A novel cell-penetrating peptide derived from WT1 enhances p53 activity, induces cell senescence and displays antimelanoma activity in xeno- and syngeneic systems

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j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / f e b s o p e n b i o

A

novel

cell-penetrating

peptide

derived

from

WT1

enhances

p53

activity,

induces

cell

senescence

and

displays

antimelanoma

activity

in

xeno-

and

syngeneic

systems

夽

Mariana H. Massaoka, Alisson L. Matsuo, Carlos R. Figueiredo, Natalia Girola, Camyla F. Faria, Ricardo A.

Azevedo,

Luiz R. Travassos

*

ExperimentalOncologyUnit(UNONEX),DepartmentofMicrobiology,ImmunologyandParasitology,FederalUniversityofS˜aoPaulo(UNIFESP),S˜aoPaulo,SP04023-062,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received10December2013

Receivedinrevisedform7January2014 Accepted14January2014

Keywords:

Malignantmelanoma Wilmstumor1(WT1) Senescence p53

a

b

s

t

r

a

c

t

TheWilmstumorprotein1(WT1)transcriptionfactorhasbeenassociatedinmalignantmelanoma withcellsurvivalandmetastasis,thusemergingasacandidatefortargetedtherapy.Alysine–arginine richpeptide,WT1-pTj,derivedfromtheZFdomainofWT1wasevaluatedasanantitumoragentagainst A2058humanmelanomacellsandB16F10-Nex2syngeneicmurinemelanoma.PeptideWT1-pTjquickly penetratedhumanmelanomacellsandinducedsenescence,recognizedbyincreasedSA-β-galactosidase activity,enhancedtranscriptionalactivityofp53,andinductionofthecellcycleinhibitorsp21andp27. Moreover,thepeptideboundtop53andcompetedwithWT1proteinforbindingtop53.WT1-pTj treatmentledtosustainedcellgrowthsuppression,abrogationofclonogenicityandG2/Mcellcycle arrest.Notably,invivo_studies_showed_that_WT1-pTj_inhibited_both_the_metastases_and_subcutaneous

growthofmurinemelanomainsyngeneicmice,andprolongedthesurvivalofnudemicechallenged withhumanmelanomacells.The27-aminoacidcell-penetratingWT1-derivedpeptide,dependsonC3

andH16_for_effective_antimelanoma_activity,_inhibits_{proliferation}_of_{WT1-expressing}_human_tumor_cell

lines,andmayhaveaneffectiveroleinthetreatmentofWT1-expressingmalignancies.

C

2014TheAuthors.PublishedbyElsevierB.V.onbehalfofFederationofEuropeanBiochemical Societies.Allrightsreserved.

1. Introduction

TheWilmstumorprotein1(WT1)isatranscriptionfactorthat playsanimportantroleincellulardevelopmentandcellsurvival.It isabnormallyexpressedinseveralhumancancersandcouldbea targetoftherapeuticagents.Structurally,theWT1proteincontains fourC2 H2 Kr¨upple-likezinc-ﬁngers(ZF)intheC-terminaldomain, whichareimportantforDNAbinding,RNAbindingandinteraction withotherproteins[1].Originallydescribedasatumorsuppressor geneinpediatricnephroblastoma[2,3],theoverexpression, confor-mationalchangesandcytoplasmiclocalizationofWT1indifferent malignanciessuchasmesothelioma,ovarian cancer,leukemia, os-teosarcomas,glioblastomasandmalignantmelanomas[4,5] demon-stratedoncogenicpropertiesofthisprotein.Inmalignantmelanoma,

夽

Thisisanopen-accessarticledistributedunderthetermsoftheCreative Com-monsAttribution-NonCommercial-NoDerivativeWorksLicense,whichpermits non-commercialuse,distribution,andreproductioninanymedium,providedtheoriginal authorandsourcearecredited.

Abbreviations:CPEP,controlpeptide;CL-ELISA,chemiluminescenceELISA;CPP, cell-penetrating peptide;pTj, Trojan peptide;SA-βGal,senescence-associatedβ -galactosidase;TMZ,Temozolomide;WT1,Wilmstumorprotein1;ZF,zinc-ﬁnger.

* Correspondingauthor.Address:UnidadedeOncologiaExperimental(UNONEX), UniversidadeFederaldeSao˜ Paulo(UNIFESP),RuaBotucatu862,8andar,S˜aoPaulo,SP 04023-062,Brazil.Tel.:+551155764551;fax:+551155715877.

E-mailaddress:travassos@unifesp.br(L.R.Travassos).

themostaggressiveandlethalformofskincancer,WT1expression hasbeenreportedinmorethan80%tumorcellsbutnotinepidermal keratinocytes,melanocytesandbenignmelanocyticneviinvivo[6]. Notably,RNAisilencingofWT1inducesapoptosisinB16F10murine melanomacells[7]anddisplaysantimetastaticactivity[8].

TheoncogenicroleofWT1incancerstimulatesattemptsat neu-tralizingthistumor-associatedantigen.Recently,theanticancer ther-apythatemployspeptides,whichcandirectlytargetcancercells,has emergedasanalternatestrategytorestraintheprogressionoftumor growthandmetastases[9].Antitumorpeptidesmayactbindingto andinhibitingoncogenesorproteinswithaberrantexpressionin tu-morcells.Theycausecellcyclearrestand

/

orinduceapoptosis,block signalingmediatorsandreceptors,inhibitangiogenesis,andmediate tumorenvironmenthomingofcytotoxicpeptidesequences[10–15]. Certainpeptidesarecell-penetrating(CPPs)orTrojanpeptides,with shortamphipathicandcationicsequencesthatpermit their pene-trationacrossthecellmembrane,andthusexertdirectanticancer activity[16].Thesepeptidesmaybecarriersofavarietyofantitumor molecules[17,18].

Inthe presentwork,weshowthat a novelWT1-derived pep-tide(WT1-pTj)isacell-penetratingantitumoragentthatsuppresses bothproliferationandclonogenicityofB16F10-Nex2melanomacells

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throughanirreversibleG2

/

Mcellcyclearrestandinductionof cel-lular senescence. In addition to morphological changesand irre-versiblegrowthinhibition,senescentcellsexpressedthe senescence-associated

β

-galactosidaseandformedhetero-chromatinfoci[19], associatedwithenhancedtranscriptionalactivationofp53,and accu-mulationofcyclin-dependentkinaseinhibitorsp21Cip1 andp27Kip1 , whichhavebeenusedasmarkersofsenescence[20].

Mostimportantly,WT1-pTjdisplayedaremarkableantimetastatic activityinthesyngeneicB16F10-Nex2melanomamodeland pro-longedsurvivalofnudemicesubcutaneouslychallengedwithhuman A2058melanomacells.Bothresultsemphasizethepotentialofthis novelantitumorpeptidetobedevelopedasatherapeuticdrug.

Thepotentialuseofbioactivepeptidesasanti-cancerdrugshas beeninvestigatedinourlaboratoryandconsiderableprogresshas beenmadeusingpeptidesderivedfromimmunoglobulinsandfrom transcriptionfactors[21,22].

2. Materials and methods

2.1. Peptides

A27-residuesyntheticpeptide(WT1-pTj)correspondingtoamino acids349–375ofthehumanWT1protein(GenBank:CAI95758)and thecontrol peptide(CPEP,withC3AandH16A)weresynthesized byPeptide2.0Inc.(Chantilly,VA)at90–99%purity,withamidated C-terminalaminoacid,andwerecompletelysolubilizedinPBSor culturemedium.TheWT1-pTjpeptideis100%identicaltotherelated sequenceofmouseWT1protein,correspondingtoaminoacids426– 452(GenBank:NP659032).Structuresandmolecularmassesofthe peptidesaredepictedonTable1.

