Metastasis suppressor proteins in cutaneous squamous cell carcinoma

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Pathology

–

Research

and

Practice

j o ur na l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / p r p

Original

article

Metastasis

suppressor

proteins

in

cutaneous

squamous

cell

carcinoma

Onder

Bozdogan

_,

_Ibrahim

_Vargel

_,

_Tarik

_Cavusoglu

_,

_Ayse

_A.

_Karabulut

_,

Gurbet

Karahan

_,

_Nilufer

_Sayar

_,

_Pınar

_Atasoy

_,

_Isik

_G.

_Yulug

a_{AnkaraNumuneEducationandResearchHospital,DepartmentofPathology,Ankara,Turkey}

b_{HacettepeUniversity,MedicalFaculty,DepartmentofPlasticSurgery,ScienceInstitute,DepartmentofBioengineering,Ankara,Turkey} c_{PrivatePractice,PlasticSurgery,Ankara,Turkey}

d_{KırıkkaleUniversity,FacultyofMedicine,DepartmentofDermatology,Kırıkkale,Turkey}

e_{BilkentUniversity,FacultyofScience,DepartmentofMolecularBiologyandGenetics,Ankara,Turkey} f_{IstanbulMedipolUniversity,InternationalSchoolofMedicine,DepartmentofPhysiology,Istanbul,Turkey} g_{KırıkkaleUniversity,FacultyofMedicine,DepartmentofPathology,Kırıkkale,Turkey}

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r

t

i

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Received9August2015 Receivedinrevisedform 15November2015 Accepted3December2015 Keywords:

Skin

Squamouscellcarcinoma Metastasissuppressorproteins A-431

HaCaT

a

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s

t

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Cutaneoussquamouscellcarcinomas(cSCCs)arecommonhumancarcinomas.Despitehaving metasta-sizingcapacities,theyusuallyshowlessaggressiveprogressioncomparedtosquamouscellcarcinoma (SCC)ofotherorgans.Metastasissuppressorproteins(MSPs)areagroupofproteinsthatcontroland slow-downthemetastaticprocess.Inthisstudy,weestablishedtheimportanceofsevenwell-defined MSPsincludingNDRG1,NM23-H1,RhoGDI2,E-cadherin,CD82/KAI1,MKK4,andAKAP12incSCCs.

ProteinexpressionlevelsoftheselectedMSPsweredetectedin32cSCCs,6insituSCCs,andtwoskincell lines(HaCaT,A-431)byimmunohistochemistry.Theresultswereevaluatedsemi-quantitativelyusing theHSCOREsystem.Inaddition,mRNAexpressionlevelsweredetectedbyqRT-PCRinthecelllines.

TheHSCOREsofNM23-H1weresimilarincSCCsandnormalskintissues,whileRGHOGDI2,E-cadherin andAKAP12weresignificantlydownregulatedincSCCscomparedtonormalskin.ThelevelsofMKK4, NDRG1andCD82werepartiallyconservedincSCCs.InstageISCCs,nuclearstainingofNM23-H1 (NM23-H1nuc)wassignificantlylowerthaninstageII/IIISCCs.OnlynuclearstainingofMKK4(MKK4nuc)showed significantlyhigherscoresininsitucarcinomascomparedtoinvasiveSCCs.

Inconclusion,similartootherhumantumors,wehavedemonstratedcomplexdifferential expres-sionpatternsfortheMSPsinin-situandinvasivecSCCs.ThiscomplexMSPsignaturewarrantsfurther biologicalandexperimentalpathwayresearch.

©2016ElsevierGmbH.Allrightsreserved.

1. Introduction

Non-melanomaskin cancers (NMSC) are themost common humanmalignantneoplasms,andcreatesignificantmedical, eco-nomical,andsocialproblemsforthehealthcareservicesworldwide

[1–3].AlthoughthereareothertypesofNMSC,thistermcommonly referstothetwocommonneoplasmsofcutaneoussquamouscell carcinoma(cSCC)andbasalcellcarcinoma(BCC)[1].BCCsareslow growing,malignant,yetrarelymetastasizingcarcinomas [4].On thecontrary,cSCCsaremoreaggressiveinbehavior,andhave con-siderablyhighermetastaticcapacitiesthanBCCs[4].

