www.bjorl.org
Brazilian
Journal
of
OTORHINOLARYNGOLOGY
ORIGINAL
ARTICLE
Overexpression
of
CDC7
in
malignant
salivary
gland
tumors
correlates
with
tumor
differentiation
夽
Zohreh
Jaafari-Ashkavandi
a,∗,
Mohammad
Javad
Ashraf
b,
Ali
Asghar
Abbaspoorfard
a aShirazUniversityofMedicalSciences,SchoolofDentistry,DepartmentofOralandMaxillofacialPathology,Shiraz,Iran bShirazUniversityofMedicalSciences,SchoolofMedicine,DepartmentofPathology,Shiraz,IranReceived5September2017;accepted8November2017 Availableonline26December2017
KEYWORDS Salivarygland; CDC7; Adenoidcystic carcinoma; Mucoepidermoid carcinoma; Pleomorphic adenoma Abstract
Introduction:Celldivisioncycle-7proteinisaserine/threoninekinasethathasabasicrolein cellcycleregulationandisapotentialprognosticortherapeutictargetinsomehumancancers. Objectives:Thisstudyinvestigatedtheexpressionofcelldivisioncycle-7proteininbenignand malignantsalivaryglandtumorsandalsoitscorrelationwithclinicopathologicfactors. Methods:Immunohistochemicalexpressionofcelldivisioncycle-7wasevaluatedin46cases, including15adenoidcysticcarcinoma,12 mucoepidermoidcarcinoma,14 pleomorphic ade-noma,and5normalsalivaryglands.Celldivisioncycle-7expressionrateandintensitywere comparedstatistically.
Results:Theproteinwasexpressedinalmostalltumors.Theintensityandmeanofcelldivision cycle-7expressionwerehigherinmalignanttumorsincomparisonwithpleomorphicadenomas (p=0.000).Theproteinexpressionwascorrelatedwithtumorgrades(p=0.000).
Conclusions:Thepresentstudydemonstratedcelldivisioncycle-7overexpressioninmalignant salivaryglandtumorsincomparisonwithpleomorphicadenomas,andalsoacorrelationwith tumordifferentiation.Therefore,thisproteinmightbeapotentialprognosticandtherapeutic targetforsalivaryglandtumors.
© 2017 Associac¸˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Published by Elsevier Editora Ltda. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).
夽 Pleasecitethisarticleas:Jaafari-AshkavandiZ,AshrafMJ,AbbaspoorfardAA.OverexpressionofCDC7inmalignantsalivaryglandtumors
correlateswithtumordifferentiation.BrazJOtorhinolaryngol.2019;85:144---9.
∗Correspondingauthor.
E-mail:jaafariz@sums.ac.ir(Z.Jaafari-Ashkavandi).
PeerReviewundertheresponsibilityofAssociac¸ãoBrasileiradeOtorrinolaringologiaeCirurgiaCérvico-Facial.
https://doi.org/10.1016/j.bjorl.2017.11.004
1808-8694/©2017Associac¸˜aoBrasileiradeOtorrinolaringologiaeCirurgiaC´ervico-Facial.PublishedbyElsevierEditoraLtda.Thisisanopen
PALAVRAS-CHAVE Glândulasalivar; CDC7; Carcinomaadenoide cístico; Carcinoma mucoepidermoide; Adenomapleomórfico
SuperexpressãodeCDC7emtumoresmalignosdeglândulassalivarescorrelaciona-se comadiferenciac¸ãodostumores
Resumo
Introduc¸ão: Acelldivisioncycle-7éumaserina/treoninaquinasequetemumpapelbásicona regulac¸ãodociclocelular eéumpotencialmarcadorprognósticoouterapêuticoem alguns tiposdecâncerhumano.
Objetivos: Esteestudoinvestigouaexpressãodecelldivisioncycle-7emtumoresdeglândulas salivaresbenignosemalignosetambémsuacorrelac¸ãocomfatoresclínico-patológicos. Método: A expressão imuno-histoquímica decell divisioncycle-7 foi avaliada em 46 casos, incluindo 15 carcinomas adenoidecísticos,12 carcinomas mucoepidermoides, 14 adenomas pleomórficose5glândulassalivaresnormais.Ataxadeexpressãoeaintensidadedaproteína celldivisioncycle-7foramcomparadasestatisticamente.
Resultados: Aproteínafoiexpressaemquasetodosostumores.Aintensidadeeamédiada expressãode celldivisioncycle-7 forammaioresem tumoresmalignosem comparac¸ãocom adenomapleomórfico(p=0,000).Aexpressãodaproteínafoicorrelacionadacomosgrausdo tumor(p=0,000).
