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RESEARCHOpenAccessSmoothenedgenealterationsinkeratocysticodontogenictumorsZhangRui1,PengLi-Ying1,QuJia-Fei1,HongYing-Ying1,ChenFeng2*andLiTie-Jun1*AbstractBackground:Ithasbeenwidelydemonstratedthatthehedgehogpathwayisstronglyassociatedwithbasalcellcarcinomaoftheskin(NBCCS).
ToassesspotentialDNAalterationsrelatedtokeratocysticodontogenictumors(KCOTs),wesequencedsmoothened(SMO)genesin12sporadicKCOTs.
Methods:Polymerasechainreaction(PCR),capillaryelectrophoresisanddideoxychain-terminationsequencingwereusedtoexaminepotentialDNAalterationsinsporadicKCOTs.
Results:FivealterationsinSMOgenesweredetected.
Fourofthesemutationsconsistedoftwosynonymousandthreemissensemutations;twoofwhichhavenotbeenreportedtodate(c.
T776A,c.
T1281G).
Conclusions:SMOgenesmayplayanimportantroleinthesonichedgehog(SHH)pathwayandcouldalsoberesponsibleforgeneratingKCOTsandNBCCS.
However,theirinfluenceonSHHsignalingremainstobeelucidated.
Keywords:Keratocysticodontogenictumor(KCOT),Hedgehog(HH)signalingpathway,Genemutation,SMOgeneBackgroundOdontogenickeratocyst(OKC)isanaggressive,cysticjawlesionwithstronggrowthpotentialandahighrecurrencerate.
Inrecentyears,theWorldHealthOrganization(WHO)reviseditsnametokeratocysticodontogenictumor(KCOT).
Thisreclassificationisbasedonitsaggressivebehaviorandhighrecurrencerate,emphasizingthatKCOTisabenigntumorratherthanacyst[1-3].
Althoughthegreatmajorityofkerato-cystsoccurinisolationassingle,non-syndromiccysts,theymayalsopresentasmultiplecystsasafeatureofthenevoidbasalcellcarcinomasyndrome(Gorlinsyndrome,OMIM#109400)[4].
Atpresent,therearemanymanuscriptsthatfocusontherelationshipbetweenKCOTandPTCH1(patched)genemutations,demonstratingthatPTCH1,thegeneresponsibleforNBCCS,mayalsoplayanimportantroleinsporadicKCOTs[5-8].
ThePTCH1geneisatumorsuppressorgenelocatedat9q22.
32[9].
Astudyof14patientswithNBCCS-associatedKCOTsand29patientswithsporadicKCOTsindicatedthatmutationsintrans-membrane2(TM2)arecloselyrelatedtothedevelopmentofsporadicKCOTs[5].
Thehedgehog(HH)signalingpathwayisakeyregulatorofembryonicdevelopment,controllingbothcellularpro-liferationandcellfate.
Bindingofsonichedgehog(SHH)toitsreceptor,patched(PTCH1),isbelievedtorelievenormalinhibitionbyPTCH1ofsmoothened(SMO),aseven-spantransmembraneproteinwithhomologytoaG-protein-coupledreceptor[10].
SMOisatumor-relatedgenelocatedat7q32.
3,con-tains12exonsspanningapproximately24kb,anden-codesa787-amino-acidtransmembraneglycoprotein[11].
ItsreceptorisaGprotein-coupledreceptorthatinteractswithPatched,animportantpartoftheHHsignalingpathwayduringembryogenesisaswellasadult-hood[12,13].
TheHHpathwayhasbeendemonstratedtoplayanimportantroleindifferentdevelopment-relatedcancers[14-18],buttheexactmechanismofactionhasnotyetbeenelucidated.
TheproteingeneratedbySMOisdownstreamofPTCH1;thatis,theexpressionofPTCH1restrainstheactivationofSMO,andtherebyinhibitsactivationoftheHHpathway[19-22].
