ofFebruary16,2013.
Thisinformationiscurrentas
Bcl2SignalingPathways
DendriticCellsbyTargetingYWHAZand
ModulatetheSurvivalandLongevityof
MultipleTumor-AssociatedMicroRNAs
XingranJiang,YongzheCheandRongcunYang
Cao,XueqingZhong,YuemingLi,JiatanSun,QiaofeiLiu,
ShiyueMei,JinzheLiu,JingyiLiu,XiaominSu,Shuisong
SipingMin,XueLiang,MiaomiaoZhang,YuanZhang,
ol.1202282
http://www.jimmunol.org/content/early/2013/01/25/jimmun
publishedonline25January2013JImmunol
Material
Supplementary
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http://www.jimmunol.org/content/suppl/2013/01/25/jimmunol.120228
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TheJournalofImmunology
MultipleTumor-AssociatedMicroRNAsModulatethe
SurvivalandLongevityofDendriticCellsbyTargeting
YWHAZandBcl2SignalingPathways
SipingMin,
,1
XueLiang,
,1
MiaomiaoZhang,YuanZhang,ShiyueMei,JinzheLiu,
JingyiLiu,XiaominSu,ShuisongCao,XueqingZhong,YuemingLi,
?
JiatanSun,
?
QiaofeiLiu,XingranJiang,YongzheChe,andRongcunYang
,?
Tumorsuseawidearrayofimmunosuppressivestrategies,suchasreducingthelongevityandsurvivalofdendriticcells
(DCs),todiminishimmuneresponsesandlimittheeffectofimmunotherapy.Inthisstudy,wefoundthattumorsupregulate
theexpressionofmultiplemicroRNAs(miRNAs),suchasmiR-16-1,miR-22,miR-155,andmiR-503.Thesetumor-associated
miRNAsinfluencedthesurvivalandlongevityofDCsbyaffectingtheexpressionofmultiplemoleculesthatareassociatedwith
apoptoticsignalingpathways.Specifically,miR-22targetedYWHAZtointerruptthePI3K/AktandMAPKsignalingpathways,
andmiR-503downregulatedBcl2expression.TheresultoftheincreasedexpressionofmiR-22andmiR-503inthetumor-
associatedDCswastheirreducedsurvivalandlongevity.Thus,tumor-associatedmiRNAscantargetmultipleintracellular
signalingmoleculestocausetheapoptosisofDCsinthetumorenvironment.UseofmiR-22andmiR-503asinhibitorsmay
thereforerepresentanewstrategytoimproveDC-basedimmunotherapiesagainsttumors.TheJournalofImmunology,
2013,190:000–000.
D
endriticcells(DCs),includingconventionalDCsand
plasmacytoidDCs,areprofessionalAPCsthatarecritical
fortheinductionofadaptiveimmunityandtolerance.
However,matureDCsalsoundergoapoptosisinthedifferenttissue
andorgans,especiallythelymphnode(1).DCapoptosisisan
importanteventthatregulatesthebalancebetweentoleranceand
immunity;manymoleculesparticipateinthisprocess,including
TNF-relatedactivation-inducedcytokine(TRANCE),CD154,
FASL,amyloidpeptide,TRAIL,LPS,typeIIFN,leptin,and
CCR7(reviewedinRef.2).DefectsinDCapoptosiscantrigger
autoimmunity,whereasenvironmentsinwhichtheapoptosisof
DCsoccursareimmunosuppressivebecausetheypromotereg-
ulatoryTcellgenerationandfunctionalimpairmentofDCs.
SeveraldifferentdeathreceptorshavebeenidentifiedonDCs,
includingFas(CD95)andtheTNFandTRAILreceptors.Al-
thoughregulatoryDCshavebeenshowntobeinducedbytumor-
derivedfactors(3),DCapoptosishasalsobeenobservedincan-
cers,andtheprocessparallelstheinductionofimmunosuppres-
sion.AmassivereductioninDCnumbershasbeenobservedin
lymphnodedrainagefromtumors,butnototherlymphnodes,
inmultiplecancers(4–7).Intumorlymphnodes,tumor-derived
TGF-bcaninduceDCapoptosis(8),andregulatoryTcellscan
alsodirectlyinteractwithtumorAg-bearingDCstoinducetheir
apoptosis(9).
TheapoptosisofDCscanberegulatedbyextrinsicandintrin-
sicpathways(10).TheratiosbetweentheantiapoptoticBcl2/
Bcl-x
L
moleculesandtheproapoptoticBax/Bakmolecules
determinethelifespanofdifferentDCsubsets.Alowerratioof
antiapoptoticBcl2/Bcl-x
L
toproapoptoticBax/Bakhasbeen
observedinshorterlivedmDCscomparedwithlongerlived
plasmacytoidDCs(11).TransfectionwithBcl2orBcl-x
L
pro-
longsthesurvivalofmouseprimarymDCsinvitro,anddeletion
ofBcl2acceleratesDCapoptosisinvivo(11).BecauseAKT
downregulationcorrelateswithBcl2downregulationandDC
death,thePI3K/AKTpathwayalsoplaysanimportantroleinthe
DClifespan.Indeed,AKTdeficiencyleadstodefectiveDCac-
tivationandsurvival(12),andinhibitionofPI3Kantagonizes
DCsurvivalmediatedbyCD40,CpG,LPS,TRANCE,TNF
superfamilymember11,andPGE
2
(2,13).Cellsurvivalhasalso
beenfoundtobedependentontheERKpathwayinseveral
cellularmodels,whereastheactivationofp38andJNKpromotes
apoptosis(14).Interestingly,theseapoptoticpathwaysmaybe
regulatedbymicroRNAs(miRNAs),includingmiR-16-1(15),
miR-155(16),miR-21(17),andmiR-451(18).miRNAsare
noncodingsmallRNAsthatregulategeneexpressionandcell
growthanddifferentiation.ItisthoughtthatmiRNAshavea
centralroleinregulatingtheapoptosisofDCs.Inthisstudy,we
demonstratethatthetumor-mediatedmiRNAsmiR-22andmiR-
503affectthesurvivalandlongevityofDCs.Specifically,in
tumorenvironments,thesemiRNAstargettheYWHAZandBcl2
pathwaysandpromoteDCapoptosis.
DepartmentofImmunology,NankaiUniversitySchoolofMedicine,NankaiUni-
versity,Tianjin300071,China;
?
KeyLaboratoryofBioactiveMaterials,Ministryof
Education,NankaiUniversity,Tianjin300071,China;and
?
Tianjin“254”Hospital,
NankaiUniversity,Tianjin300071,China
1
S.M.andX.L.contributedequallytothiswork.