Metastasisisaverycomplexandmultistepbiologicalprocess directed byvarious proteinsand pathways[5]. Though various

∗ Correspondingauthorat:BilkentUniversity,FacultyofScience,Departmentof MolecularBiologyandGenetics,TR-06800Ankara,Turkey.

E-mailaddress:yulug@fen.bilkent.edu.tr(I.G.Yulug).

proteinssupportmetastasis,agroupofproteinscalledmetastasis suppressorproteins(MSPs)specificallyinhibitorslowmetastasis

[6,7].Asadefinition,pureMSPsshouldonlysuppressmetastasis withoutanyeffectontumorigenicity(e.g.proliferation).However, in the complex environment of a cell, theyusually have other importantpropertiesastumorsuppressoractivities,affecting car-cinogenesis, besidestheseMSP functions[6].cSCCs differfrom internalidenticalorgancancersinthattheyhavelowermetastatic ratesandresultinbetterprognosis[4].Thus,cSCCsareinteresting biologicalmodelsfortheresearchof metastasissuppressors.To establishtheimportanceoftheMSPsinnon-melanomaskin can-cer,weselectedsevenessentialandwell-definedMSPs(NDRG1, NM23-H1,RhoGDI2,E-cadherin,CD82/KAI1,MKK4,andAKAP12) thataffectdifferentstepsofmetastasis.

The main aim of this study was to analyze the expression patternsofthesesevenimportantMSPsthat maycontributeto the inhibition of metastasis pathways in cSCCs, as well as in squamouscelllines.Wealsoestablishedtheassociationbetween

http://dx.doi.org/10.1016/j.prp.2015.12.018 0344-0338/©2016ElsevierGmbH.Allrightsreserved.

theseproteins and important clinicopathological parameters in cSCC.

2. Materialsandmethods 2.1. Studygroups

Atotalof38SCCscomposedof32tissuesamplesofclassical squamouscellcarcinoma(SCC-NOS)oftheskinand6insitu carci-nomatissues,obtainedfrom37patients(26M/11F),wereincluded inthisstudy.AllpatientswereCaucasians,andthedetailed char-acteristicsofthestudygrouparesummarizedinAppendix1.SCCs weregradedbyfour-tieredsystemaswell(Grade1),moderately (Grade2)poorlydifferentiated(Grade3)andanaplasticor undif-ferentiatedtumors(Grade4)[8].DatafortheSCCswerecollected byusingtheCAP(TheCollegeofAmericanPathologists)protocols forsquamouscarcinomaoftheskin(www.cap.org).

2.2. Normalskincontrolgroup

Tennormalskintissues(4M/6F)fromreconstructiveoperations, confirmedasnormalbymicroscopy,wereincludedasthenormal tissuegroup.Thenormalgroupincludedskintissuesfromdifferent localizations;twofromtheface,threefromtheextremities,three fromthebreast,andtwofromtheabdomen.Thiscontrolgroupwas thesameastheoneusedinanotherpreviousstudyofourgroup

[9].Normal,non-lesionalepidermisadjacenttoSCC(N-SCC)was alsointegratedinthisstudy.

2.3. Celllines

Thenormal immortalizedkeratinocyte cellline(HaCaT) and thevulvarsquamous carcinomacellline(A-431) wereusedfor immunohistochemistryandquantitativereversetranscriptase-PCR (qRT-PCR)studies[10,11].

2.4. Immunohistochemistrytechniqueandanalysis

Theimmunostainingprocedurewasperformedbytheclassical labeledstreptavidin-biotinimmuno-enzymaticantigendetection system(UltraVision-ThermoScientific;Waltham,MA,USA)with DABchromogen,intheThermo-ShandonSequenza®_manual

stain-ingstation(Waltham,MA,USA).Theprimaryantibodystepwas skippedforthenegativecontrol.Thesourcesofprimaryantibodies andthetechnicaldetailsaredemonstratedinAppendix2.

