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REVIEWOpenAccessTargetingtheWntpathwaysfortherapiesArtemBlagodatski1,DmitryPoteryaev2andVladimirLKatanaev3*AbstractTheWnt/β-cateninsignalingpathwayiscrucialinanimaldevelopmentfromspongestohumans.
Itsactivityintheadulthoodislessgeneral,withexceptionshavinghugemedicalimportance.
Namely,improperactivationofthispathwayiscarcinogenicinmanytissues,mostnotablyinthecolon,liverandthebreast.
Ontheotherhand,theWnt/β-cateninsignalingmustbere-activatedincasesoftissuedamage,andinsufficientactivationresultsinregenerationfailureanddegeneration.
Thesebothmedicallyimportantimplicationsareunifiedbytheemergingimportanceofthissignalingpathwayinthecontrolofproliferationofvarioustypesofstemcells,crucialfortissueregenerationand,incaseofcancerstemcells–cancerprogressionandrelapse.
ThisarticleaimsatbrieflyreviewingthecurrentstateofknowledgeinthefieldofWntsignaling,followedbyadetaileddiscussionofcurrentmedicaldevelopmentstargetingdistinctbranchesoftheWntpathwayforanti-cancerandpro-regenerationtherapies.
Keywords:Wnt,Frizzled,Cancer,Regeneration,DrugdiscoveryIntroduction:theWntsignalingpathwaysTheWntsignalingplaysinstrumentalrolesinanimalde-velopment[1].
Thistypeofintracellularsignalingappar-entlywas'invented'togetherwith(orinrequirementfor)animalmulti-cellularity,asitsarchitectureandcompo-nentsarealreadypresentinthesimplestmetazoanssuchasspongesandctenophores[2,3].
ThepathwayisinitiatedbysecretedlipoglycoproteinsoftheWntfamily,ofwhich19membersexistinhumans.
Giventhenumerouspost-translationalmodificationsandtheneedforatightlycon-trolledmannerofWntdiffusionthroughthetissue,thesecretionapparatuswithintheWnt-producingcellsisra-thercomplex[4].
Inthesignal-receivingcells,Wntacti-vatesareceptoroftheFrizzled(FZD)family(10membersinhumans)andaLRP5/6co-receptor(2membersinhumans).
WhileLRP5/6aresingle-transmembranepro-teins,FZDspossessseventransmembranedomainsandbelongtotheGprotein-coupledreceptor(GPCR)super-family[5].
AlthoughinitiallyquestionedasfunctionalGPCRs,FZDsinrecentyearshavebeenclearlydemon-strated,bygeneticandbiochemicalmeans,tosignalthroughheterotrimericGproteins[6-11].
Togetherwiththelatter,acytoplasmicproteinDishevelled(Dvl)actsasanimmediatetransducerofthesignalfromthereceptors[12].
BothtypesoftransducersactonAxin[13,14]–akeycomponentoftheβ-catenin-destructioncomplexalsoincludingtheproteinAPCandkinasesGSK3βandcaseinkinase.
Thefunctionofthiscomplexistophosphorylatecytoplasmicβ-catenin,targetingitforubiquitin-dependentproteasomaldegradation[15].
WithdysfunctionalAxin,thedestructioncomplexbe-comesinactive,leadingtoaccumulationofβ-catenin,itstranslocationtothenucleus,andactivationofLEF/TCF-dependenttranscription[16](Figure1).
AmongWnt-targetgenesarepro-proliferativec-MycandcycD1[17].
Inadditiontothepathwaydirectlyinitiatedfromtheplasmamembrane,Wnt-receptorcomplexescanalsobeinternal-ized,inaheterotrimericGprotein-andRab5-dependentmanner,intosignalingendosomes,whichisrequiredfortheamplificationofthesignalingstrength[18,19].
Theβ-catenin-dependentWntsignalingpathwayde-scribedaboveisoftenreferredtoasthe"canonical"Wntsignaling.
Inadditiontoit,agroupof"non-canonical",β-catenin-independentpathwayscanbeinitiatedbyWnt/FZDcomplexes[20].
Theycanincreaseconcentra-tionofintracellularcalciumionsandregulatethecyto-skeletonandultimately–cellpolarityandmotility.
Theycanalsoantagonizetheβ-catenin-dependentsignalingincertaincontexts[21].
ApartfromheterotrimericGpro-teinsandDvl,noothercomponentsoftheβ-catenin-dependentpathwayareinvolvedin"non-canonical"sig-naling.
Ourreviewwillonlyepisodicallytouchupon*Correspondence:vladimir.
katanaev@unil.
chEqualcontributors3DepartmentofPharmacologyandToxicology,UniversityofLausanne,Lausanne,SwitzerlandFulllistofauthorinformationisavailableattheendofthearticle2014Blagodatskietal.
;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/4.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycredited.
TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecommons.
org/publicdomain/zero/1.
0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Blagodatskietal.
MolecularandCellularTherapies2014,2:28http://www.
molcelltherapies.
com/content/2/1/28thesetypesofWntsignaling,mostlyconcentratingontheWnt/β-cateninpro-proliferativebranch.
TheWnt/β-cateninsignalingisrepeatedlyusedduringanimaldevelopment.
Giventhisimportantdevelopmen-talfunction,theroleofthispathwayinregulationofstemcellproliferationanddifferentiation,whichemergesasaunifyingfunctionofthispathwayintheadult,bothinthephysiologicalandpathologicalcontexts,isnotsurpris-ing.
InitiallyWnt/β-cateninsignalinghasbeenshowntobecrucialforthemaintenanceandself-renewalofhematopoieticcells[22].
Thisdiscoverywascorroboratedbysubsequentfindingsofthenecessityofthispathwayforproliferationofneuronal[23],embryonic[24],mammary[25],intestinal[26]andothertypesofstemcells.
Finally,theroleofWnt/β-cateninsignalingincancerstemcells(CSCs)hasalsoemerged[27].
Thesediscoveriesformthebasisforthetranslationaleffortsoftargeting(suppressingoractivating)theWnt/β-cateninsignalinginanti-cancerandregenerationtherapies,discussedbelow.
ReviewDevelopmentofanti-cancerdrugstargetingtheWnt/β-cateninpathwayTheWnt/β-cateninsignalingpathwayasatargetofanti-cancerdrugshasattractedattentionofbiotechcompaniesrelativelyrecently.
ThisfieldreceivedaspecialattentionwhenitbecameclearthatthissignalingplaysamajorroleinCSCproliferation.
CSCshavebeenimplicatedintumormaintenanceandrelapseaftersurgicalresection.
TheCSCpoolisself-renewingandthisprocessislargelydrivenbyre-activationofembryonicprogramsmediatedbyWnt,HedgehogandNotchsignalingpathwaysandthemTORsignalinghub[27,28].
Alargenumberofpreclinicalexperimentsdemon-stratedthatinhibitionofWnt/β-cateninsignalingcanaffectcancercellgrowthandsurvival(forreviewsee[29]).
WhilemutationsintheWnt/β-cateninpathwayareresponsibleforcertaintypesofcancers,mostnotablyAPCmutationsincolorectalcancer[30],manycancersdrivenbyoverstimulationofthissignalingdonotharbormutationsinitscomponents.
Forexample,thestudyof245invasivebreastcarcinomashasidentifiedasubgroupwithtriple-negativephenotype(ER-,PgR-,HER2-)whereβ-cateninwasaccumulatedinthenucleus,whichisahall-markofWnt/β-cateninpathwayactivity.
However,noβ-cateninmutationhasbeenfoundinalltriple-negativecarcinomasanalyzed[31].