ReceivedforpublicationAugust21,2012.AcceptedforpublicationDecember27,
2012.
ThisworkwassupportedbyNationalScienceFoundationofChinaGrants91029736,
30771967,and30872315,theMinistryofScienceandTechnology(863program,
Grant2008AA02Z129),theNationalThemeProgramofChina(863Program,Grant
2011AA020116),andNationalKeyScientificProgramGrant2011CB964902.
AddresscorrespondenceandreprintrequeststoRongcunYang,DepartmentofIm-
munology,NankaiUniversitySchoolofMedicine,NankaiUniversity,NankaiDistrict,
WeijingRoadNo.94,Tianjin300071,China.E-mailaddress:ryang@nankai.edu.cn
Theonlineversionofthisarticlecontainssupplementalmaterial.
Abbreviationsusedinthisarticle:BMDC,bonemarrow–deriveddendriticcell;DC,
dendriticcell;miRNA,microRNA;moDC,monocyte-deriveddendriticcell;qRT-
PCR,quantitativereal-timePCR;siRNA,smallinterferingRNA;TRANCE,TNF-
relatedactivation-inducedcytokine;UTR,untranslatedregion.
CopyrightC2112013byTheAmericanAssociationofImmunologists,Inc.0022-1767/13/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202282
PublishedJanuary25,2013,doi:10.4049/jimmunol.1202282
atEasternKentuckyUniversityonFebruary16,2013
http://jimmunol.org/
Downloadedfrom
MaterialsandMethods
Mice
Four-tosix-wk-oldfemaleC57BL/6andBALB/cmice(BeijingAnimal
Center)weremaintainedinapathogen-freeanimalfacilityforatleast1wk
priortouse.Experimentswereperformedinaccordancewiththeinstitu-
tionalguidelines.Variouss.c.tumormodels,includingLewislungcarci-
noma(AmericanTypeCultureCollection)andB16melanomainC57BL/6
miceandCT-26coloncarcinoma(AmericanTypeCultureCollection)in
BALB/cmice,wereusedinthisstudy.Thenumberoftumorcellsinjected
s.c.foreachmodelwasdeterminedbasedontheabilityofthecellstoform
atumor1.5cmindiameterwithin3–4wkinjection.
Celllines,humanmonocyte-deriveddendriticcells,and
murinebonemarrow–deriveddendriticcells
ThefollowingcelllineswerepurchasedfromtheAmericanTypeCulture
Collection:murinemonocyte/macrophageRAW264.7,murinemelanoma
B16,murinecoloncarcinomaCT-26,mousefibroblastL,humanlung
carcinomaPG,humanbreastcarcinomaMCF-7,humancervicalcarcinoma
HeLa,humanmonocyteU937,humanembryonickidney293T,andhuman
lungfibroblastWI38.Ovariancarcinoma1D8wasprovidedbyK.F.Ruby
(UniversityofKansasMedicalCenter).Allcellsweremaintainedin
completemedia,whichconsistedofRPMI1640supplementedwith10%
FCSand1%penicillinandstreptomycin.
Humanmonocyte-derivedDCs(moDCs)werepreparedfrommonocytes
aspreviouslydescribed(19).Briefly,afterFicoll-Hypaqueseparation,pe-
ripheralbloodcellswereresuspendedincompletemedia.Thecellswere
incubatedfor1hat37?C,andthenonadherentcellswereremovedbygentle
pipetting.TheadherentcellswereculturedinRPMI1640containing10%
FCS,1000U/mlGM-CSF(R&DSystems),and1000U/mlIL-4(R&D
Systems)for7d.
Murinebonemarrow–deriveddendriticcells(BMDCs)werepreparedas
describedinourpreviouswork(20).Briefly,bonemarrowcellswerecol-
lectedbyremovingthefemursofmice,cuttingoffeachend,andflushing
outthebonemarrowwithRPMI1640usingasyringe.Thepooledcells
wereharvestedbycentrifugationat6003gfor10minandresuspendedin
2mlACKbufferfor5minatroomtemperaturetolyseRBCs.Thecells
werethenwashedincompletemedia,andDCsweregeneratedbyculturing
thecellsinmediacontaining500U/mlGM-CSF(R&DSystems)for7d.
Flowcytometricanalysis
MurineBMDCswerecollectedinice-coldPBSandincubatedwiththe
followinganti-mouseAbs:FITC-,PE-orallophycocyanin-conjugated
anti-mouseCD11c(N418),B220(RA3-6B2),CD80(16-10A1),CD86
(GL1),CD40(5C3),CD11b(M1/70),CD4(L3T4),orCD8a(Ly-2)Abs.
HumanmoDCswerecollectedinice-coldPBSandincubatedwiththe
followinganti-humanAbs:FITC-,PE-orallophycocyanin-conjugated
anti-humanCD11c(3.9),CD86(IT2.2),CD80(2D10.4),CD40(HB14),or
CD11b(ICRF44).AlloftheAbsusedinthestudywerepurchasedfrom
BDBiosciences.ThecellswerestainedandresuspendedinPBSwith1%
paraformaldehydeand1%FCSandkeptat4?Cpriortoflowcytometric
analysis(FACScan;BectonDickson).Isotype-matchedcontrolmAbswere
usedasnegativecontrolsforallanalyses.
Forapoptoticanalysis,thecellswerestainedwithannexinVandpro-
pidiumiodideaccordingtothemanufacturer’sinstructions.Sampleswere
examinedbyFACScan.
RNAisolationandquantitativereal-timePCR
TotalRNAwasextractedusingTRIzolreagent,andreversetranscription
wasperformedwiththeMoloneymurineleukemiavirusreversetran-
scriptaseaccordingtothemanufacturer’sprotocol.Forgeneexpression
analysis,quantitativereal-timePCR(qRT-PCR)wasperformedusingthe
QuantiTectSYBRPCRkitwithaspecificsetofprimersaccordingtothe
manufacturer’sinstructions(Qiagen).FormaturemiRNAs,qRT-PCRwas
performedusingastandardTaqManPCRkitandanAppliedBiosystems
7900HTsequencedetectionsystem.AmplificationofU6smallRNAwas
performedtodetectmaturemiRNAs,andamplificationofGAPDHwas
performedoneachexperimentalsampleasanendogenouscontrol.Fold
changeswerecalculatedusingtheΔΔC
t
methodaccordingtothemanu-
facturer’sprotocol(AppliedBiosystems).Allreactionswererunintrip-
licate.TheprimersequencesusedarelistedinSupplementalTableI.
