JMJD1A, H3K9me1, H3K9me2 and ADM expression as prognostic markers in oral and

16 Pages·2017·10.8 MB·English

RESEARCHARTICLE
JMJD1A, H3K9me1, H3K9me2 and ADM
expression as prognostic markers in oral and
oropharyngeal squamous cell carcinoma
LucasdeLimaMaia1*,GabrielaToniniPeterle1,MarcelodosSantos2,Leonardo
OliveiraTrivilin1,SuzannyOliveiraMendes1,MayaraMotadeOliveira1,Joaquim
GasparinidosSantos1,ElaineStur1,LidianePignatonAgostini1,CinthiaVidalMonteiro
daSilvaCouto1,JulianaDalbo´1,AriciaLeoneEvangelistaMonteirodeAssis1,Anderson
BarrosArchanjo1,AnaMariaDaCunhaMercante3,RossanaVeronicaMendozaLopez4,
a1111111111 Fa´bioDaumasNunes5,MarcosBrasilinodeCarvalho6,EloizaHelenaTajara7,Iu´ri
a1111111111 DrumondLouro1,AdrianaMadeiraA´lvares-da-Silva1
a1111111111
1 ProgramadePo´s-Graduac¸ãoemBiotecnologia,UniversidadeFederaldoEsp´ıritoSanto,Vito´ria,Esp´ırito
a1111111111
Santo,Brazil,2 EscolaMulticampideCiênciasMe´dicasdoRioGrandedoNorte,UniversidadeFederaldo
a1111111111
RioGrandedoNorte,Caico´,RioGrandedoNorte,Brazil,3 DepartamentodeAnatomiaPatolo´gica,Hospital
Helio´polis,SãoPaulo,SãoPaulo,Brazil,4 CentrodeInvestigac¸ãoTranslacionalemOncologia,Institutodo
CaˆncerdoEstadodeSãoPaulo,SãoPaulo,Brazil,5 DepartamentodePatologiaBucal,Faculdadede
Odontologia,UniversidadedeSãoPaulo,SãoPaulo,SãoPaulo,Brazil,6 Laborato´riodeBiologiaMolecular,
HospitalHelio´polis,SãoPaulo,SãoPaulo,Brazil,7 DepartamentodeBiologiaMolecular,Faculdadede
Medicina,SãoJose´doRioPreto,SãoPaulo,Brazil
OPENACCESS
Citation:MaiaLdL,PeterleGT,dosSantosM, *[email protected]
TrivilinLO,MendesSO,deOliveiraMM,etal.
(2018)JMJD1A,H3K9me1,H3K9me2andADM
expressionasprognosticmarkersinoraland Abstract
oropharyngealsquamouscellcarcinoma.PLoS
ONE13(3):e0194884.https://doi.org/10.1371/
journal.pone.0194884
Editor:DajunDeng,BeijingCancerHospital,CHINA Aims
Received:September9,2017 JumonjiDomain-Containing1A(JMJD1A)proteinpromotesdemethylationofhistones,
Accepted:March12,2018 especiallyatlysin-9ofdi-methylatedhistoneH3(H3K9me2)ormono-methylated
(H3K9me1).IncreasedlevelsofH3histonemethylationatlysin-9(H3K9)isrelatedtotumor
Published:March28,2018
suppressorgenesilencing.JMJD1AgenetargetAdrenomeduline(ADM)hasshowntopro-
Copyright:©2018Maiaetal.Thisisanopen
motecellgrowthandtumorigenesis.JMJD1AandADMexpression,aswellasH3K9meth-
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,which ylationlevelhavebeenrelatedwithdevelopmentriskandprognosisofseveraltumortypes.
