10isalsopromoting gliomacarcinogenesis.FAT10seemstoexertitseffectonglioma- 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 influencetheprogressionofgliomabyenhancingthelevelofp- Smad2(Daietal.,2016).
6. Summaryandfutureprospects
Growing evidence speaks for a direct involvement of the ubiquitin-like modifier FAT10 in tumorigenesis. Recent data strongly suggests that FAT10itself promotescarcinogenesis by directlyincreasingsurvival,proliferation,invasion,andmetastasis formationoftumorcellsassummarizedinFig.1.GiventhatFAT10 isapotentsignalforproteasomaldegradation,itisremarkablethat otherthantheFAT10ylationofp53nocovalentsubstratesofFAT10
conjugationwithpotentialfortumorpromotion(e.g.tumorsup- pressorsorapoptosisinducers)havebeendescribed.SinceFAT10 getsisopeptidelinkedtohundredsofproteins,thisisclearlyafield for future investigations.Anothercentralopenquestion iswhy FAT10,whenectopicallyoverexpressedmainlyinnontransformed cells,potently inducesapoptosiswhileitseemstopromotecell growthandsurvivalinothercells,inparticularincancerderived celllines.ThestrongincreaseinFAT10expressionin anumber ofdifferenttumortypesmightbeusefulasaprognosticmarker for thesurvival rateoftheaffectedpatientsand forestimating theprobabilityofrecurrence.Potentialtherapeuticavenuesaim- ingatFAT10-inducingpathwayssuchasNF-B/STAT3signaling aswellastargetingtheFAT10conjugationpathwayseemtohold promiseinparticularbecausethegeneticablationofFAT10inmice doesnotcausesevere phenotypes.Apossiblestarting pointfor targetingtheFAT10conjugationpathwaymightbeaninhibitor of the FAT10 E1 activating enzyme UBA6, developed by Gavin andcolleagues,whichwasalreadysuccessfully testedtoinhibit ubiquitinactivationbyUBA6(Gavinetal.,2012).However,since UBA6isabispecificE1activatingenzymeforFAT10andubiqui- tin,thisoptionmustbehandledwithcaretominimizeputative sideeffects.Targetingtheubiquitin-proteasomesystemincanceris alreadyclinicallyeffectiveatleastviainhibitionoftheproteasome whileinhibitorsoractivatorsofE3ligasesorDUBswithabetter perspectiveforselectivityarebeingdeveloped(Weathingtonand Mallampalli,2014).ThesearchforFAT10-specificE3ligasesaswell asforFAT10-specificdeconjugatingenzymesisstillongoingand theidentificationoftheseenzymeswillenableinvestigationson whethertargetingoftheseenzymeswillprovidenewoptionsfor thedevelopmentoffuturepharmaceuticalsagainstcancer.
Conflictofinterest
Theauthorsdeclarenoconflictofinterest.
Acknowledgements
ThisworkwasfundedbygrantsfromtheCollaborativeResearch Center969,ProjectC01,oftheGermanResearchFoundationand bygrantNrs.855and1029fromtheVeluxfoundation.Thefund- ingsourceshadnoinfluenceonwritingofthisarticleandonthe publicationdecision.
References
Abate-Shen,C.,2002.Deregulatedhomeoboxgeneexpressionincancer:causeor consequence?Nat.Rev.Cancer2(10),777–785.
Aichem,A.,Pelzer,C.,Lukasiak,S.,Kalveram,B.,Sheppard,P.W.,Rani,N., Schmidtke,G.,Groettrup,M.,2010.USE1isabispecificconjugatingenzymefor ubiquitinandFAT10,whichFAT10ylatesitselfincis.Nat.Commun.1,13.
Aichem,A.,Kalveram,B.,Spinnenhirn,V.,Kluge,K.,Catone,N.,Johansen,T., Groettrup,M.,2012.TheproteomicanalysisofendogenousFAT10substrates identifiesp62/SQSTM1asasubstrateofFAT10ylation.J.CellSci.125, 4576–4585.
Aichem,A.,Catone,N.,Groettrup,M.,2014.Investigationsintothe
auto-FAT10ylationofthebispecificE2conjugatingenzymeUBA6-specificE2 enzyme1.FEBSJ.281(7),1848–1859.
Amit,S.,Hatzubai,A.,Birman,Y.,Andersen,J.S.,Ben-Shushan,E.,Mann,M., Ben-Neriah,Y.,Alkalay,I.,2002.Axin-mediatedCKIphosphorylationof beta-cateninatSer45:amolecularswitchfortheWntpathway.GenesDev.16 (9),1066–1076.
Balkwill,F.,2009.Tumournecrosisfactorandcancer.Nat.Rev.Cancer9(5), 361–371.
Bates,E.E.,Ravel,O.,Dieu,M.C.,Ho,S.,Guret,C.,Bridon,J.M.,Ait-Yahia,S.,Briere,F., Caux,C.,Banchereau,J.,Lebecque,S.,1997.Identificationandanalysisofa novelmemberoftheubiquitinfamilyexpressedindendriticcellsandmature Bcells.Eur.J.Immunol.27(10),2471–2477.
Bett,J.S.,Kanuga,N.,Richet,E.,Schmidtke,G.,Groettrup,M.,Cheetham,M.E.,van derSpuy,J.,2012.TheinheritedblindnessproteinAIPL1regulatesthe ubiquitin-likeFAT10pathway.PLoSOne7(2),e30866.