2.2. Celllinesandcultureconditions

CelllineswereoriginallyobtainedfromLudwigInstitutefor Can-cerResearch,Sao˜ Paulo,Brazil,ordonatedbyProf.LuisF.LimaReis, HospitalSirio-Libanez,Sao˜ Paulo,Brazil.Thesearelongestablished celllines,acquiredfrompublicculturecollectionsortransferredfrom LudwigInstituteinNewYork,andmaintainedinappropriate condi-tionstoserveasstandardtumorcelllinesforlocalstudiesand collabo-rativeresearch.Animalexperimentswerecarriedoutusingprotocols approvedbytheEthics CommitteeforAnimalExperimentationof FederalUniversityofSaoPaulo,Brazil(CEPNo.1280

/

10).

ThemurinemelanomaB16F10-Nex2sublinewasestablishedat theExperimental Oncology Unit(UNONEX), Federal University of Sao˜ Paulo,UNIFESP,asdescribed[23]andusedeversincein sub-cutaneousandmetastaticsyngeneicmodelsinmice.Thehuman tu-morcelllinesA2058andSK-MEL-28(melanoma),MCF-7and MDA-MB231(breastcarcinoma),OVCAR-3(ovariancarcinoma)andHL-60 (acuteleukemia)weremaintainedincompletemediumconsistingof RPMI-1640(Gibco,GrandIsland,NY)supplementedwith10mM N-2-hydroxyethylpiperazine-N₂ethanesulphonicacid(HEPES;Sigma– Aldrich,St.Louis,MO),24mMsodiumbicarbonate,40mg

/

l gentam-icin(Hipolabor,MinasGerais,Brazil),pH7.2,and10%fetalbovine serum(FBS;Gibco,GrandIsland,NY).

ThehumanﬁbroblastcelllineHFFandmouseembryonic ﬁbrob-lasts(MEFs)wereagiftfromLuisF.LimaReis,HospitalSirio-Libanez, Sao˜ Paulo.TheHFFcelllinewasmaintainedinminimumessential mediumEagle(Gibco,GrandIsland,NY)with2mMl-glutamineand Earle’sbalancedsalinesolutionadjustedtocontain1.5g

/

lsodium bicarbonate,0.1mMnon-essentialaminoacids,and1.0mMsodium pyruvate,and10%FBS.MEFcelllinewasmaintainedinDMEM(Gibco, GrandIsland,NY)mediumsupplementedasdescribedabove.Allcell lineswereculturedat37◦_C,_in_humid_atmosphere_and_5%_CO

2 .

2.3. Confocalmicroscopy

A2058humanmelanomacells(4 × 104 )wereplatedinround coverslipsandincubatedat37◦_C_for₂₄_h._Cells_were_treated_with

0.5mMﬂuoresceinisothiocyanate(FITC)-labeled-WT1-pTjfor1hat 37◦_C._After_incubation,_tumor_cells_were_ﬁxed_and_{permeabilized}_with

methanolfor15minatroomtemperature,followedbyblockingwith 0.2%gelatinfor10minat37◦_C._Nuclear_staining_was_carried_out_with

10

µ

g

/

mlofDAPI(Invitrogen,Eugene,OR).Cellswerethenwashed inPBS andcoverslips mountedonto slideswith4

µ

lVectashield (Sigma,St.Louis,MO)andimagedusingaCarlZeissLSM780confocal microscope(Jena,Germany).

FortimedependentWT1-pTjlocalization,A2058cellswere incu-batedfor15min,1and24hwith0.5mMbiotinylated-WT1-pTj (b-WT1-pTj),washed3timesinPBS,andﬁxedwith3.7% paraformalde-hydefor30min.Cellswerethenpermeabilizedin0.1%TritonX-100 for30minfollowedbyblockingfor1hwith150mMNaCl,50mMTris and0.25%BSA,allfromSigma–Aldrich,MO.AfterwashinginPBS,cells wereincubatedwith10

µ

g

/

mlDAPIandstreptavidin-FITC(1:200 fromstocksolution)usedassecondaryﬂuorophoreforbiotinylated peptide,andwerebothincubatedfor15minat37◦_C._Stained_cells_on

coverslipswereexaminedforpeptideintracellularlocalizationusing aConfocalLeicaSP5microscopewitha100× oilimmersion objec-tive(Leica,Wetzlar,Germany).TheZserieswasacquiredaccording tosamplingcriteriabuiltintothesoftwareforsequential imaging ofDAPI,whichstainsthenucleus(blue,excitation

/

emission=350

/

470nm)andFITC, whichmonitors peptidelocalization(green, at excitation

/

emission= 488

/

525nm).Imageswereprocessedwith theImageJsoftware(http:

//

rsb.info.nih.gov

/

ij

/

).

2.4. Cellviability

Theeffects ofWT1-pTj andCPEPoncellviabilitywere deter-minedbyTrypanblueexclusionassay.ForEC₅₀determination,cells (5×103

/

well)wereseededandcultivatedin96-wellplatesfor12h at37◦_C._Cells_were_incubated_with_increasing_{concentrations}_of_the

peptides(0–1mM)for24h,weredetachedwithTrypsin–EDTA0.25% solution(Sigma–Aldrich,St.Louis,MO)andthenumberofviablecells countedusingaNeubauerchamber(ElectronMicroscopySciences, Hatﬁeld,PA).ThegrowthkineticsofA2058cells(103

/

well)during 96hinpresenceofWT1-pTjorCPEPatdifferentconcentrationswas determinedbycellcountingevery24h.

2.5. Colonyformation

Anchorage-independent growth of peptide-treated melanoma cellswasdeterminedinsoftagar.A2058cells(103 )inculturemedium with10%FBS,0.35%agarand0.5mMWT1-pTjorCPEP,wereplated onabottomagarlayercontainingmedium,10%FBSand1%agarina 6-wellcultureplate.After6daysofcellplating,morepeptide(0.5mM WT1-pTjorCPEP)andculturemedium(300

µ

l)wereaddedtothe correspondingwells.Colonieswerestainedwith0.5%crystalviolet in70%ethanolandcounted11daysafterinitialtreatmentusinga verticalmicroscope.

Alternatively,clonogenicitywastestedinpeptide-treatedA2058 cells(103 )seededin6-wellplates.After5days,500

µ

lof0.5mM WT1-pTjorCPEPorculturemediumwereaddedinthecorresponding wells.10daysafterplating,colonieswerestainedwith0.5%crystal violetin70%ethanolfor10minandcountedinastereomicroscope.

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Table1.

Peptidesequencesandmolecularmass.

Name Sequence Mass(Da)

WT1-pTj KDCERRFSRSDQLKRHQRRHTGVKPFQ-NH2 3395.84

b-WT1-pTj biotin-CGGKDCERRFSRSDQLKRHQRRHTGVKPFQ-NH2 3839.38

FITC-WT1-pTj KDCERRFSRSDQLKRHQRRHTGVKPFQK-FITC 3914.53

CPEP KDAERRFSRSDQLKRAQRRHTGVKPFQ-NH2 3297.71

2.6. ChemiluminescenceELISA(CL-ELISA)

Binding ofp53toWT1-pTj wasexaminedbyCL-ELISA. Recom-binantp53(NovusBiotechnology,Littleton,CO),100ngin50

µ

lof carbonatebuffer (15mM Na2 CO3 ,35mMNaHCO3 ,0.2g

/

lNaN3 , pH9.6)waslefttoadhereovernightat4◦_C_onto_wells_of_maxisorp

opaqueELISAplates(NuncTM ,NalgeNuncInternational,Dusseldorf, Germany).Plateswereblockedfor1hat37◦_C_with_1%_BSA_in

PBS-Tween0.05%.After3washesinPBS-Tween0.05%,1

µ

M biotinylated-WT1-pTj was incubatedfor1 hat37 ◦_C. _{Streptavidin-peroxidase}

(1:2000;Sigma–Aldrich,St.Louis,MO)wasaddedandthereaction wasevaluatedbychemiluminescenceusingECLsolution(Millipore, Billerica,MA)inaluminometer(SpectraMax,MolecularDevices Soft-warePro5.2,Sunnyvale,CA)at470nm.Chemiluminescencereadings areexpressedasRelativeLuminescenceUnits(RLU).