All immunohistochemically stained slides were evaluated by external and internal controls. Stained slides were semi-quantitatively evaluated using a specific immunohistochemical histologicalscoretechnique,(HSCORE),withminormodifications. Theanalysistechniquewasdescribedindetailintheliteratureand inourpreviousstudy[9,12].

2.5. Cellculture

TheA-431celllinewasculturedinDulbecco’sMinimal Essen-tial Medium (DMEM-low glucose, Hyclone-Thermo Scientific, Waltham,MA,USA), andHaCaTcelllinewasgrownin DMEM-Highglucose(Hyclone),supplementedwith10%fetalbovineserum (FBS)and50mg/mlpenicillin/streptomycin,at37◦C,andin5% car-bondioxide.

2.6. Real-timePCR(qRT-PCR)

500ng of total RNA was reverse-transcribed using oligo-dT primers with the First Strand cDNA Synthesis Kit (Fermentas,

Thermo,Waltham,MA,USA).AllqRT-PCRexperimentswere per-formedusingtheSYBR®_{Greenreagent(Thermo)inanMX3005P}

thermocycler (Strategene®_{, Agilent, Santa Clara, CA, USA). The}

GAPDH(glyceraldehyde-3-phosphatedehydrogenase)andHPRT1 (hypoxanthinephosphoribosyltransferase1)referencegeneswere selectedfornormalization.DatawasanalyzedusingthefreeREST©

2009software(Qiagen,Hilden,Germany)[13].Thesequencesof primersusedareaccesiblefromourpreviouslypublishedpaper

[9].

2.7. Statisticalanalysis

StatisticalanalyseswereperformedusingthePASW®

Statis-tics18software(Chicago,IL,USA).Thedifferencesbetweenthe HSCOREsofthegroupswerefirstanalyzedwiththenon-parametric Kruskal–Wallisone-wayanalysistest;followedbyMann–Whitney Utestwasapplied.p≤0.05wasacceptedassignificant.The “Bon-ferronicorrection”wasusedtoreducetypeIerrors.Thecorrelation betweentheparameterswasanalyzedbySpearman’scorrelation test,wherer≥0.25andp≤0.05wereacceptedassignificant. 2.8. Ethicsstatement

This study was financially supported by the Scientific and Technical Research Council of Turkey (TUBITAK, grant number SBAG-108S184).TheprojectwasapprovedbytheKırıkkale Uni-versityLocalEthicsCommittee(07.04.2008/2008-039).

3. Results

3.1. Immunohistochemicalstainingresults 3.1.1. Tissuestudy

CytoplasmicpositivityofNM23-H1(NM23-H1cyt)andNDRG1

(NDRG1cyt)wasdetectedtobestrongandhomogeneousinallof

theinsituandinvasivecSCCs(Fig.1A,B,F,G).However,nuclear NM23-H1(NM23-H1nuc)expressionwassignificantlyweakerand

wasdetectedinonlytwoofinsituSCCs(IS-SCC,33.3%)and15cSCCs (46.8%) compared tonormal skin.Nuclear positivity of NDRG1 (NDRG1nuc)wasseenin4of6IS-SCCs(66.6%),and29of32cSCCs

(90.6%).

E-cadherin,CD82and AKAP12proteinsshowonlyweakand medium cytoplasmic-membranous staining (Figs. 2A, B, F, G and3A,B).E-cadherinandCD82positivitywasdetectedinallof IS-SCCs,butAKAP12positivitywasobservedin4IS-SCCs(66.6%). E-cadherin,CD82,andAKAP12expressionsweredetectedin84.3%, 68.7%and40.6%ofthecSCCs,respectively.

