Genetics and epigenetics of liver cancer

NewBiotechnology_{Volume30,Number4}_{May2013 RESEARCHPAPER}

Genetics

and

epigenetics

of

liver

cancer

Cigdem

Ozen

,

Gokhan

Yildiz

,

Alper

Tunga

Dagcan

,

Dilek

Cevik

,

Aysegul

Ors

,

Umur

Keles

,

Hande

Topel

and

Mehmet

Ozturk

1_{BilkentUniversity,BilGenGeneticsandBiotechnologyCenter,DepartmentofMolecularBiologyandGenetics,06800Ankara,Turkey} 2_{Universite´ JosephFourier–Grenoble1,INSERMInstitutAlbertBonniot,U823,SiteSante´-BP170,38042GrenobleCedex9,France}

Hepatocellular

carcinoma

(HCC)

represents

a

major

form

of

primary

liver

cancer

in

adults.

Chronic

infections

with

hepatitis

B

(HBV)

and

C

(HCV)

viruses

and

alcohol

abuse

are

the

major

factors

leading

to

HCC.

This

deadly

cancer

affects

more

than

500,000

people

worldwide

and

it

is

quite

resistant

to

conventional

chemo-

and

radiotherapy.

Genetic

and

epigenetic

studies

on

HCC

may

help

to

understand

better

its

mechanisms

and

provide

new

tools

for

early

diagnosis

and

therapy.

Recent

literature

on

whole

genome

analysis

of

HCC

indicated

a

high

number

of

mutated

genes

in

addition

to

well-known

genes

such

as

TP53,

CTNNB1,

AXIN1

and

CDKN2A,

but

their

frequencies

are

much

lower.

Apart

from

CTNNB1

mutations,

most

of

the

other

mutations

appear

to

result

in

loss-of-function.

Thus,

HCC-associated

mutations

cannot

be

easily

targeted

for

therapy.

Epigenetic

aberrations

that

appear

to

occur

quite

frequently

may

serve

as

new

targets.

Global

DNA

hypomethylation,

promoter

methylation,

aberrant

expression

of

non-coding

RNAs

and

dysregulated

expression

of

other

epigenetic

regulatory

genes

such

as

EZH2

are

the

best-known

epigenetic

abnormalities.

Future

research

in

this

direction

may

help

to

identify

novel

biomarkers

and

therapeutic

targets

for

HCC.

Introduction

Themostfrequentprimarylivercancersarehepatocellular carci-noma(HCC)andcholangiocarcinomainadults,and hepatoblas-tomainchildren.Morethan80%oflivertumoursareHCCs[1].This review will focus primarily on HCC, one of the most frequent cancers worldwidewithmorethan 500,000newcases observed eachyear.Almostthesamenumberofdeathsisobservedbecause ofthiscancercouldnotbeeasilytreated.Themostefficient treat-mentforHCCislivertransplantation,providedthatitisdetected earlyenough.Surgicalremovalandchemo-embolisationoftumour nodulesareotheralternatives.Thesetumoursareusuallyresistantto chemo-or radiotherapy[1–3].Targetedtherapy ofHCCis inits infancy. The onlyclinically relevantdrug is a kinase inhibitor, Sorafenib,hasonlyamodesteffectonpatientsurvival[4].

TheaetiologyofHCCiswellknown.Chronicliverinjury asso-ciatedprimarilywithhepatitisB(HBV)andC(HCV)virusinfection

constitutesthemostimportantcauseofHCC.Otherfactors,suchas alcoholabuseanddietaryexposuretoaflatoxins,arealsoestablished causes,buttheircontributiontothediseaseaetiologyismuchless thanthecontributionsofviralagents.Theunprecedentedincrease inobesityratesinbothdevelopedanddevelopingcountriesisa risingconcernforHCCriskthatmayaccountfortheunexpected increaseinHCCincidenceintheWesternworld[1].

Molecularmechanismsofhepatocellularcarcinogenesisremain ill-defined,mainlyduetodiseaseheterogeneity.The heterogene-ityofagentsthatcausechronicliverinjury(HBV,HCV,aflatoxins andalcohol)andthewaystheyinteractwiththehostDNAand epigeneticplayersarethemostprobableparameterscontributing toHCCheterogeneity.