ThereforeconstitutivereceptorstimulationcanaccountforhyperactiveWntsignalingintheabsenceofactivatingmutationsinthecomponentsofthepathway.
Mostcurrentanti-cancerdrugs,smallmoleculeinhibi-torsandmonoclonalantibodies(mAbs),aredesignedtotargetrapidlyproliferatingcellswhichrepresentcom-mittedcancercellsbutnotCSCs.
Sorafenib,asmallmoleculeinhibitorofmultipletyrosinekinasesinvolvedintumorproliferation,isusedinthetreatmentofacutemyeloidleukemia(AML).
ThereitissupposedtoinhibitFMS-likekinaseoverexpressedinalmostallcasesofFigure1SchematicrepresentationoftheWnt/β-cateninsignalingpathwayandtheoncology-indicationdrugcandidatesdiscussedinthepaper.
Themoleculartargets(whereknown)ofthesmallmoleculeandantibody-baseddrugcandidatesareshown.
Blagodatskietal.
MolecularandCellularTherapies2014,2:28Page2of15http://www.
molcelltherapies.
com/content/2/1/28AML.
Ashasbeenrecentlydemonstrated,sorafenibef-fectivelyreducesthenumberofmatureAMLprogeni-torsbutfailstoeradicateAMLstemcellsandprimitiveprogenitors[32].
TrastuzumabisanexampleofamAb.
IttargetstheHER2receptoroverexpressedinonequarterofbreastcancers.
Trastuzumabmonotherapyhasonlya30%responserateandacquiredresistancetotrastuzumaboccursfrequently.
Trastuzumabhasbeenshowntobeef-fectiveonlyinthecontextofPI3KsignalingandinthepresenceofPTEN,butCSCsdisplaytheaberrantformerandtheabsenceoflatter[33].
Sincecurrentcancerther-apiesfailtoeradicateCSCs,selectivetargetingofCSCswouldbeapromisingtherapeuticstrategy.
SmallmoleculestargetingtheWnt/β-cateninpathwaySignificanteffortsaremadeworld-widetodeveloppotentinhibitorsoftheWnt/β-cateninsignaling,butonlyfewofthesehavemadeittoreachclinicaltrials.
Small-moleculeinhibitorsidentifiedinanumberofhigh-throughputscreenscanbeclassifiedintofourgroups:β-catenin/TCF-antagonists,modulatorsoftranscriptionco-activator,Dvlbinders,andothermechanism-basedinhibitors[34](Figure1andTable1).
AmongsmallmoleculestheleadingclassisthePorcu-pineinhibitors,asexemplifiedbyLGK974(Novartis)[35].
Porcupineisamembrane-boundO-acetyltransferasere-quiredforacylationofWntmolecules,andinhibitionofthisenzymeresultsinreducedorabolishedWntsecretion[36].
LGK974isnowinclinicalPhaseIforthefollowingconditions:melanoma,breastcancer(triple-negative),pancreaticadenocarcinoma,colorectalcancer,headandneckcancers.
AdditionallypatientswithothertumortypeswithdocumentedgeneticalterationsupstreamintheWnt/β-cateninsignalingpathwayarebeingrecruited(www.
clinicaltrials.
gov).
Anotherclassofcompoundsinhibitstankyrases(TNK1and2)–enzymesamongotherfunctionsfoundtodestabilizeAxin.
TNKinhibitionresultsinpreventionofβ-cateninaccumulation.
ExamplesareXAV939fromNovartis[37]andG007-LKfromRoche[38].
TheactivedevelopmentofTNKinhibitorsispursuedfortworeasons:first,Axinistherate-limitingcomponentoftheβ-catenindestructioncomplex[39];second,Axinmuta-tionsandincreasedβ-cateninlevelsareassociatedwithvarioustypesofcancer[40].
IncancerswithmutatedAxinorAPC,theupstreamantagonistsactingonWntsortheirreceptorsmaybelesseffective.
However,thefullpotentialoftheantitumoractivityofTNKinhibitorsmaybelimitedbyintestinaltoxicityassociatedwithin-hibitionofWnt/β-cateninsignalingandcellprolifera-tioninintestinalcrypts[38].
Duringactivationofβ-catenin-dependentgenetran-scription,thecomplexofβ-cateninandLEF/TCFrecruitsadditionalfactorsforchromatinremodeling,likeCBPandp300,whichpossessthehistoneacetyltransferaseactivity.
Associationofβ-cateninwithhistoneacetylasescanbeantagonizedbyseveralcompounds.
Oneofthem,PRI-724(PrismPharma)hasreachedclinicaltrialsinAMLandad-vancedsolidtumors.
ItisasmallmoleculethatselectivelyinhibitsthehistonedeacetylaseCBP/β-catenincomplex,blockingexpressionoftheWnt/β-cateninpathway-dependentpro-growthandpro-survivalgenes.
PRI-724exhibitsaselectiveantiproliferativeeffect,inhibitingvari-ouscancercelllinesinvitroandsubstantiallyinhibitingtumorgrowthinanimalstudies(http://clinicaltrials.
gov/show/NCT01302405).
Alreadyapproveddrugswithwell-establishedsafetyprofilefindafareasierwaytoclinicaltrialsforadiffer-entindication.
Niclosamideisananti-helminthicdrugusedinhumansfornearly50years.
In2009,itemergedasacompoundthatinhibitsWnt3a-stimulatedβ-cateninstabilizationandTCF/LEFreporteractivityinosteosar-comacellline.
ThiswasaresultofscreeningofanFDA-approveddruglibraryforcompoundsthatwouldpromoteendocytosisofFZD1[41].
Invitro,niclosamidetreatmentreducedthelevelsofLRP6andβ-catenin,andinvivoithadasuppressiveeffectonbasalbreastcancerxenografts[42,43].
Despitetheseobservations,niclosamideisnotreadyyetforclinicaltrialsforoncologyindications.
Asanapproveddrugitisgivenorallyandisonlypartiallyabsorbedfromthegastrointestinaltract,thereforenovelderivativesareneededtoimprovethebioavailabilityofTable1CurrentstatusofclinicaltrialsofbiologicsspecificallytargetingtheWnt/β-cateninpathway(ligandsorreceptors)Name/companyTargetAgentConditionsClinicalphaseOTSA101(CentreLéonBérard,OncoTherapyScience)FZD10mAbSynovialsarcoma.
Antibody-radionuclideconjugate(90Y)IOMP-54F28(OncomedPharma)WntFzd8-Fc(scavengingreceptor)HCC,livercancer,ovariancancer,pancreaticcancer,othersolidtumorsIVantictumab(OncomedPharma)FZD1,2,5,7,8mAbSolidtumors(completed),NSCLC,metastaticbreastcancer,pancreaticcancer,(active,asacombinationwithchemiotherapy)IFoxy-5(WntResearchAB)FZD5PeptideMetastaticbreastcancer,colorectalcancer,prostatecancerIBlagodatskietal.
MolecularandCellularTherapies2014,2:28Page3of15http://www.
molcelltherapies.
com/content/2/1/28niclosamide.
Thealternativeintravenousrouteofniclosa-mideadministrationrequirescomprehensiveinvestigationregardingthesafetyandthepossibilityofsystemicappli-cation[43].
Otherpotentialanti-WntdrugcandidatesemergefromscreeningofFDA-approvedcompounds;theanti-leprosydrugclofaziminehasrecentlybeendiscoveredasapotentinhibitorofWnt/β-cateninsignalingandproliferationofWnt-dependenttriplenegativebreastcancercells[44].
