Exposure of neuroblastoma cell lines to imatinib results in the upregulation of the CDK inhibitor p27KIP1 as a consequence of c-Abl inhibition

29 July 2021

AperTO - Archivio Istituzionale Open Access dell'Università di Torino

Original Citation:

Exposure of neuroblastoma cell lines to imatinib results in the upregulation of the CDK inhibitor p27KIP1 as a

consequence of c-Abl inhibition

Published version:

DOI:10.1016/j.bcp.2014.09.016

Terms of use:

Open Access

(Article begins on next page)

Anyone can freely access the full text of works made available as "Open Access". Works made available under a

Creative Commons license can be used according to the terms and conditions of said license. Use of all other works

requires consent of the right holder (author or publisher) if not exempted from copyright protection by the applicable law.

Availability:

This is the author's manuscript

Exposure

of

neuroblastoma

cell

lines

to

imatinib

results

in

the

upregulation

of

the

CDK

inhibitor

p27

KIP1

as

a

consequence

of

c-Abl

inhibition

Elisa

Lupino,

Cristina

Ramondetti,

Barbara

Buccinna`,

Marco

Piccinini

*

DepartmentofOncology,SchoolofMedicine,UniversityofTorino,Italy

1. Introduction

TheCDKinhibitorp27KIP1_{isakeyregulatorofG1toSphase}

transitionineukaryoticcells.InnormalcellsduringG0andearly G1phasehighlevelsofp27KIP1_{bindtoandinhibittheactivityof}

nuclearcyclinD-CDK4/6andcyclinE-CDK2complexes.DuringG1

toSphasetransition,p27KIP1_{abundancedecreases,mainlybecause}

of enhanced degradation,allowingtheactivation of cyclin-CDK

complexestosupportS-phaseentry[1,2].Theinstabilityofp27KIP1

in lateG1 istriggered bycyclinE-CDK2-dependent

phosphory-lation on Thr-187 which resultsin recruitment of p27KIP1 _toa

S-phasekinase-associatedprotein2(SKP2)-containingubiquitin ligaseanddegradationbythe26Sproteasome[3].Reducedp27KIP1

proteinlevelscausedbyacceleratedproteolysischaracterizemany humancancers[1,2].

Previous studieshavedemonstrated thattyrosine

phosphor-ylation triggersthetransitionof p27KIP1frominhibitor to

non-inhibitor of cyclin-CDK complexes and converts p27KIP1 _{into a}

substrateofcyclinE-CDK2complexes[4,5].Ofthethreetyrosine residuesinp27KIP1_{(Tyr-74,Tyr-88,andTyr-89),thetwoadjacent}

residuesTyr-88andTyr-89arepartoftheinhibitory310helixof

ARTICLE INFO Articlehistory: Received25July2014 Accepted19September2014 Availableonline26September2014 Chemicalcompoundsstudiedinthisarticle: Imatinib(PubChemCID:123596) Cycloheximide(PubChemCID:6197) 5-bromo-20_{-deoxyuridine(PubChemCID:}

6035)

Hoechst33258(PubChemCID:16218619) Keywords: Imatinib Neuroblastoma p27KIP1 c-Abl CDK2 pRb ABSTRACT

Imatinibmesylateisatyrosinekinaseinhibitorwithselectivityforabelsontyrosine-proteinkinase1

(c-Abl), breakpoint clusterregion (Bcr)-Ablfusion protein(Bcr-Abl), mast/stemcell growth factor

receptorKit(c-Kit),andplatelet-derivedgrowthfactorreceptor(PDGFR).Previousstudiesdemonstrated

thatimatinibinthelowmicromolarrangeexertedantiproliferativeeffectsonneuroblastomacelllines.

However,althoughneuroblastomacellsexpressc-KitandPDGFR,theimatinibconcentrationsrequired

toachievesignificantgrowthinhibitoryeffects(10mM)aresubstantiallyhigherthanthoserequired

forinhibitionofligand-inducedphosphorylationofwildtypec-KitandPDGFR(1mM),suggestingthat

additionalmechanismsareresponsiblefortheantitumoractivityofimatinibonthesecells.Inthisstudy,

weshowthattreatmentofneuroblastomacelllineswith1–15mMimatinibresultedinadosedependent

inhibition of 5-bromo-20_{-deoxyuridine (BrdU) incorporation into newly synthesized DNA. The}

antiproliferativeeffectofimatinibwasdependentontheupregulationofthecyclin-dependentkinase

(CDK)inhibitorp27KIP1_{inthenuclearcompartmentasaresultofincreasedp27}KIP1_{proteinstability.}

We demonstrate that the mechanism of p27KIP1 _{stabilization relied on inhibition of p27}KIP1

phosphorylationontyrosineresiduesbyc-Abl.Weprovideevidencethatinneuroblastomacelllines

asignificantfractionofcellularc-AblisphosphorylatedonTyr-245,consistentwithanopenandactive

conformation.Notably,exposuretoimatinibdidnotaffectTyr-245phosphorylation.Giventhelow

affinityofactivec-Ablforimatinib,thesedataprovideamolecularexplanationfortherelativelyhigh

imatinibconcentrationsrequiredtoinhibitneuroblastomacellproliferation.

ß2014ElsevierInc.Allrightsreserved.

Abbreviations:CDK, cyclin-dependent kinase; c-Abl, abelson tyrosine-protein kinase1;Bcr-Abl,breakpointclusterregion(Bcr)-Ablfusionprotein;c-Kit,mast/ stem cell growth factor receptor Kit; PDGFR, platelet-derived growth factor receptor;SKP2,S-phasekinase-associatedprotein2;HRP,horseradishperoxidase; pRb,retinoblastomaprotein;PARP,poly-(ADP-ribose)-polymerase;siRNA,short interferingRNA;CHX,cycloheximide;BrdU,5-bromo-20_{-deoxyuridine;df,degree}

offreedom.

* Correspondingauthorat:Universita` diTorino,DipartimentodiOncologia,via MichelangeloBuonarroti27/B,10126Torino,Italy.Tel.:+390116705303; fax:+390116705310.

E-mailaddress:marco.piccinini@unito.it(M.Piccinini).

ContentslistsavailableatScienceDirect

Biochemical

Pharmacology

j our na l ho me p a ge : w ww . e l se v i e r . com / l oc a te / b i och e mph a rm

http://dx.doi.org/10.1016/j.bcp.2014.09.016

p27KIP1 _{which, by inserting into the catalytic cleft of CDK2,}

competeswithATPbinding[6].Upontyrosinephosphorylationby Abl-orSrc-familynon-receptortyrosinekinases,theinhibitory310

helix is ejected from the ATP-binding site of CDK2, partially

restoringCDK2activity.ThiseventwouldallowcyclinE-CDK2to phosphorylateThr-187leadingtop27KIP1_{proteolyticdegradation}

andcompleteCDK2activation[4,5].c-Ablisa130kDaprotein

localized to the plasma membrane, cytosol and nucleus. The

activityofc-Ablistightlyregulated.Inactivec-Ablisinaclosed auto-inhibitedconformationthatinvolvesseveralintra-molecular interactions.Uponphosphorylationbyupstreamsignalingkinases

orauto-phosphorylationevents,c-Ablundergoesconformational

changes resulting in extensive domain rearrangements and

increasedcatalyticactivity[7,8].Tyr-412andTyr-245aremajor sitesofregulatorycontrolinc-Abl.PhosphorylationonTyr-245,by

disrupting the intra-molecular engagement between the Src

homology (SH)3 domain and the SH2-kinase linker domain of

theauto-inhibitedstructure,stabilizesc-Ablinanopenandactive conformation[9].Thesmallmoleculeinhibitorimatinib(STI-571)

specifically recognizes the inactive conformation of c-Abl. By

binding with high affinity (IC50 0.2

m

M) to the ATP-binding

pocketofnon-phosphorylatedc-Abl,imatinibpreventsbindingof

ATP to the kinase domain, resulting in c-Abl inactivation.

Transitionintotheactiveconformationgreatlyreducestheaffinity ofc-Ablforimatinib(IC507

m

M)[10,11].

Previousstudieshavedemonstratedthatimatinib,atyrosine kinaseinhibitorwithselectivityforc-Abl,Bcr-Abl,c-KitandPDGFR

[12],exerts antiproliferativeeffects onneuroblastoma celllines

[13,14]. Although neuroblastoma cell lines express c-Kit and PDGFR,theimatinibconcentrationsrequiredtoachievesignificant growthinhibitoryeffects(10

m

M)aresubstantiallyhigherthan thoserequiredforinhibitionofligand-inducedphosphorylationof

wildtype c-Kit andPDGFR(1

m

M),suggestingthat additional

mechanismsareresponsiblefortheantitumoractivityofimatinib onthesecells.Inthepresentstudytheeffectsofimatinibonthe expressionof theCDK inhibitor p27KIP1_{were investigated in a}

panelofneuroblastomacelllines.