miRNA,smallinterferingRNA,gene,and39untranslated
regionexpressionconstructs
miRNAmimics,miRNAinhibitors,andcontrololigonucleotideswere
purchasedfromDharmacon.YWHAZandBcl2smallinterferingRNAs
(siRNAs)andthenegativecontrolsiRNAwerepurchasedfromSantaCruz
Biotechnology.BLOCK-iTfluorescentoligonucleotideswerepurchased
fromInvitrogen.The39untranslatedregion(UTR)ofYWHAZandBcl2
wasclonedfrommousespleencellgenomicDNAviaPCR.Primers
containingtherestrictionenzymesXhoIandNotIwereusedsothatthe
sequencescouldbeclonedintothesiCheck-2luciferasereportervector
(Promega).TheYWHAZandBcl239UTRmutantsweregeneratedusing
fourprimersaccordingtoapreviousmethod(21).Mutationswerecon-
firmedbyDNAsequencing.YWHAZandBcl2weredirectlyclonedinto
pcDNA3.1fromtotalspleencellRNAusingthepcDNA3.1TOPOkit.
ThesequencesofthemiRNAswereobtainedfromthemiRbase(http://
microrna.sanger.ac.uk/sequences/).The39UTRsequenceswereobtained
fromtheNationalCenterforBiotechnologyInformation(http://www.ncbi.
nlm.nih.gov/sites/entrez/).Primer3softwarewasusedforprimerdesign
(http://frodo.wi.mit.edu/).TheprimersusedarelistedinSupplemental
TableI.
Transfection
ForthemiRNAmimics,inhibitors,siRNAs,andnegativecontrololi-
gonucleotides,cellsweretransfectedwiththeindicatedoligonucleotides
(100nM)usingtheEntranster-Rsystem(EngreenBiosystem)accordingto
themanufacturer’sinstructions.Forsomeoftheconstructs,thecellswere
transfectedusinganucleofectionapproachaccordingtothemanufacturer’s
protocol(AmaxaBiosystems,Berlin,Germany).miRNAmimics,inhib-
itors,siRNAs,andnegativecontrololigonucleotideswerepurchasedfrom
RiboBio(Guangzhou,China).Silencingofthetargetmoleculeswascon-
firmedbyRT-PCRaftertransfectionfor24h.Theprimersusedinthe
siRNAexperimentsarelistedinSupplementalTableI.
miRNAarray
ExpressionlevelsofmiRNAsin1D8mouseovariantumor–andCT-26
coloncancer–associatedBMDCsorcontrolBMDCswereanalyzedusing
ExiqonmiRNAarrays(Vedbaek,Denmark).TotalRNAwasisolatedusing
TRIzolreagent(InvitrogenLifeTechnologies).Theconcentrationand
purityoftheRNAweredeterminedusingNanoDropND-1000.ThemiR-
NAswerelabeledusingthemiRCURYarraypowerlabelingkit(Exiqon).
miRNAarrayhybridizationwasperformed,andunboundmiRNAlabels
werewashedawayusingthemiRCURYarraywashbufferkit(Exiqon).The
arrayswerescannedonanAxon4000Bscanner(MolecularDevices),and
thesignalintensitywasdeterminedusingGenePixPro6.0software(Mo-
lecularDevices).miRNAswitha1.5-foldincreaseordecreaseinexpression
wereregardedasdifferentiallyexpressed.
Genechiparray
AgenechipanalysiswasperformedonRNAfromuntreatedBMDCsand
BMDCscoculturedwith1D8tumorcellsusingatranswellplate.Total
RNAwasisolatedfromDCsusingTRIzolreagent(Invitrogen)followed
byRNAcleanupwithanRNeasyMinikit(Qiagen).Processingofthe
sampleswasperformedfollowingAffymetrixspecifications.Fluores-
cencewasdetectedusingtheHewlett-PackardG2500GeneArrayscanner,
andimageanalysisofeachgenechipwasperformedwiththeMicroarray
Suite5.0softwarefromAffymetrixusingthestandarddefaultsettings.
Transcriptsthatwereconsistentlysignificantinatleasttwoofthefour
iterativecomparisonswereselectedforthefinalcandidatelist.Any
transcriptthatshowedatleasta2-foldchangeinexpressionlevel(ratios
arepresentedaslog
2
)betweentheexperimentalsampleandthecontrol
samplewasconsideredsignificant.
Two-dimensionalgelelectrophoresis
Two-dimensionalgelelectrophoresiswasperformedbytheTEDASchool
ofBiologicalScienceandBiotechnology,NankaiUniversity.Totalprotein
concentrationsfromRAW264.7cellstransfectedwithdifferentmiRNAsor
controloligonucleotidesweredeterminedaccordingtotheBradfordmethod
(Bio-RadLaboratories,Hercules,CA)followingthemanufacturer’spro-
tocol.Proteinsonthetwo-dimensionalgelswerevisualizedbysilverstaining
forpatterncomparisons,andproteinspotswereselectedforquantitative
analysiswhentheyshowedareduction$2-foldthatofthecontrolsample.
Selectedproteinspotsweredigested,desaltedwithZipTip(Millipore),and
subjectedtoanalysisbyMALDI-TOFMS.Thepeptidemassfingerprintings
obtainedforeachproteinofinterestweresearchedagainsttheNational
CenterforBiotechnologyInformationnonredundantdatabaseusingthe
MASCOTsearchengine(http://www/matrixscience.com).
Luciferasereporters
Cellswereplatedina48-wellplateatadensityof4310
4
cells/wellin
250mlculturemedium24hpriortotransfection;thecellswerethen
2MODULATIONOFDCSURVIVALBYTUMOR-ASSOCIATEDmiRNAs
atEasternKentuckyUniversityonFebruary16,2013
http://jimmunol.org/
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cotransfectedwiththeindicatedexpressionplasmids,thesiCHECK-2re-
porterplasmid,andcontrolplasmids(Promega)usingLipofectaminereagent
(Invitrogen)orNucleofectortechnology(AmaxaBiosystems)according
tothemanufacturer’srecommendations.Renillaandfireflyluciferase
activitiesweremeasured24haftertransfectionusingadualluciferase
kit(Promega).