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited. Methodsandresults
DataAvailabilityStatement:Allrelevantdataare WeaimedtoevaluateJMJD1A,ADM,H3K9me1andH3K9me2expressioninparaffin-
withinthepaperanditsSupportingInformation
embeddedtissuemicroarraysfrom84oralandoropharyngealsquamouscellcarcinoma
files.
samplesthroughimmunohistochemistryanalysis.OurresultsshowedthatnuclearJMJD1A
Funding:Thisworkwassupportedbythe
expressionwasrelatedtolymphnodemetastasisrisk.Inaddition,JMJD1Acytoplasmic
Fundac¸ãodeAmparoàPesquisadoEstadodeSão
Paulo(FAPESP)(http://www.fapesp.br/)grants04/ expressionwasanindependentriskmarkerforadvancedtumorstages.H3K9me1cyto-
12054-9;ConselhoNacionaldeDesenvolvimento plasmicexpressionwasassociatedwithreduceddisease-specificdeathrisk.Furthermore,
Cient´ıficoeTecnolo´gico(CNPq)(http://cnpq.br/);
highH3K9me2nuclearexpressionwasassociatedwithworsespecific-diseaseanddis-
Fundac¸ãodeAmparoàPesquisadoEstadodo
ease-freesurvival.Finally,highADMcytoplasmicexpressionwasanindependentmarkerof
Esp´ıritoSanto(FAPES)(https://fapes.es.gov.br/);
andtheCoordenac¸ãodeAperfeic¸oamentode lymphnodemetastasisrisk.
PLOSONE|https://doi.org/10.1371/journal.pone.0194884 March28,2018 1/16
JMJD1A,H3K9me1,H3k9me2andADMexpressioninoralandoropharyngealcancer
PessoaldeN´ıvelSuperior(CAPES)(http://capes. Conclusion
gov.br/).
JMJD1A,H3K9me1/2andADMexpressionmaybepredictormarkersofprogressionand
Competinginterests:Theauthorshavedeclared
prognosisinoralandoropharynxcancerpatients,aswellasputativetherapeutictargets.
thatnocompetinginterestsexist.
Introduction
Despitealladvancesintheunderstandingofmolecularmechanismsinvolvedintumordevel-
opmentandprogression,aswellasnewtreatmentprotocols,headandnecksquamouscell
carcinoma(HNSCC)isstillthesixthcauseofdeathandcancerrelatedmorbidity,withover
600,000newcasesdiagnosedeveryyear[1,2,3].
HNSCCisacomplexdisease,causedbymultiplefactorssuchassmokinganddrinkinghab-
its,HPVinfection,dietaryandgeneticfactors.Itisalsoadiversediseaseinrelationtoclinical
presentation,treatmentresponseandprognosis.Incontrastwithsuchdiversity,therearecom-
monfeaturesthatcanleadtolocalandregionalrecurrence,aswellaspredictpatientsurvival
tothedisease.ThemainprognosticfactorforHNSCCislymphnodemetastasis,decreasingby
50%patientsurvivalchances.[4,5].Presenceoftumorhypoxiaisanotherimportantprognos-
ticfactor[6]
Tumorcellresponsetohypoxiainvolvesactivationofover100genes[7].Currently,littleis
knownabouttheepigeneticmodulationthatresultsfromHIFsystemtranscriptionalactiva-
tion[8].However,suchchangesprobablyincludeepigenetichistonemodifications[9].
TheproteinJumonjiDomain-Containing1A(JMJD1A,JHDM2AorKDM3A)isregulated
byHIF1aunderhypoxicconditions.JMJD1Ageneisactivatedviaitshypoxiaresponseelement
inthepromoterregion,resultingindemethylationofgenesthathelpcelladaptationinlow
oxygenconditions[10].DemethylationoccursatlysinandarginineresiduesofhistonesH3in
anoxygen-dependentreactionthatneedsFe(II)ionandα-ketoglutarateascofactors[11].
Thiscanaltertumorcellbehaviorduetochromatinstructuralchanges,geneexpressionand
DNArepair[9,12].
Epigeneticregulationofgeneexpressionthroughhistonemethylationhasanimportant
roleindiversebiologicalprocesses,includingcellcyclecontrol,DNAdamageresponse,cellu-
larstressresponse,embryogenesisandcelldifferentiation[13,14,15].Histonemethylation
changesarerelatedtocancerduetoitsinfluenceintumorphenotype,suchasdifferentiation,
apoptosisandtreatmentresponse.HistoneH3methylationatresidue9(H3K9)isspecifically
associatedwithtranscriptionalrepressionduetoinductionofheterochromatinformationand
tumorsuppressorgenesilencinginvarioustypesofcancer[16,17].