Chromosomal

aberrations

and

hepatitis

B

virus

integration

into

the

host

genome

Chromosomal aberrations such as deletions and copy number gains are frequent in HCC. Initial studies identified that HCC

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aper

Correspondingauthor:Ozturk,M.(ozturk@fen.bilkent.edu.tr)

harbours multiple chromosomal abnormalities, predominantly losses,withincreased chromosomalinstabilityin tumours asso-ciatedwithHBVinfection.Commonalterationsincludegainof chromosomes1q,8qand17q,and lossof4q[5].Recently,data from whole genome analysis techniques showed that chromo-somes 1q,5,6p,7,8q,17qand20displaychromosomalgains, while1p,4q,6q,8p,13q,16,17pand21exhibitlossesinHCC[6]. Inaddition,HBVDNAisoftenintegratedintothehostgenome inpatientswithHBV-relatedHCCs[7].Thisintegrationmayhave cis and trans effects. Viral DNA integration into or near gene sequences mayalter gene expression aswell as gene integrity. Inaddition,integratedviralDNAmayencodewild-typeor trun-catedviralproteinsactingintransonthehostgenome,eitherby deregulatinggeneexpressionorbyinteractingwithhostproteins

[8].Recentlyreported wholegenome studiesindicated that the viral integration isassociatedwith breakpointswithinthe HBV genomethatprimarilylocalisedtothedownstreamregionofthe HBX gene. HBV genome integration was observed within or upstreamofthe TERT(telomerasereversetranscriptase)genein fourHBV-relatedHCCs.However,HBVintegrationsiteswithinthe sameordifferenttumoursdidnotshowspecificpatterns, suggest-ingthatthevirusdoesnottargetspecifichostsequences.[9].Based onthesefindings,itishighlyprobablethatlandscapechangesin the structuralintegrityofchromosomes, aswellasrandombut multipleintegrationsofHBVgenomesintohostgenomes,cause highlevelsofinstabilityinthe chromosomalintegrityofHCC. Some of theseaberrations mayhit crucialgenes suchasTERT, whichmaydirectlycontributetotumourdevelopmentby inap-propriate activation orinactivation ofthe genesthemselves.In addition,theintegrationofviralenhancersequencesinthe vici-nityofcrucialgenesmayleadtoaberrantgeneexpressioninHCC.

Gene

mutations

SincethediscoveryofTP53asthefirstmutatedgeneinHCCover20 yearsago[10]anduntilveryrecently,onlyfourgeneswereknownto displayfrequentalterationsinlivercancers.WhileTP53,CTNNB1 (encodingb-catenin)andAXIN1genesusuallydisplaypoint muta-tionsandsmalldeletions,CDKN2A(encodingp16INK4a)undergoes homozygousdeletionsandepigeneticsilencing[11,12].

Duringthepasttwoyears,thefirstreportsofwhole-genomeor exomesequencingdataforHCChaveappeared[6,9,13].Thisisthe beginningofaneweraofHCCgenetics,becauseofthefactthat these newtechniques will allow the visualisationof the muta-tionallandscapeofHCC.Figure1showsasummary ofprimary findingsgatheredbyourselvesfromtworecentlypublishedreports

[6,9].Eachstudyfirstanalysedasmallsetoftumours(n=20–25) fora genome-widesearch ofsomaticallymutatedgenes; signifi-cantlymutatedgeneswerethenfurthertestedformutationsusing alargersetoftumours(n>100).

AcloseexaminationofthedataofFig.1indicatesthatTP53and CTNNB1 represent the two most frequently mutated genes. A secondgroupofgenes(AXIN1andARID1A)wasfoundtopresent lessfrequentmutations,butstillpresentinmorethan10%ofHCC samplesstudied.Thethirdgroupisthelargestwith22genes dis-playingrecurrentmutationsinlessthan10%oftumours.Guichard et al.[6] reportedthatWnt/b-catenin, p53,PI3K/Rassignalling, oxidative,endoplasmicreticulumstresspathwaysandchromatin remodellingwerefrequentlyaffectedbythesemutations.

Wholegenomesequencingallowedthedetectionofrecurrent somaticmutationsinseveralgenesannotatedasassociatedwith chromatin regulation, such as ARID1A, ARID1B, ARID2, MLL, MLL3,BAZ2B,BRD8,BPTF,BREandHIST1H4B.Notably,14out ofthe27tumours(52%)hadeithersomaticpointmutationsor indelsinatleastoneofthesechromatinregulators.Inbothsetsof experiments(wholegenomesequencingandthevalidationsets), the numberofindels in chromatinregulatorgenes was signifi-cantlyhigherthan thosein genesbelonging tothe other cate-gories.Thissuggeststhatloss-of-functionmutationsareenriched inthesechromatin-regulatorgenesinHCCgenomes[9].