RecentstudieshavehighlightedthetherapeuticvalueofSMO*Correspondence:moleculecf@gmail.
com;litiejun22@vip.
sina.
comEqualcontributors2CentralLaboratory,PekingUniversitySchoolandHospitalofStomatology,22ZhongguancunAvenueSouth,HaidianDistrict,Beijing100081,China1DepartmentofOralPathology,PekingUniversitySchoolandHospitalofStomatology,22ZhongguancunAvenueSouth,HaidianDistrict,Beijing100081,ChinaHEAD&FACEMEDICINE2014Ruietal.
;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/4.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycredited.
TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecommons.
org/publicdomain/zero/1.
0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Ruietal.
Head&FaceMedicine2014,10:36http://www.
head-face-med.
com/content/10/1/36antagonistsforthetreatmentofHH-linkedcancers[22,23].
SMO,themainactivatoroftheHHpathwaymayserveasacatalystduringthegenerationofcysts,andtherefore,geneticmutationsofSMOareofgreatimportance.
MethodsTumorsamplesandclinicalbackgroundFourteenKCOTsampleswithadefinitediagnosiswereacquiredfromclinicalsourcesatPekingUniversitySchoolofStomatology,OralandMaxillofacialSurgeryDepartment.
DiagnoseswerebasedonWHOclassifica-tionoftumors:pathologyandgeneticsoftumorsoftheheadandneck[24].
AllsampleswerefromChinesepa-tients(eightmalesandsixfemales).
Agesvariedfrom10to58years,withanaverageof29.
2years.
ExperimentalprotocolsusedinthisstudywerereviewedandapprovedbytheEthicsCommitteeofthePekingUniversityHealthScienceCenter(Peking,China).
Informedconsentwasobtainedfromallsubjects.
DNAisolationandmutationanalysisGenomicDNAsfromepithelialcellsandinterstitialcellsoftumorswereisolatedusingaQIAampDNAMinikit(Qiagen,Hilden,American)accordingtothemanu-facturer'sinstructions.
DNAwasquantifiedusingaNanodrop(ThermoFischer,Wilmington,DE).
SMO(NM_005631.
4)codingsequencesweredeterminedusingpolymerasechainreaction(PCR)and1%agarosegelelectrophoresis.
ThirteenpairsofprimerscoveringtheentirecodingsequencesandafewnucleotidesintotheintronsequencesonbothendsofSMOarede-scribedinTable1.
PCRswereperformedonaPTC-100(MJResearch,Watertown,MA,USA)inafinalvolumeof30μLcontainingapproximately100-ngtemplateDNA,200μmol/LdNTPs,0.
2–0.
4μmol/Leachprimer,1.
25UExTaqpolymerase(TaKaRa,Dalian,China),and10XPCRbuffer(50mmol/LKCl,10mmol/LTris–HCl,pH9.
0,and1.
5mmol/LMgCl2).
Exon1,exon12aandexon12boftheSMOgenewereamplifiedbyatouch-down(TD)PCRtechnique(Table1).
Forexample,TD65-56°Cwasperformedasfollows:aninitialdenaturationstepat95°Cfor5min,followedby40cyclesat94°Cfor30s,30satanannealingtemperaturedecreasingfrom65°Cto56°C(decreaseof1°Ceach2cycles,20cyclesat55°C),30sat72°C,andafinalextensionstepat72°Cfor10min.
Thespecificityofamplifiedproductswasconfirmedby2%agarosegelelectrophoresis.
PCRproductswerepurifiedbyLifeTechnologies(AppliedBiosystems,USA)andsequencedbyanABI3730xlDNAsequencer(AppliedBiosystems,USA).
Bothforwardandreversestrandsweresequenced.
Anymutationde-tectedwasconfirmedusingbothforwardandreverseprimers.
StatisticalanalysisExperimentaldatawereanalyzedusingtheSPSSver.
10.
0softwareandarepresentedasmeans±standarddeviation(SD)usingstatisticalmethodssuchasat-test,analysisofbivariatecorrelation,etc.
APvalue<0.
05wasconsideredtoindicatestatisticalsignificance.
Resultsarerepresenta-tiveoftwoindependentexperiments.