Adrenomedulin(ADM)isoneofJMJD1A’stargets.ADMgeneproduct,undernormalcon-
ditions,ismultifunctional,playingrolesincellularprocessessuchasregulationofprolifera-
tion,differentiation,migration,growth,anti-apoptosis,angiogenesis,immunesuppressionand
hypoxia,suggestingitsroleincarcinogenesis[18,19].
Histonemethylationlevels,aswellasexpressionofJMJD1AandADMhavebeenassociated
withdevelopmentandprognosisofseveraltumortypes,suchascolorectal[15,20,21],naso-
pharyngeal[22],hepatocellular[17,23],renal[13,24,25].ItsroleinHNCisstillamatterof
debate.
Therefore,wehaveaimedtostudytheassociationofJMJD1A,histonesmonoanddi-meth-
ylated(H3K9me1eH3K9me2,respectively)andADMwithclinicopathologicalfeaturesand
prognosisofpatientswithHNCorcanceroftheoralandoropharyngealcavities.
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JMJD1A,H3K9me1,H3k9me2andADMexpressioninoralandoropharyngealcancer
Materialandmethods
Ethics
ThisstudywasapprovedbytheCommitteeofEthicsinResearchoftheHelio´polisHospital
(CEP#619)andawritteninformedconsentwasobtainedfromallpatientsenrolled.
Samples
SampleswerecollectedbytheHeadandNeckGenomeProject(GENCAPO),acollaborative
consortiumcreatedin2002withmorethan50researchersfrom9institutionsinSãoPaulo
State,Brazil,whoseaimistodevelopclinical,geneticandepidemiologicalanalysisofHNSCC.
Inthisstudy,84tumoraltissuesampleswereobtainedandusedforimmunohistochemical
analysisoftheJMJD1A,withinatotalof84patientswithoralandoropharyngealsquamous
cellcarcinomas,surgicallytreatedattheHeadandNeckSurgeryDepartmentofHelio´polis
Hospital,SãoPaulo,Brazil,duringtheperiodofJanuary/2002toDecember/2008.Theclinical
follow-upwasatleast24monthsaftersurgery.Previoussurgicalorchemotherapictreatment,
distantmetastasis,noremovalofcervicallymphnodesandpositivesurgicalmarginswere
exclusioncriteria.Histopathologicalslideswerereviewedbyaseniorpathologisttoconfirm
thediagnosisandselectappropriateareasforimmunohistochemicalanalysis.Tumorswere
classifiedaccordingtotheTNMsystem(7rdedition)[26].
Amonganalyzedindividuals,meanagewas54.2years(df±10.3)being72(86%)formen
and12(14%)forwomen.Accordingtotumoranatomicalsites,64(76%)wereintheoraland
20(24%)intheoropharyngealcavity(Table1;S1Table).
Tissuemicroarray
Tissuemicroarraysweremadeaspreviouslydescribed[27]usingbufferedformalin-fixedpar-
affin-embeddedtissuesectionsfrom84primaryoralandoropharyngealsquamouscellcarci-
nomastreatedattheHeadandNeckSurgeryDepartmentofHelio´polisHospital,SaãoPaulo,
SP.Eachslidewasexaminedbyapathologistwhomarkedtheentiretumorcircumference
withapen,afterwhich,two1mmdiametercylinderswerepunchedfromeachblockand
reembeddedinrecipientparaffinblocksbyatissuemicroarrayer(BeecherInstruments1,Sil-
verSpring,MD,USA).SectionswerethentakenfromTMA,mountedinmicroscopeslides,
tissuemicroarrayslideswereevaluatedbyHEtoconfirmthattumorrepresentativeareaswere
extractedfromallblocks.Everystepoftheprocesswassupervisedby2independentexperi-
encedpathologists,andafterbothofthemapprovedtheTMAprocedure,IHCwasperformed
ontheslides.