RHOGDI2showedcytoplasmicpositivityin5 (83.3%)and 30 (93.7%)oftheinsituandinvasivecases,respectively,thoughwith weaktomediumstrengthonly(Fig.3F,G).Nuclearpositivityof RHOGDI2(RHOGDI2nuc)wasweaker,yetmoreheterogeneous,in4

of6IS-SCCs(66.6%)and14of32cSCCs(43.7%).

MKK4cytwasdetectedinallofIS-SCCs,andweakMKK4nucwas

detectedin5of6IS-SCCs(83.3%)(Fig.4A).MKK4immunostaining ofcSCCsshowedweaktomediumcytoplasmicpositivityin25cases (78.1%),andweaknuclearpositivityin7cSCCs(21.8%)(Fig.4B). 3.1.2. Celllines

NM23-H1 showed strongcytoplasmic positivity in the both HaCaTandA-431celllines(Fig.1C,D).However,focalnuclear posi-tivitywasstrongerintheHaCaTcellline.Significantpositivitywas observedwithNDRG1antibodyinbothofthecelllines(Fig.1H, I).E-cadherinexpressionwasstrongerintheHaCaTcellline com-paredtoA-431(Fig.2C,D).CD82/KAI1showedmediumlevelsof positivityinbothcelllines(Fig.2H,I).RHOGDI2showedstrongbut heterogeneouspositivityintheHaCaTandA-431celllines(Fig.3H,

Fig.1. NM23-H1expressionininsitu(A)andinvasivecarcinoma(B).Besidesstrongcytoplasmicexpression,weaknuclearexpressionofNM23-H1isoccasionallydetectedin invasivecarcinoma(B).HaCaT(C)andA-431cells(D),bothshowcytoplasmicNM23-H1expression,butstaininginA-431isstronger.Thereisnostatisticaldifferencebetween HSCOREsofthegroups(E).NDRG1expressionininsitucarcinoma(F).Althoughcytoplasmicexpressionisheterogeneous,itisgenerallyhigh.Focalnuclearexpressionis alsodetected.Similarly,cytoplasmicexpressionofNDRG1isverystronginSCCs,butnuclearstainingisdownregulated(G).HaCaTcellsshowcytoplasmicexpression(H) whereasnuclearpositivityisdominantinA-431cellline(I).HSCOREsshowsignificantdownregulationinSCCscomparedtonormalgroups(J).*Showspvalues;*p<0.05; **p<0.01.Originalmagnification:A;B;F;G×100,C;D;H;I×200.

I).AKAP12andMKK4stainingwaseitherveryweakorcompletely lostinbothcelllines(Figs.3C,Dand4C,D).

3.1.3. Comparativestatistics

WhenHSCOREs of normal epidermis(N) were compared to insitucarcinomas,E-cadherin(p=0.016),RHOGDI2nuc(p=0.002),

and NDRG1nuc (p=0.003) stainings showed significantly lower

scoresin insitu carcinomas. However,whencompared to nor-malepidermisadjacenttoSCC(N-SCC),onlyHSCOREsofNDRG1nuc

weresignificantlylowerininsitucarcinomas(p=0.011).

NDRG1nuc/cyt (p=0.001/p=0.007), E-cadherin (p=0.001),

RHOGDI2nuc/cyt (p=0.001/p=0.001), MKK4nuc (p=0.001), and

AKAP12 (p=0.001) expressions were decreased in cSCC com-paredtoN.WhenHSCOREs ofN-SCCwere comparedtocSCCs,

Fig.2.MembranousE-cadherinexpressionininsitu(A)andinvasive(B)carcinoma.ThedownregulationiseasilydetectedinSCC.A-431,squamouscarcinomacellline, showsveryweakpositivity(D)whencomparedtoHaCaT(C).ThedifferencebetweenthenormalskinandinsitucarcinomaisstatisticallysignificantforE-cadherinstaining. Ininsitucarcinomas,theHSCOREsofE-cadherinarelowerthanthenormalepidermis(E).Howeverthereisnostatisticaldifferencebetweeninsitucarcinomaandnormal epidermisadjacenttoneoplasia(N-SCC).InvasiveSCCshowslowerlevelsofE-cadherinexpressionthannormalepidermisandN-SCC.CD82showsmembranocytoplasmic stainingininsitu(F)andinvasiveSCCs(G).InsituSCCsamplesshowmoreheterogenousstainingthaninvasiveSCCs.A-431cellsshowweakerstaining(I)thanHaCaTcells (H).NostatisticaldifferenceisdetectedbetweentheHSCOREsofthegroups(J).*showspvalues.*p<0.05;**p<0.01.Originalmagnification:A×40,B;D;F;G×100,C;H;I ×200.