2. Materialsandmethods

2.1. Cellcultureanddrugtreatments

The N-mycproto-oncogene protein (MYCN) amplified IMR5

[15]andMYCNsinglecopySJ-N-KP[16],SK-N-AS[17],and SK-N-F1[17]celllineswereusedthroughoutthisstudy.IMR5,SK-N-AS,

andSK-N-F1cellshavethewild-typeandunamplifiedALKgene

[18,19].IMR5andSK-N-F1areofN-type[20,21],SK-N-AScellsare ofS-type[22].IMR5,SK-N-AS,andSK-N-F1celllineswereobtained inOctober2012fromBancaBiologicaeCellFactory,IRCCSAzienda OspedalieraUniversitariaSanMartino–ISTIstitutoNazionaleperla RicercasulCancro(Genova,Italy).TheSJ-N-KPcelllinewasakind giftofProf.Pession(PoliclinicoS.Orsola–Malpighi,Bologna,Italy) andwasreceivedfromhislaboratoryinOctober2012.Cellswere passagedevery2–3daysanddiscardedafter8–10passages.Cells

were maintained in monolayer cultures at 378C in a 5% CO2

humidifiedatmosphere.IMR5andSJ-N-KPcellswereculturedin

RoswellParkMemorialInstitute(RPMI-1640medium)(R-8758,

Sigma, Milan, Italy) whereas SK-N-AS and SK-N-F1 cells were

culturedinDulbecco’smodifiedEagle’smedium(DMEM)(D-5796,

Sigma,Milan,Italy).RPMI-1640andDMEM weresupplemented

with10%(v/v)fetalbovineserum(F-7524,Sigma,Milan,Italy)and 1% (v/v) antibiotic-antimycotic solution (A-5955,Sigma, Milan,

Italy)(completemedium).Imatinibmesylate(ALX-270-492)was

fromAlexis(Florence,Italy)andwassolubilizedinDMSO(drug

vehicle, 41639 Fluka, Milan, Italy) at a final concentration of

10mM,whichwasusedasthestocksolutionforallexperiments.

Final dilutions were made in culture medium. Exponentially

growing neuroblastoma cells (2106) cultured in complete

mediumin6-wellplateswereexposedtoimatinibattheindicated concentrationsfor24h(SJ-N-KP,IMR5,SK-N-F1)or48h

(SK-N-AS). For cycloheximide chase analysis neuroblastoma cells

(2106_{) cultured in complete medium in 6-well plates were}

exposed to 15

m

M imatinib for 24 or 48h, washed with fresh

culturemediumandthentreatedwith25

m

g/mLcycloheximide

(CHX)(C-4859,Sigma,Milan,Italy)addedwithDMSOor15

m

M

imatinib for further 4 or 8h [23]. Whole cell extracts were

preparedat0,4and8handsubjectedtoimmunoblotting. 2.2. BrdUincorporationassays

Theeffectsofimatiniboncellproliferationweretestedusing

5-bromo-20_{-deoxyuridine (BrdU) incorporation assay}

(11669915001,Roche, Milan,Italy).Briefly, neuroblastomacells (2104/well)wereplatedin96wellcultureplatesandculturedin

complete medium. Increasing concentrations of imatinib were

addedfor24h(SJ-N-KP,IMR5,SK-N-F1)or48h(SK-N-AS).BrdU labelwasaddedatafinalconcentrationof10

m

Mforthelast16h.

Cells were treated with fixative/denaturing solution and then

incubated with anti-BrdU-peroxidase conjugate. BrdU

incorpo-rationwasquantifiedaccordingtothemanufacturer’sinstructions usingaGloMaxluminometer(Promega,Milan,Italy).Toevaluate theeffectsofp27KIP1_{genesilencingorexpressionofp27}Y88F/Y89F_or

wildtype(WT)p27KIP1_{on BrdUincorporation,cellstransfected}

withp27KIP1-targetingsiRNAoraplasmidcodingforp27Y88F/Y89F orWTp27KIP1_{wereseededintoa96-wellcultureplate(2}

104

cells/well) and cultured for 24h in complete medium before

additionofBrdUlabelatafinalconcentrationof10

m

Mfor16h.

BrdUincorporationwasquantifiedasdescribedabove.

2.3. Proteinextraction,immunoblottingandimmunoprecipitation Polyclonalantibodiestop27KIP1_{(sc-528),CDK4(sc-260),CDK6}

(sc-177),poly-(ADP-ribose)-polymerase(PARP)(sc-7150),cyclinE (sc-481),cyclinD1(sc-753),monoclonalantibodiestoCDK2

(sc-6248), cyclinA (sc-56300),pan-phosphotyrosine(sc-7020),and

horseradish peroxidise (HRP)-conjugated secondary antibodies

were from Santa Cruz Biotechnology (Heidelberg, Germany).

Polyclonal antibodies to c-Abl (2862), phospho-c-Abl tyrosine

(Tyr)-245(2868)andphospho-pRbserine(Ser)-780(3590)were

fromCellSignaling(Rome,Italy).Polyclonalantibodyto

phospho-pRbthreonine(Thr)-821(9307)wasfromBiomol(Rome,Italy).

Monoclonalantibodyto

b

-actin(A-5441)wasfromSigma(Milan,

Italy). Monoclonal antibody to SKP2 (32-3300) and polyclonal

antibodytophospho-p27KIP1 _{Thr-187(71-7700) werefromLife}

Technologies(Milan,Italy).IMR5,SJ-N-KP,SK-N-AS,andSK-N-F1 cellswerecollectedusingacellscraper,washedwithPBS,counted

manually, lysed with cell extraction buffer (FNN0011, Life

Technologies, Milan, Italy) (30

m

L/1106 _{cells) supplemented}

with1% (v/v) protease inhibitor mixture(P8340, Sigma, Milan,

Italy)and4%(v/v)phosphataseinhibitormixture(P-0044,Sigma, Milan, Italy) and centrifuged at 15,000g for 20min at 48C. ProteinswerequantifiedusingProteinAssayKitII(500-0002, Bio-Rad, Milan, Italy). Proteins (20

m

g/lane)were resolved by

SDS-polyacrylamidegelelectrophoresis(PAGE)on9or12%gelsand

electro-transferred(16v,12h)at48Ctopolyvinylidenefluoride

membranes (PVDF) (IPVH00010, Millipore, Milan, Italy)

equili-bratedinTowbinbuffer[24].Membraneswereblockedin5%(w/v) BSA(sc-2323,SantaCruzBiotechnology,Heidelberg,Germany)in

20mMTrispH7.6,140mMNaCl,0.02%(v/v)Tween-20(blocking

buffer)andprobedwiththeindicatedprimaryantibodiesdiluted

in blocking buffer. The commercial antibodies were used at

employedat a dilution of 1:20,000. After washing, membranes

wereincubatedwithHRP-conjugatedsecondaryantibodies

dilut-edin blocking buffer.Immunoreactive bands wererevealedby

enhanced chemiluminscence (ECL) (Millipore, Milan, Italy) and

visualizedusing G:Box Chemi-XT CCD gel-imaging system and

GeneSnapimageacquisitionsoftware(SynGene,Cambridge,UK).

ImmunoreactivebandswerequantitatedusingGeneToolsoftware

(SynGene,Cambridge,UK).Immunoprecipitationofp27KIP1_or

c-Ablfromwholecellextractswerecarried-outusingtheindicated

antibodies conjugated to Dynabeads protein-A (100.01D, Life

Technologies,Milan,Italy)accordingtomanufacturer’s

specifica-tions. Dynabeads-IgG conjugates were combined with 100

m

L

wholecellextractcontaining500

m

gprotein(DMSOtreatedcells

or cells transfected withnon-targeting controlsiRNA or empty

Fig.1.ImatinibinhibitsBrdUincorporationandpromotestheupregulationofp27KIP1

inthenuclearcompartment.

Neuroblastomacellswereincubatedfor24h(IMR5,SJ-N-KP,SK-N-F1)or48h(SK-N-AS)intheabsence(DMSO)orpresenceofimatinib(Im)attheindicatedconcentrations.(A) EvaluationofBrdUincorporation.ValuesaremeansSDofthreeindependentexperimentsandareexpressedaspercentofDMSO(control).Absolutechemiluminescencevaluesof controls:1.9106_(IMR5),1.5

106_{(SJ-N-KP),2.1}

106_{(SK-N-AS),2.9}

106_{(SK-N-F1).(B)Immunoblottinganalysisusinganti-p27}KIP1_{antibody.Histogramsrepresentp27}KIP1

relativebandintensityquantifiedbydensitometricanalysisandnormalizedusingb-actinbandsasreference.Bandintensityincellsexposedto15mMimatinibissetto1(control). ValuesaremeansSDoffiveindependentexperiments.Onerepresentativeimmunoblotisshown.(C)Immunofluorescencemicroscopyanalysis.Permeabilizedneuroblastomacells werestainedwithantip27KIP1

antibody(red).NucleiwerestainedwithHoechst-33258(blue).Mergedcolorimagesareshowninthelastcolumn.Representativeresultsofthree independentexperimentsareshown.Statisticalsignificance:*

P<0.05,** P<0.01,*** P<0.001(versusDMSO),df=4;# P<0.05,## P<0.01,### P<0.001(versus15mMimatinib),df=8.

plasmid) or 150

m

g protein (imatinib treated cells or cells

transfectedwith siRNAs or plasmid DNA) andincubated with

rotationfor1hat 48C.Supernatantwasremoved,beadswere

washedtwicewith500

m

LPBScontaining0.02%(v/v)Tween-20,

andproteinswereelutedwith30

m

L0.1McitratepH3.1. Fol-lowingneutralizationwith20

m

L1MTrispH9,elutedproteins

were mixed 1:1 with Laemmli sample buffer and subjected

toimmunoblotanalysis.Toverifythespecificityof

phospho-c-Abl Tyr-245antibody, c-Abl immunoprecipitateswere treated

for2hat 378Cwith10Ualkalinephosphatase (AP+)(P-4252,

Sigma,Milan,Italy)orvehicle (AP), mixed1:1with Laemmli

sample buffer, heated at 958C for 5min, before immunoblot

analysis.