Immunohistochemistry
Anti-JMJD1Amousemonoclonalantibody(1:400)(107234,Abcam1),Anti-HistoneH3–
MonomethylK9rabbitmonoclonalantibody(1:600)(9045,Abcam1),Anti-HistoneH3–Di
methylK9monoclonalmousemonoclonalantibody(1:800)(1220,Abcam1)andAnti-Adre-
nomedullinrabbitpolyclonalantibody)(1:200)(69117,Abcam1)wereusedintheimmuno-
histochemistryreactionusingREVEALPolimer-HRP,mouse/rabbit(SpringBioscience),
accordingtothemanufacturer’sprotocol[28–30].Positiveandnegativecontrols(absenceof
primaryantibody)wereusedforreactionqualitycontrol.Samplescoringwasperformedby
semi-quantitativemicroscopicanalysis,consideringthenumberofstainedtumorcellsand
signalintensity.Twospotswereevaluatedforeachsampleandameanscorewascalculated.
Consideringthepercentageofimmune-positivetumorcells,ascoreof1wasgivenwhen
(cid:20)10%ofcellswerepositive;2when11–50%ofcellswerepositiveand3when>50%ofcells
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JMJD1A,H3K9me1,H3k9me2andADMexpressioninoralandoropharyngealcancer
Table1. Epidemiologicalandprognosticfeatures.
Features Total
No. (%)
Gender
Female 12 14.3
Male 72 85.7
Age,yr
mean54.2,df±10,3
Smoker 64 76.2
Alcoholuser 48 57.1
Tumorsites
Oralcavity 64 76.2
Oropharingeal 20 23.8
Tumorsize(T)¥
T1,T2,T3 54 64.3
T4 30 35.7
Lymphnode(N)¥
Negative 34 40.5
Positive 50 59.5
Tumorstage
I,II,III 41 48.8
IV 43 51.2
Diseaserelapse
No 27 32.1
Yes 55 65.5
Notavailable§ 2 2.4
Diseasespecificdeath
No 31 36.9
Yes 46 54.8
Notavailable§ 7 8.3
Total 84 100.0
¥TNMclassification7thedition.
§Notavailable(notconsideredinthestatisticalcalculations).
https://doi.org/10.1371/journal.pone.0194884.t001
werepositive.Signalintensitywasscoredasnegative(0),weak(1),moderate(2)andstrong
(3).Bothscoresweremultiplied[31,32,33]andtheresultingscorewasusedtocategorize
expressionoftheproteinsasnegative(0),positivelow(1–3)andpositivehigh((cid:21)3).Two
pathologists/investigatorsanalyzedtheslides,withnopriorknowledgeordiscussionaboutthe
cases.Afterwards,theindependentreportswerecomparedforconcordance,inwhicha98.1%
concordanceratewasobtained.Non-concordantresultswerereanalyzedtogetherinorderto
achieveaconsensusbetweenthe2investigators.
Statisticalanalysis
ThechisquareandFisherexacttestswereusedforassociationanalysisandconfirmationwas
obtainedbytheLillieforstest(significanceconsideredwhenp<0.05).Multivariatelogistic
regressionwasusedtoobtainoddsratio(OR)andconfidenceintervals(CI(cid:21)95%).Survival
wascalculatedbythenumberofmonthsbetweensurgeryanddeathforeachpatientorthelast
appointmentincasethepatientwasalive.Tocalculatedisease-freesurvival,thetimeendpoint
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JMJD1A,H3K9me1,H3k9me2andADMexpressioninoralandoropharyngealcancer
Fig1.Immunohistochemistry.(A)NegativeJMJD1Anuclearandcytoplasmicexpression.(B)LowJMJD1Anuclearandcytoplasmicexpression.(C)
HighJMJD1Anuclearandcytoplasmicexpression.(D)NegativeJMJD1Anuclearandcytoplasmicexpression.(E)LowH3K9me1nuclearand
cytoplasmicexpression.(F)HighH3K9me1nuclearandcytoplasmicexpression.Thescalebarindicates10μμM.Magnificationwas400x.
https://doi.org/10.1371/journal.pone.0194884.g001
wasthedateofdiseaserelapse.TheKaplan-Meiermodelwasusedforsurvivalanalysis,using
theWilcoxonp-valueandtheCoxProportionalHazardstoadjustp-valuesandobtainhazard
ratio(HR).StatisticalcalculationswereperformedusingtheIBMSPSSSTATISTICS1v.20,
2011softwares.