Westernblotanalysis
Westernblotanalysiswasperformedaccordingtoourpreviousprotocol
(21).HybridizationswithprimaryAbswereperformedfor1hatroom
temperatureinblockingbuffer.Theprotein/Abcomplexesweredetected
usingperoxidase-conjugatedsecondaryAbs(BoehringerMannheim)and
ECL(AmershamBiosciences).Rabbitanti-YWHAZ(Sigma-Aldrich),
anti-Bcl2(CellSignalingTechnology),anti-Bax(Sigma-Aldrich),anti-
AKT(CellSignalingTechnology),anti-FOXO3(Sigma-Aldrich),anti–
phospho-AKT(CellSignalingTechnology),andanti–phospho-FOXO3
(CellSignalingTechnology)wereusedinadditiontothefollowingAbs
purchasedfromBiosBiosynthesisBiotechnology(Beijing,China):anti-
actin,anti-p38,anti-JNK,anti-ERK,anti–phospho-p38,anti–phospho-JNK,
anti–phospho-ERK,andanHRP-labeledgoatpolyclonalAbagainstrabbit
IgG(H+L).
Invitroexperiments
Forinvitropreparationoftumor-associatedDCs,2310
5
murineorhuman
tumorcellswerecoculturedwith2310
6
murineBMDCsorhuman
moDCsina24-welltranswellplateinRPMI1640mediumsupplemented
with3%FCS(LifeTechnologies)and1%penicillinandstreptomycin
(LifeTechnologies)for24h.Forinvitroapoptosisanalysis,murine
BMDCsorhumanmoDCswereharvested,transfected,andthencoincu-
batedwiththemurineorhumantumorcellsinatranswellplatefor3d.
Specifically,2310
6
DCswereplacedinto24-wellplatesin1mlmedium,
and2310
5
tumorcellswereplacedintotheinsertsonthetopofeach
well.Ascontrols,DCswerecoincubatedwithmurineorhumanfibroblasts
ormediumaloneandplacedintheinserts.Thecellswerealsocultured
withTGF-b,TNF-a,orunderconditionsofGM-CSFwithdrawal.Apo-
ptoticcellswereassessedusinganannexinVbindingassaywithpro-
pidiumiodidestainingandanFITC-conjugatedTUNELreactionmixture
thatwasprovidedwithaninsitucelldeathdetectionkit(RocheDiag-
nostics,Indianapolis,IN).
Invivoexperiments
Togeneratethemousetumormodel,B16cells(0.5310
6
in100mlPBS)
wereinoculateds.c.intotheinguinalregionbacksofC57BL/6mice.
Tumorswereallowedtodevelopfor9d,andmicewerethenrandomly
dividedintodifferentexperimentalgroups(sixmicepergroup).
ToanalyzeDCsurvivalinvivo,BMDCsweretransfectedwithdiffer-
entmiRNAs,labeledwithCFSEandinjecteds.c.(1310
6
cells/mouse)
intothefootpadsofsyngeneicC57BL/6micebearingB16tumors.The
drainingpopliteallymphnodeswereharvestedatvarioustimepointsafter
injection.Thetotalnumbersoflymphnodecellswerecounted,andthe
percentageofCFSE
+
DCswasanalyzedbyflowcytometry.Theproportion
ofCFSE
+
DCsinthedraininglymphnodesofeachmousewascalculated.
Theeffectoftumor-associatedmiRNAsonDC-basedimmunotherapy
againsttumorswasassessedaccordingtothepreviouslyreportedprotocol
FIGURE1.TumorcellsinduceDCapoptosis.(AandB)MousetumorcellsinducedtheapoptosisofmurineBMDCs.BMDCswerecoculturedwith
murinemelanomaB16F10cells,coloncarcinomaCT-26cells,ovariancarcinoma1D8cells,RAW264.7monocytes,ormouseLfibroblastcells(mFB)in
atranswellfor72h.B16F10inFACSdataindicatedtheBMDCscoculturedwithB16F10.ThecellswerestainedwithannexinVandpropidiumiodideand
aFITC-conjugatedTUNELreactionmixturethatwasprovidedwithaninsitucelldeathdetectionkit(RocheDiagnostics)accordingtothemanufacturer’s
instructions.Originalmagnification340.SampleswereexaminedbyFACScanandfluorescencemicroscopy,respectively.(C)MouseB16tumorcells
inducedtheapoptosisofBMDCsinvivo.ThenumbersinthegatesindicatetheproportionofCFSE
+
cellsinthedraininglymphnodespooledfromB16
tumormice(sixmice/group)injectedwith1310
6
CFSE-labeledBMDCsattheindicatedtimes.ThemouseB16tumormodelwasgeneratedaccordingto
theprotocoldescribedinMaterialsandMethods.B16,thecellsfromthepooleddraininglymphnodesofmicebearingB16tumors;tumor-free,thecells
fromthedraininglymphnodesoftumor-freemice.(DandE)HumantumorcellsinducedtheapoptosisofhumanmoDCs.HumanmoDCswerecocultured
withhumanbreastcarcinomaMCF-7cells,lungcarcinomaPGcells,cervicalcarcinomaHeLacells,U937monocytes,orWI38humanfibroblastcells
(hFB)inatranswellfor72h.Originalmagnification340.HeLainFACSdataindicatedthemoDCscoculturedwithHeLa.Theresultsshownare
representativeofthreeindependentexperiments.p,0.05.Ctr.,Cellsfrompooleddraininglymphnodesfrommice(sixmice/group)injectedwith13
10
6
unlabeledBMDCs.
TheJournalofImmunology3
atEasternKentuckyUniversityonFebruary16,2013
http://jimmunol.org/
Downloadedfrom
(20).MicebearingB16tumorswererandomlydividedintodifferent
experimentalgroups(sixmicepergroup)andtreatedviaintratumoral
injectionwiththetransducedDCs.GroupstreatedwithPBSonlyor
untreatedDCswereusedascontrols.Thediameteroftheperpendicular
tumorwasmeasuredwithacalipertoassesstumorsize.Miceweresac-
rificedwhentheyexhibitedsignsofdistressorwhenthetotaltumorvol-
umewas.3000mm
3
.
Statisticalanalysis
Datawerepresentedasmean6SDormean6SEM.Statisticalcom-
parisonsweremadebyone-wayANOVAfollowedbytheposthocTukey–
KramermultiplecomparisontestandaStudentttest.Atwo-sidedpvalue
,0.05wasconsideredstatisticallysignificant.
Results
Tumor-associatedmiRNAsregulatethesurvivalandlongevity
ofDCs
Cancerssuchasbreastcancer(22,23),hepatocellularcarcinoma
(24),andmelanoma(25)areassociatedwithDCapoptosis.Inthis
study,whenDCswerecoculturedwithdifferenttypesofmouse
orhumantumors,thenumberofapoptoticanddeadDCssignif-
icantlyincreasedcomparedwithDCsculturedwithonlymedium
orfibroblasts(p,0.05);however,thetumorcellsshoweddif-
ferentabilitiestoinduceapoptosis(Fig.1A,1B).Ininvivoex-
periments,B16tumorsalsopromotedtheapoptosisofBMDCs
ascomparedwithcontrol(mean6SEM,p,0.01),consistent
withotherreports(26)(Fig.1C).Similarly,humanmoDCswere
sensitivetoMCF-7humanbreastcarcinoma,PGlungcarcinoma,
andHeLacervicalcarcinomacell–mediatedapoptosis(Fig.1D,
1E),consistentwithotherreports(27).