2.4. Immunofluorescencemicroscopy

Cells (1105_{)werefixedin 4% (w/v) paraformaldehydein}

PBS for 30min, washed with PBS, permeabilized with PBS

containing0.1%(v/v)TritonX-100and15mMNaN3(PBS-Triton

buffer),andblockedinPBS-Tritonbuffercontaining2%(w/v)BSA for1h.Cellswereincubatedovernightat48Cwiththeprimary

antibody (sc-528) diluted 1:500 in PBS-Triton buffer, washed

with PBS-Triton buffer, and incubated for 1h with

Cy3-conjugatedgoatanti-rabbitsecondaryantibody(AP132C,

Milli-pore, Milan,Italy) diluted 1:1000 in PBS-Triton buffer. Nuclei

were stained for 20min in the dark with Hoechst-33258

(861405, Sigma, Milan, Italy) to a final concentration of

500ng/mL. Images were acquired using a Leica DMI 4000 B

invertedmicroscope(Milan.Italy). 2.5. RNAinterference

siRNAstargetingthehumanformsofCDK2orp27KIP1_orc-Abl

and control nontargeting siRNA (1027280, AllStar negative

control) were purchased from Qiagen, Milan, Italy. Two CDK2

targetingsiRNAs(SI00299775;SI02654638),twop27KIP1_targeting

siRNAs(SI02621990;SI02621997)andfourc-AbltargetingsiRNAs

(SI00288316;SI00288323;SI00299082;SI00299089)weretested

individually.Neuroblastomacells(2106_{)weretransfectedina}

volumeof100

m

Lwith3pmolsiRNAusingAmaxaNucleofector

device(Lonza,Rome,Italy).NucleofectorkitVandprogramC-005

wereused for IMR5 and SJ-N-KP cells; nucleofector kit V and

programX-005wereusedforSK-N-F1cells;nucleofectorkitTand

programB-013wereusedforSK-N-AScells.Following

transfec-tion,cells were cultured for 24h before further treatments or expressionanalysis.

2.6. Real-timePCR

TotalRNAwasextractedfrom2106_{cells,andconvertedto}

cDNAaspreviouslydescribed[24].Primersweredesignedusing

Primer3 (http://frodo.wi.mit.edu) and synthesized by MWG

(Martinsried,Germany).SKP2mRNA(NM_005983.3)was

ampli-fiedfrom749to962withprimersforward50

-catttcagcccttttcgtgt-30_andreverse50_{-gggcaaattcagagaatcca-3}0_{.CCNA2(cyclinA)mRNA}

(NM_001237.3)wasamplified from1366to1587withprimers

forward50_{-ttattgctggagctgccttt-3}0_andreverse50

-ctggtgggttgagga-gagaa-30_{.CDKN1B (p27}KIP1_{)(NM_004064.3)wasamplifiedfrom}

836to1008withprimersforward50_{-ccggctaactctgaggacac-3}0_and

reverse50_{-ggggaaccgtctgaaacatt-3}0_{.ACTB(betacytoskeletalactin)}

mRNA (NM_001101.3) was amplified from 698 to 969 with

primersforward 50_{-agcgggaaatcgtgcgtgacatta-3}0 _{and reverse 5}0

-ggcgtacaggtctttgcggatgtc-30_{. Relative transcript abundance was}

calculatedbyCFXManagersoftware(Bio-Rad,Milan,Italy)using

the

DD

Ct methodwithACTBasthereferencegene.Allsamples

wereassayedintriplicate.

2.7. Cloningandsite-directedmutagenesis

Plasmids encoding human full-length pRb (IMAGE ID:

5267622)andp27KIP1_{(IMAGEID:3458141)wereobtainedfrom}

Source Bioscience(Nottingham,UK) andpurified frombacterial

culturesusingaPlasmidMidiKit(12143,Qiagen,Milan,Italy).The

human p27KIP1 _{coding sequence was amplified using Pfu}

thermostableDNApolymerase(600670-51,Agilent,Milan,Italy)

inthepresenceoftheforwardprimer50

-caccatgtcaaacgtgcgagtgtc-taac-30_{, in which the 5}0_-cacc-30 _{sequence comprising the}

topo-isomerase I recognition site was fused to the sequence

corresponding to positions 473–496 of human p27KIP1 _mRNA

(NM_004064.3),andthereverseprimer50

-ttacgtttgacgtcttctgaggc-cag-30 _{correspondingtopositions1044–1069.Asequencecoding}

foratruncatedformofhumanpRb(pRb

D

882)comprisingamino

acids 373–882 was amplified using Pfu thermostable DNA

polymeraseinthepresenceoftheforwardprimer50

-caccatgactc-cagttaggactgttatg-30_,inwhichthe50_{-cacc-3’sequencewasfusedto}

thesequencecorrespondingtopositions1283–1303ofhumanpRb

mRNA(NM_000321.2),andthereverseprimer50

-tcatgatccttcaa-tatcaaagcgtagtt-30 _{corresponding to positions 2787–2812. The}

amplificationproducts weregel-purifiedusingtheQiaquick Gel

Extraction Kit (28704, Qiagen, Milan, Italy), subcloned into

pcDNA3.1 directional TOPO expression vector (K4900-01, Life

Technologies,Milan,Italy),andtransformedintoTop10chemically

Fig.2.Knockdownofp27KIP1

impairstheantiproliferativeeffectofimatinib. Neuroblastoma cells were transfected with p27KIP1

-targetingsiRNAs or non-targetingcontrolsiRNA(scramble).After24hfromtransfection,cellsweretreated with15mMimatinibforadditional24h(IMR5,SJ-N-KP,SK-N-F1)or48h (SK-N-AS).(A)Evaluationofp27KIP1_{expression byimmunoblotanalysis.Histograms}

representp27KIP1_{relativebandintensityquantifiedbydensitometricanalysisand}

normalizedusingb-actinbandsasreference.Bandintensityinscrambleexposedto 15mMimatinibissetto1(control).ValuesaremeansSDofthreeindependent experiments. One representative immunoblot is shown.(B) Evaluation ofBrdU incorporation.Values aremeansSDofthreeindependentexperimentsand are expressedaspercentofBrdUincorporationincellstransfectedwithscrambleand exposedtoDMSO(control).Absolutechemiluminescencevaluesofcontrols:1.2106

(IMR5), 1.1106 _{(SJ-N-KP),1.2}

106 _{(SK-N-AS), 1.2}

106 _{(SK-N-F1).Statistical}

significance: ##

P<0.01, ###

P<0.001 (versusscramble+15mMimatinib), df=4;

***

P<0.001(versusscramble+DMSO),df=4.

Comparableresultswereobtainedwhencellsweretransfectedwithapooloftwo p27KIP1

competent Escherichia coli cells. After isolation of positive

transformants, plasmids were purified from bacterial cultures

using EndoFree Plasmid Kit (12362, Qiagen, Milan, Italy) and

sequencedatMWG.Thepurifiedplasmidsweredissolvedinsterile

TEbuffer(10mMTrispH7.6,1mMEDTA).Plasmidcodingfora

mutantnonphosphorylatableformofp27KIP1_(p27Y88F/Y89F_)was

obtainedusing QuikChangeMultiSite-Directed MutagenesisKit

(200514,Agilent,Milan,Italy)inthepresenceoftheprimer50

-agcttgcccgagttcttcttcagacccccgc-30_{. Automated sequencing}

con-firmed that the expected mutations were introduced into the

sequence. For recombinant protein expression, neuroblastoma

cells(2106)weretransfectedwith3

m

goftheindicatedplasmid oremptyvectorusingAmaxaNucleofectorasdescribedinsection 2.5.

2.8. Caspase3/7activityassay

Cells(2104_{/well)wereseededin100}

_m

_{Lcompletemedium}

intoa black96wellcultureplate,treatedwith15

m

Mimatinib ordrugvehicle(DMSO)for24h(IMR5,SJ-N-KP,SK-N-F1)or48h

Fig.3.Imatinibpromotesp27KIP1

upregulationasaresultofp27KIP1

proteinstabilization.