ResultsanddiscussionHedgehog(HH)signalingpathwayHedgehog(HH)isasignaltransductionpathwaycloselyrelatedtocellgrowthanddifferentiation,andplaysavitalroleinembryonicdevelopment.
MutationorabnormalTable1PCRconditionsofSMOexonsExonSize(bp)Forwardprimersequence(5′to3′)Reverseprimersequence(5′to3′)ReferenceAnnealingtemperature(°C)1391GGGCTGCTGCTGCTGCTGGTCCCCGCCCTCTCCAAAACTSunetal.
,2008[25]TD60~502283AAGAACTGTCCTGCCCAGATGCCACTGGACCCTGCCCTATACWangetal.
,2013[12]633305AATAATTTGCCAAGCCAGCCCTTCTGATCATGACCCTTCCCWangetal.
,2013[12]634256AGGGTCATGATCAGAAGGGTCAGTATGCAGTAGGGCAGAGCCWangetal.
,2013[12]635302CTGACTTCTGGGAACCTCCAGGACAGAAGGTGGGTTACTGGCWangetal.
,2013[12]636206GTGGCGCAGGTATAGTGACTGGCCCTATAGGAGCTAGCTGGGWangetal.
,2013[12]637175GACTCCAGAGCCTTAGGACCCTCCCTATGGCTAACTTGTCCCWangetal.
,2013[12]638196AAGCAGTTCTTGGACTGAGCCCCATCCATTGAATCTGCTGTCWangetal.
,2013[12]559379AGTTGGAAGCTGCAGTGGGCAAGGCTGTGCTAGAGGCAGWangetal.
,2013[12]6310228CTCTGGAAAGAATGGCATCGTTCCAAATAATCTGTGTGCCCWangetal.
,2013[12]5511215AATGGCACTGACTATGGGAGGCCACTCTTCAGATCCTCTGGGWangetal.
,2013[12]6312a292GAGCCAGGGCCCCAGGCTCGTACGCTCCCTGTCGGCAAGAGTSunetal.
,2008[25]TD65-5612b315AGTACCATTCCTCGACTGCCTGGTATTGGTTCCTCTCTTTCCSunetal.
,2008[25]TD65-56Ruietal.
Head&FaceMedicine2014,10:36Page2of7http://www.
head-face-med.
com/content/10/1/36expressionofcomponentsofthispathwaywillleadtovariousdevelopmentaldefectsand/ortumors.
AlteredHHsignalingisimplicatedinthedevelopmentofapproxi-mately20-25%ofallcancers,especiallythoseofsofttissues[26].
ThesefindingsalsosuggestthatproteinsoftheHHsignalingpathwayarepredominantlylocatedwithintheepithelialcomponentsofglandularodontogeniccysts(GOCs)anddentigerouscysts(DCs).
Therefore,theHHsignalingpathwaymayplayanimportantroleintheformationofepitheliallining[27].
ThediscoveryofoncogenicmutationsintheHHandmitogen-activatedproteinkinase(MAPK)pathwaysinover80%ofame-loblastomas,locallydestructiveodontogenictumorsofthejaw,wasreportedin2014bygenomicanalysisofarchivalmaterial.
MutationsinSMOarecommoninameloblastomasofthemaxilla,whereasBRAFmuta-tionsarepredominantintumorsofthemandible[28].
Inaddition,SHHsubgroupmedulloblastomasaregen-eticallydistinctininfants,childrenandadults[29].
Mostmeningiomashavesimplegenomes,withfewermutations,rearrangementsandcopy-numberalter-ationsthanreportedinotheradulttumors.
However,severalmeningiomasharbormorecomplexpatternsofcopy-numberchangesandrearrangements,includingonetumorwithchromothripsis.
AsubsetofmeningiomaslackingNF2alterationsharboredrecurrentoncogenicmu-tationsinAKT1(p.
Glu17Lys)andSMO(p.
Trp535Leu),andexhibitedimmunohistochemicalevidenceofacti-vationofthesepathways[11].