AsshowninTable1,thefrequentmutationsthatidentifiedso farinHCCarelikelytoresultinlossoffunctionwiththenotable exceptionof CTNNB1mutations.It willbeinterestingtostudy whylossoffunctionratherthangainoffunctionofcrucialgenesis associatedwithHCC.Bycontrast,thispatternofmutationdoes notoffera broad spectrumoftherapeuticintervention applica-tions.Cancercellscaneasilybetargetedbyblockinggenesthatare aberrantlyoveractiveinthesecells.Therestorationofalostgene activity to achieve a therapeutic intervention is difficult to achieve.Thus,althoughthegenome-wideanalyseshavebeenvery helpfulinestablishingthelistofalargesetofmutatedgenesin HCC,this will most probably serve diagnosticneeds whilethe chanceoftheirtherapeuticuseismorelimited.

Epigenetic

deregulation

EpigeneticregulationofgeneexpressioninvolvesDNA methyla-tion,post-translationalhistonemodifications,chromatinchanges andnon-codingRNAsthatareoftenaffectedincancercells[14,15]. TheroleofepigeneticderegulationinHCCisbeingincreasingly recognised[16].InadditiontochangesinDNAmethylation, micro-RNAexpression,mutations affectingepigeneticregulatorygenes haverecentlybeendiscoveredinHCC[6,9,13].

HCCcellsdisplayglobalhypomethylationaswellaspromoter hypermethylationof a largeset ofgenes [17].Promoter hyper-methylationappearstoaffectmainlytumoursuppressorand anti-proliferativegenesresultingindownregulationofgeneexpression (Fig. 2). Aberrations in microRNA expression have also been observed with several of them being linked to metabolic and phenotypicchangesinHCCcells[14,18–20].

RESEARCHPAPER NewBiotechnology_{Volume30,Number4}_May2013

0 5 10 15 20 25 30 35 40 TP53

CTNNB1 AXIN1 ARID1A WWP1 _RPS6KA3

ATM

ARID1B CDKN2A NFE2L2 IGSF10 ERRFI1 ARID2 ZIC3 ZNF226 ALB MLL3 IRF2 BAZ2B UBR3 GXYL1 OTOP1 USP25 MLL1 BPTF

Liver CancerGenome Mutations (%)

FIGURE1

Mostfrequentlymutatedgenesinhepatocellularcarcinoma.

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Several genes encoding epigenetic regulatory proteins are involvedinhepatocellularmalignancy.TheEZH2(KMT6)encodes thecatalyticcomponentofthePolycombRepressiveComplex2 (PRC2),creatingthetranscriptionally repressiveH2K27Me3 his-tonemarkwhichresultsintranscriptionalsilencing[21].EZH2is over-expressedinHCCandmostlyassociatedwiththeprogression andaggressivebiologicalbehaviourofHCC[22,23].EZH2protein silences Wnt pathway antagonists and constitutively activates Wnt/b-cateninsignallingcausingcellproliferationinHCCcells

[24].EZH2alsoexertsaprometastaticfunctionthroughepigenetic silencingofmultipletumoursuppressormiRNAsincluding miR-139-5p,miR-125b,miR-101,let-7candmiR-200b[25].Yangetal. identified an lncRNA calledlncRNA-HEIH (High Expressionin HCC)thatassociateswithEZH2torepressEZH2targetgenessuch

asp16Ink4a_andp21Cip1_{inHBV-relatedHCC[26].BMI1isanother}

PRC2member overexpressedinHCC.Effendi etal. determined thatBMI1isupregulatedinearlyandwell-differentiatedHCCand thisexpressioncorrelateswithABCB1expression[27].

Expressionofhistone deacetylases(HDACs) isderegulated in differentcancers[28],andsomeofthemarealsoderegulatedin HCC. HDACs-1, -2 and -3 are over-expressed in HCC [29,30]. LC3B-II-induced inactivation of HDAC1 caused regression of HCCcellproliferationand triggeredcaspaseindependent autop-hagy.p21Cip1_andp2Kip1_{wereselectivelyinducedwhilecyclinD1}

andCDK2weresuppressedbyinactivationofHDAC1.Asaresult, HDAC1inactivationresultedinhypophosphorylationofpRbinthe G1/S checkpoint to inactivate E2F/DP1 transcriptional activity. Also, p21(WAF1/Cip1) _{transcriptional activity was suppressed by}

NewBiotechnology_{Volume30,Number4}_{May2013 RESEARCHPAPER}

TABLE1

Mostfrequentgenemutationsinhepatocellularcarcinomaarepredictedtoleadtoaloss-of-function

Genes %mutationrates Proteinfunction Known/expectedoutcome

TP53 35 DNAdamageresponse,other Loss-of-function

CTNNB1 19 PositiveregulatorofWntsignalling Gain-of-function

AXIN1 13 NegativeregulatorofWntsignalling Loss-of-function

ARID1A 12 Chromatinremodelling Loss-of-function

WWP1 9 E3ubiquitinligase Loss-of-function?