WntsastargetsAlthoughtherehasbeenanumberofreportswhereWntproteinsweretargeteddirectlybyantibodies(seeforexample[45,46]),noneoftheanti-WntmAbsiscur-rentlyvisibleeveninthepre-clinicalpipelinesofpharmacompanies.
AnotherwaytoneutralizeWntligandsischosenbythecompanyOncoMedPharmaceuticals.
ItscandidatebiologicOMP-54F28isafusionbetweentheWnt-bindingCRDdomainofFZD8andtheFc-fragmentofIgG.
OMP-54F28worksasascavengerforWntpro-teins(apparentlyseveralofthefamily)preventingthemfrombindingtoendogenousmembrane-boundFZDs[47].
Surprisingly,despitepronouncedreductionofxenografttumorgrowthinmice,OMP-54F28treatmentdidnotvis-iblychangethelevelsorcellularlocalizationofβ-catenininxenografttissues.
Thissuggeststhatalthoughchangesinβ-cateninmayhavebeentoosmalltodetectbyimmu-nohistochemistry,theattenuationofWnt/β-cateninsig-nalingwassufficienttoinhibitthetumorgrowth.
Moreimportantly,thisstudyhasshownnoadverseeffectsintheskinandintestine(http://www.
oncomed.
com/Pipeline.
html)(butseesection"SafetyofWntpathwaytargeting"below).
FZDsastargetsTheFZDfamilyofGPCRsprovidesalargeandpractic-allyuntappedsourceofpotentialtargetsfortherapeuticinterventions[48].
AnumberofpharmacompaniesaresearchingfornovelGPCR-interactingmolecules.
Themosthigh-throughputapproachisthescreeningofsmallmoleculechemicallibrariestoidentifycandidatethera-peutics.
Yet,inthepastdecadethenumberofsmallmole-culestargetingGPCRsthatwereapprovedastherapeuticshasbeenverylow.
Thehighattritionrateinpreclinicalandclinicalstudies,creditedtotoxicity,lowefficacyorselectivityputsanenormousburdenondrugdiscoverybudgets.
Incontrasttothat,proteinbiologics,suchasmonoclonalantibodies(mAbs),haveseveraladvantagesastherapeutics.
Theyarehighlyselectiveandhavemuchlon-gerhalf-livesthansmallmolecules[49,50].
PeptidefragmentsofWntligands,bindingtotheCRDdomainofFZDreceptors,havebeenproposedaspoten-tialtherapeuticagents.
Indeed,invitroexperimentsindi-catethatthesepeptidescancompetewithfull-lengthWntsandattenuatecanonicalsignaling.
Howeveronecandoubttheirvalueeveninanimalmodelpreclinicalstudies,sincetherapidclearanceofnon-modifiedpeptideswouldpreventanylastingeffectoncancercells.
Suchan-tagonistmimeticsofWntswouldneedtobemodified,forexamplebyPEGylationorformylation,toeffectivelyin-creasetheirhalf-life,beforeconsideringthemasthera-peuticcandidates.
AhexapeptideBox5,derivedfromWnt5aandstabilizedbytheN-butyloxycarbonylgroup,hasbeendevelopedtoantagonizeWnt5a-stimulatedme-tastasisinmelanoma[51].
Incontrasttoitsactivityinmelanoma,Wnt5ashowstumor-suppressingactivityinthebreast,andrestoringthisproteincansuppressmi-grationofbreastcancercells–activityrecapitulatedbyaformylatedhexapeptideFoxy-5alsoderivedfromWnt5a[52,53];thisdrugcandidateiscurrentlyinphaseIclinicaltrials.
FZD10hasaveryrestrictedexpressionpattern;itisun-detectableinnormalhumantissuesexceptplacenta,butup-regulatedinsynovialsarcomas.
Takingthisoppor-tunity,OncoTherapySciencehasdevelopedachimerichumanizedmAbagainstFZD10,namedOTSA101.
Non-radiolabeledOTSA101antibodyhasonlyweakantagonis-ticactivityonsynovialsarcomacellgrowth.
However,Yttrium90-radiolabeledOTSA101(OTSA101-DTPA-90Y)showedsignificantantitumoractivityfollowingasingleintravenousinjectioninmousexenograftmodel[54]andisnowinPhaseIclinicalstudies.
FZD7istheWntreceptormostcommonlyup-regulatedinavarietyofcancersincludingcolorectalcancer,hepato-cellularcarcinoma(HCC)andtriplenegativebreastcan-cer[55].
IthasbeendemonstratedthatsiRNA(smallinterferingRNA)knockdownofFZD7displayedanti-canceractivityinvitroandinvivoduetotheinhibitionofthecanonicalWntsignalingpathway[56].
WhilesiRNAapproachremainstobeproblematicinclinicforanumberofreasons,anti-FZDmAbnamedvantictumabisoneoftheleadproteinbiologicsintheOncomedPharmaceuti-calspipelineforoncologyindications.
ThisantibodywasinitiallyidentifiedbybindingtoFZD7'CRDdomainandabilitytoinhibitWnt3a-inducedsignalinginacell-basedassay.
InadditiontoFZD7,vantictumabalsobindstofourotherFZDreceptors(outoftenencodedbythehumangenome)–restrictingitsselectivity(seebelow).
Inmousexenograftmodelsofseveraltypesofsolidtumors(amongthempancreatic,coloncancers,andtriple-negativebreastcancer),vantictumabasamonotherapydemonstratedjustadecreaseinthetumorgrowthrate.
Ontheotherhand,combinedtreatmentwithcytotoxicchemotherapypro-ducedinsometumortypesnotonlyadramatictumorshrinkagebutalsotumorcelldifferentiationandreductionofCSCnumbers[57].
Theinterimdataofvantictumabclinicaltrials(PhaseI)havebeenreportedin2013–2014atvariousconferences(acollectionofposterscanbeBlagodatskietal.
MolecularandCellularTherapies2014,2:28Page4of15http://www.
molcelltherapies.
com/content/2/1/28foundatOncomedPharmaceuticals'website).
Vantictu-mabhasclearpharmacodynamicseffectsonexpressionofstemcellanddifferentiationgenesinthetumor,aswellasinhairfolliclesandbones.
Theinitialenthusiasmhoweverhasrecentlydiminishedwhenclinicaltrialswereputonhold(seesection"SafetyofWntpathwaytargeting"below).
SecretedFrizzled-relatedproteins(sFRPs)havealsobeeninfocusastargetsforcancertherapy.
ThereareconflictingreportsintheliteratureastowhethersFRPsareantagonistsoragonistsofWnt/β-cateninsignaling[58-60].
Forexample,sFRP2isover-expressedinhumanangiosarcomaandbreastcancerandstimulatesangiogen-esisviaactivationofthecalcineurin/NFATc3pathway.
Recently,sFRP2hasbeenassessedasaviabletherapeutictarget[61].
sFRP2mAbhasbeenshowntoinduceanti-tumorandanti-angiogeniceffectsinvitroandinhibitacti-vationofβ-cateninandnuclearfactorofactivatedT-cellsc3(NFATc3)inendothelialandtumorcells.
sFRP2mAbtreatmentofangiosarcomaallograftsorMDA-MB-231breastcarcinomaxenograftsinnudemicesignificantlyreducedtumorsvolume.
GiventhatthismAbpreferen-tiallyaccumulatesinsFRP2-positivetumorsandislongcirculatingintheblood,itmaybeagoodcandidateforclinicalstudies.
Evenmoreperspectivewouldbeanapproachcombin-ingtwoormoretargetedtherapies.
InthisrespectarecentstudybyBirchmeierandcolleagues[62]isofaparticularinterest.