Inanotherstudy,SMOmutations,whichactivatetheSHHsignalingpathway,wereidentifiedin~5%ofnon-NF2mutantmeningiomas.
Collectively,thesefindingsidentifydistinctmeningiomasubtypes[30].
Moreover,somefindingsprovideara-tionaletoexploretheuseofSMOandBCL2inhibitorsasadjuvanttherapyfortreatmentofDLBCLoftheGCtype[31].
TheexpressionofSMOinNPCisgenerallyhigh,whereasexpressionofPTCH-1isrelativelylow.
DownregulationofPTCH1andupregulationofSMOmaycauseabnormalactivationoftheHHsignalingpathwayinNPC,albeitthatthegenesisanddevelop-mentofNPCmaybeassociatedwithabnormalactiva-tionofHHsignaling[32].
TheHHsignalingpathwayiscontrolledbyPTCH1andSMO,whicharelocatedonthetargetcell'smembrane.
Asthereceptorofsonichedgehog(SHH),thePTCH1gene(51kb)encodesa12-transmembrane-domainprotein(total,1,447aminoacids).
Therearetwohomologousgenesinhumans,PTCH2andPTCH1,whichnegativelyregulateSMO,aG-protein-coupledreceptor,throughSHH.
SMObelongstotheGprotein-coupledreceptorFZ/SMOsuperfamily,containingafieldthatcrossesthecellmembraneseventimes.
Withoutaligand(SHH),PTCHandSMOformaninhibitorycompound,restrainingsignaltransductionthroughouttheentirepathway.
However,whenSHHbindstoPTCH1,SMOescapesfromtheinhibitionbyPTCH1,resultinginenhancedexpressionofdownstreamtargetgenes,includingtheGLIsuperfamily[33].
Asshownbynumerousresearchers,abnormalactivationoftheHHsignalingpathwayiscloselyrelatedtothedevelopmentofavarietyoftumors,andbothPTCH1andSMOplayacriticalroleinthispathway.
TheHHsignalingcascadeishighlyconservedandinvolvedinthedevelopmentofdiseasethroughoutevolution.
Nevertheless,comparedwithotherpathways,ourmechanisticunderstandingofHHsignaltransductionisremarkablyincomplete.
Intheabsenceofligand,theHHreceptorPatched(Ptc),repressesthekeysignaltransducerSmoothened(Smo)throughanunknownmechanism.
HHbindingtoPtcalleviatesthisrepression,causingSmoredistributiontotheplasmamembrane,phosphorylationandsubsequentopeningoftheSmocyto-plasmictailandSmooligomerization.
However,theorderandinterdependenceoftheseeventsarepoorlyunder-stood.
WehavemathematicallymodeledandsimulatedSmoactivationfortwoalternativemodesofactivation,withPtcprimarilyaffectingeitherSmolocalizationorphosphorylation.
Here,weshowthatSmolocalizationtotheplasmamembraneissufficientforphosphorylationofthecytoplasmictailinthepresenceofPtc.
Usingfluor-escencecross-correlationspectroscopy(FCCS),wealsodemonstratedthatinactivationofPtcbyHHinducesSmoclusteringirrespectiveofSmophosphorylation.
Ourob-servationsthereforesupportamodelofHHsignaltrans-ductionwherebysubcellularlocalizationofSmo,andnotphosphorylation,istheprimarytargetofPtcfunction[31].
Severalstudies[34-36]haveconfirmedthatthePTCH1geneisatumorsuppressor;thissuggeststhatitsmutationincreasesthelikelihoodofdevelopingcancer,althoughfewreportsregardingtherelationshipbetweenSMOandKCOThavebeenpublished.
SMOmutationsCodingvariantsofSMOwereidentifiedfollowingouranalysis,andthemutationsarelistedinTable2.
ThefourSMOmutationsdetectedweredistributedonfiveexons(exons2,3,5,6,and10),ofwhichthereweretwosynonymousmutationsandthreemissensemutations.
Thefirstalterationwaslocatedatnucleotideposition776ofexon2,aheterozygousmutationfromTtoA(Figure1A);thisalterationhasnotyetbeenreportedbyotherresearchers.