Conclusões: O presente estudo demonstrou a superexpressão de cell division cycle-7 em tumoresmalignosdeglândulassalivaresquandocomparadacomoadenomapleomórfico,além de uma correlac¸ão com adiferenciac¸ão de tumores. Portanto, essa proteína pode ser um potencialmarcadorprognósticoeterapêuticoparatumoresdeglândulassalivares.
© 2017 Associac¸˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Publicado por Elsevier Editora Ltda. Este ´e um artigo Open Access sob uma licenc¸a CC BY (http:// creativecommons.org/licenses/by/4.0/).
Introduction
Salivaryglandtumors(SGT)arerelativelyrareanddiverse tumors which account for 3---6% of all head and neck neoplasms.1 These tumors consist ofdifferent benign and
malignant subtypeswith a wide histopathologic spectrum which may overlapwith each other; however, witha dif-ferent clinical behavior and management, pleomorphic adenoma(PA),mucoepidermoidcarcinoma(MEC)and ade-noidcysticcarcinoma(AdCC)arethemostcommonbenign and malignant SGTs. Surgery is the main treatment and inmalignanttumorsadjuvant chemo-radiotherapymaybe required.Today,thereismuchpromiseinfindingnovel anti-cancertreatmentstothebasisofmoleculartarget-therapy. The keymolecules thatparticipateinthe cellgrowthand divisionare promising candidates for this goal. They may affect a broad range of various tumor types with a high proliferationrate.2
Celldivisioncycle-7protein(CDC7)isaserine-threonine kinase,originallyintroduced inbuddingyeast,andplaysa keyrolein DNAreplication, principallybyactivating MCM complex,andregulationofS-phasecheckpointinthecell cycle.3,4 The regulator subunit of CDC7 is Dbf4/activator
ofS-phases.5CDC7overexpressionwasalsocorrelatedwith
P53inactivation5andhasbeenfoundinmanyhumantumor
celllinesandtissues,includingbreast,colonandlung can-cers,melanomaandoralsquamouscellcarcinoma(OSCC), butthisproteinhasveryloworundetectableexpressionin normaltissues.2,5---10
It has been shown that CDC7 overexpression was correlated with poor prognosis in patients with B-cell lymphoma.11 Also,itcontributedtotheresistancetoDNA
damagingagentsandincreasingsurvivalinOSCCcancercell line.9CDC7wasatherapeutictargetinovariancarcinoma.12
Therefore,CDC7isapromisingandpotentcandidatetarget forcell-growthinhibitionbecauseitpointsDNAreplication beforeit starts.4,6,13 Therefore, evaluation of CDC7
func-tionin anyspecifictumor issuggested. Tothebestofour knowledge,thereis noresearch focused onCDC7 expres-sionanditssignificanceinSGTs.Thisstudyaimedtoevaluate CDC7expressionrateanditscorrelationwith clinicopatho-logicparametersofthemostcommonbenignandmalignant SGTs.
Methods
Tissuesamples
Inthiscross-sectionalretrospectivestudy,46cases consist-ingof14PA,15AdCC,12MECand5normalsalivaryglands (NSG)were included. The cases obtained fromarchive of Pathology Department from 2009 to 2014. All cases had definitivediagnosisand adequateepithelial tissue.Severe inflamedcaseswereexcluded.Thebaselinedataincluding patient’sageandgender,aswellastumorsite,size,grade andstagewererecorded,usingthepatient’smedicalfiles.
Immunohistochemistry
4mtissuesectionswereprovidedfromformalin-fixedand paraffin-embeddedblocks.Afterdeparaffinizationand rehy-dration, antigenretrieval wasperformed byTris-buffer in PH=8at121◦Cfor20min.Endogenousperoxidaseactivity
wasblockedusing3%hydrogenperoxidefor30min.Then, the sections were incubated by primary antibody (anti-CDC7polyclonalantibody,1:50,GenetexCompany,USA)for 60min.Envisionsystemwasappliedassecondaryantibody andthesectionwerewashedin PBS.Thechromogen solu-tion was 3,3-diaminobenzidine tetrahydrochloride (DAB). Finally,theslideswerecounterstainedwithMayer’s hema-toxylin.Asectionofnormallymphnodewasusedaspositive controlandthesamesectionbyomittingprimaryantibody asthenegativecontrol.
