Clinical implications in the shift of syndecan-1 expression from the cell membrane to the cytoplasm in bladder cancer

MISSING IMAGE

Material Information

Title:
Clinical implications in the shift of syndecan-1 expression from the cell membrane to the cytoplasm in bladder cancer
Series Title:
BMC Cancer
Physical Description:
Mixed Material
Creator:
Makito Miyake
Adrienne Lawton
Yunfeng Dai
Myron Chang
Lourdes Mengual
Antonio Alcaraz
Steve Goodison
Charles J Rosser
Publisher:
BMC Cancer
Publication Date:

Subjects

Subjects / Keywords:
Syndecan
Bladder
Cancer biomarker
Specificity

Notes

Abstract:
Background: To determine the diagnostic and prognostic capability of urinary and tumoral syndecan-1 (SDC-1) levels in patients with cancer of the urinary bladder. Methods: SDC-1 levels were quantitated by enzyme-linked immunosorbent assay (ELISA) in 308 subjects (102 cancer subjects and 206 non-cancer subjects) to assess its diagnostic capabilities in voided urine. The performance of SDC-1 was evaluated using the area under the curve of a receiver operating characteristic curve. In addition, immunohistochemical (IHC) staining assessed SDC-1 protein expression in 193 bladder specimens (185 cancer subjects and 8 non-cancer subjects). Outcomes were correlated to SDC-1 levels. Results: Mean urinary levels of SDC-1 did not differ between the cancer subjects and the non-cancer subjects, however, the mean urinary levels of SDC-1 were reduced in high-grade compared to low-grade disease (p < 0.0001), and in muscle invasive bladder cancer (MIBC) compared to non-muscle invasive bladder cancer (NMIBC) (p = 0.005). Correspondingly, preliminary data note a shift from a membranous cellular localization of SDC-1 in normal tissue, low-grade tumors and NMIBC, to a distinctly cytoplasmic localization in high-grade tumors and MIBC was observed in tissue specimens. Conclusion: Alone urinary SDC-1 may not be a diagnostic biomarker for bladder cancer, but its urinary levels and cellular localization were associated with the differentiation status of patients with bladder tumors. Further studies are warranted to define the potential role for SDC-1 in bladder cancer progression.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
All rights reserved by the source institution.
System ID:
AA00020099:00001

Full Text

PAGE 1

RESEARCHARTICLEOpenAccessClinicalimplicationsintheshiftofsyndecan-1 expressionfromthecellmembranetothe cytoplasminbladdercancerMakitoMiyake1,AdrienneLawton2,YunfengDai3,MyronChang3,LourdesMengual4,AntonioAlcaraz4, SteveGoodison1,5,6andCharlesJRosser1,5,7*AbstractBackground: Todeterminethediagnosticandprognosticcapabilityofurinaryandtumoralsyndecan-1(SDC-1) levelsinpatientswithcanceroftheurinarybladder. Methods: SDC-1levelswerequantitatedbyenzyme-linkedimmunosorbentassay(ELISA)in308subjects(102 