Theauthorshavegeneratedacompoundmutantmousemodelofaggressivebasalbreastcancer,combiningtheactivationofβ-cateninandHGF(Wnt-Metsignaling).
TheyidentifiedthechemokinesystemCXCL12/CXCR4asacrucialdriverofthesetumors.
Moleculartherapytar-getingWnt,MetandCXCR4significantlydelayedtumordevelopmentinmice.
Moreover,thegenesignatureidenti-fiedinthesemodeltumorswasfoundtobepredictiveofpoorsurvivalinhumanpatientswithER-negativebreastcancers.
Althoughthesmallmoleculeinhibitorsusedinthisstudyareonlyprototypesofthepossiblefuturedrugs,theoutlookispromising.
Insuchacombinationtherapy,inhibitionoftheWnt/β-cateninpathwaywouldstopself-renewalprogramofCSCs,whilesuppressingCXCR4signalingwouldleadtodifferentiationofcancerpropagatingcells.
RegenerationTheWnt/β-cateninpathwayisknowntobeessentialforstemcellproliferation.
Whilelife-threateninginthecon-textofCSCs,thisfactunderliestheimportanceofthepathwayinregenerativeprocessesinmanytissues,suchastheliver,brain,muscle,skinandbone.
ThismakesWntpathwayagonistsdesirablecandidatesforregeneration-enhancingtherapies,andsomehavealreadyshowntheirregenerativeeffects.
WewillreviewtheroleofdifferentbranchesoftheWntpathway(butmostlyWnt/β-catenin)separatelyfordifferentorgansandtissues,payingspecialattentiontothetherapeuticpotentialandsuccessfulpracticalapplicationsofactivatorsoftheWntsignaling(Figure2andTable2).
LiverTheWnt/β-cateninsignalingisknowntobeinvolvedinliverorganogenesisduringembryonicdevelopment[63]andisactiveatallstagesoftheorganogenesis,activatingcellproliferationthroughc-MycandcycD1andregulatinghepatocytedifferentiationviaaninterplaywithHNF4,C/EBPa,BMP4,andsomeotherpathways[64,65].
InthenormaladultlivertheWnt/β-cateninpathwayisgenerallyinactive,andβ-cateninundergoesphosphoryl-ationandsubsequentdegradation[66].
Nevertheless,re-activationofthepathwayhasbeenshownasoneofthedrivingforcesessentialforliverregeneration,intermsofliverstemcellsproliferationanddifferentiation[67].
Intheliver,theroleofstemcellsisknowntobeplayedbyadulthepaticprogenitorcellsalsoknownasovalcells[68].
Theovalcellshavebeenshowntoparticipateinre-generationandinarangeofhumanliverdiseases,suchasHCC.
Thenatureofliverovalcellsisbi-potentialastheyhavebeenshowntodifferentiatetowardboththehepaticandbileductularepitheliallineagesintheliver[69].
IthasbeendemonstratedthatactiveWnt/β-cateninsignalingintheliveroccurspreferentiallywithintheovalcellpopula-tion,andforcedover-expressionofaconstitutivelyactiveβ-cateninmutantdrivesexpansionoftheovalcellpopula-tionintheregeneratingliver[70].
ThedualroleoftheWnt/β-cateninpathwayinregulatingthefateoftheovalstemcellshasbeenshownbydemonstrationthatWnt1caninducetheirdifferentiationtohepatocytesinaratliverregenerationmodel[71].
Thisexampleindicatesthetissue-specificimportanceofcertainWntligandsascandi-datesfortheuseinregenerativemedicine.
TheimportanceofWnt/β-cateninsignalingfortheliverregenerationhasbeenshowninaseriesofotherobservationsfocusingondifferentlevelsofregulationoftheWntcascade.
Forexample,ithasbeendemonstratedthatWnt-dependentregenerationinthelivercanbecon-trolledbyanendogenouslongnon-codingRNA,whichactivatesWnt/β-cateninsignalingbyinhibitingexpressionofAxin.
Thisactionofthenon-codingRNAleadstoen-hancedhepatocyteproliferationduringliverregeneration[72],showingthepotentialforthetherapeuticuseofWntpathway-relatednon-codingregulatoryRNAsinthetreat-mentofliverdiseases.
SeveralexamplesdemonstratingtheinterplayoftheWnt/β-cateninpathwaywithothersignalingcascadesduringliverregenerationhaverecentlybeendescribed.
TheTGFβfamily-relatedproteinSMAD6isabletosup-presstheWnt/β-cateninsignalingintheliveractingattheβ-catenin-TCF-promoterinteraction;down-regulationofBlagodatskietal.
MolecularandCellularTherapies2014,2:28Page5of15http://www.
molcelltherapies.
com/content/2/1/28SMAD6leadstoincreasedWnt-dependentproliferationandself-renewalofthehepaticprogenitorcells[73].
TheWnt/β-cateninpathwayalsointeractswithHedgehogsignalingintheliver,beingabletostimulateexpressionofthetranscriptionfactorGli1whichisadownstreameffectoroftheHedgehogpathway.
Gli1onitsturnup-regulatescycD1causingenhancedhepatocyteprolif-eration[74].
GiventheimportanceofWnt/β-cateninsignalingforhepatocyteproliferationanddifferentiation,therapeuticinterventionintheWntsignalingpathwayappearsasapromisingtoolforliverregeneration.
Thefirstsuccessfulattemptshavebeenalreadymade.
LivercellsexpressingLgr5,amarkerofactivelydividingstemcellsinWnt-drivenself-renewingtissues,areabletoclonallyexpandinvitrofromsinglecellsandformtransplantable"orga-noids",retainingmanycharacteristicsoftheoriginalepithelialarchitecture,undertheinfluenceofaWntsyn-ergistR-spondin1whichactsasaDkk1competitor[75].
Suchapproachesarepavingthewaytofuturedenovogrowthoftransplantableorgans.
AtherapeuticeffectofasmallmoleculeWnt/β-cateninpathwayagonist,2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine(2-AMBMP),hasbeendemonstratedinthehepaticischemiamodelinrats.
Thedrugbluntedtheischemia-inducedelevationofaspartateaminotransferaseandalanineaminotransfer-aselevels,increasingcellproliferationrateanddecreasingnegativeeffectssuchasinflammationandapoptosis,andreducedthedeathrateingeneral[76].
Assmallmoleculesareoftenacheapandconvenientsubstitutionfornaturaleffectorproteins,suchthetherapeuticapproachrepresentsapromisingstrategyfortheliverregenerationtherapy.
BoneWntsignalingisknowntobeakeyregulatorofbonetissuegrowthinembryosandintheadult[77].
Itises-sentialforosteocyteformationfromstemcells[78]andFigure2SchematicrepresentationoftheWnt/β-cateninsignalingpathwayandtheregenerationtherapydrugcandidatesdiscussedinthepaper.
Themoleculartargets(whereknown)ofthesmallmoleculeandantibody-baseddrugcandidatesareshown.
Table2Currentstatusofregeneration-relatedclinicaltrialsofbiologicsandsmallmoleculestargetingtheWnt/β-cateninpathwayName/companyTargetAgentConditionsClinicalphaseWnt3a(ChinaMedicalUniversityHospital)FZDsNativeWnt3aPrimaryDiseaserecruitingAMG785(Amgen)SclerostinmAbBoneFractureHealing,OsteoporosisIIValproicacid(SeoulNationalUniversityHospital)GSK-3βValproicacidAndrogeneticAlopeciaIIMalePatternBaldnessAMG162(Amgen)Dkk1mAbOsteoporosisIIHSC(Histogen)multipleWnt7a-containingcomplexAndrogeneticAlopeciaIIBlagodatskietal.