CompetitiveELISAwasperformedtoinvestigatewhetherWT1-pTj competeswithWT1proteinforbindingtop53.Tothatend,100ngof recombinantp53proteinincarbonatebufferwasdispensedinwells ofELISAmicroplate(96-well) andleftovernight at4◦_C._Blocking

andwashingprecededtheadditionof100ngofrecombinantWT1 (JenaBioscience,Jena,Germany),followedby1-hincubationat37◦_C.

Afterwashing3timesinPBS-Tween0.05%,WT1-pTjorCPEPwere incubatedatdifferentconcentrationsfor1hat37◦_C._Binding_of_WT1

top53wasdetectedusinganti-WT1monoclonalantibody(1:1000, Millipore,Billerica,MA).Afterincubationfor1hat37◦_C,_the_plate

wasextensivelywashedinPBS-Tween0.05%andthesecondary anti-mouseIgGcoupledtoperoxidase(1:2000;Sigma–Aldrich,St.Louis, MO)wasadded.Theplatewaswashed3timesinPBSandthereaction wasevaluatedbychemiluminescenceasdescribedabove.

2.7. Luciferasereporterassay

Detection ofp53 activitywasperformed usingthe Cancer 10-pathwayReporterArray(SABiosciences,Fredrick,MD)followingthe manufacturer’s instructions.AfterA2058melanoma cells transfec-tion,theywereleftuntreatedortreatedwith0.5mMWT1-pTjor 0.5mMCPEP,andfurtherincubatedfor24h.Luciferaseactivitywas measuredusingtheDualLuciferaseAssaysystem(Promega, Madi-son,WI)onaluminometer(SpectraMax,MolecularDevicesSoftware Pro5.2,Sunnyvale,CA).Fireﬂyluciferasewastheexperimental re-porterandRenillaluciferasewastheinternalcontrolfornormalizing transfectionefﬁciencies.

2.8. SA-

β

-galactosidaseactivity

A2058cells(103 )werecultivatedin6-wellplatesandthenleft untreatedortreatedwith0.5mMWT1-pTj,0.5mMCPEPor0.5mM TMZ(Positivecontrol)[24]for4days.Cellswerewashedtoremove the peptides andTMZ, following incubation withcomplete fresh mediumforadditional3days.SA-

β

-galactosidaseactivitywas de-tectedwiththeSenescence

β

-galactosidaseKit(CellSignaling, Bev-erly,MA),followingthemanufacturer’sinstructions.Toquantify

SA-β

-galactosidaseactivity,atleast100cellswerecountedinthree ran-domﬁeldsusinganinvertedlightmicroscope.

2.9. Lysosomalvacuolationobservedwithacridineorange

LysosomesfromA2058cells(103

/

well)platedin4-wellchambers andtreatedornotwith0.5mMWT1-pTjor0.5mMCPEPfor4days at37◦_C,_were_observed_following_incubation_with₁

_µ

_g

_/

_ml_acridine

orange(Sigma–Aldrich,St.Louis,MO)for15min.ANikonBioStation IM-Qinvertedmicroscope(red,excitation

/

emission=490

/

650nm) wasused.

2.10. Cellcycleanalysis

A2058cellswereseededin12-wellplatesat2 × 105 cellsper wellandincubatedovernightwithoutserumforcellcycle synchro-nization.Cellsweretreatedornotwith0.5mMWT1-pTjor0.5mM CPEPfor24h.Afterincubation,cellswereharvestedwithtrypsin– EDTA0.25%,washedwithPBSandcentrifuged.Pelletswerestained withpropidiumiodide(PI)in0.1%TritonX-100,0.1%sodiumcitrate, 50

µ

g

/

mlPI(Sigma–Aldrich,St.Louis,MO)and300

µ

g

/

mlRNAseA (Invitrogen,Eugene,OR)for30minat4◦_C_in_the_dark._Flow_cytometric

analysisusedaFACSCantoIIﬂowcytometer(BectonDickinson,San Jose,CA).Post-acquisitionanalysisusedtheFlowJosoftware(Tree StarInc.,Ashland,OR).ForevaluationofDNAcontent,20,000events wereacquiredfromatleastthreeindependentexperiments.

2.11. Westernblotting

A2058cells(106 )treatedwith0.5mMWT1-pTjor0.5mMCPEP for24haftercellcyclesynchronizationoruntreatedcells(Control) werelysedforproteinextractionwithRIPAbuffer(50mMTris–Cl,pH 7.5,150mMNaCl,1%NonidetP-40,0.5%sodiumdeoxycholate,and 0.1%SDS)supplementedwithproteaseandphosphataseinhibitors (Sigma–Aldrich,St.Louis,MO).After20minincubationonice,thecell lysateswerecollectedaftercentrifugationat1500rpm.Theprotein concentrationoflysateswasdeterminedbyBradfordmethod (Bio-Rad,Hercules,CA).Proteins(15

µ

g)wereseparatedbySDS–PAGE andtransferredtoa nitrocellulosemembrane(Millipore,Billerica, MA).Immunoblottingwasrunwithantibodiesagainstp53, phospho-p53(Ser15,Ser392),p27,p21,cyclinB1,phospho-cdc2(Tyr15),and

β

-actin,allpurchasedfromCellSignalingTechnology(Beverly,MA).

β

-actinwasusedastheproteinloadingcontrol.Secondaryantibodies conjugatedwithIgGhorseradishperoxidasewerepurchasedfrom Sigma–Aldrich(St.Louis,MO)andimmunoreactivitywasdetected usingtheImmobilonsolution(Millipore,Billerica,MA).Proteinbands weredetectedusingtheUVItecAlliancegeldocumentationsystem (UVItec,Cambridge,UK).

2.12. Experimentalmelanomamodelsinvivo

TheEthicsCommitteeforanimalexperimentationofFederal Uni-versityofSao˜ Pauloapprovedallexperimentsusingmice,CEPNo. 1280,2010.Allinvivoexperimentswereperformedatleasttwice.

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werecollectedfromanimalsofeachgroup,andinspectedforlung colonization.

Inthesyngeneicsubcutaneous(s.c.)tumormodel,B16F10-Nex2 cells(105

/

animal)wereinoculatedinto therightﬂank ofC57BL

/

6mice. Oncenodulesreached200mm3 , mice(n= 5per group) received5i.p.dosesof300

µ

gofWT1-pTj,300

µ

gofCPEPorPBSon alternatedays.ThehumanA2058melanomacells(4× 106

/

animal) wereinjectedsubcutaneouslyinathymicnudemice(n=5pergroup), treatedwith300

µ

gofCPEP,300

µ

gofWT1-pTjorvehicle(PBS)for5 consecutivedays.Peritumortherapystartedthedayafterinoculation ofmelanomacells. Thetumor volume (V), measured dailywitha caliper,wascalculatedbytheformulaV=0.52 × d2 × D,where

dandDaretheshortandlongdiametersofthetumor,respectively. Miceweresacriﬁcedwhenthetumorsizereached3000mm3 .