cancersubjectsand206non-cancersubjects)toassessitsdiagnosticcapabilitiesinvoidedurine.Theperformance ofSDC-1wasevaluatedusingtheareaunderthecurveofareceiveroperatingcharacteristiccurve.Inaddition, immunohistochemical(IHC)stainingassessedSDC-1proteinexpressionin193bladderspecimens(185cancer subjectsand8non-cancersubjects).OutcomeswerecorrelatedtoSDC-1levels. Results: MeanurinarylevelsofSDC-1didnotdifferbetweenthecancersubjectsandthenon-cancersubjects, however,themeanurinarylevelsofSDC-1werereducedinhigh-gradecomparedtolow-gradedisease( p <0.0001), andinmuscleinvasivebladdercancer(MIBC)comparedtonon-muscleinvasivebladdercancer(NMIBC)( p =0.005). Correspondingly,preliminarydatanoteashiftfromamembranouscellularlocalizationofSDC-1innormaltissue, low-gradetumorsandNMIBC,toadistinctlycytoplasmiclocalizationinhigh-gradetumorsandMIBCwasobservedin tissuespecimens. Conclusion: AloneurinarySDC-1maynotbeadiagnosticbiomarkerforbladdercancer,butitsurinarylevelsandcellular localizationwereassociatedwiththediffe rentiationstatusofpatientswithbladde rtumors.Furtherstudiesarewarranted todefinethepotentialroleforSDC-1inbladdercancerprogression. Keywords: Syndecan,Bladder,Cancerbiomarker,SpecificityBackgroundSyndecan1(SDC-1)isoneoffourmembersofatransmembraneheparansulfateproteoglycanfamily.SDC-1is themajorsyndecanexpressedinepithelia,anditplaysa criticalroleincellularprocessesincludingdifferentiation, celladhesion,migrationandinvasion,andangiogenesis [1-3].Functionshavebeenascribedtotheextracellular domainthatcarriesglycosaminoglycan(GAG)sidechains, tothetransmembranedomainandtothecytoplasmicdomainthattransducessignalsfromextracellularligand binding[3].AlteredSDC-1expressionhasbeenreported inanumberofmalignanttumortypesandhasbeenassociatedwithdifferentiationstatusandsurvival[4-6]. Aaboe etal .,identifiedSDC-1asabladdercancer(BCa) biomarkerusinggeneexpressionprofiling[7].Through proteomicanalysesofvoidedurinesfromBCapatients, SDC-1hasalsobeenidentifiedasapotentialdiagnostic biomarker[8].However,inasubsequentmultiplexbiomarkerstudyof127subjects,urinarySDC-1proteincould notbeconfirmedtobesignificantlyelevatedinpatients withBCa[9].Theobservedinconsistencyasadiagnostic biomarkermayberelatedtothestudycohortsemployed todate,butitmayalsobedueinparttothetransmembranenatureofSDC-1.ReleaseofSDC-1intothesoluble fractionoftheurineisdependentonanumberoffactors including:cellularturnover,aberrantprocessingindisease *Correspondence: crosser@cc.hawaii.edu1CancerResearchInstitute,OrlandoHealth,Orlando,FL32827,USA5NonagenBioscienceCorp,Orlando,FL32827,USA Fulllistofauthorinformationisavailableattheendofthearticle 2014Miyakeetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycredited.Miyake etal.BMCCancer 2014, 14 :86 http://www.biomedcentral.com/1471-2407/14/86