(A)Neuroblastomacellswereincubatedfor24h(IMR5,SJ-N-KP,SK-N-F1)or48h(SK-N-AS)intheabsence(DMSO)orpresenceof15mMimatinib(Im)andp27KIP1

mRNA levelsevaluatedbyreal-timePCR.Resultsareexpressedasarbitraryunits.p27KIP1

expressioninDMSO-treatedIMR5cellsissetto1(control).ValuesaremeansSDofthree independentexperiments.(B)Neuroblastomacellswereexposedto15mMimatinibasdescribedin(A),washedwithfreshculturemedium,addedwithCHX+DMSOor CHX+15mMimatinibandfurtherincubatedfortheindicatedtimes.Wholecellextractswerepreparedandanalyzedbyimmunoblotanalysisusingap27KIP1

specificantibody. Histogramsrepresentp27KIP1_{relativebandintensityquantifiedbydensitometricanalysisandnormalizedusingb-actinbandsasreference.Theintensityofthebandsattime0isset}

to1(control).ValuesaremeansSDofthreeindependentexperiments.Onerepresentativeimmunoblotisshown.Statisticalsignificance:##_P<0.01,###_{P<0.001(versusIMR5}

cells),df=4;*

P<0.05,**

P<0.01,***

(SK-N-AS)andculturedat378Cfor24hina5%CO2humidified

atmosphere.Aspositive control, cells weretreated with20nM

epoxomicin(EPX) (sc-201298, SantaCruz,Heidelberg, Germany)

for24h(IMR5,SJ-N-KP,SK-N-F1)or48h(SK-N-AS)[25].Atthe

endofincubation100

m

LofApo-ONEHomogeneousCaspase-3/7

Reagent(Promega,Milan,Italy)wasaddedineachwell.Theplate

was mixed for 5min on a plate shaker at 300–500rpm and

incubate at room temperature for 10h. Fluorescence was

measured in each well at 485nm (excitation) and 530nm

(emission) using a Fluoroskan Ascent-Thermo microplate

fluo-rometer(Thermo-FisherScientific,Milan,Italy). 2.9. Statisticalanalysis

Student’s t-test was used for statistical comparison of

differences.P<0.05wasconsideredsignificant.

Fig.4.Effectsofimatinibonp27KIP1

phosphorylationontyrosineresiduesandThr-187.

Neuroblastomacellswereincubatedfor24h(IMR5,SJ-N-KP,SK-N-F1)or48h(SK-N-AS)intheabsence(DMSO)orpresenceofimatinib(Im)attheindicatedconcentrations. (A)Evaluationofp27KIP1_{phosphorylation.p27}KIP1_{wasimmunoprecipitatedwithanti-p27}KIP1_{antibodyandblottedwiththeindicatedantibodies.(B)EvaluationofpRb}

phosphorylationstatus.Wholecellextractswereanalyzedbyimmunoblotanalysisusingantiphospho-pRbThr-821antibody.HistogramsinpanelsAandBrepresent relativebandintensityoftheindicatedproteinsquantifiedbydensitometricanalysisandnormalizedusingp27KIP1

(A)orb-actin(B)bandsasreference.Bandintensityin DMSO-treatedcellsissetto1(control).ValuesaremeansSDofthreeindependentexperiments.Onerepresentativeimmunoblotisshown.Statisticalsignificance:*

P<0.05,

**

P<0.01,***

3. Results

3.1. ImatinibinhibitsBrdUincorporationandpromotesthe upregulationofp27KIP1_{inthenuclearcompartment}

ExposureofIMR5,SJ-N-KP,SK-N-F1andSK-N-AS

neuroblasto-macelllinesto1–15

m

Mimatinib,resultedinadosedependent

inhibition of BrdU incorporation into newly synthesized DNA

(Fig.1A).A24hperiodofdrugexposurewasusedforIMR5, SJ-N-KPandSK-N-F1 cells. Atvariance, exposureof SK-N-AScells to

imatinib was prolonged to 48h to reach an inhibitory effect

comparablewiththatoftheotherthreecelllines.Noactivationof caspase-3/7orPARPdegradationwasdetectedinimatinibtreated cells(notshown),thusexcludingapoptosisasthecauseofreduced

BrdU incorporation. Taken together, these results suggest an

impairment in G1 to S phase transition. No changes in the

expressionofcyclinEandCDK2orcyclinD1andCDK4/6which

regulateSphaseentry[26]wererevealedinimatinibtreatedcells

(not shown). At variance, a dose dependent increase in the

expressionof theCDK inhibitor p27KIP1_{was detected (Fig. 1B).}

Fluorescence microscopy analysis provided evidenceof nuclear

localizationofp27KIP1_{inimatinibtreatedcells(Fig.1C).Tofurther}

Fig.5.CDK2knockdownrevealsconstitutivephosphorylationofp27KIP1

ontyrosineresidues.

NeuroblastomacellsweretransfectedwithCDK2-targetingsiRNAsornon-targetingcontrolsiRNA(scramble)andculturedforadditional24h.(A)Immunoblotanalysiswith anti-CDK2oranti-p27KIP1

specificantibodies.(B)Immunoprecipitationwithanti-p27KIP1

antibodyfollowedbyimmunoblottingwiththeindicatedantibodies.Histograms representrelativebandintensityoftheindicatedproteinsquantifiedbydensitometricanalysisandnormalizedusingb-actin(A)orp27KIP1

(B)bandsasreference.Band intensityinscrambleissetto1(control).ValuesaremeansSDofthreeindependentexperiments.Onerepresentativeimmunoblotisshown.Statisticalsignificance:***_P<0.001

(versusscramble),df=4.

Fig.6.Effectsofc-Ablknockdownonp27KIP1

expressionandphosphorylationstatus.

Neuroblastomacellsweretransfectedwithc-Abl-targetingsiRNAsornon-targetingcontrolsiRNA(scramble)andculturedforadditional24h.(A)Immunoblotanalysiswith anti-c-Abloranti-p27KIP1_{antibodies.Histogramsrepresentc-Ablandp27}KIP1_{relativebandintensityquantifiedbydensitometricanalysisandnormalizedusingtheb-actin}

bandsasreference.Bandintensityinscrambleissetto1(control).ValuesaremeansSDoffiveindependentexperiments.(B)Real-timePCRevaluationofp27KIP1_mRNAlevels.

Resultsareexpressedasarbitraryunits.ExpressioninIMR5cellstransfectedwithscramblesiRNAissetto1(control).ValuesaremeansSDofthreeindependentexperiments.(C) Immunoprecipitationwithanti-p27KIP1

antibodyfollowedbyimmunoblottingwiththeindicatedantibodies.Equalproteinloadingwasverifiedbystrippingandreprobingeachblot withanantibodytop27KIP1

.Onerepresentativeimmunoblotofthreeindependentexperimentsisshown.(D)Evaluationoftheinteractionbetweenp27KIP1

andc-Abl.p27KIP1

was immunoprecipitatedwithanti-p27KIP1

investigate the dependence of the antiproliferative effect of imatinibonp27KIP1,theexpressionofp27KIP1wasdownregulated

by siRNA-mediated gene silencing (Fig. 2A). Noteworthy, in

neuroblastomacells depleted ofp27KIP1_{theinhibitory effectof}

15

m

Mimatinibon BrdUincorporation didnotreach statistical significance,thusexcludingadirectinhibitoryeffectofimatinibon

BrdU incorporation (Fig. 2B). To determine the mechanism of

p27KIP1_{upregulation,mRNAexpressionandproteinstabilitywere}

investigated. In all cell lines, no significant changes in p27KIP1

mRNA levels were detected by real-time PCR assays between

DMSOandimatinibtreatedcells(Fig.3A).Incontrast,

cyclohexi-mide chase analysis evidenced an increased p27KIP1 _protein

stabilityin cells exposed to imatinib(Fig. 3B). Taken together, these results support the conclusion that the antiproliferative

effect of imatinib on neuroblastoma cells relies on p27KIP1

upregulation in the nuclear compartment caused by increased

p27KIP1_{proteinstability.}

3.2. Exposuretoimatinibdecreasesp27KIP1_{phosphorylationon}

Thr-187andtyrosineresidues

CyclinE-CDK2-dependentphosphorylationatThr-187playsa

wellestablishedroleintheregulationofp27KIP1_{proteinstability} [27].Moreover,recentresultssuggestthatp27KIP1

phosphoryla-tiononThr-187isprimedbyphosphorylationontyrosineresidues bynon-receptortyrosinekinases[4].Tostudythis,p27KIP1_was

immunoprecipitatedfromneuroblastomacells exposedto10or

15

m

Mimatinibordrugvehicleandblottedusinganti phospho-p27KIP1_{Thr-187orantiphospho-tyrosinespecificantibodies.As}

showninFig.4A,adosedependentdecreaseinp27KIP1

phospho-Thr-187 and phospho-tyrosine content was detected in cells

exposedtoimatinib.Moreover,inagreementwiththenotionthat bindingofp27KIP1_{tocyclin-CDK2isinverselyrelatedtop27}KIP1

tyrosinephosphorylation[28],adosedependentincreaseinthe

amount of CDK2 was observed in p27KIP1 _{immunoprecipitates}

fromimatinibtreatedcells.Collectively,theseresultsare

consis-tent with a model in which imatinib by inhibiting p27KIP1

phosphorylationontyrosineresiduesrestoresp27KIP1_inhibitory

activityonCDK2,resultingindecreasedThr-187phosphorylation. Inagreementwiththishypothesis,inneuroblastomacellsexposed toimatinib,thephosphorylationofpRbattheCDK2specific Thr-821[29]wasconsistentlyreduced(Fig.4B).

3.3. Inneuroblastomacellsp27KIP1isconstitutivelyphosphorylated ontyrosineresidues

Toinvestigatethemechanismofp27KIP1tyrosine phosphory-lation,neuroblastomacellsweretransfectedwithCDK2-targeting siRNAs,inordertosuppressThr-187phosphorylation.Asshownin

Fig.5A, depletion ofCDK2resulted in theupregulationp27KIP1

whichcontainedagreatlydecreased amountofphosphorylated

Thr-187butunchangedlevelsofphosphotyrosine(Fig.5B).Taken together,theseresultsareconsistentwiththeconclusionthatin neuroblastomacells p27KIP1_{is constitutivelyphosphorylatedon}

tyrosineresiduesand indicatethatp27KIP1_tyrosine

phosphory-lationisindependentofThr-187phosphorylation.