The stained tissues evaluated by light microscopyand thecellswithbrownnucleiwereconsideredaspositive.In eachcase,atleast1000cellswerecountedin3microscopic fieldsandthepercentageofpositivecellswasnoted.The intensity of staining wasassessed andscored as1 --- mild or 2 --- moderate/severe, in comparison withthe positive control.ThemeanofCDC7expressionwasscoredas:(1) pos-itivecells<5%,(2)5---10%and(3)>10%.Thefinalscorewas obtainedbymultiplyingtheintensityandpercentagescores. DatawereanalyzedbyKruskal---Wallis,TukeyandDunntests andSpearman’scorrelation.p<0.05wasconsideredas sig-nificant.
Results
Thepatientswere18malesand28femaleswithameanage of49.4±15.Thebaselinedatarelatedtoallstudygroups areillustratesinTable1.
InNSGs,twospecimensexhibitedalimitednuclear stain-ing in acinar and ductal cells, with weak to moderate intensity.Almostalltumors,exceptoneMEC,showed posi-tivenuclearCDC7expression.
PAsshowedCDC7expressionintheepithelialandductal cells(Fig.1AandB)withamean of2.3±1.2.71% ofthe casesrevealedweakstaining.
The epidermoid cells of MEC showed CDC7 staining (Fig.2A and B) and most of the cases (74%) had moder-ate/severeexpressionwithameanof 32.1±14.3.Mucous andclearcellswerenotstained.AllcasesofAdCCexhibited moderate/severe CDC7 expression witha meanof 9.7±3 (Fig.3AandB).
Details about mean expression of CDC7, intensity of staining and final scores are shown in Table 2. Kruskal---Wallistest showeda significant differenceamong groups in CDC7 expression (p=0.000). Dunn test showed this difference between PA and MEC, as well as PA and AdCC (p=0.000 and p=0.004). However, there
Figure1 Nuclear,weakCDC7expressioninpleomorphic ade-noma(A,×200andB,×400).
was not any significant difference between MEC and AdCC groups. Also, final scores of the groups were significantly different, according to Kruskal---Wallis test (p=0.000).
PAshowedsignificantlowerscoresofCDC7mean,in com-parison withMEC and also with AdCCgroups, using Dunn test(p=0.000andp=0.02,respectively).However,MECand AdCCgroupswerestatisticallysimilar(Dunntest,p=0.26). Finalscoreswerealsodifferentamongthegroups accord-ingtotheresultsofKruskal---Wallistest(p=0.000)(Table3). Dunn test showed that finals scores of the PA group was significantlylowerthanbothmalignanttumors(p=0.000); however,MECandAdCCwerenotdifferent(p=1).
Table1 Basicinformationofallstudygroups.
Group(n) PA(14) MEC(12) AdCC(15) NSG(5) Total(46)
Age 39.4±14 60.6±12 49.7±12 49±14 49.4±15.1 (M/F) 5/9 7/5 4/11 2/3 18/28 Site(Major/Minor) 11/2 10/2 6/8 1/4 28/16 Grade(1,2,3) --- 3,0,8 5,10,0 --- 8,10,8 Stage(1,2,3,4) --- 1,3,2,5 1,3,4,5 --- 2,6,6,10 Size(1,2,3,4) --- 1,5,2,3 1,4,3,5 --- 2,9,5,8
PA,pleomorphicadenoma;MEC,mucoepidermoidcarcinoma;AdCC,adenoidcysticcarcinoma;NSG,normalsalivaryglands. Clinicdataofsomecaseswerenotavailable.
Figure2 SeverenuclearCDC7expressioninepidermoidcells ofhigh-grademucoepidermoidcarcinoma(A,×200;B,×400).
Table2 ScoresofintensityandCDC7meanexpressionin allstudygroups.
PA MEC AdCC NSG CDC7meanscore 1 5 0 0 2 2 9 1 7 0 3 0 10 7 0 Intensityscore 1 10(71.4) 2(18.2) 0 0 2 4(28.6) 9(81.8) 15(100) 2(100) PA,pleomorphic adenoma;MEC, mucoepidermoidcarcinoma; AdCC,adenoidcysticcarcinoma;NSG,normalsalivaryglands.
Table3 Finalscoresofallstudygroups(Intensity×Mean scores). Scores 1 2 3 4 6 MEC 1 0 2 0 9 AdCC 0 0 1 5 7 PA 0 7 0 2 0 NSG 0 2 0 0 0 Total 1 9 3 7 16
PA,pleomorphic adenoma;MEC, mucoepidermoidcarcinoma; AdCC,adenoidcysticcarcinoma;NSG,normalsalivaryglands.
Figure3 Severenuclear CDC7expressioninadenoid cystic carcinoma(A,×200;B,×400).