PAGE 2

states,releasebyinflammation-associatedshedding[10], andashiftofexpressionfromepithelialtostromalcellsin tumors[11]. Herein,wereportfurtherevaluationofthepotential utilityofSDC-1asadiagnosticandprognosticbiomarker inBCabyanalysisofalargediversetestcohortthrough enzyme-linkedimmunosorbentassay(ELISA),andtheinvestigationofSDC-1proteinexpressionpatternsinbladdertumorsthroughimmunohistochemical(IHC)analysis ofarchivaltissuespecimens.MethodsUrinarySDC-1levelsAfterInstitutionalReviewBoardapprovalbyMDAnderson CancerCenterOrlandoandHospitalClnicofBarcelona andwritteninformedconsent,voidedurineswerecollected intoinstitutionaltissuebanks.Fromthesetissuebanksin theDepartmentsofUrologyfromOrlandoHealthand HospitalClnicofBarcelona,308voidedurinesamplesand associatedclinicaldatawereidentified.Thestudycohort consistedof206adultsubjectswithnoactiveBCaorprevioushistoryofBCa(47withvoidingsymptoms,44with urolithiasis,9withgrosshematuria,14withurinarytract infectionand92withoutanyoftheabovediagnoses)and 102subjectsdiagnosedwith denovo urothelialcarcinoma. Medianfollow-upofthepatientswithBCawas14months. Inourcancergroupandhematuriagroup,imagingofthe upperurinarytractandcystoscopywereperformed.Furthermore,thehistologicsubtype,urothelialcarcinoma,was confirmedbyhistologicalexaminationofexcisedtissuein thecancergroup. Voidedurinesampleswerecentrifugedtoseparate thesupernatantfromthecellularpellet.Thesupernatantwasdecantedandaliquoted,andtheurinarypelletwassnapfrozen.Boththesupernatantandpellet werestoredat-80Cpriortoanalysis.Urinesupernatant proteinconcentrationwasde terminedusingPierce660nmProteinAssayKit(ThermoFisherScientificInc., Waltham,MA,USA).ThelevelofhumanSDC-1(Cat# ab46507Abcam,Cambridge,MA,USA)wasmonitoredin urinesamplesusingacommercialELISAassay.Theassay wasconductedaccordingtothemanufacturer ’ sinstructions.Acalibrationcurvewaspreparedusingpurified standardsforSDC-1.Curvefittingwasaccomplishedby eitherlinearorfour-parameterlogisticregressionfollowingmanufacturer ’ sinstructions.Laboratorypersonnel wereblindedtofinaldiagnosis.Syndecan-1expressioninhumanbladdertumorsUnderInstitutionalReviewBoardapprovalwithawaiver ofconsent,185bladdertumorparaffinblocksandeight benignbladderparaffinblocksdatingfrom2002-2009 wereidentifiedinthepathologicarchivesofOrlando HealthDepartmentofPathology.Theeightbenignbladder paraffinblockswerefromau topsycasesinwhichthere wasnorecordofBCa,hematuriaortobaccouse.Medianfollow-upofthepatientswas18months.AllparaffinblockswereexaminedbyH&Eforhistological verificationofurothelialcarcinomaonlyhistology.Paraffinblockswerecut5 msectionsandplacedona SuperfrostPlusMicroslide.Sectionsweredeparaffinized followedbyantigenretrievalusingcitricacidbuffer (pH6.0,95Cfor20min).Slidesweretreatedwith1% hydrogenperoxideinmethanoltoblockendogenous peroxidaseactivity.After20minblockinginphosphate bufferedsaline(PBS)containing1%bovineserum albumin(BSA),slideswereincubatedovernightat4C withanti-humanSDC-1antibody(mousemonoclonal – Abcamab34164,dilution1/400inPBScontaining 1%BSA).Next,slideswereincubatedwith2 g/mLofbiotinylatedanti-mouseIgGsecondaryantibody(Vector Laboratories,Burlingame,CA)for30minatroomtemperature.Subsequently,thes ectionswerestainedusing StandardUltra-SensitiveA BCPeroxidaseStainingkit (Pierce/ThermoFisherScientific,SanJose,CA)and3, 3 -diaminobenzidine(DAB;VectorLaboratories),counterstainedbyhematoxylin ,dehydrated,andmounted withacoverslide.Humanlivertissue,knowntostain stronglyforSDC-1,wasusedasapositivecontroland omittingtheprimaryantibodyservedasthenegative control.TheaboveimmunostainingforSDC-1aswell astheinterpretationoftheimmunostainingforSDC-1 werebasedonapreviousreportbyMukunyadzi, etal [12].Briefly,thelocati onofimmunoreactivity( e.g. nuclear,cytoplasm,cellmembrane,andstroma)was noted.Thesectionswereanalyzedandstainingassessed usingasemiquantitativegradingsystemasfollows: negative(-),completelackofstainingorstainingin <10%oftumorcells;weak(+),stainingin10to20%of tumorcells;mild(++),stainingin20to50%oftumor cells;moderate(+++),stainingin50to70%oftumor cellsand;strong(++++),stainingin>70%oftumorcells. Usinglightmicroscopy,twoinvestigators(MMandAL), whowerebothblindedtopatients ’ data,interpretedimmunostainingresults.Athirdinvestigator(CJR)reviewed discrepanciesandrenderedafinalscore.DataanalysisTheWilcoxonranksumtestwasusedtodeterminethe associationbetweenurinarySDC-1andBCa.Nonparametricreceiveroperatingcharacteristic(ROC)curves wereplottedandtheabilityoftheurinarySDC-1biomarkertoindicateBCawasestimatedbycalculatingthe areaundertheROCcurves(AUC).Thesensitivityand specificityofthebiomarkerattheoptimalcutoffvalue wasdefinedbycalculatingtheYoudenindex[13].The agreementbetweeninterpre tingSDC-1immunohistochemistryresultsbetweenthetwo investigatorswasanalyzedMiyake etal.BMCCancer 2014, 14 :86 Page2of7 http://www.biomedcentral.com/1471-2407/14/86