MolecularandCellularTherapies2014,2:28Page6of15http://www.
molcelltherapies.
com/content/2/1/28forboneregenerationafterinjuryordisease[77].
En-hancedWnt/β-cateninsignalingincreasesbonevolumeandcauseshyperostosisandpathologicalbonethicken-ing(sclerosingbonedysplasia)[79-82].
Sucheffectsareachievedbystimulatingthebone-formingosteoblastactivity[83,84],byinhibitingthebonedisassemblingosteoclastfunction[85,86],andbydifferentiationofdi-versepluripotentstemcellstowardosteoblasts[87-89].
IthasbeenreportedthatanumberofcomponentsoftheWnt/β-cateninpathway,suchastheligandsWnt4,Wnt5a,Wnt10b,Wnt11,andWnt13,thereceptorsFZD1,2,4and5,theco-receptorsLRP5andLRP6,β-catenin,andtheWnttargetgenes,suchastheosteoblastdifferentiation-associatedtranscriptionfactorRunx2,areup-regulatedinthefracturecalyxduringboneregener-ation[90-94].
Itneedsbehighlightedthatnotonlyβ-catenin-dependentWntsignalingtakespartinthebonetissuegrowth,butotherbranchesofthepathwaycontrolitaswell.
Ithasrecentlybeenshownthatβ-catenin-independentsignalingthroughWnt5a-FZD9issignifi-cantlyup-regulatedduringtheearlystagesofosteoblastdifferentiation[95]andisreactivatedintheregenerat-ingbone,beingessentialforthefracturehealinginafemurosteotomymodelinFzd9/mice[96].
Theβ-catenin-independentWnt5asignalinghasbeenreportedtoefficientlypromotetrans-differentiaonofadipopro-genitorsintoosteoblastsinculturebysuppressingper-oxisomeproliferator-activatedreceptor(PPAR)-γ,akeyadipogenesis-stimulatingtranscriptionfactor,andbyactivatingRunx2[97].
Wnt5a+/micedemonstratede-creasedtrabecularbonemassinthefemurs,andmicehomozygousfortheloss-of-functionmutationinWnt5ashowtruncationoftheproximalskeletonandlackingofdistaldigits[98].
Theβ-catenin-independentWnt5asig-nalinghasbeenalsoshowntoinhibitapoptosisofdiffer-entiatedosteoblastsandtheirprogenitors,comparabletotheanti-apoptoticeffectsofsignalingbyWnt3aandWnt1involvingβ-catenin[99].
Takentogether,thesedatasuggestthatWntsignalingmodulatorscouldbeusedastherapeuticagentstostimu-lateboneformationafterinjuryordisease.
CurrentlytwostrategiesareexploitedtofindaneffectiveWnt-basedboneregenerativetherapy.
ThefirstinvolvesreductionoftheactionofnativeWntsignalinginhibitorsendogenouslypresentintheorganism,whichinturnwouldenhancetheintrinsicWntactivity.
Inthisregard,activationofWntsignalingbyneutralizingantibodiesagainsttheWntin-hibitorsDkk1[100]andsclerostin[101-104]hasbeenshowntoimprovebonehealinginmice.
sFRP1isalsoaWntinhibitorandapromisingtargetforboneregener-ationtherapy[105].
AnotherwaytopromoteWntsignalinginordertoboostthebonetissuegrowthisadirectuseofexogen-ousWntsorotherpathwayagonists.
AlthoughnativeWntproteinsarehardtouseduetotheirlowsolubility,asuccessfulattempthasbeenrecentlymadewithliposome-loadedWnt3a,whichuponapplicationtotheinjurysitehascausedaca.
3.
5-foldincreaseintheboneregenerationrateinmice[106].
Aclinicaltrial,sponsoredbytheChinaMedicalUniversityHospital(Taiwan),isbeinginitiatedtostudytheosteogeniceffectsofhumanmesenchymalstemcellsenhancedbyWnt3achargedonhydroxyapatitenano-particles(http://clinicaltrials.
gov/show/NCT01323894).
StimulationofmurineboneregenerationhasbeenalsoobtainedbyapplicationofLiCl[92],andsimilareffectsonratboneregenerationhavebeenobservedusingLi2CO3[107].
Li+isawell-knowninhibitorofGSK3β–akeyen-zymetargetingβ-cateninforproteosomaldegradation–andthusactivatoroftheWntpathway;however,itislessspecificthanWntproteinsorotherpathwayagonistsandthereforemaycausemoresideeffects.
IngeneralitcanbestatedthatWntsignalinginthebonetissueiswellstud-ied,leadingtoabigvarietyofpossibleartificialmodula-tionsofthepathwaytoimprovebonehealingandtotreatbonediseases.
SkeletalmuscleTheroleofWnt/β-cateninsignalingintheskeletalmuscleremainscontroversial.
Intheadult,theβ-catenin-dependentWntpathwayhasbeensuggestedtocontrolmyogeniclineageprogressionbylimitingNotchsignal-ingandthuspromotingdifferentiation[108,109],inpar-ticularthroughMyf5andMyoDgrowthfactors[110].
OtherdatademonstratethatWnt/β-cateninsignalingintheadulttissue,e.
g.
throughWnt1andWnt3a,pro-motesonlyslowmyofibertypesgenerationandinhibitsmyogenesisingeneral[111].
ButtheroleofWntproteinsinregenerativeprocessesintheskeletalmusclediffersfromthatinmostothertissues,duetoincreasedsignifi-canceoftheβ-catenin-independentWntactivity.
IthasbeenshownthatWnt7a,signalingthroughitsreceptorFZD7,iscapableofactivationofdistinctpathwaysatdif-ferentstagesofmyogenesis.
First,theWnt7a-FZD7inter-actionleadstoactivationoftheWnt-PCP(planarcellpolarity)pathwaywhichisresponsibleforsymmetricexpansionofsatellitecellsinthemuscletissue[112]–asmallsub-populationofmusclecellsthatarecapableofself-renewalandactasmusclestemcellsduringregener-ation[113].
Over-expressionofWnt7aenhancesmuscleregenerationbymeansofincreasingthesatellitecellnum-ber,whereasmuscleswithdown-regulatedWnt7aexhibitasignificantdecreaseinsatellitecellnumbers,impairingtheregenerationcapacity.
Atthislevelofaction,Wnt7adoesnotaffectthegrowthordifferentiationofmyoblasts.
Next,stimulationofFZD7byWnt7aindifferentiatedmyo-fibersdirectlyactivatestheAkt/mTORgrowthpathwaythroughGαsandPI3K,therebyinducingmyofiberhyper-trophy[114].
ThusWnt7aservesasamusclegrowth-Blagodatskietal.
MolecularandCellularTherapies2014,2:28Page7of15http://www.
molcelltherapies.
com/content/2/1/28promotingfactorattwolevels,andatbothitactsinde-pendentlyfromβ-catenin.
RecentlyathirdlevelofWnt7aactivityinregeneratingmuscleshasbeendescribed,wheretheWnt7a/FZD7interactionshavebeenshowntoin-creasethepolarityanddirectionalmigrationofmurineandhumanmyogenicprogenitorsthroughactivationofDvl2andthesmallGTPaseRac1,resultinginimprovedmusclestrength[115].
Takentogether,thesefindingsidentifyWnt7aasanimportantdrugcandidateagainstmusclewastingdis-easeslikesarcopenia,cachexiaormusculardystrophies,andforimprovementofstemcell-basedmuscleregenera-tivetherapy.
TherapeuticpotentialofWnt7ahasindeedbeentestedinseveralsystems,likethemousemodelofDuchennemusculardystrophy,wheretheWnt7atreat-mentstimulatedsatellitecellexpansionandmyofiberhypertrophyandevenledtoasignificantincreaseinmusclestrength.