Wedeterminedthat9of12KCOTpatientshavethisalteration.
Aftercomparingtheproteinaminoacidsequencesofhuman,rice,mouse,andDros-ophila(Figure2),weconcludedthatthisaminoacidisrelativelyconserved.
Meanwhile,followingGenomicsonlinesoftwareSIFT[37](http://sift.
jcvi.
org/)analysis,weconcludedthattheaminoacidchangeresultsinanegativeanddamagingeffectontheprotein(ProveanPreduction:Ruietal.
Head&FaceMedicine2014,10:36Page3of7http://www.
head-face-med.
com/content/10/1/36Deleterious;SiftPreduction:Damaging).
Throughfurtheranalysis,wediscoveredthattheaminoacidislocatedinacysteine-richdomain(CRD)oftheSmoothenedreceptor(Smo)integralmembraneprotein.
TheCRDisoneofthekeyplayersintheHHsignalingpathway,andiscriticalfordevelopment,cellgrowthandmigration,aswellasstemcellmaintenance.
TheCRDofSmoisconservedinverte-bratesandcanalsobeidentifiedininvertebrates.
Thepre-cisefunctionoftheCRDinSmoisunknown.
MutationsintheDrosophilaCRDdisruptSmoactivityinvivo,whiledeletionoftheCRDinmammaliancellsdoesnotappeartoaffectSmooverexpression[38-40].
Therefore,webe-lievethatthisaminoacidmaytremendouslyimpactdevel-opment,cellgrowthandmigration,aswellasstemcellmaintenance.
Itisimportanttonotethatthepeakofthisalterationisdifferentfrommutationsatotherpositions.
Forexample,thepeakofAisrelativelylowerthanT,butnotashighashalfofT.
Accordingtoarecentreport[41],thismaybeduetotheheterogeneityoftumorsinissueswedetected,indicatingthatsomecellshavemutatedwhileothersremainunchanged.
Thesecondalteration,aheterozygousmutationfromAtoG,waslocatedatnucleotideposition862ofexon3(Figure1B)butdoesnotresultinanaminoacidchangeatp.
G194.
Thethirdmutation(c.
G1444C,withG/Calleles)wasidentifiedinseventumors(Figure1C),butdoesnotresultinanaminoacidchangeatp.
G388.
Oneinterestingfactaboutthissynonymousmutationis,withtheexceptionofonesporadicKCOT,allothersamples(includingsporadicandNBCCS-associated)wereiden-tifiedwiththismutation,ofwhich17werehomozygousand13wereheterozygous.
ThefrequencyofGreachedahighof0.
7581,whichisfarhigherthanthestatisticre-portedinthesingle-nucleotidepolymorphism(SNP)databasecuratedbytheNationalCentreforBiotechnol-ogyInformation(NCBI)andthe1000GenomesProject(minorallelefrequency[MAF]=0.
2277).
Thismutationhasbeenreportedinmanyothercelllinesassociatedwithmesothelioma,suchasMSTO-211H,NCI-H28,JU77,LO68,NO36,ONE58,andSTY51[42].
Therefore,itishighlylikelythatthismutationplaysasignificantroleintheoccurrenceoftumors.
However,whetherthismutationisrelatedtoKCOTsshouldbedetermined.
Oneofthemissensemutations(c.
G2049C,withG/Calleles),whichhasbeenreportedpreviously,hasun-knownfunctionalimplications(Figure1D).
Thismuta-tionleadstoanaminoacidchangeatp.
Ser590Thr;itisimportanttonotethatbothValandGlycanbephos-phorylated.
Meanwhile,SIFTsoftwareanalysisrevealedthataninteractivemutationbetweentheseaminoacidsisneutralandtolerated(Table3).
Infact,thisalteredaminoacidislocatedwithintheintracellularloopsofSmoothened,whichmaybecriticallyrelatedtodown-streamproteins,andsubsequentlyimpactthefunctioningofSMO,therebyimpactingactivationoftheHHsignalingpathway[43].