Results
PositiverelationshipbetweenJMJD1Aexpressionwithlymphnodestatus
andtumorstage
JMJD1Anuclearexpressionpositivitywasstudiedin84tumors,ofwhich27werenegative
(32.1%),21werelowpositive(25.0%)and29werehighlypositive(34.6%).RegardingJMJD1A
cytoplasmicexpression,11werenegative(13.1%),59(70.3%)werelowpositiveandonly7
(8.3%)werehighpositive(Fig1A,1Band1C;S2Table).Sevenofthe84cases(8.3%)couldnot
beanalysed.
PositiveJMJD1Anuclearexpressionshowedasignificantassociationwithtumorstageand
lymph-nodestatus(p=0.033andp=0.001,respectively,Table2).Multivariateanalysis
showedthatpositiveJMJD1Anuclearexpressionwasanindependentmarkerforlymph-node
positivity,yieldinganapproximately10-foldincreasedrisk(OR=10.086,CI=2.02–50.35,
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JMJD1A,H3K9me1,H3k9me2andADMexpressioninoralandoropharyngealcancer
Table2. ClinicalandpathologicaltumorfeaturesandtheirassociationwithJMJD1Aexpression,accordingtocelllocalization.
Features JMJD1Aexpression
Nuclear Cytoplasmic
Negative Positive p Negative Positive p
No. (%) No. (%) No. (%) No. (%)
Tumorsize(T)¥
T1,T2,T3 20 74.0 30 60.0 0.317 11 100.0 39 59.1 0.007
T4 7 26.0 20 40.0 0 0.0 27 40.9
Lymphnode(N)¥
Negative 18 66.7 13 26.0 <0.001 10 90.9 21 31.8 <0.001
Positive 9 33.3 37 74.0 1 9.1 45 68.2
Tumorstage
I,II,III 18 66.7 20 40.0 0.033 10 90.9 28 42.4 0.003
IV 9 33.3 30 60.0 1 9.1 38 57.6
Diseaserelapse
No 7 25.9 14 28.0 0.764 3 45.5 18 27.3 0.954
Yes 20 74.1 34 68.0 8 54.5 46 69.7
Notavailable§ 0 0.0 2 4.0 0 0.0 2 3.0
Diseasespecificdeath
No 9 33.3 18 36.0 0.946 3 27.3 24 36.4 0.729
Yes 14 51.9 29 58.0 6 54.5 37 56.0
Notavailable§ 4 14.8 3 6.0 2 18.2 5 7.6
Total 27 35.0 50 65.0 11 14.3 66 85.7
¥TNMclassification7thedition.
§Notavailable(notconsideredinthestatisticalcalculations).
https://doi.org/10.1371/journal.pone.0194884.t002
Table3).Moreover,JMJD1Anuclearexpressionlevelsdidnotshowasignificantrelationship
withtumorcharacteristics(Table2).
PositiveJMJD1Acytoplasmicexpressionwassignificantlyassociatedwithtumorsize
(p=0.007),lymph-nodestatus(p<0.001)andtumorstage(p=0.003,Table2).Multivariate
analysisshowedthatpositiveJMJD1Acytoplasmicexpressionwasanindependentmarkerfor
tumorstage,yieldinganreducedrisk(OR=0.092,CI=0.009–0.930,Table3).Inaddition,
JMJD1Acytoplasmicexpressionlevelsdidnotshowasignificantrelationwithtumorcharac-
teristics(Table2).
H3K9monomethylationexpressionincytoplasmreducesdiseasespecific
deathrisk
H3K9me1nuclearexpressionpositivitywasstudiedin84tumors,ofwhich16(19.1%)were
negative,44(52.4%)werelowpositiveand19(22.6%)werehighlypositive.Regarding
H3K9me1cytoplasmicexpression,12(14.3%)werenegative,58(69.1%)werelowpositiveand
only9(10.7%)werehighlypositive(Fig1D,1Eand1F;S2Table).Fiveofthe84cases(5.9%)
couldnotbeanalysedduetoIHCfailure.