PAGE 3

usingkappastatisticswiththestrengthofagreement 0.81-1.00interpretedasalmostperfect.Theresultsare presentedasweightedkappawith95%confidenceinterval(CI).ComparisonofimmunohistochemicaldistributiondatawasperformedusingChisquaretest. Disease-specificsurvival(DSS)curveswereobtained usingtheKaplan-Meiermethod,andcomparedbythe logranktestforeachprognosticvariable[14].Multivariateanalysiswasperformedtoidentifyindependent prognosticvariablesusingastepwiseCoxproportional hazardsregressionmodel.Statisticalsignificanceinthis studywassetat p <0.05andallreported p valueswere 2-sided.AllanalyseswereperformedusingSASsoftwareversion5.00(SanDiego,CA). Table1Demographicandclinicopathologiccharacteristicsof308subjectscomprisingELISAstudycohortand193 subjectscomprisingIHCstudycohortELISACohortIHCCohort BCa(%)n=102Controls(%)n=206BCa(%)n=185Controls(%)n=8 MedianAge(range,y)69(20 – 93)56(18 – 89)73(30 – 94)26(21 – 43) Male:Femaleratio84:18152:54143:424:4 Race White91(89%)135(66%)156(84%)N/A AfricanAmerican5(5%)20(10%)8(4%)N/A Other6(6%)51(24%)19(12%)N/A PositiveFISH40/74(54%)2/22(9%)N/AN/A Suspicious/positivecytology37/94(39%)2/22(9%)N/AN/A Medianfollow-up(months)14418N/A Clinicalstage Tis6(6%)17(9%) Ta41(40%)45(24%) T114(14%)63(34%) T241(40%)60(33%) Tumorgrade Low38(37%)27(15%) High64(63%)158(85%) Mediantumorsize(cm)3.03.0IHC immunohistochemistry, BCa bladdercancer, N/A notavailable. Figure1 UrinarySyndecan-1levels. ComparisonofurinaryconcentrationsofSDC-1betweenthecancerandnon-cancergroups.Inthe box-and-whiskerplotofurinaryconcentrationofSDC-1,thecentralboxrepresentsthevaluefromthelowertoupperquartile.Significance( p<0.05 ) wasassessedbytheWilcoxonranksumtest. Miyake etal.BMCCancer 2014, 14 :86 Page3of7 http://www.biomedcentral.com/1471-2407/14/86

PAGE 4

ResultsUrinarySDC-1ELISACharacteristicsofthestudycohortof308subjects(102 subjectswithactiveBCaand206subjectswithnoevidenceofactiveBCaorahistoryofBCa)arepresented intheTable1.Themedianurinaryconcentrationof SDC-1wasnotsignificantlyhigheroverallinsubjects withBCacomparedtosubjectswithoutBCa(71.25ng/ ml vs .36.10ng/ml, p =0.23)(Figure1).Neitherdid SDC-1levelsdifferamongthegroupsthatmadeupthe diversecontrolcohort( p =0.562,datanotshown).However,adifferenceinurinarySDC-1levelwasnotedbetweenpatientswithtumorsofdifferinggradeandinvasive subtype.Specifically,low-gradebladdertumorswerenoted tohavehighermedianurinarySDC-1levelscomparedto high-gradebladdertumors(64.55ng/ml vs .26.1ng/ml, p <0.0001),andnon-muscleinvasivebladdercancer (NMIBC)hadhighermedianurinarySDC-1levels comparedtomuscleinvasivebladdercancer(MIBC) (58.23ng/ml vs .28.53ng/ml, p =0.0049)(Figure1).Immunohistochemicalstainingofbladdertissue specimensCharacteristicsofthestudycohortof193subjects(185 subjectswithurotheliacarcinomahistologyonlyand8 subjectswithbenignbladderhistology)arepresentedin theTable1.Thepathologists ’ intra-observeragreement onSDC-1interpretationandscoringwas  good ’ witha Figure2 ExpressionofSyndecan-1proteininhumanbladdertissue.a) Representativestainingofbenignbladderepithelium(left)and cancerousbladder(right)showingmembranousstainingofepithelialcells. b) Representativestainingoflow-gradebladdercancer(left)and high-gradebladdercancer(right).High-gradecancerswerenotedtohavecytoplasmicstainingwhilelosingtheirmembranousstaining. c) Representativestainingoflowpathologicstage(pTa)bladdercancer(left)andhighpathologicstage(pT2)bladdercancer(right).Allimageswere capturedat400magnification.ColumnbargraphsillustratethepopulationofsubjectswithSDC-1membranestainingandSDC-1cytoplasmic stainingin (d) benignbladderepithelium vs .non-muscleinvasivebladdercancer(NMIBC) vs .muscleinvasivebladdercancer(MIBC), (e) low-grade tumor vs .high-gradetumorand (f) ,Ta-1tumor vs .T2-4tumor. Miyake etal.BMCCancer 2014, 14 :86 Page4of7 http://www.biomedcentral.com/1471-2407/14/86