2.13. Statisticalanalysis

Dataareexpressedasmeanvalues± standarddeviations(SD)of multiplereplicates.Alldataarerepresentativeofatleasttwo indepen-dentexperiments.p-ValueswerecalculatedbyStudent’st-testand consideredsigniﬁcantiflessthan0.05.Statisticaldifferencesamong treatedgroupsinthes.c.tumormodelwereassessedbytheGehan– Breslow–Wilcoxontest.TheKaplan–Meiermethodwasusedto cal-culatesurvivalcurves,andlog-ranktestwasusedtocomparethe survivalratesofdifferentgroups.Allstatisticalanalysesweredone usingPrismGraphpadSoftware(SanDiego,CA).

3. Results

3.1. WT1-pTjdisplayseffectiveantimelanomaactivityinvivo

TheWT1-pTjpeptidebutnottheCPEPcontrolpeptide,withtwo aminoacidsreplacedbyalanine,signiﬁcantlyreducedthenumberof lungmetastaticnodulesinthesyngeneicB16F10-Nex2melanoma model (Fig. 1A). Melanoma cells were injected endovenously in C57Bl

/

6miceandtreatmentconsistedin5daily i.p.injectionsof 300

µ

gofeitherpeptideorvehicle(PBS),startingthedayaftertumor cellchallenge.

ByusingsubcutaneousB16F10-Nex2melanomagraftinsyngeneic mice,thetumor waslefttogrow untilitreached200mm3 . Pep-tidesweretheninjectedi.p.,with5dosesof300

µ

g

/

animalin al-ternatedays.WT1-pTjratherthantheCPEPcontrolclearlydelayed melanomagrowth,evenaftertumorestablishment(Fig.1B).

TheWT1-pTjprotectiveactivitywasalsoevaluatedusingahuman melanomaxenograftinnudemice.Animalswerechallengedwith humanmelanomacellss.c.,andtreateddaily,for5days,with300

µ

g WT1-pTjorCPEP.Peritumortherapystartedonedayafterinoculation ofmelanomacells.AsobservedinFig.1C,WT1-pTjwaseffectivein delayingthegrowthofhumanmelanoma-bearingnudemiceusingin situinjections.Moreover,thesurvivalofmicetreatedwithWT1-pTj wassigniﬁcantlyprolongedincomparisontothatofthePBSandC PEPcontrolgroups(Fig.1D).

Itshould be notedthat inall in vivo experiments mice main-tainedhealthyphysicalappearance,normalactivitylevelsandnormal weightthroughoutthestudyperiod,showingnotoxiceffectsof pep-tides.Notwithstanding,aneffectiveantimetastaticactivityanda re-markabletherapeuticefﬁcacyofWT1-pTjpeptideagainstmelanoma wereshown.

3.2. WT1-pTjpeptiderapidlytranslocatesintohumanmelanomacells

Thedirect effectsofWT1-pTj onhumanmelanoma cellswere investigated.Consideringthecharacteristicsofthepeptide, Trojan-likeproperties,andeffectsrelatedtozinc-ﬁngerinteractionscouldbe predicted.Infact,WT1-pTjisa27-merlysine–argininerichpeptide, fromtheC-terminalregionofWT1,whichcontainspartofthesecond

Fig.1. ProtectiveeffectofWT1-pTjinvivo.(A)LungcolonizationofB16F10-Nex2 endovenouslyinoculatedinC57BL/6mice.Animalsreceived5i.p.dosesof300µgof CPEP,300µgofWT1-pTjorvehicle(PBS)onconsecutivedays;*_p₌_0.02_versus_C PEP.(B)EffectofWT1-pTjonsubcutaneousgrowthofB16F10-nex2melanomatumor. Oncenodulesreached200mm3_,_mice_were_randomized_and_received₅_alternate_i.p. injectionsof300µgofWT1-pTjorCPEP;*_p_<_0.05_versus_C_PEP._(C)_Effect_of_WT1-pTj onA2058tumorgrowthinnudemice.Animalsweretreatedwithperitumorinjections of300µgofWT1-pTj,300µgofCPEPorvehicle(PBS)for5consecutivedays,starting onday1post-tumorcellchallenge;*_p_<_0.05_in_comparison_with_C_PEP._(D)_Survival ofnudemicegraftedwithhumanmelanomaaftertreatmentwithWT1-pTj,CPEPor PBS;n=5micepergroup;*_p₌_0.0185_in_comparison_with_C_PEP.

zinc-ﬁnger(ZF)domainofWT1.Theanalogouspeptide,withalanine substitutionsatcysteine-3andhistidine-16,bothinvolvedintheZF motif,wasusedasasuitablecontrol peptide(CPEP). Viable non-permeabilizedA2058humanmelanomacellswereexposedto0.5mM FITC-labeledWT1-pTjfor1h.Usingconfocalmicroscopy,weobserved thatthepeptideenteredthecellsshowingadiffusedistributioninthe cytoplasmandwasalsoseeninthenucleuswithin1h,conﬁrming theTrojannatureofthepeptide(Fig.2A).

A2058cellswerealsoincubatedwithbiotinylated-WT1-pTj (b-WT1-pTj)for15min,1and24h,thenwereﬁxedandpermeabilized, followedbyincubationwithstreptavidin-FITCandDAPI.Imagestaken 15minafterexposureto0.5mMofb-WT1-pTjshowedpredominantly membraneandcytoplasmicstaining.After1and24hthepeptidewas completelydistributedinthecells,particularlyinthenucleuswhere itcolocalizedwithnuclearDNA(Fig.2B).

3.3. WT1-pTjinhibitstheproliferationofWT1-expressingtumorcells

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Fig.2. IntracellulardistributionofWT1-pTjinA2058humanmelanomacells.(A)Cells weretreatedwithFITC-labeledWT1-pTjfor1hat37◦_C_and_visualized_by_confocal

mi-croscopy.Sequence(Z-stacks)showingthedistributionofFITC-labeledpeptide(green) andthenucleistainedwithDAPI(blue).Bar=10µm.(B)Co-localizationofWT1-pTj andnuclearDNAinhumanmelanomacells.A2058cellsweretreatedwithbiotinylated WT1-pTjfor24h,fixedwithparaformaldehyde,andpermeabilizedwithTritonX-100. (a)Differentialinterferencecontrast(DIC)image;(b)CellsstainedwithDAPI;(c)Cells treatedwithstreptavidin-FITC;(d)Merge,showingnuclearcolocalizationofWT1-pTj. Inserts:highmagnificationofcellsindicatedbyarrows.Scalebar=10µm.(For inter-pretationofthereferencestocolourinthisfigurelegend,thereaderisreferredtothe webversionofthisarticle.)

Table2.

AntiproliferativeactivityofWT1-pTjonWT1-expressingtumorcelllinesandnontumor formingcelllines.

Celllines EC50a(mol/lx10−3)

A2058humanmelanoma 0.455±0.032 SK-MEL-28humanmelanoma 0.680±0.020 B16F10-Nex2murinemelanoma 0.466±0.067 MCF-7humanbreastcancer 0.247±0.058 MDA-MB231humanbreastcancer 0.759±1.40 OVCAR-3humanovariancancer 0.208±0.02 HL-60humanacuteleukemia >1 HFFhumanforeskinﬁbroblast >1 MEFmurineembryonicﬁbroblast >1

a_EC50_is_the_{concentration}_that_decreases_viability_by_50%_in_a_{dose-dependent}_survival

curve.

3.4. WT1-pTjbutnotCPEPdisplaysstrongantiproliferativeeffectson humanmelanomacells

SinceWT1geneisessentialformelanomacellsproliferationand metastasis[26,27,8]weexaminedwhetherWT1-pTjandmore specif-ically,thetruncatedZF-motifofWT1-pTj,displayedanti-melanoma proliferativeeffects.HumanA2058melanomacellswereincubated withWT1-pTjorCPEP(A3 C,A16 H)at0.125to0.5mMandthecell numberofviablecellsdeterminedevery24hfor4days.Wefound thattreatmentwithWT1-pTjresultedinadose-dependentinhibition ofproliferationofmelanomacellswhencomparedtountreatedcells.