ToinvestigatethemiRNAsthatmaybeinvolvedinregulating
thesurvivalandlongevityofDCsintumormicroenvironments,
wefirstanalyzedtheexpressionofmiRNAsintumor-associated
BMDCs.GenoExplorermiRNAarrays(http://www.ncbi.nlm.
nih.gov/geo/query/acc.cgi?acc=GSE42722,GPL16352,National
CenterforBiotechnologyInformationtrackingsystemno.
16691611)showedthatsomemiRNAs,suchasmiR-16-1,miR-
FIGURE2.ExpressionofmiRNAsintumor-associatedmurineBMDCs.(A)miRNAexpressionintumor-associatedBMDCs.BMDCsweregenerated
andcoculturedwiththeCT-26mousecoloncanceror1D8mouseovariancancercelllinesina24-welltranswellplatefor24h.TheexpressionofmiRNAs
inthetumor-associatedBMDCswasanalyzedusingaGenoExplorermiRNAarraychipaccordingtotheprotocoldescribedinMaterialsandMethods;16,
21,22,142-5p,146a,155,and503aremiR-16-1,miR-21,miR-22,miR-142-5p,miR-146a,miR-155,andmiR-503respectively.(B)Tumor-mediated
miRNAexpressionwasfurtherconfirmedinCT-26–andB16-associatedBMDCsbyqRT-PCR.BMDCswerecoculturedwithB16orCT-26cells,and
miRNAexpressionwasdetectedattheindicatedtimesaccordingtotheprotocoldescribedinMaterialsandMethods.(C)ExpressionofmiRNAsinthe
draininglymphnodesfromtheB16andCT-26mousetumormodels.TheDCswereisolatedfromthedraininglymphnodesofCT-26andB16orcontrol
(Ctr.)miceusingDynabeads(Invitrogen)coatedwithananti-mouseCD11cAb(MBLInternational,Woburn,MA)accordingtothemanufacturers’
instructions.TheCT-26andB16mousetumormodelsweregeneratedaccordingtotheprotocoldescribedinMaterialsandMethods.p,0.05,p,
0.01.R.E.,Relativeexpression.
4MODULATIONOFDCSURVIVALBYTUMOR-ASSOCIATEDmiRNAs
atEasternKentuckyUniversityonFebruary16,2013
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21,miR-22,miR-142-5p,miR-146a,miR-155,andmiR-503,
wereupregulatedwhentheBMDCswereculturedwithmouse
CT-26coloncancercellsor1D8ovariancancercells(Fig.2A).
TheseupregulatedmiRNAswerealsodetectedbyqRT-PCRin
BMDCsculturedwithmousetumorB16cells.Notably,except
formiR-22andmiR-142-5p,thekineticsoftheexpression
ofthesemiRNAswasdifferentfromtheBMDCsculturedwith
CT-26cells(Fig.2B),implyingdifferentmechanismsthatare
involvedinthe1D8-andCT-26–mediatedmodulationonmiRNAs.
Importantly,theupregulatedmiRNAswerealsofoundinDCs
isolatedfromthedraininglymphnodesofmicebearingB16and
CT-26tumors(Fig.2C).Significantdifferenceswereapparent
ascomparedwithcontrol(mean6SD,p,0.01).Thus,our
resultssuggestthattheexpressionofmiR-16-1,miR-21,miR-22,
miR-142-5p,miR-146a,miR-155,miR-503,andthepreviously
reportedmiR-17-5pandmiR-20(21)maybeupregulatedbytumor-
associatedfactors.
Althoughthereexistsapossibilityfortherolesofthedown-
regulatedmiRNAsinDCapoptosis,wenextonlyassessedeffects
ofthetumorupregulatedmiRNAsonthetumor-inducedapoptosis
invitrobycoculturingmurineB16tumorcellswithmiRNAsor
theirinhibitor-transfectedBMDCswiththedemonstratedtrans-
fection(notshown).Theresultsshowedthattumor-associatedmiR-
22andmiR-503andthepreviouslyreportedmiR-16-1(15)and
miR-155(16)significantlyincreasedthenumberofapoptoticand
deadDCs,whereastheirinhibitorsresistedtumor-inducedapo-
ptosisofDCs(Fig.3Aandnotshown).Becauseothershave
showntheeffectofmiR-16-1andmiR-155onmediatingcellular
apoptosis(15,16),wenextfocusedontheeffectsofmiR-22and
miR-503onmodulatingDCapoptosis.AsshowninFig.3,after
s.c.deliveryofBMDCstransfectedwithmiR-22andmiR-503
inhibitors,thepercentageoflivecellsfromthedraininglymph
nodeofmurineB16tumormodelwassignificantlyhigherthan
thatobtainedfromthelymphnodesofmiceinjectedwithcontrol
DCs(Fig.3B,3C).Thisdisproportionatepercentageoflivecells
wassustainedforatleast5dafterdelivery(Fig.3B,3C).These
datasuggestthatthemiR-22andmiR-503inhibitorsprolongDC
lifespan.
WealsoemployedapoptoticmodelsthataremediatedbyTNF-
a(28),TGF-b(29),orGM-CSFwithdrawal(30)tofurtherinves-
tigatetheeffectsofthetumor-associatedmiRNAsontheapoptosis
ofDCs.miR-22andmiR-503mimicspromotedtheapoptosisof
DCsuponTNF-a,TGF-b,andGM-CSFwithdrawal,whereas
miR-155promotedDCapoptosisonlywhenexposedtoTNF-a
(notshown).miR-16didnothaveaneffectonDCsinthese
apoptoticmodels(notshown),implyingthatmultiplesignaling
pathwaysareinvolvedinthetumor-associatedmiRNAregulation
ofDCapoptosis.