PAGE 5

notedkappascoreof0.64(0.8 – 1.0,excellent;0.6 – 0.8, good;0.4 – 0.6,moderate;0 – 0.4,poor),95%CI0.61 – 0.68. Thepercentageagreementwas82.0%.Innormaltissue,as wellaslow-gradediseaseandNMIBC,over70%ofSDC-1 immunostainingwaslocatedwithinthecellularmembrane(Figure2a)andwasgradedasmoderate(+++)to strong(++++).Minimalimmunoreactivitywasnotedin thestroma.Withinbladdertumors,55%ofhigh-grade tumors(comparedtolow-gradetumors, p <0.0001) werenotedtohaveincreasedcytoplasmicexpressionand reducedmembranousexpressionofSDC-1(Figure2b). Inthesameway,higherstagetumors(T2-4 vs .Ta-1, p <0.0001)werenotedtohaveincreasedcytoplasmic expressionandreducedmembranousexpressionof SDC-1(Figure2c).Thoughthelocationofstaining changedfrommembranoustocytoplasmicamongst high-gradeandhighstagetumors,immunostaining grading,weak(+)tostrong(++++),didnotchange, illustratingashiftoftheubiquitouslyexpressedSDC-1 fromthecellularmembraneinwell-differentiated,low stagetumorstothecytoplasminpoorly-differentiated, higherstagetumors.Analysesofprognosticparametersassociatedwith diseasespecificsurvivalUnivariateanalysisrevealedthatNMIBCandmembranousimmnostainingforSDC-1representfavourableprognosticfactorsassociatedwithdisease-specificsurvival (DSS)( p <0.0001and p =0.0004,respectively)(Figure3). Howeveronmultivariateanalysis(Table2),onlyMIBC (hazardratio[HR]=21.1,95%confidenceinterval[CI]= 4.24 – 105.1, p =0.0001)provedtobeanindependentrisk factorforDSS,resultinginasignificantreductionin survival.Furthermore,MIBCwasassociatedwithasignificantreductioninoverallsurvival(HR=9.60,CI: 2.59-35,5, p =0.001).DiscussionSDC-1isexpressedmainlyinepithelialtissues,hence, studiesaimingtoaddressitsroleinmalignancieshave focusedoncarcinoma.Inanumberofmalignancies,the expressionofSDC-1correlateswithtumorstageand grade[15-18],buttheassociationbetweenSDC-1status andBCahasnotbeenextensivelystudied.OtherinvestigatorshavereportedapositivecorrelationofSDC-1 withfibroblastgrowthfactors(FGFRs)inbladdertumors,thesefactorsarethoughttobekeymoleculesin low-gradeBCa[19].OnlyShimada etal .,haveinvestigatedthebiologicroleofSDC-1inhumanBCacells.In theirstudy,theBCacelllines,UMUC2andUMUC3 hadSDC-1expressionsilencedbysiRNAtransfection, whichledtoaninductionofapoptosis invitro andareductioninmouseorthotopicbladdertumorgrowth[20]. Toourknowledge,ourstudyisthelargeststudyto datetoevaluateSDC-1inhumanbladdertumorsboth invoidedurineandintumorsections.Weusedtwo complimentaryapproachestoclassifySDC-1expression inhumanbladdertumors.First,urinarySDC-1levels weremonitoredbyELISAinacohortof308subjects. WhiletherewasnodifferenceinurinarySDC-1levels betweenBCa-bearingsubjectsandnon-BCabearing subjects( p =0.23),lowerurinarylevelsofSDC-1were associatedwiththepresenceofhigh-gradetumorsand/ orMIBC.TheprognosticcapabilityofSDC-1in Figure3 Kaplan-Meiercurvesfordisease-specificsurvival. Disease-specificsurvivalstratifiedby (a) membranous vs .cytoplasmic SDC-1, (b) low-grade vs .high-gradeand (c) non-muscleinvasivebladder cancer(NMIBC) vs .muscleinvasivebladdercancer(MIBC).HR,hazard ratio;95%CI,95%confidenceinterval. Miyake etal.BMCCancer 2014, 14 :86 Page5of7 http://www.biomedcentral.com/1471-2407/14/86