Fig.3. EffectsofWT1-pTjonproliferationandclonogenicabilityofhumanmelanoma cells.(A)A2058cells(103_/_well)_were_treated_with_WT1-pTj_(left)_or_C_PEP_(right)_at theindicatedconcentrationsfor96h,andcellproliferationwasdailymonitoredby Trypanblueexclusionassay;(B)A2058cellsweresuspendedin300µlof0.5mM WT1-pTj,0.5mMCPEP,orunsupplementedculturemedium(Control)andcultivated onsoft-agarmedium.Thenumberofcolonieswasdetermined11daysaftertheﬁrst treatment;*_p_<_0.02._(C)_{Representative}_{photomicrographs}_of_colony_formation_assay. A2058cellswereplatedon(a)untreatedculturemedium(Control),(b)mediumtreated with0.5mMCPEP,or(c)treatedwith0.5mMWT1-pTj.After5days,culturemedia wereaspiratedandfreshmediumwiththerespectivepeptidesat0.5mMwasadded. After10daysincubation,colonieswerestainedwithcrystalvioletandcountedona stereomicroscope.Datarepresentmeans±SDofthreeindependentexperiments.(For interpretationofthereferencestocolourinthisﬁgurelegend,thereaderisreferredto thewebversionofthisarticle.)

Thenumberofcellsremainedstablefrom24to96hwhentreated withWT1-pTjat0.5mM,suggestingthatthepeptidehadasustained cytostaticeffectonthesecells.Nosigniﬁcantchangesonproliferation wereobservedinCPEP-treatedcells,exceptatthehighestdoseafter 96hofincubation(Fig.3A).

Hanetal.[28]reportedonthetransfectionofMCF-7humanbreast cancercellswithconstructscontainingonlytheZFdomainsofWT1 andconsequentreductionintheexpressionofproteinsimportantfor thesurvivalandtumorcellproliferation,suchasc-myc,Bcl-2and amphregulin,alsointerferingwiththeclonogenicabilityofbreast cancercells.Basedonthesefindingswetestedtheinfluenceof WT1-pTjontumorcellclonogenicity.Asobserved,treatmentwith0.5mM WT1-pTjdrasticallydecreasedthenumberofA2058coloniesbothin semi-solidsoft-agarmedium(Fig.3B)anddirectlyincultureplates (Fig.3C),whencomparedtountreatedorCPEP-treatedcells.Inthe anchorage-independentgrowth assayinsoft-agar, treatmentwith WT1-pTjreducedcolonyformationby70.8%,ascomparedto25.4% withCPEP.Quantificationofcellgrowthonplatesshowedonly20 coloniesafterWT1-pTjtreatmentfor9days,whereascellincubation inunsupplementedcompleteculturemediumorwith0.5mMPEPC, resultedin300and248colonies,respectively.

3.5. WT1-pTjenhancesthetranscriptionalactivityofp53inmelanoma cellsandcompeteswithWT1proteinforbindingtop53

WT1bindstoDNAandcaninteractalsowithseveral intracellu-larproteins[29].AphysicalandfunctionalassociationbetweenWT1 andtheproductofp53tumorsuppressorgene[30]hasbeen demon-strated.Suchinteractionwasshowntocross-modulate transactiva-tionproperties.WT1interactswithp53throughzincﬁngers1and 2,stabilizesp53,enhancesp53-mediatedtranscriptionalactivation, andantagonizesp53-mediatedapoptosistriggeredbyultraviolet ra-diation[31].

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Fig.4. BindingofWT1-pTjtop53,displacementofp53boundtoWT1,and WT1-pTjmodulationoftranscriptionalactivityofp53.(A)Theinteractionbetweenp53 andWT1-pTjwasdeterminedusingbiotinylatedpeptideandCL-ELISA;*_p_<_0.01._(B) CompetitiveinhibitionofWT1associationtop53wasevaluatedinwellscoatedwith recombinantp53,incubatedwithWT1andthenexposedtoWT1-pTjorCPEPat indi-catedconcentrations.Bindingofp53toWT1wasquantifiedwithanti-WT1monoclonal antibody;*_p_<_0.02_compared_to_C_PEP_effect._(C)_A2058_cells_were_transfected_with_the p53-responsiveluciferasereporterconstruct,andthentreatedwith0.5mMWT1-pTj or0.5mMCPEPfor24h.TheratiooffireflytoRenillaluciferasewasdeterminedand normalizedtothevalueobtainedforuntreatedcells(=100%).Dataarerepresentative oftwoindependentexperiments.*p<0.02comparedtoControl(untreated)andC PEPsystems.

inthep53interaction [31] weconsidered thepossibilitythat the peptidemightalsobindtop53.AsshowninFig.4A,CL-ELISAusing recombinantp53andbiotinylatedWT1-pTjconfirmedtheassociation betweenthepeptideandtheprotein.Inaddition,usingcompetitive ELISAweverifiedthat WT1-pTj dose-dependentlyinhibited WT1-p53complexformation(Fig.4B).Further,inA2058melanomacells transfectedwithap53-responsiveluciferasereporterconstruct, WT1-pTj(butnotCPEP)enhancedp53transcriptionalactivation(Fig.4C). Together,theseresultssuggestthatWT1-pTjinteractswithp53,and maydecreasetheassociationbetweenp53andWT1.Inparallel,it causedp53accesstospecifictranscriptionalresponseelementson DNA.

3.6. WT1-pTjinducesG2

/

Mcellcyclearrestandsenescencefeaturesin humanmelanomacells

Thecellcycleofmelanomacellswasexaminedbyﬂowcytometry tobettercharacterizethemechanismbywhichWT1-pTjsuppresses tumorcellproliferation.TreatmentwithWT1-pTjeffectivelyarrested thecell-cycleprogressioninA2058cells,depletingtheS-phase com-partment(from20.2%to6%whencomparedtoControl)and increas-ingtheG2

/

M(from10.5%to27%whencomparedtoControl)phase compartment(Fig.5A).A2058cellstreatedwithCPEPshowedno sig-niﬁcantdifferencesregardingthecellcycleproﬁlewhencompared totheControlsystem,suggestingthattheZF-coordinatingregionis essentialfortheantiproliferativeeffects.

Inaddition,weevaluatedthepredominantsignalpathways ar-bitratingcellcyclearrestinhumanmelanomacellsunderpeptide treatment. As shownin Fig. 5B,by Westernblottinganalysis we determinedthattotal p53levelswerenot signiﬁcantlyaffectedin cellstreatedwithWT1-pTj.Nevertheless,adecreaseinserine392 phosphorylationandanincreaseinserine15phosphorylationwere observeduponWT1-pTjtreatment.Additionally,increasedlevelsof bothp27Kip1 andp21Cip1 ,knowntoregulatebothG1–SandG2–M transitions,weredetectedinWT1-pTj-treatedcells.Furthermore,in

Fig.5. EffectofWT1-pTjoncellcycleprogressionandmodulationofp53,and down-streammediators,inhumanmelanomacells.(A)A2058cellsweretreatedeitherwith 0.5mMWT1-pTjor0.5mMCPEP,for24h,andthecellcycleprofilewasexaminedby flowcytometry.Control,unsupplementedculturemedium.Resultsarerepresentative ofthreeindependentexperiments.(B)CellularextractsfromA2058cellsexposedto 0.5mMWT1-pTjor0.5mMCPEPweresubjectedtoimmunoblottingwithantibodies specificforp53,phospho-p53,p27,p21,phospho-cdc2andcyclinB1.Proteinloadwas normalizedtoβ-actin.

ordertoelucidatethenatureofthecell-cyclearrestcausedby WT1-pTj,weexaminedtheexpressionofcellcycle-regulatingfactorsat theG2

/

Mphase.WefoundthattheproteinlevelsofcyclinB1and phospho-cdc2(Tyr15)weremarkedlydownregulatedin WT1-pTj-treatedA2058cells.Theseresultsassociatetheinhibitoryeffectsof WT1-pTjtoblockingofthecellcycleprogressionatG2

/

Mphaseand modulationofp53anddownstreamsignalingcascades.