Furthermore,Wnt7adecreasedthelevelofcontractiledamage,mostlikelybyinducingashiftinthefibertypetowardtheslow-twitch[114].
Inotherex-perimentsashorttreatmentwithWnt7anotablyincreasedmuscletissuedispersalandengraftment,significantlyim-provingthemusclefunction[115].
Interestingly,atruncatedWnt7avariantconsistingofonly137C-terminalaminoacidsanddevoidofthecon-servedpalmitoylationsiteshasrecentlybeenshowntopreservethefullbiologicalactivityofthenativeproteininskeletalmuscles[116].
Itretainedthecapabilityofinter-actionwithitsreceptorFZD7andstimulationofsymmet-ricexpansionofsatellitestemcellsthroughthePCPpathway,aswellasinductionofmyofiberhypertrophybysignalingthroughtheAKT/mTORpathway[116].
ThisfindingisofaspecialimportancefortheWnt7a-basedmuscletherapy,becausenatural,palmitoylatedWntproteinsarehardtouseinmedicalandbiotechno-logicalapplicationsduetotheirlargesize,lowproduct-ivityandpoorsolubility.
EvidencethattruncatedWntsmayinsomecasesretainthetherapeuticpotentialmaybeafirststeptoproductionofactiveWnt-relatedpep-tides,easilyapplicabletomusclediseasetreatment.
SkinandhairfolliclesIntheskin,β-catenin-dependentWntsignalingisoneofthedominantpathwaysregulatingthepatterninganddeterminingthefateofembryonicandadultstemcellsduringtheirdifferentiation,aswellassubsequentlycon-trollingthefunctionofdifferentiatedskincells[117].
Animportantelementoftheskinarehairfollicles,whosemor-phogenesisalsodependsonWnt,alongwithShh,Notch,BMPandothersignalingpathwaysinteractingwitheachother.
TheWnt/β-cateninpathwayisakeyplayerduringhairfollicleinduction,actingthroughtheEDA/EDAR/NF-κBsignaling[118].
ThereisevidencepointingoutaninvolvementofWntsignalingintheskinrepairandhairfollicleregeneration.
Forexample,aWntsynergistR-spondin2promotescellproliferationintheadultepidermis[119]whichisdirectlylinkedtotheskinwoundhealingrate.
Wntsalsoactasnichesignalsforskinstemcellslocatedinthebulgeregionofthehairfollicles[120,121].
AlthoughtheeffectsofWnt/β-cateninsignalingwerepreviouslyregardedtobeoppositeontheepidermalandthehairfolliclestemcells[122],ithasrecentlybeenshownthatβ-catenin-dependentsignal-ingactuallypromotesproliferationofthesebothstemcellpopulations,thusbeingadrivingforceforregenerationofboththeskinandhairs[117,123].
InterfollicularepidermalstemcellshavebeenshowntoexpresstheWnttargetgeneAxin2andtorequireWnt/β-cateninsignalingforprolif-eration,producingautocrineWntsaswellaslong-rangesecretedWntinhibitors,thussuggestinganautocrinemechanismofstemcellself-renewalintheepidermis.
Thesecellsareabletopromoteskinwoundhealing,withnodemandforaquiescentstemcellsubpopulation[117].
Inanotherwork,experimentsonβ-catenindele-tionandLRP5/6inhibitorDkk1over-expressionhaveindicatedanecessityofWnt/β-cateninsignalingforfol-licularstemcellproliferation;thesameworkconfirmsthedataontheWnt-dependentrenewaloftheinter-follicularepidermis[123].
Activationofβ-cateninspecificallywithinmurinehairfolliclestemcellshasbeenfoundsufficienttoinducehairgrowthindependentlyofmesenchymaldermalpapillanichesignalsnormallyrequiredforthehairregen-eration[124].
Artificialup-regulationofWnt/β-cateninsignalinginregeneratinghairfolliclesthroughadenoviralinfectionofmicewithWnt10bhasledtoanincreaseinthesizeofregeneratingfolliclesandtoexcessiveproliferationoffolliclecells.
Theobservedeffectswerereducedwhenthemicewereco-infectedwithDkk1andWnt10b[125].
ThesefindingssuggestthatectopicWnt10bandDkk1canbeusedtomodulatethefolliclesizeandproliferationdur-inghairregeneration.
AnotherexampleofeffectiveuseofexternalWnttreatmenttoimprovetheskinwoundhealingrateistheinjectionofWnt3a-carryingliposomesintotheskinofinjuredmouseears.
TheliposomalWnt3ahasdemon-stratedtheabilitytoenhanceWntsignalinginthedam-agedtissueandtodramaticallyimprovethewoundhealingrate[126].
Hairgrowth(seenashairfollicleprogressionfromtelogentoanagenandbyoverallup-regulationofhairinduction-relatedgenes)couldalsobeenhancedinmiceupontreatmentwithWnt1-conditionedmedia[127].
Interestingly,thereareclinicaldatashowingthatec-topicapplicationofWnt7aasacomponentofagrowthfactorcocktailenhanceshairgrowth,possiblysuggest-ingthatWnt7acouldactinaβ-catenin-dependentwayinthiscase[128].
Blagodatskietal.
MolecularandCellularTherapies2014,2:28Page8of15http://www.
molcelltherapies.
com/content/2/1/28Finally,morerobustwaystoup-regulateWnt/β-cateninsignalinghavebeensuccessfullyappliedtoskinandhairregeneration.
Valproicacid,aninhibitiorofGSK3β,hasshownitsefficacyinboostingthecutaneouswoundrepairinmice[129]aswellaspromotinghairgrowthincellculture[130]andinducinghairregenerationinmurinesystems[131]andinclinicaltrials[132].
Alongwiththebone,theskinandhairregenerationisthefieldwheretheWnt-basedtherapyiswelladvanced,ascomparedtootherorgansandtissues.
NeuroprotectionandAlzheimer'sdiseaseWntsignalingisactivelyinvolvedinneurogenesisandmodulationofthesynapticfunctionintheadult[133].
Further,associationoftheAβ-peptidesandAlzheimer'sdiseaseetiologyingeneralwiththeWntsignaling-inducedprocesseshasbeenreportedmanytimes[134,135].
TheAβ-peptideshavebeenshowntodirectlyinhibittheWnt/β-catenincascadebyinteractingwithFZDsandimpairingthenormalactivationofthepathway[136].
Suchimpair-mentcaninturnleadtosynapticdegeneration.
Ontheotherhand,additionofexogenousWnt3aorLiClactivat-ingtheWnt/β-cateninpathwayhasbeenshowntopre-venttheAβ-peptide-mediatedsynapticdegenerationandcelldeathinaneuronalcellculture[137].
IthasalsobeenshownthatneuroprotectiveeffectsofWnt3aaremediatedbythereceptorFZD1[138].
RecentstudiesconfirmtheimportanceoftheWnt/β-cateninsignalingasananti-Alzheimeractingforce,asactivationofthecascadeusingBromoindirubin-30-Oxime(6-BIO),aninhibitorofGSK3β,protectshippocampalneuronsfromAβ-oligomersandreducestherateofneuronalapoptosis[139].
Moreover,thesamestudysuggeststhattheβ-catenin-independentWnt5a-mediatedCa2+-dependentsignalingcouldmodulatemitochondrialdynamicsandpreventthechangesinducedbytheAβ-peptideoligo-mersinmitochondrialfissionandfusionusuallypresentinneurodegenerativediseases[139].