PAGE 6

predictinghighergradeandhigherstagediseasepriorto patientsundergoingcystoscopyandtransurethralresectionofbladdertumorhasthepotentialtoimprovepatients ’ outcomes.Second,wedeterminedtheexpression patternofSDC-1proteininacohortof193bladdertissuespecimens.ThoughadifferenceinSDC-1expressionpatternwasnotseenbetweenbladdertumorsand benignbladderhistology,possiblyduetothesmallsamplesizeofthebenigncohort,asignificantshiftincellularlocalizationofSDC-1wasassociatedwithhigh-grade tumorsandMIBC.Thesetumorstendedtolosethedistinctmembranousstainingobservedinnormalurothelia. Thetwocomplimentaryapproachesutilizedinthecurrent studyyieldedsimilarinferences, i.e. ,moreaggressiveor moreadvanceBCahaslessmembraneboundSDC-1.If lessmembraneboundSDC-1ispresentinatumormass, thenitmightbeexpectedthatlessshedorreleasedSDC-1 wouldbepresentinthesolublefractionofvoidedurine frompatientswithmoreaggressiveoradvancedBCa. ShiftsinSDC-1expressionpatternshavebeenalluded toinpreviousreports,butnoneinBCa.Astudyby Mennerich etal .,describedashiftofSDC-1expression fromtheepithelialcomponenttothestromalcomponentinsolidtumors[11].Anobservedoverallincrease intumorSDC-1mRNAwasdemonstratedby insitu hybridizationandproteinlevelsconfirmedbyimmunohistochemistryintumor-associatedstromalcellsin breast,lungandcoloncarcinoma.Wedidnotobserve thisphenomenoninourstudy,themajorityofSDC-1 expressionwasintheepithelialcomponentofthebladder tumors.Theexpressionpatternshiftthatouranalysesrevealedwasfromdistinctlymembranoustodiffuselycytoplasmicinhigh-gradeandhigh-stagebladdertumors.This associationwithdiseaseprogressionsuggeststhattheloss ofSDC-1functionatthecell-surfaceorcellmembrane andthusmayfacilitatecancerpr ogressionandthedevelopmentofinvasiveandmetastaticdisease.Severalstudies haveshowntheinvolvementofcell-surfaceSDC-1incellcellandcell-matrixadhesion,possiblythroughtheregulationofintegrinactivities[21].ThelossofSDC-1atthecell surfacebyextracellularcleavagecandecreasethestrength oftumorcelladhesionwithinthetissuearchitecture, resultinginanincreaseincellularmotility.Thisinturn mayallowcancercellstocrossthebasementmembrane andinvadesurroundingtissuesaswellasdistantsites[11]. ThelossofSDC-1atthecell-surfacecouldalsooccur throughaswitchtotranslat ionofalternative,nonmembranousisoforms,orbyaberrantprocessinginanadvancedtumor.Thisconceptexistsforthewell-known tumorsuppressorgeneE-cadherin.SimilartoSDC-1,cellsurfaceE-cadherinassists incelladhesionandlossof E-cadherinisassociatedwith moreaggressiveBCathatpossessagreaterpotentialtoinvadeandmetastasize[22,23]. Thoughthepresentstudiesarequiteintriguing,theyonly eludetoabiologicphenomenonwhichnowmustbefurtherexploredtoa)reportassociatedcellularandmolecular changes,b)confirmtheELISAandimmunohistochemistry resultsinalargecohort,c)determinewhichdomain(cytoplasmic,transmembraneorext racellular)isshedinvoided urineandd)determineinadditiontochangesinlocation inexpressioniftherearechangesinthequantityofSDC-1 expressionbetweenthevariousd iseasestates.Furthermore, thepreliminarynatureofourimmunohistochemicalresults shouldbeconfirmedinalargercohort.ConclusionsInsummary,decreasedurinarylevelsofSDC-1inBCa patientswereassociatedwithhigh-gradeorhigh-stage disease,andthisphenomenoncorrelatedwithashiftof SDC-1proteincellularlocal izationfromthecellular membranetothecytoplasminthesehigh-gradeand highstagebladdertumors.On univariateanalysis,loss ofmembranouslocalizationofSDC-1wasassociated withasignificantreductioninDSS.ThisisthefirstreporttodescribespecificSDC-1expressionchangesas beingassociatedwithmoreag gressive,lethalBCa.Furtherstudiesareunderwaytounderstandtheroleof SDC-1inBCaandtoinvestigatetheprognosticpotentialofSDC-1monitoringinhumanbladdertumors.Competinginterests DrGoodisonandCharlesJ.Rosserhaveacompetinginterestinthattheyare officersofNonagenBioscienceCorp,asmallbiotechcompanywithan interesttodevelopurinarybiomarkers.Nootherauthorspossessa competinginterest. Table2MultivariateanalysisofdiseasespecificsurvivalandoverallsurvivalVariables N Disease-specificsurvivalOverallsurvival HR95%CI p HR95%CI p Stage NMIBC12511 MIBC6021.104.24 – 105.10.00019.602.59 – 35.50.001 SDC-1expression Membrane9611 Cytoplasm890.870.21 – 3.600.850.990.27 – 3.600.99HR Hazardratio, 95%CI 95%confidenceinterval, NMIBC Non-muscleinvasivebladdercancer, MIBC Muscleinvasivebladdercancer.Miyake etal.BMCCancer 2014, 14 :86 Page6of7 http://www.biomedcentral.com/1471-2407/14/86