Themissensemutation(c.
T1281G,withT/Calleles)hasnotbeenreportedpreviously(Figure1E).
Thismu-tationleadstoanaminoacidchange(p.
Val334Gly)lo-catedonthefirstextracellularloopofSmoothened,whichiscloselyassociatedwiththefunctionofSMO.
Functionalstudiesontransmembraneregionsarere-strictedbytechnologicalconditions,butaccordingtotheSIFTsoftware(Table3),mutationofthisaminoacidleadstodamagingchangesintheprotein;therefore,webelievethatthismutationstronglyimpactsdiseaseoccurrence.
AdditionalstudiesarerequiredtodeterminewhetherthismutationcausesfunctionalchangesintheSMOgene.
ThemodeofactionbetweentheSMOandPTCHgeneshasyettobeelucidated,butrecentstudieshaveshownthatanymissinggeneinyeastwillimposepressureonthecelltocompensate,therebyleadingtoadd-itionalgeneticmutations[44].
MostSMOgenemutationsTable2SMOmutationsin12sporadicand19NBCCS-associatedKCOTsHistologySampleIDExon/intronNucleotidedefinitionAminoaciddefinitiondbSNPrs#clusterIDMAFClassFunctionS*K1,K2,K4,K5,K6,K7,K8,K10,K11Exon2c.
T776Ap.
Phe166Ile——MissenseUnknownSK7Exon3c.
A862Gp.
=(194)Glurs563342500.
0266SynonymousPolymorphismSK4Exon5c.
T1281Gp.
Val334Gly——MissenseUnknownSK2,K3,K4,K5,K6,K7,K8,K9,K10,K11,K12Exon6c.
G1444Cp.
=(388)Glyrs22286170.
2277SynonymousPolymorphismN*N1,N2,N3,N4,N5,N6,N7,N8,N9,N10,N11,N12,N13,N14,N15,N16,N17,N18,N19,Exon6c.
G1444Cp.
=(388)Glyrs22286170.
2277SynonymousPolymorphismSK1Exon10c.
G2049Cp.
Ser590Thrrs1144068350.
010MissensePolymorphism*S=sporadic,N=NBCCS-associated.
Ruietal.
Head&FaceMedicine2014,10:36Page4of7http://www.
head-face-med.
com/content/10/1/36detectedareseriousalterations,suchasmissenseandsynonymousmutations,whileframeshiftandnonsensemutationshavenotbeendetected.
Therefore,webelievethattheSMOgenemutationmayserveasadrivingforceinpatientswithKCOTs.
TocompensateforthedefectscausedbytheSMOmutation,PTCH1causesthesamesignalingpathwaytomutate,leadingtomuta-tion(s)inthePTCHgene.
Thisconjectureneedsfurtherworktoconfirm.
Inmostcases,peoplebelievethattumorgrowthisdrivenbythe"latest"cancercellsubsetsbecausetheycarrymostcancermutations.
However,manymutationsexistatalowfrequency,suggestingthattumorscontainmanysubclonestherelationshipsamongwhicharestillunclear.
Arecentreportexaminingthehet-erogeneityofbreastcancer[41]showedthatstemcellsinbreastepitheliacoulddifferentiateintoluminalandbasalcells,whichconstitutetheepithelia.
SomealsobelievethatbreastneoplasmsinducedthroughWnt1overexpressionFigure1SMOmissensemutationinsporadicKCOTs.
A.
Thisalternationatnucleotideposition776ofexon2isaheterozygousmutationfromTtoAandcausesthep.
Phe166Ilemutation.
B.
Thisalternationatnucleotideposition862ofexon3isaheterozygousmutationfromAtoG.
However,thisnucleotidechangedoesnotresultinaminoacidchange(p.
=(194)Glu).
Minorallelefrequency(MAF)=0.
0266.
C.
Thisalternationatnucleotideposition1444ofexon6wasidentifiedinseventumors.
Among31patientssequenced,17arehomozygousand13areheterozygousatthissite.
Thisnucleotidechangedoesnotresultinanaminoacidchange(p.