NDRG1nuc(p=0.001), E-cadherin(p=0.001),and RHOGDI2nuc/cyt

(p=0.001/p=0.001)showedsignificantlylowerscoresincSCCs. NM23-H1nucshowedsignificantlylowerscoresinstageIcSCCs

thanstageII/IIISCCs(p=0.049).MKK4nuc wastheonlyonethat

showedhigher scoresin insitu carcinomas when comparedto invasiveSCCs(p=0.011).

3.1.4. Correlationanalysis

There were limited numbers of positive significant corre-lations (p=0.01 level) between several markers as follows: E-cadherin-RHOGDI2cyt (r=0.452), RHOGDI2cyt-MKK4cyt

(r=0.486), and MKK4nuc-MKK4cyt (r=0.481) in invasive

3.1.5. RealtimePCRresults

NM23-H1 and E-cadherin expression levels did not differ betweenA-431andHaCaTcelllines.Yet,NDRG1expressionwas upregulated (34.4-fold, p=0.001), whereas ARHGDIB (RHOGDI2, −4.7-fold,p=0.001),MKK4(−2.1-fold,p=0.001),CD82/KAI( −2.4-fold,p=0.001)andAKAP12(−9.7-fold,p=0.001)expressionswere downregulatedinA-431cellscomparedtoHaCatcells.

4. Discussion

Inthisstudy,wedemonstrateddifferentexpressionpatternsof MSPsincutaneousSCCs.NM23-H1levelswereconservedininsitu andinvasivecarcinomas,and insquamous carcinomacelllines. AlthoughtheroleofNM23-H1inmetastasissuppressioniswell describedinothercarcinomas,itisnotclearinNMSC[6,7].Similar

Fig.3. AKAP12positivityininsitucarcinoma(A).AKAP12stainingisclearlylostinatypicalcells,andpositivityisseenonlyinmaturebenignkeratinocytes.LowAKAP12 expressioninSCC(B).Veryweak,hardlydetectedpositivityintheHaCaTcellline(C).AKAP12isnegativeintheA-431cellline(D).InvasiveSCCshowsdownregulation comparedtoN(E).RHOGDI2positivityininsitucarcinoma(F).Thereisnosignificantdifferenceininsitucarcinomacomparedtoadjacentepidermis.However,asignificant decreasecanbeseenininvasiveSCCscomparedtothenormalgroup.Inflamatorycellsshowstrongpositivity(G).Strong,mainlycytoplasmicRHOGDI2positivityinHaCaT (H).DownregulatedbutnuclearpositivityinA-431(I).IninsituSCCs,onlyRHOGDI2nucshowssignificantlyreducedlevelscomparedtoN.InvasiveSCCsshowsreduced levelsofRHOGDI2nuc/cytcomparedtobothNandN-SCC(E).*showspvalue.*p≤0.05;**p≤0.01.OriginalMagnification:Fx40,A;B;G×100,D;E;H;I×200.