PAGE 7

Authors ’ contributions Allauthorshavereadandapprovedthefinalmanuscript.MM,AL:acquisition ofdata.YD,MC:statisticalanalysis.LM,AA:clinicalsamples,draftingof manuscript.SG:studyconceptanddesign,draftingofmanuscript.CJR:study conceptanddesign,draftingofmanuscript,funding. Acknowledgements Theauthorsaregratefultothe501subjectswhoparticipatedinthisstudy. ThisworkwassupportedbyresearchgrantsfromFlightAttendantMedical ResearchInstitute(CJR),FloridaDepartmentofHealthJamesandEstherKing TeamScienceAward10KT-01(CJR),FloridaStateJamesandEstherKing BiomedicalResearchAwardTechnologyTransferFeasibility1KF06(SG)and NationalCancerInstituteRO1CA116161(SG).SGandCRareemployeesof NonagenBioscienceCorp. Authordetails1CancerResearchInstitute,OrlandoHealth,Orlando,FL32827,USA.2DepartmentofPathology,OrlandoHealth,Orlando,FL32806,USA.3DepartmentofBiostatistics,TheUniversityofFlorida,Gainesville,FL32610, USA.4LaboratoryandDepartmentofUrology,HospitalClnic,Universitatde Barcelona,Barcelona,Spain.5NonagenBioscienceCorp,Orlando,FL32827, USA.6DepartmentofHealthSciencesResearch,MayoClinic,Jacksonville,FL 32224,USA.7UniversityofHawaiiCancerCenter,ClinicalandTranslational ResearchProgram,701IlaloStreet,Honolulu,HI96813,USA. Received:3April2013Accepted:11February2014 Published:13February2014 References1.CouchmanJR,PatakiCA: Anintroductiontoproteoglycansandtheir localization. JHistochemCytochem 2012, 60 (12):885 – 897. 2.McQuadeKJ,RapraegerAC: Syndecan-1transmembraneandextracellular domainshaveuniqueanddistinctrolesincellspreading. JBiolChem 2003, 278 (47):46607 – 46615.Epub2003Sep14. 3.TengYH,AquinoRS,ParkPW: Molecularfunctionsofsyndecan-1in disease. MatrixBiol 2012, 31 (1):3 – 16.Epub2011Oct18. 4.ConejoJR,KleeffJ,KoliopanosA,MatsudaK,ZhuZW,GoeckeH,BichengN, ZimmermannA,KorcM,FriessH,BchlerMW: Syndecan-1expressionis up-regulatedinpancreaticbutnotinothergastrointestinalcancers. IntJ Cancer 2000, 88 (1):12 – 20. 5.ContrerasHR,LedezmaRA,VergaraJ,CifuentesF,BarraC,CabelloP,GallegosI, MoralesB,HuidobroC,CastellnEA: Theexpressionofsyndecan-1and-2is associatedwithGleasonscoreandepithelial-mesenchymaltransition markers,E-cadherinandbeta-catenin,inprostatecancer. UrolOncol 2010, 28 (5):534 – 540.Epub2009May17. 6.ShahL,WalterKL,BorczukAC,KawutSM,SonettJR,GorensteinLA, GinsburgME,SteinglassKM,PowellCA: Expressionofsyndecan-1and expressionofepidermalgrowthfactorreceptorareassociatedwith survivalinpatientswithnonsmallcelllungcarcinoma. Cancer 2004, 101 (7):1632 – 1638. 7.AaboeM,MarcussenN,JensenKM,ThykjaerT,DyrskjtL,OrntoftTF: Gene expressionprofilingofnoninvasiveprimaryurothelialtumoursusing microarrays. BrJCancer 2005, 93 (10):1182 – 1190. 8.YangN,FengS,SheddenK,XieX,LiuY,RosserCJ,LubmanDM,Goodison S: Urinaryglycoproteinbiomarkerdiscoveryforbladdercancerdetection usingLC/MS-MSandlabel-freequantification. ClinCancerRes 2011, 17 (10):3349 – 3359. 9.UrquidiV,ChangM,DaiY,KimJ,WolfsonED,GoodisonS,RosserCJ: IL-8as aurinarybiomarkerforthedetectionofbladdercancer. BMCUrol 2012, 12: 12. 