OthersmallmoleculeWnt/β-cateninsignalingago-nistshavealsodemonstratedanabilitytoincreaseadultneurogenesisorinhibittheneurodegenerativeeffectsoftheAβ-peptides.
SimvastatinhasbeenshowntosynergizewithWnt/β-cateninsignalinginvivoandinvitro,enhan-cingitthroughdepletionofisoprenoidsynthesis(whichisinvolvedintheregulationofmembrane-locatedproteinslikesmallGTPases)andimprovingtherateofadulthippo-campalneurogenesis,makingitapotentialneuroprotectivedrug[140].
InamousemodelofAlzheimer's,treatmentwithLiClreducedamyloid-inducedmemoryimpairmentanddecreasedAβ-peptideaggregation.
Theseeffectswereparalleledbystabilizationofβ-catenin[141].
AnotherinterestingexampleofsmallmoleculeagonistsofWnt/β-cateninsignalingabletoactinthebrainiscur-cumin.
Cucrumin-coatednanoparticlescouldactivatetheWnt/β-cateninpathwayinratbrainsandreverselearningandmemorydysfunctionsintheAβ-inducedratmodeloftheAlzheimer'sdiseasebystimulationofneurogenesis.
InsilicomoleculardockingstudiessuggestthepossiblemechanismsofcurcuminactionthroughinteractionwithWntantagonistsWif-1,Dkk,andtheβ-catenininhibitorGSK3β[142].
TheseobservationsmakethetherapiesbasedupontheWnt-cascadeactivationapromisingwaytotreattheAlzheimer'sdiseaseandotherneurodegen-erativediseases.
However,incaseofthebraintissue,over-activationofWntpathwayscanalsobecounterproductiveinthera-peuticapplications,asWntsareknowntostimulatepro-inflammatoryprocessesinmicroglia,thuscomplicat-ingtheAlzheimer'sdiseasetreatment.
AnincreaseinWnt/β-cateninsignalinghasbeenobservedinmicrogliaofmicewithAlzheimer's-likepathology;inculturedmicroglia,treatmentwithWnt3aactivatedtheβ-catenin-dependentpathwayandledtoincreasedexpressionofpro-inflammatorygenes[143].
Experimentsontheaxoninjurymodelinmicehavedemonstratedthatdown-regulationofβ-catenin-dependentsignalingbyatissue-specificgeneknockoutinoligodendrocyteprecursorcellsfacilitatesaxonalregenerationindamagedtissueandreducestheglialscarring[144].
Interestingly,β-catenin-independentWnt5asignalingisalsoimplicatedinmicroglialpro-inflammatorytransformationviatheERK1/2pathway[145].
SuchcontroversialinvolvementofdistinctWntpathwaybranchesinregenerationandinflammationoftheneuraltissuewillrequireamoredetailedstudyoftheinterplayofdifferentsignalingpathwaysandaveryaccurateapplicationofWntsignalingactivatorsincaseoffuturetherapeuticAlzheimer'sdiseasetreatment.
DiscussiononregenerationGenerally,theregenerationprocessesinmosttissuesareregulatedbyre-activatingtheWnt/β-cateninsignaling,whichleadstostemcellproliferationand/ordifferenti-ation,althoughinsomecasesβ-catenin-independentWntstakeontheleadingrole,asitisthecasewiththeskeletalmuscle.
InspiteofacomplexinterplayofWntsig-nalingwithothersignalingcascades,straightforwardap-proacheslikeectopictreatmentoftheinjuredorgansortissueswithWntagonistshaveshowntheireffectivenessindifferenttissues,improvingtheregenerationrate.
Com-biningthesefindingswithpossibilitiestofurtherenhancetherapeuticpotentialofWntsthroughviralorliposomaldeliveryorbyengineeringofsmallWnt-relatedpeptidesallowsustolookoptimisticallytowardsthedirectuseofWntcascadeagonistsinregenerativemedicine.
InadditiontoapplicationofthetruncatedWnt7ainthemuscle,shortpeptideanalogsofWnt5aortheirmorestableformylatedderivativeshaveshowntomimicthebiologicalactivityofthenativeWnt5aproteinbyBlagodatskietal.
MolecularandCellularTherapies2014,2:28Page9of15http://www.
molcelltherapies.
com/content/2/1/28blockingcellmigrationinbreastepithelium[52,53].
Structure-assisteddesign[44,146]anddirectedproteinevolutionapproaches[147]canhelpingenerationofnovelWnt-derivedpro-regenerativeagents.
Otherap-proachesaretheindirectmethodstoenhanceintrinsicWntactivityintheinjuredordiseasedtissueandhavealsoproventheirapplicability.
Forexample,down-regulationofthenaturalWntinhibitorshasbeenshowntoworkwellinaidingthebonerepair.
SeveralregenerativetherapeuticapproachestargetingtheWnt/β-cateninpathwayintheskinandthebonearealreadyinclinicaltrials(Table2).
Liver,brain,skinandbonesarethewell-studiedorgansforWnt/β-cateninregenerativeapplications,buttheydonotlimitthepotentialofpossibletargetsfortheWnt-basedregenerationtherapy.
ItisknownthatWnt/β-cateninsig-nalingisalsoinvolvedinangiogenesis[148],indicatingthatmodulationofthispathwaymightbeusedasaninstrumenttotreatcardiovasculardiseasesincludingmyocardialinfarc-tion[149]ortargetangiogenesisintumors.
Practicalat-temptsinthisdirectionhavealreadybeenmade:recentstudiesindicatethatUM206,asmallpeptidehomologofWnt3a/Wnt5acapableofblockingFZDsignaling,canim-provehearttissueregenerationafterinfarction[150].
Approachesforthefutureregenerativemedicinedonotstopjustattheincreasingofthenaturalregenerationrate.
Ahighlydesiredgoalistodeveloptechnologiesoftissueengineeringandofgrowthandtransplantationofartificialorgans.
Althoughnocompletefunctionalorgansreadyfortransplantationhavebeengrownsofar,firsteffortshavealreadybeenmadeinthisdirection.
Suchcomplextaskswouldinvolvemultiplestepsanddemandcompoundcocktailsofdifferentgrowthfactors,whereWntsignalingactivatorsareessentialcomponentsinmostcases.
Asuccessfulattempttogrowanopticcup(retinalprimordium)structurefromathree-dimensionalcultureofmouseembryonicstemcellaggregateshasbeenper-formed,whichinvolvedatreatmentwithWnt/β-cateninpathwayagonistsatcertainsteps[151].
Asaconclusion,itcanbeclaimedthatdevelopmentandproductionofhighlyactiveandtissue-specificagonistsoftheWnt/β-cateninsignaling,eitherpeptide-basedorsmall-moleculeanalogs,isagoalofhighimportanceforregenerativemedicineinthenextdecades.
SafetyofWntpathwaytargetingAsexemplifiedabove,theWntpathwaysareinvolvednotonlyinmanydevelopmentalprocessesbutalsointhemaintenanceofadulttissuehomeostasis.
Thusacarefulsafetyassessmentofdrugsdirectlyornon-directlytarget-ingtheWntsignalingisrequired.
ItisclearthatageneralinhibitionoftheWnt/β-cateninpathwayispotentiallyun-safe,mainlybecauseof:(i)broadWnt/FZDexpressionpattern;(ii)theroleofthepathwayinthemaintenanceofthedifferentiatedepitheliumanditsinteractionwithmesenchymalcells;(iii)itsinvolvementinstemcellpluripotentstatemaintenance;(iv)itsroleinbonehomeostasis.
Arecentreview[152]providesaveryde-taileddiscussionofsafetyconcerningtargetingoftheWntpathways,bothinregenerativemedicineandon-cology.