PositiveH3K9me1nuclearexpressiondidnotshowasignificantassociationwithtumor
characteristics(Table4).
PositiveH3K9me1cytoplasmicexpressionshowedasignificantassociationwithdisease
relapse(p=0.028)anddiseasespecificdeath(p=0.039,Table4).Multivariateanalysisshowed
thatpositiveH3K9me1cytoplasmicexpressionwasanindependentmarkerfordiseasespecific
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JMJD1A,H3K9me1,H3k9me2andADMexpressioninoralandoropharyngealcancer
Table3. MultivariateanalysisoftherelationshipbetweenlymphnodestatusandJMJD1AandADMexpression.
Features MultivariateAnalysis
Lymphnodes(N)¥ Tumorstage¥ Diseasespecificdeath Diseaserelapse
OR(IC95%) p OR(IC95%) p OR(IC95%) p OR(IC95%) p
JMJD1ANuclearexpression
Negative 1
Positive 10.08(2.02–50.35) 0.005
JMJD1ACytoplasmicexpression
Negative 1 1
Positive 10.74(0.63–182.30) 0.100 0.09(0.00–0.93) 0.043
H3K9me1Cytoplasmicexpression
Low 1 1
High 0.25(0.03–1.70) 0.157 0.06(0.00–0.79) 0.032
H3K9me2nuclearexpression
Low 1 1
High 3.50(0.82–14.81) 0.089 4.15(0.99–17.31) 0.050
ADMCytoplasmicexpression
Low 1
High 9.16(1.14–73.47) 0.037
Tumorsize(T)¥
T1,T2,T3 1 1 1
T4 8.82(1.60–48.41) 0.012 3.88(0.99–15.14) 0.050 3.78(0.92–15.50) 0.064
Necrosis
Absent 1 1
Present 4.81(0.73–31.76) 0.102 4.81(0.73–31.76) 0.102
Smoking
No 1
Yes 13.46(2.14–84.52) 0.006
Alcoholism
No 1 1
Yes 3.92(1.09–14.09) 0.036 2.53(0.79–8.08) 0.117
Age
(cid:20)55 1
>55 2.89(0.84–9.87) 0.090
Anatomicalsite
Oralcavity 1
Oropharynx 8.46(1.36–52.49) 0.022
¥TNMclassification7thedition.
https://doi.org/10.1371/journal.pone.0194884.t003
death(OR=0.068;IC=0.006–0.793,Table3).Inaddition,cytoplasmicexpressionlevelsdid
notshowasignificantrelationwithtumorcharacteristics(Table4).
H3K9dimethylathioninfluencesdiseasefreeanddiseasespecificsurvival
H3K9me2nuclearexpressionpositivitywasstudiedin84tumors,ofwhich7(8.3%)wereneg-
ative,50(59.5%)werelowpositiveand25(29.8%)werehighlypositive.However,H3K9me2
cytoplasmexpressionwaspositiveonlyin3(3.5%)cases(Fig2A,2Band2C;S2Table).Twoof
the84cases(2.4%)couldnotbeanalysed.
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JMJD1A,H3K9me1,H3k9me2andADMexpressioninoralandoropharyngealcancer
Table4. ClinicalandpathologicaltumorfeaturesandtheirassociationwithH3K9me1expression,accordingtocelllocalization.