10.PruessmeyerJ,MartinC,HessFM,SchwarzN,SchmidtS,KogelT,HoetteckeN, SchmidtB,SechiA,UhligS,LudwigA: Adisintegrinandmetalloproteinase 17(ADAM17)mediatesinflammation-inducedsheddingofsyndecan-1 and-4bylungepithelialcells. JBiolChem 2010, 285 (1):555 – 564. 11.MennerichD,VogelA,KlamanI,DahlE,LichtnerRB,RosenthalA,Pohlenz HD,ThierauchKH,SommerA: Shiftofsyndecan-1expressionfrom epithelialtostromalcellsduringprogressionofsolidtumours. EurJCancer 2004, 40 (9):1373 – 1382. 12.MukunyadziP,LiuK,HannaEY,SuenJY,FanCY: Inducedexpressionof syndecan-1inthestromaofheadandnecksquamouscellcarcinoma. ModPathol 2003, 16 (8):796 – 801. 13.FlussR,FaraggiD,ReiserB: EstimationoftheYoudenIndexandits associatedcutoffpoint. BiomJ 2005, 47: 458 – 472. 14.PepeMS,FengZ,JanesH,BossuytPM,PotterJD: Pivotalevaluationofthe accuracyofabiomarkerusedforclassificationorprediction:standards forstudydesign. JNatlCancerInst 2008, 100: 1432 – 1438. 15.AnttonenA,KajantiM,HeikkilP,JalkanenM,JoensuuH: Syndecan-1 expressionhasprognosticsignificanceinheadandneckcarcinoma. BrJCancer 1999, 79 (3 – 4):558 – 564. 16.AltemeierWA,SchlesingerSY,BuellCA,BrauerR,RapraegerAC,ParksWC, ChenP: Transmembraneandextracellulardomainsofsyndecan-1have distinctfunctionsinregulatinglungepithelialmigrationandadhesion. JBiolChem 2012, 287 (42):34927 – 34935.doi:10.1074/jbc.M112.376814.Epub 2012Aug30. 17.BarbareschiM,MaisonneuveP,AldoviniD,CangiMG,PecciariniL,Angelo MauriF,VeroneseS,CaffoO,LucentiA,PalmaPD,GalligioniE,DoglioniC: Highsyndecan-1expressioninbreastcarcinomaisrelatedtoan aggressivephenotypeandtopoorerprognosis. Cancer 2003, 98 (3):474 – 483. 18.LundinM,NordlingS,LundinJ,IsolaJ,WikstenJP,HaglundC: Epithelial syndecan-1expressionisassociatedwithstageandgradeincolorectal cancer. Oncology 2005, 68 (4-6):306 – 313.Epub2005Jul12. 19.MarzioniD,LorenziT,MazzucchelliR,CapparucciaL,MorroniM,FioriniR, BracalentiC,CatalanoA,DavidG,CastellucciM,MuzzonigroG,MontironiR: Expressionofbasicfibroblastgrowthfactor,itsreceptorsandsyndecans inbladdercancer. IntJImmunopatholPharmacol 2009, 22 (3):627 – 638. 20.ShimadaK,NakamuraM,DeVelascoMA,TanakaM,OujiY,MiyakeM, FujimotoK,HiraoK,KonishiN: Roleofsyndecan-1(CD138)incellsurvival ofhumanurothelialcarcinoma. CancerSci 2010, 101 (1):155 – 160. 21.ChenP,AbacherliLE,NadlerST,WangY,LiQ, etal : MMP7sheddingof syndecan-1facilitatesre-epithelializationbyaffecting 2 1integrin activation. PLoSONE 2009, 4 (8):e6565.22.ThoresonMA,ReynoldsAB: Alteredexpressionofthecateninp120in humancancer:implicationsfortumorprogression. Differentiation 2002, 70 (9 – 10):583 – 589. 23.HuX,RuanY,ChengF,YuW,ZhangX,LarrS: p130Cas,E-cadherinand -catenininhumantransitionalcellcarcinomaofthebladder:expressionandclinicopathologicalsignificance. IntJUrol 2011, 18 (9):630 – 637. doi:10.1111/j.1442-2042.2011.02793.x.Epub2011Jun14.doi:10.1186/1471-2407-14-86 Citethisarticleas: Miyake etal. : Clinicalimplicationsintheshiftof syndecan-1expressionfromthecellmembranetothecytoplasmin bladdercancer. BMCCancer 2014 14 :86. Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Miyake etal.BMCCancer 2014, 14 :86 Page7of7 http://www.biomedcentral.com/1471-2407/14/86