=(388)Gly).
MAF=0.
2277.
D.
Thisalternationatnucleotideposition2049ofexon10isaheterozygousmutationfromGtoCandcausesthep.
Ser590Thrmutation.
E.
Thisalternationatnucleotideposition1281ofexon5isaheterozygousmutationfromTtoCandcausesthep.
Val334Glymutation.
Figure2Conservedaminoacidsinhuman,rice,mouse,andDrosophila.
Aminoacidsequencesbetweenhuman,rice,mouse,andDrosophilaaresimilar(t),indicatingthataminoacidresiduesarerelativelyconservedatthemutationsitedetected(c.
T776A).
Ruietal.
Head&FaceMedicine2014,10:36Page5of7http://www.
head-face-med.
com/content/10/1/36arederivedfromthiscelltype.
Paracrineinteractionbe-tweentwocelltypes,whichisdrivenbysignalingmole-cules,anditsshortdistance,maintainsco-existenceoftwotypesofpedigree.
OnlyluminalcellscanproduceWnt1,whereasbasalcellsrelyonthisproteintoprolifer-ate.
Wnt1inducestheHrasgeneinbasalcellsofbreastneoplasms,whichmaycontainamutationthatdrivescancerprogression,butthismutationhasnotyetbeendetectedincancerouscellsinthelumen.
Thereisco-operationbetweenbasalandluminalcellclones;thiscooperationisnecessaryforWnt1todrivetwotypesofcellsintumors.
Inaddition,KCOTsoccurduringtheearlystagesofdentalepithelialformation,duringwhichdentallaminaanditsresidential,epitheliumandstromainteractwithothersduringgrowth,subsequentlyinducingdifferentiation.
Therefore,wehypothesizethatmutationofsomegenesinthesubcutaneousinterstitialtissuemayimposepressureonepithelialgenestocompensateforthedefect,andthattheSMOmutationmayplayacriticalroleinthisprocess.
However,additionalstudiesareneededforconfirmation.
Asweknow,thehedgehogsignalingpathwayisbeingsuggestedtobeadrugtargetforcancertherapyforitsactivationinhumancancers[45-47].
Therefore,wethinkthetwonewlyidentifiedSMOmutationsdeservetobefurtherinvestigatedfortheirtherapeuticapplicationincancertreatment.
ConclusionsFollowingtheanalysisofSMOgenemutationsandcom-biningourresultswithsimilarstudies,weconcludethatSMOplaysanimportantroleintheHHsignalingpathwayandmayberesponsibleforthedevelopmentofKCOTsandNBCCS.
However,furtherresearchfocusingonthemechanismoftheirinfluenceontheSHHsignalingpathwayisrequired.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsFCconceivedanddesignedtheresearch.
JFQandYYHprovidedsamples.
RZandLYPperformedtheexperiments,andcollected,analyzedandinterpretedthedata.
LYPwrotethemanuscript,andeditedtablesandfigures.
RZeditedthemanuscript,tablesandfigurelegends.
FCandTJLreviewedandeditedthemanuscript.
Allauthorsreadandapprovedthefinalmanuscript.
AcknowledgmentsWegratefullyacknowledgethepatientswhoparticipatedinthisstudyandDr.
QianZhangforhertechnicalassistance.
ThisstudywassupportedbygrantsfromtheNationalNaturalScienceFoundationofChina(nos.
81030018,30872900and81200762).
TheEnglishinthisdocumenthasbeencheckedbyatleasttwoprofessionaleditors,bothnativespeakersofEnglish.
Foracertificate,pleasesee:http://www.
textcheck.
com/certificate/GilDRH.
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Table3PROVEANhumanproteinbatchresultVariationProteinsequencechangePROVEANpredictionSiftpredictionRow_No.
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1166FI5.
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063Deleterious0.
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1590ST1.
487Neutral0.
219ToleratedChangesataminoacids166and344causeadeleteriousalterationandadamagingresult.
Thechangeataminoacidposition590isrelativelyneutralandtoleratedbytheprotein.
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