Features H3K9me1expression
Nuclear Cytoplasmic
Negative Positive p Negative Positive p
No. (%) No. (%) No. (%) No. (%)
Tumorsize(T)¥
T1,T2,T3 11 68.8 39 61.9 0.774 7 63.6 42 62.7 0.952
T4 5 31.2 24 38.1 4 36.4 25 37.3
Lymphnode(N)¥
Negative 6 37.5 25 39.7 0.873 4 36.4 26 38.8 0.877
Positive 10 62.5 38 60.3 7 63.6 41 61.2
Tumorstage
I,II,III 8 50.0 29 46.0 0.787 5 54.5 31 46.3 0.960
IV 8 50.0 34 54.0 6 36.4 36 53.7
Diseaserelapse
No 3 18.8 18 28.6 0.365 0 0.0 21 31.3 0.028
Yes 13 81.2 38 60.3 10 90.9 40 59.7
Notavailable§ 0 0.0 7 11.1 1 9.1 6 9.0
Diseasespecificdeath
No 5 31.3 22 34.9 0.766 1 9.1 26 38.8 0.039
Yes 10 62.5 30 47.6 9 81.8 30 44.8
Notavailable§ 1 6.2 11 17.5 1 9.1 11 16.4
Total 16 20.3 63 79.7 11 14.1 67 85.9
¥TNMclassification7thedition.
§Notavailable(notconsideredinthestatisticalcalculations).
https://doi.org/10.1371/journal.pone.0194884.t004
PositiveH3K9me2nuclearexpressiondidnotshowasignificantassociationwithtumor
characteristics(Table5).Althoughmultivariateanalysisdidnotshowasignifcantrelationship
betweencytoplasmicH3k9me2expressionandsystemicdiseaserelapse,thedatasuggestsa
tendencyofassociationbetweenthem(p=0.050,Table3).
Incontrast,H3K9me2proteinexpressionshowedanassociationwithbothdisease-free
survival(p=0.019,Table6,Fig2D)anddisease-specificsurvival(p=0.021,Table6,Fig
2E).MultivariateanalysisshowedthathighnuclearH3K9me2proteinexpressiondecreases
diseasefreesurvivalbymorethan2-fold(HR=2.034;CI=1.12–3.67,Table6),whereas
highnuclearexpressiondecreaseddiseasespecificsurvivalbyapproximately3-fold
(HR=2,620;CI=1,156–5,938,Table6).Wehaveattemptedtocompareoursurvivaldata
withtheonepresentinTCGAsurvivaldatabase,howevernosuccesswasachievedbecause
ofeitherlackofinformationintheTCGAdatabaseornosignificantstatisticalresultsafter
thecomparisons.
ADMexpressionasanindependentmarkeroflymphnodepositivity
ADMdidnotshownuclearexpression.Incontrast,ADMcytoplasmicexpressionwasnegative
in3cases(3.6%),lowpositivein64(76.2%)andhighlypositivein14(16.6%)(Fig3A,3Band
3C;S2Table).Threeofthe84casescouldnotbeanalysed(3.6%).
PositiveADMcytoplasmicexpressiondidnotshowasignificantassociationwithtumor
characteristics(Table5).ADMcytoplasmaticexpressionlevelsshowedasignificantrelation-
shipwithlymph-nodestatus(p=0.037,Table5).Multivariateanalysisshowedthatstrong
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JMJD1A,H3K9me1,H3k9me2andADMexpressioninoralandoropharyngealcancer
Fig2.Survivalplotsandimmunohistochemistry.(A)NegativeH3K9me2nuclearexpression(B)LowH3K9me2nuclearexpression.(C)High
H3K9me2nuclearexpression.(D)DiseaserelapsesurvivalaccordingtoH3K9me2nuclearexpression.(E)Diseasespecificsurvivalaccordingto
H3K9me2nuclearexpression.Thescalebarindicates10μμM.Magnificationwas400x.
https://doi.org/10.1371/journal.pone.0194884.g002
ADMcytoplasmicexpressionwasanindependentmarkerforlymph-nodepositivity,yielding
anapproximately9-foldincreasedrisk(OR=9.167;CI=1.14–73.47,p=0.037,Table3).
Discussionandconclusions
JMJD1Aproteinpromotesdemethylationofhistones,especialyatlysin-9ofdi-methylatedhis-
toneH3(H3K9me2)ormono-methylated(H3K9me1)[11].Histonedemethylationalters
chromatinstructureresultingingeneexpressionchanges,DNArepair,replication[12],as
wellascelldifferentiation[13].TheADMproteinhasitsexpressionalteredbytheactionof
JMJD1A[23].TheJMJD1AandADMexpression,aswellasthehistoneH3K9methylation
level,havebeenrelatedtodevelopmentandprognosisofdiversetumortypes[13–25].