TheYWHAZ-basedapoptoticsignalingpathwaymaybe
directlyorindirectlymodulatedbymultipletumor-associated
miRNAs
GiventhepotentialofmiRNAstoregulatealargenumberof
cellulartranscripts,wedecidedtotakeabroadapproachtoiden-
tifytargetsanddeterminethemechanismsbywhichmiRNAs
acttoregulateDCapoptosis.Theexpressionofintracellular
proteinsinthemonocytecelllineRAW264.7transfectedwith
tumor-associatedmiR-16-1,miR-20a,miR-22,miR-155,ormiR-
FIGURE3.Tumor-associatedmiRNAsinducetheapoptosisofDCs.(A)Tumor-associatedmiR-16-1,miR-22,miR-155,andmiR-503inducethe
apoptosisofBMDCsinvitro.MurineBMDCsweretransfectedwithmiR-16-1,miR-22,miR-155,andmiR-503mimicsortheirinhibitors,andapoptotic
cellsweredetectedbypropidiumiodideandannexinVstainingaftertheBMDCswerecoculturedwithB16cellsinatranswellplatefor3d.(BandC)miR-
22andmiR-503inducetheapoptosisofBMDCsinvivo.ThemicebearingB16tumorswereinjectedwith1310
6
CFSE-labeledBMDCsinthefootpads,
andthedraininglymphnodeswereanalyzedbyFACScanattheindicatedtimes.TheBMDCsweretransfectedwithmiR-22ormiR-503mimicsortheir
inhibitorsaccordingtotheprotocoldescribedinMaterialsandMethods.miR-22MandmiR-503MindicatetheBMDCsthatweretransfectedwithmiR-22
andmiR-503mimics,respectively;miR-22IandmiR-503IindicatetheBMDCsthatweretransfectedwithmiR-22andmiR-503inhibitors,respectively.
M1–M6representthedifferentmiceineachgroup.Thenumbersinthegatesin(C)indicatetheproportionofCFSE
+
cellsinthepooleddraininglymph
nodesofB16tumormice(sixmice/group)injectedwith1310
6
CFSE-labeledBMDCs.Ctr.,thepooleddraininglymphnodesoftumor-freemice(six
mice/group)injectedwith1310
6
labeledBMDCs.p,0.01.
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503wasinvestigatedusingtwo-dimensionalgelsandMALDI-
TOFMS.WefoundthatthesemiRNAscouldindeeddown-
regulatethelevelsofmultipleproteins,suchasYWHAZ,ALB,
HMOX1,LDHA,ANXA2,CCT4,ANXA5,TCP1,CCT2,ANXA1,
EEF2,VIM,PGK1,CCT7,TKT,Cofilin,CAP1,CFL1,P4HB,
TPl1,ALDOA,PGAM1,CS,GLUD1,PKM2,andENO1(Fig.
4,SupplementalFig.1).InadditiontopredictionsofmiRNA-
targetedmolecules,multiplepotentialtargetsofmiR-16-1,miR-
20a,miR-22,miR-155,andmiR-503wereidentified(Fig.4,
SupplementalFig.1,andnotshown).Forexample,miR-22
markedlyreducedtheexpressionofYWHAZ(Fig.4,Sup-
plementalFig.1).SeveralmoleculesrelatedtoYWHAZwere
alsodownregulatedbytumor-associatedmiRNAs,including
ALDOA,GLUD1,andENO1bymiR-16,ANXA2bymiR-20a,
CAP1bymiR-22,ANXA2bymiR-20a,andENO1bymiR-
503(SupplementalFig.1).Thesedatasuggestthatthetumor-
associatedmiRNAsaffectedtheYWHAZ-basedapoptoticsignaling
pathwaybyreducingtheexpressionofYWHAZ-associatedmol-
ecules.
Tumor-associatedmiR-22targetsYWHAZ
YWHAZplaysanimportantroleintheassemblyofsignaling
complexesrequiredfortheactivationofpathwaysdownstream
ofgrowthreceptors(31).Interestingly,miR-22notonlyreduced
theproteinlevelsofYWHAZbutalsoitstranscriptlevels
(Figs.4,5A).FurtheranalysesdemonstratedthatmiR-22tar-
getedthe39UTRregionofYWHAZ.Wefusedthe39UTR
sequenceofmouseYWHAZtoaluciferasereportergeneand
foundsignificantrepressionofluciferaseactivitybymiR-22,
suggestingadirecteffect(Fig.5B).Wealsoperformedexperi-
mentswithmutatedtargetmRNAsequences(Supplemental
TableII)andmiR-22inhibitors.BothYWHAZ39UTRmu-
tationandmiR-22inhibitorsabolishedtheinteractionbetween
miR-22andthe39UTRofYWHAZ(Fig.5B).Whentheex-
pressionofYWHAZintumor-associatedDCswasassessed,we
foundthattheDCshadreducedexpressionofYWHAZ(Fig.
5C).Thus,thesedatademonstratethattumor-associatedmiR-22
caninhibittheexpressionofYWHAZbybindingtoits39UTR
region.
PreviousstudieshaveshownthatYWHAZcaninteractwith
multiplemoleculesindifferentsignalingpathways,suchasAKT
(32–36).Indeed,asshowninFig.5D,transfectionwithmiR-22
reducedthephosphorylationofAKT,whereasmiR-22inhibitors
promotedthephosphorylationofAKT.Wealsoobservedthat
miR-22inhibitorsinducedthephosphorylationofFOXO3after
transfectionfor5min.Becausethesephosphorylatedresidues
subsequentlysequesteredFOXO3toYWHAZtopreventapo-
ptosis(37),miR-22maypromotetheapoptosisofDCsbyre-
ducingtheexpressionofYWHAZandaffectingtheactivityof
AKTandFOXO3.
YWHAZhasbeenshowntopromoteERK/MAPKactivation
butinhibittheactivationofJNKandp38MAPK(31).Indeed,as
showninFig.5D,administrationofmiR-22mimicsremarkably
causedthedownregulationofERKphosphorylationandtheup-
regulationofJNKandp38phosphorylation;conversely,ERKwas
activatedandthephosphorylationofJNKandp38wasinhibited
bymiR-22inhibitors.Thedifferenceofp38andERKphosphor-
ylationcouldbefoundaftertransfectionfor5min.JNKwasalso
remarkablephosphorylatedaftermiR-22transfectionascom-
paredwithcontroltransfection.BecauseAKTandMAPKac-
tivityisassociatedwiththeexpressionofapoptoticmolecules,
suchasBcl2andBax,wenextdeterminedtheeffectofmiR-22
FIGURE4.BothmiR-22andmiR-155downregulatetheexpressionofYWHAZ.(A)ThelevelsoftheYWHAZprotein(spot1201)inmiR-22–and
miR-155–transfectedRAW264.7cells.miR-22–,miR-155–,orcontrololigonucleotide(Ctr)–transfectedRAW264.7cellswereanalyzedbytwo-dimen-
sionalgelelectrophoresis.1201,proteinspotnumber.(BandC)Theselectedspot1201istheYWHAZprotein.Spot1201wasdigestedandsubjectedto
analysisbyMALDI-TOFMS.