Fig.4. MKK4expressionininsituandinvasiveSCC(A,B).MKK4cytexpressionisclearlyseenininsitu(A),butshowsweakerstaininginSCCs(B).MKK4showsveryweak

positivityinHaCaT(C),butnopositivityisdetectedinA-431cellline(D).MKK4nucshowssignificantlylowerHSCOREsthannormalepidermisininvasivecSCC(B).However, MKK4cytlevelsaremaintainedincSCCs.Ininsitucarcinomas,MKK4nuc/cytlevelsarealsopositive.MKK4nucshowshigherscoresininsitucarcinomaswithrespectto invasiveSCCs.*showspvalue.*p<0.05;**p<0.01.Originalmagnification:A×40,B×100,C;D×200.

toourfindings,Roetal.[14]andStephensonetal.[15]showed thattheexpressionofNM23-H1waswellconservedincSCCsof theskinandkeratoacanthomas(KA).However,Kanitakisetal.[16]

obtained contradictory results and showed reduced NM23-H1 levelsincSCCs.ThesustainedNM23-H1levelsincSCCsprobably contributetotheirnon-metastasizingfeaturesasobservedinother carcinomas.

Despitebeingdownregulatedincarcinomasamplescompared tonormal tissues,the cytoplasmicNDRG1expression is gener-allymaintainedininsituandinvasiveSCCs.Furthermore,mRNA expression of NDRG1 in the SCC cell line, A-431, was 34-fold increasedcomparedtonormal immortalizedcellline,HaCaT. In accordancewithourfindings,Dangetal.[17]demonstratedthat NDRG1mRNAexpressionincreased5.87-foldinactinickeratoses and7.07-foldincSCCswhencomparedtonormalskin.The impor-tance of NDRG1 expression has been examined in SCCs of the internalorgans, in theliterature.DosSantosetal. [18]showed significantupregulationofNDRG1inoralandoropharyngealSCCs, andconcludedthatNDRG1overexpressionwassignificantly corre-latedwithlong-termsurvival.Similarresultswerealsoreported by Changet al. [19]. Theprognostic importanceof NDRG1was alsodemonstratedinesophagealSCCs,aswellasinotherhuman tumors, suchas tumors of prostate, breast, and colon [20–24]. However, the MSP function of NDRG1 is probably carcinoma-dependent.NDRG1overexpressioninhepatocellularcarcinomahas beenshowntobeanindicatorofpoorprognosis[25].Innonsmall celllungcarcinoma,itsexpressionissignificantlyassociatedwith advancedstagesandweakvascularization[26].

AlthoughHSCOREvaluesofCD82ininsituandinvasive squa-mouscarcinomawerenotdifferentfromnormalskin,only68.7% ofcSCCsshowedCD82positivity,whilelimiteddownregulationof mRNAexpression(2.4-fold)wasdetectedintheA-431cellline.

Similartoourfindings,Okochietal.[27]showedsignificant down-regulation of CD82in basal cellcarcinoma of theskin, despite conserved expression inBowen disease. Thefindings regarding CD82expressioninothertypesofhumanSCCarecontroversial. CD82 downregulation has been demonstrated in oral, cervical, penile,laryngeal,headandneck,andlungSCCs[28–30]. Interest-ingly,when comparedtocSCCs,allofthesecarcinomaspossess greatermetastasispotential.RelativelyconservedlevelsofCD82 maysupportthenon-metastasizingfeaturesofcutaneousSCCs.

Cytoplasmic MKK4 expression was maintained at relatively higherlevelsinSCCsinourstudy.MKK4expressionwas previ-ouslyshowntobereducedduringcancerprogressioninprostate andovarycarcinomas[31,32].Similarresultswereshowninother epithelialcancers,includingendometrialcarcinoma,gastric carci-noma,andpancreaticcancer[33–35].MKK4isgenerallyaccepted tobeametastasissuppressor,buttherearestillconflictingreports intheliterature.Huang etal.foundhigherexpressionofMKK4 inlaryngealSCCsincomparisonwiththeirnormalcounterparts, inadditiontoapositivecorrelationbetweenhigherexpressionof MKK4andincreasedmetastasis[36].Inarecentexperimental arti-cle,Fineganetal.[37]proposedthatMKK4haspro-oncogenicroles inskincarcinoma.OurdatamaysupporttheirhypothesisincSCCs. TheHSCOREsofRHOGDI2weresignificantlydownregulatedin cSCCs.However,RHOGDI2cytoplasmicexpressionwasmaintained ininsitucarcinoma.RHOGDI2expressionhasnotbeenstudiedin skincarcinomaspreviouslyasfarasweareaware.TheMSPfunction ofRHOGDI2wasfirstshowntobeametastasissuppressorgenein bladdercarcinoma[38].Becauseofconflictingreports,the metas-tasissuppressorfunctionofthisproteincouldbeconsideredtobe tissueororgan-dependent.RHOGDI2hasbeenpreviouslyshownto beupregulatedingastricandovariancarcinoma,andprobablyhas adualroleincarcinogenesis[39].