3.4. Silencingofc-Ablrecapitulatestheeffectsofimatinibonp27KIP1

expressionlevels

Toinvestigate thepotentialinvolvement of c-Abl in p27KIP1

tyrosinephosphorylation, neuroblastoma cells weretransfected

withc-Abl-targetingsiRNAs.AsshowninFig.6AandB,depletion ofc-Ablresultedintheupregulationofp27KIP1intheabsenceof significantchangesinp27KIP1_{mRNAlevels.Importantly,inp27}KIP1

immunoprecipitates virtually no phosphotyrosine or

phospho-Thr-187 was detected, recapitulating the effects of imatinib

(Fig.6C).Takentogether,theseresultshighlightasofarunknown roleforc-Ablintheregulationofp27KIP1_{tyrosinephosphorylation}

invivoandareconsistentwiththenotionthatphosphorylationon tyrosineresiduesprimesp27KIP1_{forThr-187phosphorylation.In}

addition,thesedatasupporttheconclusionthatinneuroblastoma

cells exposed to imatinib p27KIP1 _{stabilization results from}

inhibition ofc-Abl-dependent phosphorylationon tyrosine

resi-dues. Of note, analysis of p27KIP1 _{immunoprecipitates from}

untransfected DMSO-treated neuroblastoma cells with anti

c-Ablantibody,evidencedsignificantamountsofc-Abl,indicatinga directinteractionbetweenp27KIP1_{andc-Abl(Fig.6D).}

3.5. Expressionofp27Y88F/Y89Fbutnotwildtypep27KIP1recapitulates theeffectsofimatinibonBrdUincorporation

Tyr-88andTyr-89aremajorphospho-acceptorsitesofp27KIP1

targetedbyc-Abl [4,5].Tofurthertest theeffectsofthelossof p27KIP1_{phosphorylationontyrosineresidues,neuroblastomacells}

were transfected with a plasmid coding for a mutant form of

p27KIP1_(p27Y88F/Y89F_{)whichcannotbephosphorylatedonTyr-88}

andTyr-89orwildtypep27KIP1_{.Transfectionwithp27}Y88F/Y89F_but

notwildtypep27KIP1_{,resultedintheexpressionofastableprotein}

(Fig.7A)whichwasdevoidofphosphorylatedThr-187(Fig.7B)and

recapitulated the effects of imatinib on BrdU incorporation

(Fig. 7C). In addition, expression of p27Y88F/Y89F _{recapitulated}

theeffectsofimatinibonpRbphosphorylationatThr-821(Fig.7D).

These data support theconclusion that in neuroblastoma cells

tyrosinephosphorylationnegativelyregulatesp27KIP1_stabilityand

p27KIP1_{inhibitoryactivityonCDK2andthatsuppressionofp27}KIP1

phosphorylation on tyrosine residues exerts growth inhibitory

effectsonneuroblastomacells.

3.6. Inneuroblastomacellsc-Ablisconstitutivelyphosphorylatedon Tyr-245

Toinvestigatec-Ablactivationstatus,c-Ablwas immunopre-cipitatedfromcellextractsofexponentiallygrowing

neuroblasto-macellsandimmunoblottedusinganantibodytophospho-c-Abl

Tyr-245.AsshowninFig.8AandB,Tyr-245phosphorylatedc-Abl wasdetectedinallcelllines,suggestingthatasignificantfraction ofcellularc-Ablisinanopenandactiveconformation.Thehighest

level ofTyr-245phosphorylatedc-AblwasdetectedinSK-N-AS

cells.Exposuretoimatinibdidnotaffectc-Ablphosphorylationon Tyr-245inanyofthecelllinesexamined(Fig.8C).

3.7. ExposuretoimatinibdownregulatestheexpressionofSKP2and cyclinA

Lossofphosphorylationontyrosineresiduesconvertsp27KIP1

fromaboundnon-inhibitortoaboundinhibitorofcyclinD-CDK4 complexes[5].ToevaluatetheeffectsofimatinibonCDK4activity,

thephosphorylation statusof pRbat theCDK4-specificSer-780

[30] wasinvestigated. Asshown in Fig.9A, in cells exposedto

10and15

m

MimatinibphosphorylationofSer-780wasreduced,

suggesting a decreased CDK4 activity. Collectively, the above

reported results indicate that in imatinib treated cells pRb

phosphorylation at CDK2- and CDK4-specific sites is reduced.

analysisandnormalizedusingp27KIP1

bandsasreference.BandintensityinIMR5cellsissetto1.ValuesaremeansSDofthreeindependentexperiments.Statisticalsignificance:

***

P<0.001(versusscramble),df=8;§§

P<0.01,§§§

P<0.001(versusIMR5cellstransfectedwithscramblesiRNA),df=4;#

P<0.05,##

P<0.01(versusIMR5cells),df=4. Comparableresultswereobtainedwhencellsweretransfectedwithapooloffourc-Abl-targetingsiRNAsorwheneachsiRNAwasusedindividually.

Fig.7.Effectsoftheexpressionofp27Y88F/Y89F

.

Neuroblastomacellsweretransfectedwithaplasmidcodingforp27Y88F/Y89F_{orwildtype(WT)p27}KIP1_{oremptyplasmid(vector)andculturedforadditional24h.(A)}

Immunoblotanalysisusingantip27KIP1_{antibody.(B)Immunoprecipitationwithanti-p27}KIP1_{antibodyfollowedbyimmunoblottingwithantiphospho-p27}KIP1_Thr-187

antibody.Equalproteinloadingwasverifiedbystrippingandreprobingeachblotwithanantibodytop27KIP1_{.Onerepresentativeimmunoblotofthreeindependent}

experimentsisshown.(C)EvaluationofBrdUincorporation.ValuesaremeansSDofthreeindependentexperimentsandareexpressedaspercentofBrdUincorporationincells transfectedwithvectorandexposedtoDMSO(control).Absolutechemiluminescencevaluesofcontrols:4.7106

(IMR5),2.4106

(SJ-N-KP),1.6106

(SK-N-AS),1.7106

(SK-N-F1).(D)Immunoblotanalysisusingantiphospho-pRbThr-821antibody.HistogramsinpanelsAandDrepresentp27KIP1

(A)andphospho-pRbThr-821(D)bandintensity quantifiedbydensitometricanalysisandnormalizedusingb-actinbandsasreference.Bandintensityinvectorissetto1(control).ValuesaremeansSDofthreeindependent experiments.Onerepresentativeimmunoblotisshown.Statisticalsignificance:*_P<0.05,**_P<0.01,***_{P<0.001(versusvector),df=4;}###_{P<0.001(versusvector+DMSO),df=4.}

Fig.8.Evaluationofc-AblphosphorylationonTyr-245.

(A)c-Ablwasimmunoprecipitatedfromwholecellextractsusingantic-Ablantibodyandimmunoblottedwithantiphospho-c-AblTyr-245antibody.(B)Specificityofanti phospho-c-AblTyr-245antibody.c-Ablimmunoprecipitatesweretreated(AP+)ornot(AP)withalkalinephosphatasefollowedbyimmunoblottingwithanti phospho-c-AblTyr-245antibody.(C)Effectsofimatinibonc-Ablphosphorylation.Neuroblastomacellswereexposedfor24h(IMR5,SJ-N-KP,SK-N-F1)or48h(SK-N-AS)to15mM imatiniborDMSOandc-AblphosphorylationatTyr-245evaluatedasdescribedin(A).HistogramsinpanelsA–Crepresentphospho-c-AblTyr-245bandintensityquantified bydensitometricanalysisandnormalizedusingc-Ablbandsasreference.InhistogramA,bandintensityinIMR5cellsissetto1.InhistogramB,bandintensityinAPissetto 1(control).InhistogramC,bandintensityinDMSOtreatedcellsissetto1(control).ValuesaremeansSDofthreeindependentexperiments.Onerepresentativeimmunoblotis shown.Statisticalsignificance:*

P<0.05,**

P<0.01(versusIMR5cells)df=4;###

Fig.9.EffectsofimatinibonpRbphosphorylationandSKP2andcyclinAexpression.