Severalreportshaveshownthecentralroleofp53incontrolling senescence[32],whichis characterizedby stableandirreversible lossofcellproliferation[33].Particularlyinmelanoma,telomere dys-function[34]oncogeneactivation[35]andanticanceragents[36]can activatep53anditssignalingpartners(e.g.p16Ink4A ,Rbandp21Cip1 ), whicharethusimplicatedinthesuppressionoftumorinitiationand progression.

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Fig.6. InductionbyWT1-pTjofcellularsenescenceinhumanmelanomacells.(A) QuantiﬁcationofcoloniesformedafterremovalofthepeptidesandTMZ.A2058cells weretreatedwithWT1-pTj,CPEPandTMZfor4days.Cellsweredetachedandequal numbersofuntreatedandtreatedcellswereseededandcultivatedinfreshmediafor additional4days.Thenumberofcolonieswasscoredinthreeindependentexperiments performedinduplicates;*_p_<_0.01_compared_to_Control_and_C_PEP._(B)_{Representative} photomicrographs(originalmagniﬁcation400×)ofA2058cellstreatedwith0.5mM WT1-pTj,CPEPorTMZ(positivecontrol)for4days.ThereafterthepeptidesandTMZ werewashedoutandcellswerestainedforSA-β-Galafterincubationforadditional3 daysinfreshmedia.PositivecellsforSA-β-Galwerevisualizedunderaphasecontrast microscopeandexpressedaspercentageoftotalcellscountedatrandom.(C)Acridine orangewasusedtostainacidicvesicularorganellesinuntreatedcontrolcells(a);serum starvedcellsfor24h(positivecontrol)(b);cellstreatedwith0.5mMWT1-pTj(c); cellstreatedwithCPEP(d),for4days.Imagesarerepresentativeoftwoindependent experimentsperformedinduplicates.Bars=10µM.

widely usedsenescencebiomarker[36].Incontrast,CPEP caused littlereductioninthenumberofcoloniesascomparedtountreated cells,andaminoreffectonsenescenceinduction(16%)when com-paredtoWT1-pTj.Thus,theproliferativecapacityofWT1-pTj-treated cellswaslostevenuponremovalofthepeptide.

TheSA-

β

-galactosidasehasbeenshowntobeamanifestationof residuallysosomalactivityafteralkalinization,onlydetectabledue totheincreasedlysosomalcontentinsenescentcells[37].Therefore, weexaminedthelysosomalcontentinmelanomacellstreatedeither withWT1-pTjorCPEP,usingthelysosomotropicagentacridine or-ange.StainingofA2058cellswithacridineorangeshowedamarked increaseinthenumberandsizeoflysosomesinsenescentcellsafter treatmentwith0.5mMWT1-pTjfor4daysasvisualizedby ﬂuores-cence microscopy(Fig.6C). MostoftheCPEP-treatedcells exhib-itedminimalredﬂuorescence.Theseresults,alongwiththe molecu-larmechanismunderlyingWT1-pTj-inducedgrowtharrest,strongly suggestthatWT1-pTj-treatedcellsundergocellularsenescence.

4. Discussion

Peptide-basedantitumortherapyexploresthehighafﬁnityand speciﬁcityofpeptidesforparticulartargets,thelowtoxicityandgood

tissuepenetration[38].Ourlaboratoryhasfocusedonantitumor pep-tidesderivedfrominternalsequencesofimmunoglobulinsand tran-scriptionfactors[22].Inthecurrentstudy,wereportonapeptide derivedfromWT1,endowedwithanti-melanomaproperties.Both humanmelanomaandB16F10murinemelanomaweresensitiveto theWT1-pTjTrojanpeptide.

WT1calledourattentionduetoitshighexpressionand immuno-genicityinB16F10murinemelanoma(unpublishedresults).In com-parisonwithhumancancer,WT1hasemergedasanimportanttarget expressedinhematologicandsolidtumors.Thereis,indeed,plenty ofevidencesupportingtheroleofWT1intheoncogenicprocessof melanoma.ThefunctionofWT1intranscriptionalregulation,aswell inRNAmetabolismandtranslation,hasbeenextensivelyreviewed [25,39].WT1overexpressionhasbeendetectedinmelanomapatients’ samplesandmelanomacells,whereasnoWT1staininghasbeen ob-servedinthemajorityofbenignmelanocyticnevi,epidermal ker-atinocytesandmelanocytes[4,40].RNAisilencingofWT1inhibited melanomaproliferationassociatedwithdown-regulationofnestin andzyxin[6],sensitizedB16F10cellstoconventional chemothera-peuticagents(e.g.doxorubicinandcisplatin)andreducedlung metas-tases,emphasizingWT1functioninmelanomaprogression[8].

TheWT1-pTjpeptidecontainspartofaparticularlyreactive zinc-ﬁngerdomain,andowingtoitsabundantbasicaminoacid compo-sition,exhibitscell-penetratingproperties.Thepeptidewasableto arresttumorcellgrowthandinducesenescenceinhumanmelanoma cells.Asigniﬁcantantimelanomaactivityinvivowasalsoobserved usingsubcutaneousandmetastaticmodelsinmice.Increasedlevels ofactivep53andenhancedtranscriptionalactivationwere associ-atedwithWT1-pTj-inducedsenescenceinA2058humanmelanoma cells.Cyclin-dependentkinaseinhibitorsp27Kip1 andp21Cip1 medi-atedthesustainedcellcyclearrest.Thetypicalsenescentphenotype inWT1-pTj-treatedcellsincludedenlargedcytoplasm,andcellular increaseofbothSA-

β

-galactosidaseactivityandlysosomalcontent. Toexertitsactivities,peptideWT1-pTj,likeWT1,doesnotbindto p53promoterbutphysicallyinteractswiththep53proteinitself.In fact,wehaveshownthatthepeptidecompeteswithWT1forprotein bindingtop53,supportingthehypothesisthatthepeptidemay pre-ventimportantinteractionsofWT1withitspartners,includingp53, andthereforedisturb relevantpro-tumor signalingofthe original proteinviatheZFdomain.Protein-proteininteractionofWT1with p53modulatesbindingtodifferentpromotersdetermininggene ex-pressionorrepression.AswithWT1,wedemonstratedthatpeptide WT1-pTjactivatedp53toactasatranscriptionfactorinaluciferase geneexpressionsystem.

Therapy-inducedsenescenceisanoveltherapeuticapproachto treatcancers[41].Standardchemotherapyandradiotherapy,besides inducingDNAdamageandcytotoxicity,maytriggerarobust senes-cenceresponseinvitroandinvivo[42,43].Pro-senescencetherapy mayalsobeachievedwithgreatlyreducedtoxicity.

The molecular mechanism underlying therapy-induced senes-cenceconferredby WT1-pTjtreatmentdependsonp53 transcrip-tionalactivationandiscorrelatedwithp53post-transcriptional phos-phorylationatSer15andSer392.Consistentwithpreviousﬁndings that have reportedon speciﬁc changesinp53, asustained treat-menttriggersap53-dependentsenescenceprogram.Prolonged

β

-interferonstimulationswitchedonp53intwostepsinvolvingfirst, dephosphorylationatserine392,andthen,phosphorylationofp53at serine15leadingtoitstranscriptionalactivity[44].Similar posttrans-lationalmodificationsofp53wereobservedinreplicativesenescence inhumanfibroblasts[45].