OurresultsshowedthatpositiveJMJD1Anuclearexpressionisaworseprognosticfactor
becauseincreaseslymphnodemetastasisriskbyover10-fold(Fig4).Colorectalcancerstudies
havecorrelatedhighJMJD1Aexpressionwithanaugmentedriskforlymphnodepositivityby
over6-fold[20].Otherstudieswithcervical,gastricandnasopharyngealcancerhavereported
anassociationJMJD1Aexpressionandanincreasedriskoflymphnodemetastasis[22,34,35].
OurresultssuggestthatJMJD1Acytoplasmicexpressionisrelatedwithlessaggressive
tumorstages.JMJD1Aproteinlevelsincertaincytoplasmiclocationsisdependentuponcell
PLOSONE|https://doi.org/10.1371/journal.pone.0194884 March28,2018 9/16
JMJD1A,H3K9me1,H3k9me2andADMexpressioninoralandoropharyngealcancer
Table5. ClinicalandpathologicaltumorfeaturesandtheirassociationwithH3K9me2andADMexpression,accordingtocelllocalization.
Features H3K9me2expression ADMexpression
Nuclear Cytoplasmic
Negative Positive p Negative Positive p
No. (%) No. (%) No. (%) No. (%)
Tumorsize(T)¥
T1,T2,T3 5 71.4 47 62.7 0.645 2 66.7 49 62.8 0.892
T4 2 28.6 28 37.3 1 33.3 29 37.2
Lymphnode(N)¥
Negative 3 42.9 29 38.7 0.828 1 33.3 31 39.7 0.824
Positive 4 57.1 46 61.3 2 66.7 47 60.3
Tumorstage
I,II,III 5 71.4 34 45.3 0.249 2 66.7 36 46.2 0.598
IV 2 28.6 41 54.7 1 33.3 42 53.8
Diseaserelapse
No 0 0.0 21 28.0 0.177 2 66.7 19 24.4 0.197
Yes 6 85.7 48 64.0 1 33.3 51 65.4
Notavailable§ 1 14.3 6 8.0 0 0.0 8 10.2
Diseasespecificdeath
No 1 14.3 26 34.7 0.394 1 66.7 25 32.1 0.558
Yes 5 71.4 38 50.7 2 33.7 40 51.3
Notavailable§ 1 14.3 11 14.6 0 0.0 13 16.6
Total 7 8.5 75 91.5 3 3.7 78 96.3
¥TNMclassification7thedition.
§Notavailable(notconsideredinthestatisticalcalculations).
https://doi.org/10.1371/journal.pone.0194884.t005
growthrateandHsp90chaperoneactivity[36,37,38],whichalsointerfereswithJMJD1Asta-
bilityandactivity[36].Kasioliusetal.(2014)showedthatJMJD1Adeficiencyinratsresulted
incytoskeletonabnormalities,whichinturnwasassociatedwithmetastaticpotential,more
aggressivetumorsanddecreasedglobalsurvivalanddisease-freesurvivalinbreastcancer
patients[39].ThisstudyalsoshowedanassociationbetweenADMproteinexpressionand
lymphnodemetastasis,sothathighexpressionincreaseslymphnodemetastasisriskby9fold.
Table6. Multivariateanalysisoftherelationshipbetweendiseaserelapseanddiseasespecificsurvivaland
H3k9me2expressionandtumorsize.
Varia´veis CoxProportional CoxProportional
Diseaserelapsesurvival Diseasespecificsurvival
HR(IC95%) p HR(IC95%) p
H3K9me2Nuclearexpression
Low 1 1
High 2.034(1.126–3.676) 0.019 2.620(1.156–5.938) 0.021
Tumorsize(T)¥
T1,T2,T3 1 1
T4 1.993(1.113–3.570) 0.020 2.444(1.128–5.296) 0.024
¥TNMclassification7thedition.
https://doi.org/10.1371/journal.pone.0194884.t006
PLOSONE|https://doi.org/10.1371/journal.pone.0194884 March28,2018 10/16