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onBcl2andBax.TransfectionwithmiR-22reducedtheex-
pressionofBcl2andenhancedtheexpressionofBax,whereas
themiR-22inhibitorinhibitedtheexpressionofBaxanden-
hancedtheexpressionofBcl2(Fig.5E).Thus,theERK/MAPK
signalingpathwayisinvolvedinthemiR-22–mediatedapoptosis
ofDCs.
Takentogether,boththeAKT/FOXO3andERK/MAPKsig-
nalingpathwaysmaybeinvolvedinthemiR-22–mediatedapo-
ptosisofBMDCs.Additionally,wefoundthatmiR-155,which
inducedDCapoptosis(Ref.16andFig.3),alsodownregulatedthe
expressionofYWHAZandaffectedtheexpressionofBcl2and
Bax(Fig.5E).Thus,theAKT/FOXO3andERK/MAPKsignaling
pathwaysmayalsoplayaroleinthemiR-155–mediatedapoptosis
ofBMDCs.
TheBcl2-basedapoptoticsignalingpathwaymaybe
potentiallymodulatedbymultipletumor-associatedmiRNAs
WeanalyzedthegeneexpressionprofileofBMDCsusingagene
chipafterexposureoftheDCstotumorcells.Approximately2701
genesweresignificantlydownregulatedinthetumor-associated
BMDCscomparedwiththecontrolBMDCs(notshown).Com-
binedwithanalysesusingtheTargetScanprogram,wepredicted
thepotentialtargetmoleculesoftumor-associatedmiR-16-1,miR-
17-5p,miR-20a,miR-21,miR-22,miR-142-5p,miR-146a,miR-
155,andmiR-503.AsshowninSupplementalFig.2,someof
thesedownregulatedgenes,suchasBCL2,MTF1,DNAJC7,
PRPF4B,FGD4,CDK6,MAP3K3,MAP3K9,KLF4,PPP3CA,
FKBP1A,MAPK14,DPYSL2,TRIB2,RCN2,ITGA4,TRIB2,
RGS2,PLXNA2,PLCB1,andSACS,maybepotentiallymodulated
FIGURE5.miR-22targetsYWHAZ.(A)miR-22downregulatestheexpressionofYWHAZ.MurineBMDCsweretransfectedwithdifferent
concentrationsofmiR-22oritsinhibitor.ThetranscriptandproteinlevelsofYWHAZweredetectedbyRT-PCR,qRT-PCR,andWesternblot,
respectively.R.E.,relativeexpression.(B)TheluciferaseactivitydemonstratesthatmiR-22targetsthe39UTRofYWHAZ.The39UTRofYWHAZ
(Wt.UTR)orthemutated39UTR(Mut.UTR)wereusedtoconstructluciferasereportervectorsthatwerecotransfectedwithmiR-22(miRNA)its
inhibitororcontrololigonucleotides(Oligos.ctr)into293Tcells.Luciferaseactivitywasmeasuredafter48haccordingtotheprotocoldescribedin
MaterialsandMethods.Thedataareexpressedastherelativeluciferaseactivityofthecontrolsamplesthatwerecotransfectedwithanequal
concentrationofnegativecontrolvectorsandarepresentedasthemeans6SDfromtriplicatetests.Theluciferaseactivityin293Tcells
cotransfectedwithYWHAZwild-type39UTRandcontrololigonucleotides(Oligos.ctr)wasarbitrarilysetat1.ErrorbarsrepresenttheSD.p,
0.05,p,0.01.(C)Tumor-associatedBMDCshadreducedexpressionofYWHAZ.MurineBMDCswerecoculturedwithmurine1D8ovarian
carcinomacells,CT-26coloncarcinomacells,B16melanomacells,andmurinefibroblastcells(mFB)orwithdifferentconcentrationsoftumor
supernatant.ThetranscriptandproteinlevelsofYWHAZweredetectedbyqRT-PCRandWesternblotafter24h;1/2,1/4,1/8,and1/16indicatethat
BMDCswereculturedwith50,25,12.5,and6.25%tumorsupernatant,respectively.0,withouttumorsupernatant.ThenormalizedYWHAZex-
pressionforcontrolcellswassetat1.(D)miR-22reducedtheactivityofAKT,FOXO3,andERK,butp38andJNKactivitywasenhanced.BMDCs
weretransfectedwithmiR-22oritsinhibitors,andphospho-proteinsweredetectedattheindicatedtimesbyWesternblotaccordingtotheprotocol
describedinMaterialsandMethods.(E)miR-22inhibitorsupregulatedtheexpressionofYWHAZandBcl2,butBaxexpressionwasdownregulated.
BMDCsweretransfectedwithmiR-22,miR-155,ortheirinhibitors.TheproteinlevelsofBcl2andBaxweredetectedbyWesternblotafter
transfectionfor24h.ThenormalizedYWHAZ,Bcl2,orBaxexpressionforcontrolcellswassetat1.R.E.,relativeexpression;RLU,relativelight
unit.
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bythetumor-associatedmiRNAs.Thesegenesaredirectlyor
indirectlyrelatedtoBcl2(SupplementalFig.2),whichisamol-
eculethatfavorscellsurvivalbyinhibitingcelldeath(38).Thus,
theBcl2-basedapoptoticsignalingpathwaymaybepotentially
modulatedbymultipletumor-associatedmiRNAs.
Tumor-associatedmiR-503targetsBcl2
Next,weinvestigatedtheeffectofthetumor-associatedmiRNAs
onthepredictedtargetmolecules.WefoundthatmiR-503andmiR-
16-1couldpotentiallytargetBcl2(Fig.6A).Indeed,asshownin
Fig.6Band6C,miR-503downregulatedBcl2atboththetran-
scriptandproteinlevel.miR16-1alsoreducedtheexpressionof
Bcl2(Fig.6B),consistentwithapreviousreport(39).Wethen
fusedthe39UTRsequenceofmouseBcl2toaluciferasereporter
geneandfoundasignificantrepressionofluciferaseactivityby
miR-503comparedwithcontrolvectors,suggestingadirecteffect
(Fig.6D).Weperformedfurtherexperimentswithmutatedtarget
mRNAsequences(SupplementalTableII)andmiR-503inhib-
itors;asexpected,boththeBcl239UTRmutationandmiR-503
inhibitorsabolishedtheinteractionbetweenmiR-503andthe39
UTRofBcl2.WhentheexpressionofBcl2wasassessedintumor-
associatedDCs,thesetumor-associatedDCshadreducedex-
pressionofBcl2(Fig.6E).Thesedatasuggestthatinaddition
towhathasbeenpreviouslyreportedformiR16-1(39),tumor-
associatedmiR-503mayinteractdirectlywiththe39UTRofBcl2
todownregulatetheexpressionofBcl2.Thus,tumor-associated
miR-503couldregulatetheapoptosisofDCsbyaffectingBcl2
expression.