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cellsexposedtoWT1-pTj.Ourresultsexcludedp16Ink4A -Rbsignal ac-tivationinWT1-pTj-inducedcellularsenescence(datanotshown),in linewithpreviousdatashowingthatp16isfrequentlyinactivatedin melanoma[48,49].

CellcycleanalysisrevealedthatWT1-pTjinhibitionofmelanoma cellproliferationwascausedbycellcyclearrestatG2

/

Mphase, ac-companiedbyadecreaseinthenumberofcellsintheSphase.The G2–Mtransitionispositivelyregulatedby Cdc2

/

cyclinB complex [50],whichiscontrolledbyphosphorylationatvarioussites, includ-ingtheinhibitoryphosphorylationattyrosine-15andthreonine14 byWee1andMyt1[51,52].Adecreaseinthelevelsofphospho-cdc2 (Tyr15),cdc2,cyclinAandcyclinB1hasbeenassociatedwithcell cyclearrestatG2

/

M[53].Similarly,wefoundasigniﬁcantdecrease incyclinB1andphospho-cdc2(Tyr15)expressionaftertreatment withWT1-pTj, incontrastwithCPEP-treatedcells. Inconclusion, modulationofp53activityandactivationofdownstreamsignaling isessentialforsenescenceresponseinWT1-pTj-treatedcells.These eventsdependontheZF-coordinatingregionbecauseCPEPelicited nosimilarresponse.

GiventhelimitedexpressionofWT1inadultanimals,restrictedto fewcelltypesoftheurogenitalsystem[3],thisfactormightotherwise beanoptimaltargetfortreatmentofWT1-expressingmalignancies. Intheﬁeldoftargetedtherapies,CPPs(cationiccell-penetrating pep-tides)havebeenusedtoovercomepermeabilitybarriersinthetumor andtomediatecargodeliveryintocancercells,leadingtothe de-velopmentoftumor-speciﬁcmoleculartherapeutics.CPP-mediated transductionhasbeenusedtoinhibitnuclearoncoprotein translo-cation[54],modulateoncoproteinsignaling[10],enableapoptotic cell-death[16,55],anddeliveroligonucleotides[56]tocancercells. Presently,weshowthatpeptideWT1-pTjexhibitstrojanproperties andexertsprotectiveeffectsinexperimentalmelanomatherapy.In additiontoantiproliferativeeffectsmediatedbyWT1-pTjtreatment

invitro,thepeptideinhibitedhumanmelanomaprogression,innude mice.

Conventionalchemotherapeuticdrugsactmainlythrough induc-tionofapoptosis,andtheirreducedefﬁcacyinmelanomapatients isrelatedtothehighresistanceofmelanoma cells[57]. Therapy-inducedsenescencemayrepresentanalternatefunctionalapproach toimprovecancertherapy[58,59].

Toconclude,ourpresentstudyreportsonthesenescence-inducing peptideWT1-pTjasapromisingcandidatetoeradicatetumor cell progression,includingthosethatfailtorespondtoconventional an-titumortherapies.TheWT1-pTjisuniqueinthatitderivesfroman oncoprotein,iscell-penetratingbyitself,inducescellularsenescence andeffectivelyprotectsinacancerpreclinicalmodel.

Conflict of interest statement

Theauthorshavenoconﬂictsofinteresttodeclare.

Acknowledgments

TheauthorsthanktheLudwigInstituteforCancerResearch,Sao˜ Paulobranch,Dr.O.KeithOkamotofromtheUniversityofS˜aoPaulo, andDr.LuizF.L.ReisfromSirio-LibanezHospitalforthecelllines usedinthepresentstudy.TheStateofSao˜ PauloResearchSupport Foundation(FAPESP), Brazil, supported thiswork through Grants 2010

/

51423–0and2012

/

19476–2.L.R.T.isaresearchfellowfrom theBrazilianNationalResearchCouncil(CNPq).

References

[1] Yang,L.(2007)Atumorsuppressorandoncogene:theWT1story.Leukemia21, 868–876.

[2] Call,K.M.(1990)Isolationandcharacterizationofazincﬁngerpolypeptidegene atthehumanchromosome11Wilms’tumorlocus.Cell60,509–520.

[3] Pelletier,J.(1991)WT1mutationscontributetoabnormalgenitalsystem devel-opmentandhereditaryWilms’tumour.Nature353,431–434.

[4] Nakatsuka,S.(2006)ImmunohistochemicaldetectionofWT1proteinina vari-etyofcancercells.Mod.Pathol.19,804–814.

[5] Oji,Y.(1999)ExpressionoftheWilms’tumorgeneWT1insolidtumorsandits involvementintumorcellgrowth.Jpn.J.CancerRes.90,194–204.

[6] Wagner, N.(2008) TheWilms’ tumorsuppressorWT1is associatedwith melanomaproliferation.PﬂugersArch.455,839–847.

[7] Zamora-Avila,D.E.(2007)RNAisilencingoftheWT1geneinhibitscell prolifera-tionandinducesapoptosisintheB16F10murinemelanomacellline.Melanoma Res.17,341–348.

[8] Zamora-Avila,D.E.(2009)WT1genesilencingbyaerosoldeliveryofPEI-RNAi complexesinhibitsB16-F10lungmetastasesgrowth.CancerGeneTher.16, 892–899.

[9] Thundimadathil,J.(2012)Cancertreatmentusingpeptides:currenttherapies andfutureprospects.J.AminoAcids2012,967347.

[10] Giorello,L.(1998)Inhibitionofcancercellgrowthandc-Myctranscriptional activitybyac-Mychelix1-typepeptidefusedtoaninternalizationsequence. CancerRes.58,3654–3659.

[11] Bonfanti,M.(1997)p21WAF1-derivedpeptideslinkedtoaninternalization pep-tideinhibithumancancercellgrowth.CancerRes.57,1442–1446.

[12] Rosca,E.V. (2011)Anti-angiogenic peptidesfor cancertherapeutics. Curr.Pharm. Biotechnol.12,1101–1116.

[13] Tan,M.(2006)SelectiveinhibitionofErbB2-overexpressingbreastcancer in vivo byanovelTAT-basedErbB2-targetingsignaltransducersandactivatorsof transcription3-blockingpeptide.CancerRes.66,3764–3772.

[14] Cardo-Vila,M.(2010)Fromcombinatorialpeptideselectiontodrugprototype (II):targetingtheepidermalgrowthfactorreceptorpathway.Proc.Natl.Acad. Sci.U.S.A.107,5118–5123.

[15] Matsuo,A.L.(2011)Anewphage-displaytumor-homingpeptidefusedto an-tiangiogenicpeptidegeneratesanovelbioactivemoleculewithantimelanoma activity.Mol.CancerRes.9,1471–1478.

[16] Johansson,H.J.(2008)Characterizationofanovelcytotoxiccell-penetrating peptidederivedfromp14ARFprotein.Mol.Ther.16,115–123.

[17] Niesner,U.(2002)Quantitationofthetumor-targetingpropertiesofantibody fragmentsconjugatedtocell-permeatingHIV-1TATpeptides.Bioconjug.Chem. 13,729–736.

[18] Massodi,I.(2009)Inhibitionofovariancancercellmetastasisbyafusion polypeptideTat-ELP.Clin.Exp.Metastasis26,251–260.

[19] Caino,M.C.,Meshki,J.andKazanietz,M.G.(2009)Hallmarksforsenescencein carcinogenesis:novelsignalingplayers.Apoptosis14,392–408.

[20] Bringold,F.andSerrano,M.(2000)Tumorsuppressorsandoncogenesincellular senescence.Exp.Gerontol.35,317–329.