Neuroblastomacellswereincubatedfor24h(IMR5,SJ-N-KP,SK-N-F1)or48h(SK-N-AS)intheabsence(DMSO)orpresenceofimatinib(Im)attheindicatedconcentrations. (A)Immunoblotanalysisusingantiphospho-pRbSer-780antibody.(BandC)EvaluationofSKP2andcyclinAmRNAandproteinexpressionbyreal-timePCR(B)and immunoblotanalysis(C).HistogramsinpanelsAandCrepresentphospho-pRbSer-780(A)andSKP2andcyclinA(C)bandintensityquantifiedbydensitometricanalysisand normalizedusingb-actinbandsasreference.BandintensityinDMSOtreatedcellsissetto1(control).ValuesaremeansSDofthreeindependentexperiments.One representativeimmunoblotisshown.HistogramsinpanelBrepresentrelativeSKP2andcyclinAmRNAlevels.Resultsareexpressedasarbitraryunits.ExpressioninDMSO-treated cellsissetto1(control).ValuesaremeansSDofthreeindependentexperiments.Statisticalsignificance:*_P<0.05,**_P<0.01,***_{P<0.001(versusDMSOtreatedcells)df=4.}

This led us to hypothesize that in imatinib treated cells E2F activityisdecreased. Totestthishypothesis,theexpressionof

SKP2 and cyclin A, two E2F responsive genes known to be

upregulated in neuroblastoma cells [30,31] was evaluated. As

showninFig.9BandC,incellsexposedto10and15

m

Mimatinib

theexpressionofSKP2andcyclinAwasdecreasedatthemRNA

andproteinlevels.Importantly,incellssubjectedtop27KIP1_gene

silencingtheeffectsofimatinibontheexpressionofSKP2and cyclinAwereprevented,thusexcludingadirectinhibitoryeffect of imatinibonSKP2 andcyclinAexpression (Fig.10Aand B). Expressionofp27Y88F/Y89F_{butnotwildtypep27}KIP1_(notshown)

recapitulated the effects of imatinib on SKP2 and cyclin A

expression(Fig.10CandD).Takentogether,thesedataindicate

that p27KIP1_{-dependent downregulation of E2F-controlled cell}

cycle regulatory genes is an important consequence of the

exposureofneuroblastomacellstoimatinib.

Of note, transfection of neuroblastoma cells with a plasmid

codingforaconstitutivelyactiveformofpRbthatlacksallcyclin D-CDKdockingsitesandallbutonepotentialcyclinE-CDKdocking

sites (pRb

D

882) [32], caused the downregulation of SKP2 and

cyclin A expression, demonstrating that in neuroblastomacells

SKP2 and cyclin A expression can be repressed by active pRb

(Fig.11).

4. Discussion

Inthepresentstudywefoundthatexposureof

neuroblasto-macelllinesto1-15

m

Mimatinibresultedinadosedependent

inhibition of BrdU incorporationinto newly synthesized DNA.

This effect was dependent on the upregulation of the CDK

inhibitor p27KIP1_{in thenuclearcompartmentasdemonstrated}

by siRNA-mediated depletion of p27KIP1, which prevented the

effectsofimatinibonBrdUincorporation.Regulationofp27KIP1

protein levels is mainly achieved by ubiquitin dependent

proteolysis [27,33]; however, increased p27KIP1 mRNA levels

inimatinib-treatedcellshavebeendescribed[34].Real-timePCR assaysruled-outincreasedp27KIP1_mRNAlevelsin

neuroblasto-macellsexposedtoimatinib.Incontrast,cycloheximidechase

assays demonstrated a substantialincrease in p27KIP1 _protein

stability. Taken together,these results support the conclusion

thatp27KIP1_{upregulationinthenuclearcompartmentpromoted}

by increased protein stability mediates the antiproliferative

effects of imatinib onneuroblastoma cells. The lower p27KIP1

geneexpressioninMYCN-amplifiedIMR5cells(Fig.3A)provides an explanation of the apparently less efficientstabilization of p27KIP1_{inthiscellline(Fig.3B).PhosphorylationatThr-187by}

cyclinE-CDK2playsawellestablishedroleintheregulationof p27KIP1 protein stability [3]. In addition, phosphorylation on

tyrosine residues by Abl- or Src-family non-receptor tyrosine

kinases has been demonstrated to prime p27KIP1 _{for Thr-187}

phosphorylation[4].Weprovideevidencethatinneuroblastoma

cellsexposedtoimatinib,thecontentofphospho-Thr-187and

phospho-tyrosine in p27KIP1 _{immunoprecipitates consistently}

decreased.This result supports a modelin whichimatinib by

inhibitingp27KIP1_{tyrosinephosphorylationrestoresthe}

inhibi-tory activity of p27KIP1 _{itself on cyclin E-CDK2 resulting in}

decreased phosphorylationat Thr-187.Totestthishypothesis,

Fig.10.Effectsofsilencingofp27KIP1

orexpressionofp27Y88F/Y89F

onE2Ftarget genes.

(Aand B)Cellsweretransfected with p27KIP1_{-targetingsiRNAsorwith}

non-targetingcontrolsiRNA(scramble).After24hfromtransfection,cellsweretreated with15mMimatinibforadditional24h(SJ-N-KP,IMR5,SK-N-F1)or48h(SK-N-AS) andSKP2andcyclinAmRNAandproteinexpressionevaluatedbyreal-timePCR(A) andimmunoblotanalysis(B).HistogramsinpanelArepresentSKP2andcyclinA relativemRNA levels.Results are expressedas arbitrary units.Expressionin scramble is set to 1 (control). Values are meansSD of three independent experiments.Histogram inpanel BrepresentsSKP2and cyclinAbandintensity quantifiedbydensitometricanalysisandnormalizedusingb-actinbandsasreference. Bandintensityinscrambleissetto1(control).ValuesaremeansSDofthree independentexperiments.Onerepresentativeimmunoblotisshown.(CandD)Cells weretransfectedwithaplasmidcodingforp27Y88F/Y89F

oremptyplasmid(vector). After24hfromtransfection,SKP2andcyclinAmRNAandproteinexpressionwere evaluatedbyreal-timePCR(C)andimmunoblotanalysis(D).HistogramsinpanelC representSKP2andcyclinArelativemRNAlevels.Resultsareexpressedasarbitrary units.Expressioninvectoris setto1(control).ValuesaremeansSDofthree independentexperiments.HistograminpanelDrepresentsSKP2andcyclinAband intensityquantifiedbydensitometricanalysisandnormalizedusingb-actinbandsas

reference.Bandintensityinvectorissetto1(control).ValuesaremeansSDofthree independent experiments. One representative immunoblot is shown. Statistical significance: ** P<0.01, *** P<0.001 (versus scramble), df=4; ## P<0.01, ### P<0.001(versusvector),df=4.

Comparableresultswereobtainedwhencellsweretransfectedwithapooloftwo p27KIP1

CDK2 was depleted from neuroblastoma cells by

siRNA-mediated gene silencing, which resulted in the upregulation

ofendogenous p27KIP1 _{containinga greatly decreased amount}

ofphosphorylatedThr-187butanunchangedphosphotyrosine

level.Theseresultsdemonstratethatinproliferating neuroblas-tomacellsp27KIP1_{is constitutivelyphosphorylatedontyrosine}

residuesandindicatethatp27KIP1_{phosphorylationontyrosine}

residuesdoesnotrequirepriorphosphorylationatThr-187.

Previous studies havedemonstratedthat overexpressionof

c-Ablineukaryotic cellspromotesp27KIP1 _{phosphorylationon}

tyrosine residues[35] therefore, the potential involvement of

c-Ablinp27KIP1_{tyrosinephosphorylationinneuroblastomacell}

lines was investigated. To do this, neuroblastoma cells were

transfectedwithsiRNAstoc-Abl.Depletionofc-Ablresultedin theupregulationofp27KIP1intheabsenceofchangesinp27KIP1

mRNA levels. Strikingly, virtually no phosphotyrosine or

phosphorylatedThr-187wasdetectedinp27KIP1

immunopreci-pitatesfromcellssubjectedtoc-Ablknockdown,recapitulating

the effects of imatinib. Immunoprecipitation experiments

provided evidence of the interaction between c-Abl and

p27KIP1_{,consistentwithadirectmechanismofphosphorylation}

(Fig. 6D). Collectively, these results highlight a previously unknownroleforc-Ablin theregulationofp27KIP1stabilityin

vivo and support the conclusion that inhibition of

c-Abl-dependent phosphorylation of p27KIP1 _{on tyrosine residues is}

themechanismoftheimatinib-mediatedstabilizationofp27KIP1

inneuroblastomacells.Tofurthertesttheeffectsofthelossof

p27KIP1 _{phosphorylation on tyrosine residues, neuroblastoma}

cellsweretransfectedwithaplasmidcodingforamutatedform ofp27KIP1_(p27Y88F/Y89F_{)thatcannotbephosphorylatedon}

Tyr-88 and Tyr-89, the major phospho-acceptor sites of p27KIP1

targetedby c-Abl [4,5].Transfectionwith p27Y88F/Y89F _butnot

with a plasmid coding for wild type p27KIP1, resulted in the

expression of a stable protein which lacked phosphorylated

Thr-187. Moreover, expression of p27Y88F/Y89F _{but not wild}

type p27KIP1, recapitulated the effects of imatinib on BrdU

Fig.11.EffectsoftheexpressionofpRbD882.