High-gradetumorsshowedasignificant increasedCDC7 expressionincomparisonwithlowandintermediategrades. Spearman’scorrelation test showedthat CDC7 expression wascorrelatedwithtumorgrades(p=0.000),butnotwith tumorstageorpatient’sageandgender(p>0.05).
Discussion
Inthepresentstudy,we describedCDC7expression inPA, MECandAdCC,andalsoitsoverexpressioninmalignantSGTs incomparisonwithbenignonesandnormalglands.Various studieshavepreviouslyrevealedthatCDC7hasabasicrolein cellproliferation,tumorogenesisandmalignantprogression6
byactivatingDNAreplication.5,8Ourfindingssupport
previ-ousstudiesinvarioushumancancercelllinesandtissues. Mellingetal.demonstratedoverexpressionofCDC7protein incolorectalcancerinassociationwithP53overexpression and as a favorable prognostic marker.2 Bonte et al. also
showedthatCDC7isveryloworundetectableinnormal tis-sue,butit wasover-expressedinthehumanbreast,colon and lung cancer cell lines.5 One study showed increased
CDC7 mRNAlevel inmalignant transformedbreast cancer cell line and also in hyper-proliferating cells in a lesser degreeincomparisonwithprimarynormalcells.6Increased
CDC7expression hasbeen amarkerof OSCCandhas con-tributedtotheresistancetoDNA-damagingmaterial.9CDC7
andbf4subunitformacomplexthatactsasanactiveprotein kinaseinallorganisms.14 The maintargetofthatcomplex
activityistheMCMcomplex.ThestrongMCMpositivecells indicate a high CDC7 activity.11 MCM2-7 have considered
asreplicationinitiation factorsand, asanovel diagnostic andprognosticbiomarkerforseveralhuman cancers.MCM expression has been reported in the tumors that showed CDC7overexpressionsuchasOSCC,breastcancercellline andtissueandalsoSGTs.15---18 MCM2andMCM3represented
an overexpression in malignant SGTs in comparison with benignones17,18whichindirectlysupporttheoverexpression
ofCDC7inoursamples.
IthasalsobeendemonstratedthatCDC7overexpression wascorrelatedwith TP53 genemutation. CDC7 inhibition caninducecelldeathviaaP53-dependentpathway.19Bonte
etal.foundacorrelationbetweenP53loss,andCDC7 over-expressioninsomecancercellline.5Also,inanotherstudy
CDC7 expression was linked to P53 positivity in colorec-talcancer.2CDC7wasatherapeutictargetforP53mutant
breast cancer.6 Previous studies onSGTs showed a higher
expressionofP53inmalignantSGTsincomparisonwithPA, whichindirectlysupportourfindings.20
Thepresentstudyshowedapositivecorrelationbetween CDC7expressionandtumor grades.However,ourdatadid notshowasignificantdifferencebetweenAdCCandMECin CDC7expression.AlthoughAdCCisahigh-gradetumorwith moreaggressive behavior in comparison withMEC,in our samplesmostoftheMECspecimenswerehigh-gradetumors. In agreement with our results, increased CDC7 expres-sion waslinked to loss of tumor differentiation, genomic instability and development of aggressive phenotype in breastcancer.6Also,highgradecolorectalandovarian
can-cers showed higher CDC7 expression.2,12 This association
ofCDC7expressionwithdifferentiationmakesthisprotein apotential suitabletarget for therapeutic and prognostic approaches.However,incontrasttotheseresearches,our limitedcaseswithcompletedataaboutclinicalstagedidnot showanysignificantcorrelationwithproteinexpression.
The presentstudy revealednuclearstainingof CDC7in all positive specimens which reinforced CDC7 function in DNAreplication.Previousstudieshavedemonstratedprotein accumulationinthecellnucleiduringG1phase.21
Conclusion
The present findingsshowed CDC7 expression in themost commonbenignandmalignantSGTsanditsoverexpression inmalignantones.Positivecorrelation ofthisproteinwith tumoraldifferentiationmayrepresentitasapotential prog-nosticandtherapeutictarget.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgment
TheauthorswouldliketothanktheVice-Chancelleryof Shi-razUniversityofMedicalScienceforsupportingthisresearch (Grant #10052), the Research Consultation Center (RCC) of Shiraz University of Medical Sciences for their invalu-ableassistantineditingthisarticle.Theauthorsalsowould
liketothankDrSalehi fromtheDental Research Develop-mentCentre,forthestatisticalanalysis.Thismanuscriptis relatedtoundergraduatethesisofAliasgharAbbaspoorfard.
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