Herewewouldliketoprovideonlyafewillus-trationshighlightingthissafetyissue–aswellastheissueofdifficultyinpredictingwhetheracertainWnt-targetingdrugcandidatewouldorwouldnothavepar-ticularsideeffects.
ThefirstexampleisabouttheFZDco-receptorantago-nists.
BlockadeofWnt/β-cateninsignalingbyadenovirus-mediatedexpressionofDkk1(anaturalLRP5/6antagonist)inmicehasbeenshowntosuppressepitheliumprolifera-tioninsmallintestineandcolon,accompaniedbyprogres-sivearchitecturaldegenerationwiththelossofcrypts,villi,andglandularstructureby7days[153].
Further,ectopicDkk1expressionledtoacompletefailureofhairfolliculeformationinadultmice[154].
IncontrasttothatinvivoadministrationofanLRP6antagonistMesdmarkedlysup-pressedgrowthofMMTV-Wnt1tumorswithoutcausingundesirablesideeffects[155].
AnotherexampleconcernsthepotentialofusingWntproteinsasdrugs.
Indeed,someWntshavetumorsup-pressingactivity.
Wnt5a,forexample,isoftenviewedasa"non-canonical"WntcapableofantagonizingtheWnt/β-cateninsignalingandhasaroleinlimitingB-cellprolifera-tionandfunctionsasatumorsuppressorinhematopoietictissue[156].
MicehemizygousforWnt5adevelopclonalmyeloidleukemiasandBcelllymphomasanddisplaylossofWnt5afunctionintumortissues.
Ontheotherhand,expressionofWnt3a(aprototypicalactivatoroftheWnt/β-cateninsignalingandthusapresumedpro-oncogenicfactor)inmousemelanomamodeldecreasedtheprolifera-tionofthetumorandsuppressedthemetastasis[157].
ThereforewhethertheapplicationofacertainWntwillbeanti-orpro-oncogenicmaybehighlycontext-depending.
Inmanycases,thesideeffectswhosepresenceorser-iousnesshasbeenruledoutduringpre-clinicalstudiesappearduringclinicaltrials,despiteallpre-cautionarymeasures.
Forexample,theenthusiasminitiallyshownforbothofOncomedPharmaceuticals'Wntpathwaybio-logics,vantictumabandOMP-54F28,diminishedwhenclinicaltrialsforbothwereputonhold.
Thequotedrea-sonsforhaltingpatientenrollmentdosingarebone-relatedside-effectsranging"frommildtomoderate"(http://www.
reuters.
com/article/2014/06/13/us-oncomed-study-idUSKBN0EO1B920140613).
Thesewereobservedin8outof63(13%)patientstreatedwithvantictumabandin2outof41(5%)withOMP-54F28.
Sincetheincreaseinboneturnoverhasbeenstatedasoneofthevantictumabpharmacodynamicseffects,itislogicaltoassumethatthementionedpercentageofpatientsdevelopedosteoporosis.
LoweringthedosageandfrequencyofthedrugsmightBlagodatskietal.
MolecularandCellularTherapies2014,2:28Page10of15http://www.
molcelltherapies.
com/content/2/1/28neutralizesuchsideeffects–butthiscouldalsohaveanegativeimpactonthedrugefficacy.
Therootofthecurrentproblemwithvantictumabinparticularmaylieintheinitialdesignofthedrugmolecule.
Itbindsto5outof10FZDfamilymem-bersinvitroandpotentiallyevenmoreinvivo.
How-ever,notalloftheFZDsthatbindtovantictumabareover-expressedorover-activatedinthesolidtumorsincludedintheclinicalstudy.
Conversely,onlysomeFZDmembersmaybecriticalforboneremodeling.
AFZDmAbwithlessbroadspecificitycouldstillhavebeeneffective,inhibitingsignalingoriginatingfrompro-oncogenicFZDs(orjustoneFZD)whilenotaf-fectingtheFZDsindispensableforosteogenesis.
TheseconsiderationshighlightperhapsthemostimportantissueintargetingtheWntpathways–safetyislikelytoresultfromhigherspecificityintargetingaparticularsub-pathwayimplicatedinacertainpathogeniccondition,ratherthanbluntlysuppressingallormostofthesub-pathways[158,159].
ConclusionAsdiscussedabove,Wntsignalingplaysimportantfunc-tionsincancerprogressionandtissueregeneration.
Iron-ically,whatistherapeuticallygoodforone(activationofthepathwayinregeneration)isbadinfortheother(anti-cancertreatment).
Thus,extremecaremustbetakenwhendevelopingWnt-stimulatingpro-regenerativedrugs,toexcludetherisksofcancercomplications.
Thisdemandappearsnotun-achievablethroughdesignofhighlyspe-cificand/orlocalactivatorsoftheWntpathways.
Ontheotherhand,theexpectedsideeffectsintargetingtheWnt/β-cateninpathwayinanti-cancertreatmentsaremyelo-andgastrointestinalsuppression,exactlyduetotheadverseeffectsofanti-Wnttreatmentsonprolifera-tionofhematopoeticandintestinalstemcells,aswellasprogenitorcellsinotherorgans.
Hereagain,ahopeisfordesigningspecificagentstargetingtheWnt/FZDsub-pathwaysactivatedinagivencancer,ratherthanblockingtheWnt/β-cateninsignalingaltogether[159].
Inthisregard,attackinghigher'floors'ofthesignalinghierarchy–i.
e.
theligandsandreceptors–appearsespeciallypromising[48,158].
Itisimportanttoremem-berthattheWntpathwaysmustbefine-tunedforthenormalphysiologycontrol.
Asrecentworksindicate[47],evenmoderateattenuationofWntsignalingcaneliminateitscarcinogenicpotential.
Thereforeitispos-sibletokeepthe"good"physiologicallevelofWntsig-nalingbyfindingtherighttargetandarighttooltoactonthistargetinadesiredmode.
PerhapsthefactthatWntsignalingintumortissuesisexacerbatedrelativetonormaltissueaswellasapotentialofnormaltissuetorecovercouldprovidethesafewindowofWntinhib-itionintherapy.
AbbreviationsAML:Acutemyeloidleukemia;2-AMBMP:2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine;6-BIO:Bromoindirubin-30-Oxime;CSC:Cancerstemcell;Dvl:Dishevelled;FZD:Frizzled;GPCR:G-proteincoupledreceptor;HCC:Hepatocellularcarcinoma;mAb:Monoclonalantibody;PCP:Planarcellpolarity;sFRP:SecretedFrizzled-relatedprotein;siRNA:SmallinterferingRNA;TNK:Tankyrase.
CompetinginterestsD.
P.
isanemployeeofIBCGeneriumLLC.
V.
L.
K.
isaco-founderofTNBCureLtd.
BothcompaniesareinvolvedinWnt-targetingdrugdiscoveryefforts.
Authors'contributionsABandDPwrotethemanuscript;VLKdesignedthestructureandwrotethemanuscript.
Allauthorsreadandapprovedthefinalmanuscript.
AcknowledgementsThisworkwasfundedbytheSwissCancerLeague(grantnumberKFS-2978-08-2012toV.
L.
K.
)andtheDynastyFoundation(grantnumberDP-B-14/13toA.
B.
).
WethankDr.
GonzaloSolisforcriticallyreadingthemanuscript.
Authordetails1InstituteofProteinResearch,RussianAcademyofSciences,Pushchino,RussianFederation.
2IBCGeneriumLLC,Volginsky,RussianFederation.
3DepartmentofPharmacologyandToxicology,UniversityofLausanne,Lausanne,Switzerland.
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