NeuroblastomacellsweretransfectedwithpRbD882oremptyplasmid(vector)andSKP2andcyclinAmRNAandproteinexpressionevaluatedbyreal-timePCR(A)and immunoblotanalysis(B).HistogramsinpanelArepresentrelativeSKP2andcyclinAmRNAlevels.Resultsareexpressedasarbitraryunits.Expressioninvectorissetto1 (control).ValuesaremeansSDofthreeindependentexperiments.HistograminpanelBrepresentsSKP2andcyclinAbandintensityquantifiedbydensitometricanalysisand normalizedusingb-actinbandsasreference.Bandintensityinvectorissetto1(control).ValuesaremeansSDofthreeindependentexperiments.Onerepresentativeimmunoblot isshown.Statisticalsignificance:##

P<0.01,###

incorporationandpRbphosphorylationattheCDK2-specificsite Thr-821(Fig.7CandD).Theseresultsprovidefurthersupportfor

theconclusionthatin neuroblastomacells tyrosine

phosphor-ylationnegativelyregulatesthestabilityandinhibitoryactivity ofp27KIP1.Moreover,thesedatademonstratethatinhibitionof

p27KIP1 _{tyrosine phosphorylation exerts growth inhibitory}

effects on neuroblastoma cells. Tyr-245 is a major site of

regulatory control in c-Abl. Phosphorylation on Tyr-245, by

disrupting the intra-molecular engagement between the SH3

domainandtheSH2-kinaselinkerdomainoftheauto-inhibited

structure,stabilizesc-Abl in anopen andactiveconformation

[9]. We found that a significant fraction of cellular c-Abl is

phosphorylatedonTyr-245,consistentwithanopenandactive

conformation of the enzyme. Given the low affinity of

phos-phorylatedc-Ablforimatinib(IC507

m

M) [10,11],thisresult

providesamolecularexplanationfortherelativelyhighimatinib concentrations(10

m

M)requiredtoinhibitneuroblastomacell proliferation,reportedinthisandotherstudies[13,36].

Impor-tantly,exposure toimatinibdidnotaffectTyr-245

phosphory-lation,suggestingthatanimatinibinsensitivetyrosinekinase(s) mediatesc-Ablactivationinneuroblastomacells.Onthebasisof thewellestablishedroleplayedbyc-Srcintheactivationofc-Abl mitogenicfunction[37]andthecapacityofSrc-familykinasesto renderc-Ablresistanttoimatinib[38],aroleforc-Srcinc-Abl

activationin neuroblastomacellscanbeenvisaged. Consistent

with this hypothesis,increased c-Src activity was detected in

neuroblastomacelllines[39].Furtherworkisneededtoclarify thisissue.

In high risk neuroblastoma, deregulated E2F transcriptional

activitycausedbyfunctionalinactivationofthetumorsuppressor pRbresultsinelevatedexpressionofcell-cycleregulatoryproteins,

high proliferation rate and aggressive phenotype [40]. Upon

mitogenic stimulation pRb functional inactivation is promoted

throughsequentialphosphorylationbycyclinD-CDK4/6andcyclin

E-CDK2 complexes and results in the release of active E2F1-3

transcriptionfactorscapableofinducingtheexpressionofgenes encodingproteinscrucialfortheG1toSphasetransition[41,42].In

neuroblastomacellscyclinD1andCDK4overexpressionhasbeen

reportedtoplayaroleinpRbinactivation[30].Inthisstudy,we

showthat in imatinibtreatedcells pRb phosphorylation at the

CDK2-specificThr-821andCDK4-specificSer-780wasdecreased

and in parallel the expression of two E2F responsive genes

previously reported to be upregulated in neuroblastoma cells:

SKP2 and cyclin A [30,31] was downregulated. Importantly,

silencing of p27KIP1 _{prevented the imatinib-dependent}

down-regulationofSKP2andcyclinAwhereasexpressionofp27Y88F/Y89F

recapitulatedit. Collectively,thesedata areconsistentwiththe

conclusion that downregulation of SKP2 and cyclin A is an

important effect of the exposure of neuroblastoma cells to

imatinib.Since SKP2 is reported toact as a cofactor of c-MYC

transcriptionalactivity [43], it is tempting tospeculate that in

imatinib treated cells MYCN transcriptional activity may be

downregulated[31].

The expression by neuroblastoma cells of drug efflux

transportersof theABC transporter familysuch asMDR1[44]

andMRP1[45]raisesthepossibilitythatdrugeffluxisamajor determinantofthereducedsensitivityofneuroblastomacellsto imatinib.However,whenevaluatingthispossibilitythe follow-ingpointsshouldbeconsidered.First,imatinibisnotasubstrate

for MRP1 [46] which in clinical settings [45] and in animal

models [47] has been demonstrated to be a key factor for

multidrugresistancein neuroblastoma. Second, although

ima-tinib isa substrate of MDR1,thereported IC50of imatinibfor

MDR1(3–8

m

M)[48]is1–2loggreaterthantheIC50ofimatinib

for its high affinity targets. Third, while a previous study

demonstratedthatMDR1expressionis considerablygreaterin

SK-N-F1cellsthaninSK-N-AScells[49],theinhibitionofBrdU

incorporation exerted by imatinib in the two cell lines was

comparable (Fig. 1A). For these reasons and because of the

relativelyhighimatinibconcentrationnecessarytoinhibitactive c-Abl [10,11],a major rolefor drug efflux transporters in the resistanceofneuroblastomacellstoimatinibshowninthisstudy

seems unlikely. The longer exposure time to imatinib (48h

insteadof24h)necessarytoinhibitBrdUincorporationin SK-N-AScellstoanextentcomparablewiththatoftheothercelllines

couldbeexplainedbythegreateramountofTyr-245

phosphor-ylatedc-Abldetectedinthesecells(Fig.8A).However,areduced

expressionor activityoftheOCT1transporter whichmediates

imatinibinflux[50]couldalsoexplainthisresult.

In conclusion, in this study we demonstrated that in

neuroblastoma cells active c-Abl constitutively phosphorylates

p27KIP1_{ontyrosineresidues,promotingp27}KIP1_{downregulation}

and uncontrolled cell proliferation (Fig. 12). Moreover, we

provided evidence that the mechanism of the antiproliferative

effectofimatinibonneuroblastomacellsreliesontheinhibitionof p27KIP1tyrosinephosphorylationbyactivec-Abl.Thecollaborative

interaction between c-Abl and CDK2 in controlling p27KIP1

stability, suggests a potential synergistic interaction between

imatinib and CDK2 inhibitors in inhibiting neuroblastoma cell

proliferation. Should this be the case, new treatments for the

controlofminimalresidualdiseasecouldbedeveloped.Further workisneededtotestthishypothesis.

Conflictofinterest

Theauthorsdisclosenopotentialconflictsofinterest. Acknowledgments

This study was funded by Progetti di ricerca finanziati

dall’Universita`’ degli Studi di Torino and Regione Piemonte,

Ricerca Scientifica Applicata project A189 and by the TAKTIC

consortiumFP7-SME-2012-315746-TAKTIC.

References

[1]ChuIM,HengstL,SlingerlandJM.TheCdkinhibitorp27inhumancancer: prognostic potentialandrelevanceto anticancertherapy.NatRevCancer 2008;8:253–67.

[2]LeeJ,KimSS.Thefunctionofp27KIP1duringtumordevelopment.ExpMol Med2009;41:765–71.

[3]BloomJ,PaganoM.Deregulateddegradationofthecdkinhibitorp27and malignanttransformation.SeminCancerBiol2003;13:41–7.

[4]GrimmlerM,WangY,MundT,CilensekZ,KeidelEM,WaddellMB,etal. Cdk-inhibitoryactivityandstabilityofp27Kip1aredirectlyregulatedbyoncogenic tyrosinekinases.Cell2007;128:269–80.

Fig.12.Modeloftheproposedmechanismofp27KIP1

inactivationinneuroblastoma cells.

Activec-Ablphosphorylatesp27KIP1

attyrosineresidues88and89resultingin CDK2partialactivation.FollowingCDK2-dependentphosphorylationonThr-187, p27KIP1

[5]JamesMK,RayA,LeznovaD,BlainSW.Differentialmodificationofp27Kip1 controlsitscyclinD-cdk4inhibitoryactivity.MolCellBiol2008;28:498–510.

[6]RussoAA,JeffreyPD,PattenAK,MassagueJ,PavletichNP.Crystalstructureof thep27Kip1cyclin-dependent-kinaseinhibitorboundtothecyclinA-Cdk2 complex.Nature1996;382:325–31.

[7]BrasherBB,VanEttenRA.c-Ablhashighintrinsictyrosinekinaseactivitythat isstimulatedbymutationoftheSrchomology3domainandby autopho-sphorylationattwodistinctregulatorytyrosines. JBiolChem 2000;275: 35631–37.

[8]NagarB,HantschelO,YoungMA,ScheffzekK,VeachD,BornmannW,etal. Structural basis for the autoinhibition of c-Abl tyrosine kinase. Cell 2003;112:859–71.

[9]HantschelO,Superti-FurgaG.Regulationofthec-AblandBcr-Abltyrosine kinases.NatRevMolCellBiol2004;5:33–44.

[10]NagarB,BornmannWG,PellicenaP,SchindlerT,VeachDR,MillerWT,etal. Crystalstructuresofthekinasedomainofc-Ablincomplexwiththesmall moleculeinhibitorsPD173955andimatinib(STI-571).CancerRes2002;62: 4236–43.

[11]Schindler T,BornmannW,PellicenaP,MillerWT,ClarksonB,Kuriyan J. Structural mechanism for STI-571 inhibition of abelson tyrosine kinase. Science2000;289:1938–42.

[12]DeiningerM,BuchdungerE,DrukerBJ.Thedevelopmentofimatinibasa therapeuticagentforchronicmyeloidleukemia.Blood2005;105:2640–53.

[13]VitaliR,CesiV,NicotraMR,McDowellHP,DonfrancescoA,MannarinoO,etal. c-KitispreferentiallyexpressedinMYCN-amplifiedneuroblastomaandits effect oncell proliferationis inhibitedin vitroby STI-571.IntJ Cancer 2003;106:147–52.