E-cadherinlevelsweresignificantlydownregulated incSCCs. DownregulationofE-cadherinlevelshasbeenwelldemonstrated incSCCbefore[40–42].However,wefoundthatmRNAlevelswere notchangedinthesquamouscellcarcinomacellline.Ithasalso beenreportedthatareducedlevelofE-cadherinexpressionwas moreevidentinacantholyticsubtypesofcSCCs[41].

We demonstrated thatAKAP12 wasdownregulated in cSCC, withlessthanhalfofthecSCCsbeingpositiveforAKAP12staining. SignificantmRNAdownregulationofAKAP12wasalsodetectedin theA-431SCCcellline.ThedecreaseinAKAP12levelsinhuman carcinomashasbeenreportedinvarioustypesofhumancancers

[43].Yet,theexpressionpatternofAKAP12isnotknowninskin car-cinomas.ArecentstudyfocusedonepigeneticregulationofAKAP12 in skincancer and pointed out that thepromoter methylation frequenciesweresignificantlyhigherincarcinomaswithrespect tonormalskintissues.AKAP12promotermethylationfrequencies weredemonstratedtobe89.6%,87.1%and51.2%incSCC,BCCand AK,respectively[44].

Inthisstudy,NM23-H1,NDRG1,RHOGDI2andMKK4antibodies showedbothcytoplasmicandnuclearpositivity.Itiswellknown thatthesubcellularlocalizationoftheproteinsalterstheir func-tions[45].TheimportanceofthisfactisnotclearinMSPs.However, Epstein–Barrvirusnuclearantigen3C(EBNA-3C)promotes Nm23-H1 nuclear localization and modulate Nm23-H1activities [46]. Ismailet al. [47] showedthat nuclearlocalization of NM23-H1 proteininbreastcancerisseenmorefrequentlyinnode-positive metastaticcases.Theyconcludedthatabnormalnuclear accumula-tionoftheproteinmayindicatetumorprogression[47].Similarly, wedemonstratedthat nuclearNM23-H1wassignificantly posi-tivelycorrelatedwithstage.ThenuclearsequestrationofNM23-H1 mayreduceoralter thenormal functionoftheprotein.Besides NM23-H1,theimportanceofsubcellularlocalizationofNDRG1has alsobeenemphasizedintheliterature[48–50].Althoughnuclear positivityofMKK4andRHOGDIhasbeendemonstratedinthe lit-erature,toourknowledge,itsmeaningisnotwellknown[35,51]. 5. Conclusions

Although NM23-H1 expression was maintained at constant levels, RGHOGDI2, E-cadherin, and AKAP12 were significantly downregulatedincSCCs.Furthermore,MKK4,NDRG1andCD82 werealsopartially expressedincSCCs. Similartootherhuman tumors,cSCCsshowadistinctMSPprofile.Data fromthisstudy mightalsorevealpossiblepathwaysamongMSPs,whencombined withthecurrentknowledgeonrelatedpathways.Thisrelationship betweentheseMSPswarrantsfurtherbiologicalandexperimental pathwayresearchtobetterunderstandthemetastaticeventswith regardtoSCCs.

Acknowledgements

This study was financially supported by the Scientific and Technical Research Council of Turkey (TUBITAK, grant number SBAG-108S184).TheprojectwasapprovedbytheKırıkkale Uni-versityLocalEthicsCommittee(07.04.2008/2008-039).

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.prp.2015.12.018. References

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