[21] Arruda,D.C.(2012)beta-Actin-bindingcomplementarity-determiningregion 2ofvariableheavychainfrommonoclonalantibodyC7inducesapoptosisin severalhumantumorcellsandisprotectiveagainstmetastaticmelanoma.J. Biol.Chem.287,14912–14922.

[22] Massaoka,M.H.(2013)Melanoma:perspectivesofavaccinebasedonpeptides. In:M.Giese(Ed.),MolecularVaccines:FromProphylaxistoTherapy.Wien: Springer-Verlag,pp.397–412.

[23] Dobroff,A.S.(2002)Protective,anti-tumormonoclonalantibodyrecognizesa conformationalepitopesimilartomelibioseatthesurfaceofinvasivemurine melanomacells.HybridHybridomics21,321–331.

[24] Mhaidat,N.M.(2007)Temozolomideinducessenescencebutnotapoptosisin humanmelanomacells.Br.J.Cancer97,1225–1233.

[25] Hohenstein,P.andHastie,N.D.(2006)ThemanyfacetsoftheWilms’tumour gene,WT1.Hum.Mol.Genet.15(SpecNo2),R196–R201.

[26] Zapata-Benavides,P.(2012)WT1silencingbyRNAisynergizeswith chemother-apeuticagentsandinduceschemosensitizationtodoxorubicinandcisplatinin B16F10murinemelanomacells.Oncol.Lett.3,751–755.

[27] Michiels,J.F.(2010)PPARbetaactivationinhibitsmelanomacellproliferation involvingrepressionoftheWilms’tumoursuppressorWT1.PﬂugersArch.459, 689–703.

[28] Han,Y.(2007)ThezincﬁngerdomainofWilms’tumor1suppressorgene(WT1) behavesasadominantnegative,leadingtoabrogationofWT1oncogenic po-tentialinbreastcancercells.BreastCancerRes.9,R43.

[29] Englert,C.(1998)WT1–morethanatranscriptionfactor?TrendsBiochem.Sci. 23,389–393.

[30] Maheswaran,S.(1993)PhysicalandfunctionalinteractionbetweenWT1and p53proteins.Proc.Natl.Acad.Sci.U.S.A.90,5100–5104.

[31] Maheswaran,S.(1995)TheWT1geneproductstabilizesp53andinhibits p53-mediatedapoptosis.GenesDev.9,2143–2156.

[32] Itahana,K.,Dimri,G.andCampisi,J.(2001)Regulationofcellularsenescenceby p53.Eur.J.Biochem.268,2784–2791.

[33] Larsson,L.G.(2011)Oncogene-andtumorsuppressorgene-mediated suppres-sionofcellularsenescence.Semin.CancerBiol.21,367–376.

[34] Sviderskaya,E.V.(2003)p16/cyclin-dependentkinaseinhibitor2Adeﬁciency inhumanmelanocytesenescence,apoptosis,andimmortalization:possible im-plicationsformelanomaprogression.J.Natl.CancerInst.95,723–732. [35] Wajapeyee, N. (2008) Oncogenic BRAFinduces senescence and apoptosis

throughpathwaysmediatedbythesecretedproteinIGFBP7.Cell132,363–374. [36] Cozzi,SJ.(2006)Inductionofsenescenceinditerpeneester-treatedmelanoma cellsviaproteinkinaseC-dependenthyperactivationofthemitogen-activated proteinkinasepathway.CancerRes.66,10083–10091.

(9)

Sci.113,3613–3622.

[38] Bitler,B.G.andSchroeder,J.A.(2010)Anti-cancertherapiesthatutilizecell penetratingpeptides.RecentPat.AnticancerDrugDiscov.5,99–108. [39] Roberts,S.G.(2005)TranscriptionalregulationbyWT1indevelopment.Curr.

Opin.Genet.Dev.15,542–547.

[40] Henderson,E.J.andPentland,B.(1991)Homepassassessmentin neurorehabil-itationpractice.J.Adv.Nurs.16,1439–1443.

[41] Nardella,C.(2011)Pro-senescencetherapyforcancertreatment.Nat.Rev. Can-cer11,503–511.

[42] tePoele,R.H.(2002)DNAdamageisabletoinducesenescenceintumorcellsin vitro andin vivo .CancerRes.62,1876–1883.

[43] Sidi,R.(2011)Inductionofsenescencemarkersafterneo-adjuvant chemother-apyofmalignantpleuralmesotheliomaandassociationwithclinicaloutcome: anexploratoryanalysis.Eur.J.Cancer47,326–332.

[44] Moiseeva,O.(2006)DNAdamagesignalingandp53-dependentsenescenceafter prolongedbeta-interferonstimulation.Mol.Biol.Cell17,1583–1592. [45] Webley,K.(2000)Posttranslationalmodiﬁcationsofp53inreplicative

senes-cenceoverlappingbutdistinctfromthoseinducedbyDNAdamage.Mol.Cell. Biol.20,2803–2808.

[46] Herbig,U.(2004)Telomere shorteningtriggerssenescenceofhumancells throughapathwayinvolvingATM,p53,andp21(CIP1),butnotp16(INK4a). Mol.Cell14,501–513.

[47] DiMicco,R.(2006)Oncogene-inducedsenescenceisaDNAdamageresponse triggeredbyDNAhyper-replication.Nature444,638–642.

[48] Castellano,M.(1997)CDKN2A/p16isinactivatedinmostmelanomacelllines. CancerRes.57,4868–4875.

[49] FitzGerald,M.G.(1996)Prevalenceofgerm-linemutationsinp16,p19ARF,and CDK4infamilialmelanoma:analysisofaclinic-basedpopulation.Proc.Natl. Acad.Sci.U.S.A.93,8541–8545.

[50] Innocente,S.A.(1999)p53regulatesaG2checkpointthroughcyclinB1.Proc. Natl.Acad.Sci.U.S.A.96,2147–2152.

[51] McGowan,C.H.andRussell,P.(1993)HumanWee1kinaseinhibitscelldivision byphosphorylatingp34cdc2exclusivelyonTyr15.EMBOJ.12,75–85. [52] Wells,N.J.(1999)TheC-terminaldomainoftheCdc2inhibitorykinaseMyt1

interactswithCdc2complexesandisrequiredforinhibitionofG(2)/M progres-sion.J.Cell.Sci.112,3361–3371.

[53] Deep,G.(2006)SilymarinandsilibinincauseG1andG2-Mcellcyclearrest viadistinctcircuitriesinhumanprostatecancerPC3cells:acomparisonof ﬂavanonesilibininwithﬂavanolignanmixturesilymarin.Oncogene25,1053– 1069.

[54] Lin,Y.Z.(1995)Inhibitionofnucleartranslocationoftranscriptionfactor NF-kappaBbyasyntheticpeptidecontainingacellmembrane-permeablemotif andnuclearlocalizationsequence.J.Biol.Chem.270,14255–14258. [55] Selivanova,G.(1997)Restorationofthegrowthsuppressionfunctionofmutant

p53byasyntheticpeptidederivedfromthep53C-terminaldomain.Nat.Med. 3,632–638.

[56] Kanazawa,T.(2012)Suppressionoftumorgrowthbysystemicdeliveryof anti-VEGFsiRNAwithcell-penetratingpeptide-modiﬁedMPEG-PCLnanomicelles. Eur.J.Pharm.Biopharm.81,470–477.

[57] Grossman,D.andAltieri,D.C.(2001)Drugresistanceinmelanoma:mechanisms, apoptosis,andnewpotentialtherapeutictargets.CancerMetastasisRev.20,3– 11.

[58] Jing,H.(2011)OpposingrolesofNF-kappaBinanti-cancertreatmentoutcome unveiledbycross-speciesinvestigations.GenesDev.25,2137–2146. [59] Schmitt,C.A.(2002)Asenescenceprogramcontrolledbyp53andp16INK4a