RPS6KA3 8 RibosomalproteinS6kinase ?

ATM 8 DNAdamageresponse Loss-of-function?

ARID1B 7 Chromatinremodelling Loss-of-function?

CDKN2A 6 Positiveregulatorofsenescence Loss-of-function

NFE2L2 5 Redoxhomeostasis? ?

IGSF10 5 ? Loss-of-function

ERRFI1 5 EGFR/ERB2kinaseinhibitor Loss-of-function

ARID2 5 Chromatinremodelling Loss-of-function?

0 20 40 60 80 100

NR0B2 APC _{AKAP12 RASSF1A} BLU _{PRDM2 OXGR1} ZEB2 FHIT

p16INK4A MT1G BLMH CDH1 GSTP1 CCND2 RECK DKK3 HINT1 KLK10 HHIP WIF-1 EFEMP1

FBLN1 LIFR _{DIRAS3 HTATIP2 p14ARF} CHFR _{CADM1 SOCS1} PLK3 TAT

TP53BP2 PTGS2 PLK2 MGMT p15INKB PLK3 RB1 PTEN

PPP1R13B

Methylaon Frequency (%)

FIGURE2

Thefrequencyofpromotermethylationinhepatocellularcarcinoma.

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HDAC1byinteractionwithanSp1-bindingsiteinthep21(WAF1/Cip1)

promoter[31].HDAC4alsosuppressesthepromoteractivityof miR-200a anditsexpressionand interacts withSp1in themiR-200a promoter to attenuate histone H3 acetylation levels. miR-200a repressesHDAC4expressionthroughtargetingthe30_{-untranslated}

regionofmessengerRNAofHDAC4.Inthisrespect,miR-200ahas anabilitytoinduceitsowntranscriptionandincreasethelevelsof histone H3 acetylation at its promoter. Furthermore, miR-200a inducesup-regulationofthelevelsoftotalacetyl-histoneH3and histoneH3acetylationinthep21Cip1promoter[32].

DNAmethylatingenzymesDNMT1,DNMT3AandDNMT3Bare over-expressedinHCCcomparedtononcancerousliversamples

[33,34].Finally,CENPAexpressionwasfoundtobesignificantly

elevatedinHCCtissues,andapositivecorrelationexistsbetween CENP-A expressionand HBx COOH mutations inHCC tissues. HBxmutantincreasestheexpressionofCENPAmRNA[35].

Future

perspectives

Recentadvancesingenomesequencingtechnologieswillchange radicallyourcapabilitiesforfinemappingofhepatocellularcancer genomes.Itisexpectedthatpatienttumourswillbefullyanalysed in ashorttimeata moderatecost.Therefore,thegenomic and

epigenomicstatusofthepatient’sowntumourwillbeacrucial elementfordecisionmakingintermsofdiseaseprognosis, ther-apeuticchoicesandpredictionofpatientsurvival.However,most oftheknownmutationsobservedinHCCareassociatedwithaloss offunction.Apparently,targetablegenesfoundinothercancers suchasgrowthfactorreceptorsandintracellularproteinkinases arenotmutatedatsignificantlevelsinHCC.Therefore,weneedto findother targetsfor the treatmentofliver cancers. Epigenetic characterisationofHCChasallowedthediscoveryofmany epi-geneticplayersinthisdisease.However,thesestudiesarefarfrom beingcomplete.TherarityoftargetablemutationsinHCCjustifies asystematicstudyofepigeneticchangestoidentifynewtargetsfor thetherapyofthisdisease.

Acknowledgements

TheresearchstudyissupportedbygrantsfromTU¨ BI˙TAK(109S191 and111T558)withadditionalsupportfromStatePlanningOffice (DPT-KANI˙LTEKProject),TurkishAcademyofSciences,Institut NationaldeCancerandLaLigueNationaleContreleCancerin France(Equipelabelise´e).C.O.,G.Y.andD.C.receivedfellowships fromTurkishAcademyofSciences(C.O.),TU¨ BI˙TAK(G.Y.,D.C.) andEMBO(G.Y.).

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