[14]MecoD,RiccardiA,ServideiT,BrueggenJ,GessiM,RiccardiR,etal.Antitumor activity of imatinib mesylate in neuroblastoma xenografts. Cancer Lett 2005;228:211–9.

[15]FischerM,Berthold F.Characterizationofthe geneexpressionprofileof neuroblastomacelllineIMR-5usingserialanalysisofgeneexpression.Cancer Lett2003;190:79–87.

[16]TonelliR,PurgatoS,CamerinC,FronzaR,BolognaF,AlboresiS,etal.Anti-gene peptidenucleicacidspecificallyinhibitsMYCNexpressioninhuman neuro-blastomacellsleadingtocellgrowthinhibitionandapoptosis.MolCancer Ther2005;4:779–86.

[17]ThompsonPM,MarisJM,HogartyMD,SeegerRC,ReynoldsCP,BrodeurGM, etal.HomozygousdeletionofCDKN2A(p16INK4a/p14ARF)butnotwithin 1p36oratothertumorsuppressorlociinneuroblastoma.CancerRes2001;61: 679–86.

[18]DeBrouwerS,DePreterK,KumpsC,ZabrockiP,PorcuM,WesterhoutEM,etal. Meta-analysisofneuroblastomasrevealsaskewedALKmutationspectrumin tumorswithMYCNamplification.ClinCancerRes2010;16:4353–62.

[19]George RE,SandaT,HannaM,FrohlingS, Luther2ndW,ZhangJ, etal. ActivatingmutationsinALKprovideatherapeutictargetinneuroblastoma. Nature2008;455:975–8.

[20]ToniniGP,CasalaroA,CaraA,DiMartinoD.Inducibleexpressionofcalcyclin,a genewith strong homologytoS-100 protein,during neuroblastomacell differentiationanditsprevalentexpressioninSchwann-likecelllines.Cancer Res1991;51:1733–7.

[21]VoigtA,ZintlF.Effectsofretinoicacidonproliferation,apoptosis,cytotoxicity, migration,andinvasionofneuroblastomacells.MedPediatrOncol2003;40: 205–13.

[22]BianX,GiordanoTD,LinHJ,SolomonG,CastleVP,OpipariJrAW. Chemother-apy-inducedapoptosisofS-typeneuroblastomacellsrequirescaspase-9andis augmentedbyCD95/Fasstimulation.JBiolChem2004;279:4663–9.

[23]BuschbeckM,HofbauerS,DiCroceL,KeriG,UllrichA.Abl-kinase-sensitive levelsofERK5anditsintrinsicbasalactivitycontributetoleukaemiacell survival.EMBORep2005;6:63–9.

[24]Lupino E,RamondettiC, PiccininiM.IkappaBkinase betaisrequiredfor activationofNF-kappaBandAP-1inCD3/CD28-stimulatedprimaryCD4(+) Tcells.JImmunol2012;188:2545–55.

[25]ChengB,MaffiSK,MartinezAA,AcostaYP,MoralesLD,RobertsJL.Insulin-like growthfactor-Imediatesneuroprotectioninproteasomeinhibition-induced cytotoxicityinSH-SY5Ycells.MolCellNeurosci2011;47:181–90.

[26]SherrCJ,RobertsJM.CDKinhibitors:positiveandnegativeregulatorsof G1-phaseprogression.GenesDev1999;13:1501–12.

[27]CarranoAC,EytanE,HershkoA,PaganoM.SKP2isrequiredfor ubiquitin-mediated degradationof the CDKinhibitor p27. Nat Cell Biol1999;1: 193–9.

[28]ChuI,SunJ,ArnaoutA,KahnH,HannaW,NarodS,etal.p27phosphorylation bySrcregulatesinhibitionofcyclinE-Cdk2.Cell2007;128:281–94.

[29]MolenaarJJ,EbusME,GeertsD,KosterJ,LamersF,ValentijnLJ,etal. Inactiva-tionofCDK2issyntheticallylethaltoMYCNover-expressingcancercells.Proc NatlAcadSciUSA2009;106:12968–73.

[30]MolenaarJJ,EbusME,KosterJ,vanSluisP,vanNoeselCJ,VersteegR,etal. CyclinD1andCDK4activitycontributetotheundifferentiatedphenotypein neuroblastoma.CancerRes2008;68:2599–609.

[31]WestermannF,HenrichKO,WeiJS,LutzW,FischerM,KonigR,etal.HighSkp2 expression characterizeshigh-risk neuroblastomas independent ofMYCN status.ClinCancerRes2007;13:4695–703.

[32]HarbourJW,LuoRX,DeiSantiA,PostigoAA,DeanDC.Cdkphosphorylation triggerssequentialintramolecularinteractionsthatprogressivelyblockRb functionsascellsmovethroughG1.Cell1999;98:859–69.

[33]PaganoM,TamSW,TheodorasAM,Beer-RomeroP,DelSalG,ChauV,etal. Roleoftheubiquitin-proteasomepathwayinregulatingabundanceofthe cyclin-dependentkinaseinhibitorp27.Science1995;269:682–5.

[34]AndreuEJ,LledoE,PochE,IvorraC,AlberoMP,Martinez-ClimentJA,etal. BCR-ABLinducestheexpressionofSkp2throughthePI3Kpathwaytopromote p27Kip1degradationandproliferationofchronicmyelogenousleukemiacells. CancerRes2005;65:3264–72.

[35]KardinalC,DangersM,KardinalA,KochA,BrandtDT,TamuraT,etal.Tyrosine phosphorylationmodulatesbindingpreferencetocyclin-dependentkinases andsubcellularlocalizationofp27Kip1intheacutepromyelocyticleukemia celllineNB4.Blood2006;107:1133–40.

[36]BeppuK,JaboineJ,MerchantMS,MackallCL,ThieleCJ.Effectofimatinib mesylateonneuroblastomatumorigenesisandvascularendothelialgrowth factorexpression.JNatlCancerInst2004;96:46–55.

[37]FurstossO,DoreyK,SimonV,BarilaD,Superti-FurgaG.RocheS.c-Ablisan effectorofSrcforgrowthfactor-inducedc-mycexpressionandDNAsynthesis. EmboJ2002;21:514–24.

[38]TanisKQ,VeachD, DuewelHS,BornmannWG,KoleskeAJ.Twodistinct phosphorylationpathwayshaveadditiveeffectsonAblfamilykinase activa-tion.MolCellBiol2003;23:3884–96.

[39]BolenJB,RosenN,IsraelMA.Increasedpp60c-srctyrosylkinaseactivityin humanneuroblastomasisassociatedwithamino-terminaltyrosine phosphor-ylationofthesrcgeneproduct.ProcNatlAcadSciUSA1985;82:7275–9.

[40]HernandoE,NahleZ,JuanG,Diaz-RodriguezE,AlaminosM,HemannM,etal. Rbinactivationpromotesgenomicinstabilitybyuncouplingcellcycle pro-gressionfrommitoticcontrol.Nature2004;430:797–802.

[41]Weinberg RA. The retinoblastoma protein and cell cycle control. Cell 1995;81:323–30.

[42]LundbergAS,WeinbergRA.Functionalinactivationoftheretinoblastoma proteinrequiressequentialmodificationbyatleasttwodistinctcyclin-cdk complexes.MolCellBiol1998;18:753–61.

[43]vonderLehrN,JohanssonS,WuS,BahramF,CastellA,CetinkayaC,etal.The F-boxproteinSkp2participatesinc-Mycproteosomaldegradationandactsasa cofactorforc-Myc-regulatedtranscription.MolCell2003;11:1189–200.

[44]FlahautM,MeierR,CoulonA,NardouKA,NiggliFK,MartinetD,etal.TheWnt receptorFZD1mediateschemoresistanceinneuroblastomathrough activa-tionoftheWnt/beta-cateninpathway.Oncogene2009;28:2245–56.

[45]HaberM,SmithJ, BordowSB, FlemmingC, CohnSL,London WB,etal. Associationofhigh-levelMRP1expressionwithpoorclinicaloutcomeina largeprospectivestudyofprimaryneuroblastoma.JClinOncol2006;24: 1546–53.

[46]MukaiM,CheXF,FurukawaT,SumizawaT,AokiS,RenXQ,etal.Reversalofthe resistancetoSTI571inhumanchronicmyelogenousleukemiaK562cells. CancerSci2003;94:557–63.

[47]BurkhartCA,WattF,MurrayJ,PajicM,ProkvolitA,XueC,etal.Small-molecule multidrugresistance-associatedprotein1inhibitorreversanincreasesthe therapeuticindex ofchemotherapy in mouse models ofneuroblastoma. CancerRes2009;69:6573–80.

[48]HegedusT,OrfiL,SeprodiA,VaradiA,SarkadiB,KeriG.Interactionoftyrosine kinaseinhibitorswiththehumanmultidrugtransporterproteins,MDR1and MRP1.BiochimBiophysActa2002;1587:318–25.

[49]DijkhuisAJ,DouwesJ,KampsW,SietsmaH,KokJW.Differentialexpressionof sphingolipids in P-glycoprotein or multidrug resistance-related protein 1expressinghumanneuroblastomacelllines.FEBSLett2003;548:28–32.

[50]MilojkovicD,ApperleyJ.Mechanismsofresistancetoimatiniband second-generationtyrosineinhibitorsinchronicmyeloidleukemia.ClinCancerRes 2009;15:7519–27.