InhibitorsofmiR-22andmiR-503promotetheDC-based
immunotherapyagainsttumors
BothBcl2andYWHAZplayacriticalroleinregulatingtheap-
optosisofdifferenttypesofcells(30,40–42).BecausemiR-22and
miR-503couldaffectthesurvivalandlongevityofDCsinthe
tumorenvironmentbytargetingYWHAZandBcl2,thesemiR-
NAsmightbepromisingtargetsforDC-basedimmunotherapies
againsttumors.TotesttheeffectsofmiR-22ormiR-503onan
exvivoDCvaccineagainsttumors,wetestedtheefficacyof
DCstransfectedwithmiR-22andmiR-503inhibitorsonanestab-
lishedtumor.AsshowninFig.7,tumorgrowthwasslowerin
micevaccinatedwithDCstransfectedwithmiR-22andmiR-503
inhibitors,whereasmicevaccinatedwithDCstransfectedwith
miR-22andmiR-503mimicsexhibitedfastertumorgrowth.
SignificantdifferencesintumorgrowthbetweencontrolandmiR-
22ormiR-503inhibitortransfectedgroupsorbetweencontrol
andmiR-22ormiR-503mimicstransfectedgroupwereap-
parentafter12d(p,0.05).Thereweresignificantlydifferences
intumorweightascomparedwithcontrol(mean6SEM,p,
0.01).Consistentwithotherreports(43),siRNAstargetingBcl2
andYWHAZinDCsalsosuppressedtheDC-basedimmuno-
therapyagainsttumors;conversely,Bcl2andYWHAZimproved
theDC-basedimmunotherapyagainsttumors(Fig.7).
FIGURE6.miR-503targetsBcl2.(A)Bcl2isapotentialtargetofmiR-503.Inadditiontothedownregulatedgenesintumor-associatedBMDCs
showninSupplementalTableII,miR-503andmiR-16-1potentiallytargetBcl2basedonthepredictionofTargetScan5.1(http://www.targetscan.
org/cgi-bin/vert_50/targetscan.cgi?mirg=mmu-miR-223)PICTAR(http://pictar.bio.nyu.edu/),andMIRANDA(http://cbio.mskcc.org/cgi-bin/
mirnaviewer/mirnaviewer.pl).Cyclenumber2071indicatesthe2071genesthataredownregulatedintumor-associatedBMDCs.Cyclenumbers243
and821indicatethenumberofpredictedtargetmoleculesbymiR-16-1andmiR-503,respectively.(BandC)BothmiR-503andmiR-16-1
downregulatetheexpressionofBcl2.BMDCsweretransfectedwithdifferentconcentrationsofmiR-503ormiR-16-1.Thetranscriptandprotein
levelsofBcl2weredetectedbyqRT-PCRandWesternblot,respectively.(D)miR-503cantargetthe39UTRofBcl2.TheBcl2luciferasereporter
vector(Wt.UTR)ormutantBcl2luciferasereportervector(Mut1.UTR)wascotransfectedwithmiR-503mimics(miR503),miR-503inhibitors,or
controloligonucleotides(Oligos.ctr.)into293Tcells.Luciferaseactivitywasmeasuredafter48haccordingtotheprotocoldescribedinMaterials
andMethods.Thedataareexpressedastherelativeluciferaseactivityofcontrolsamplesthatwerecotransfectedwithanequalconcentrationof
negativecontrolvectorsandarepresentedasthemeans6SDfromtriplicatetests.Theluciferaseactivityin293TcellscotransfectedwithBcl2Wt.
UTRandcontrololigonucleotideswasarbitrarilysetat1.ErrorbarsrepresenttheSD.p,0.05,p,0.01.(E)TheexpressionofBcl2was
regulatedbytumorcells.MurineBMDCswerecoculturedwithmurinemelanomaB16cells,coloncarcinomaCT-26cells,ovariancarcinoma1D8
cells,andmurinefibroblastcells(mFB).ThetranscriptandproteinlevelsofBcl2weredetectedbyqRT-PCRandWesternblot.R.E.,relative
expression.
8MODULATIONOFDCSURVIVALBYTUMOR-ASSOCIATEDmiRNAs
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Discussion
Inthisstudy,weidentifiedmurinetumor-associatedmiRNAsthat
affectedthesurvivalandlongevityofDCs.First,tumor-associated
miR-22reducedthesurvivalofDCsbytargetingYWHAZ,which
diminishedtheactivityofAKTandFOXO3topromoteapoptosis.
Second,thetargetingofmiR-22toYWHAZinhibitedERK
activity,promotedtheactivationofJNK/p38,andaffectedthe
expressionofBcl2andBax.Third,tumor-associatedmiR-503
directlytargetedBcl2topromotetheapoptosisofDCs.Finally,
tumor-associatedmiR-16,miR-22,miR-155,andmiR-503could
alsoaffecttheexpressionofmultiplemoleculesrelatedtoapoptotic
signalingpathways.Thus,tumor-associatedmiRNAscantarget
multipleintracellularsignalingmoleculestoreducethesurvivalof
DCsinthetumorenvironment.
TumorcellsmaynotonlypromotethesurvivalofDCsand
induceregulatoryDCstomediateimmunetolerance(3)butalso
causetheapoptosisofDCs.Severaltypesofsolidandblood
cancers,suchaspancreatic,breast,prostate,hepatocellular,lung,
headandneckcarcinomas,andleukemia,areaccompaniedby
impairedfunctionandreducednumbersofDCs(23,44,45).
DCsareoftendepletedfromthetumorsiteitselfandalsofrom
thecirculation.ThereducednumbersofDCsmaybefromthe
enhancedapoptosis.Indeed,ourdatashowedthattumor-mediated
miRNAssuchasmiR-22andmiR-503promotedtheapoptosis
ofDCsbytargetingYWHAZandBcl2.Othersalsofoundthat
tumor-derivedTGF-bmayinduceDCapoptosis(8).Thismay
beanimportantmechanismfortumorimmunetoleranceand
escape.
ApoptoticpathwaysmayberegulatedbymiRNAs,including
miR-16(15),miR-155(16),miR-21(17),andmiR-451(18).The
variousmediatorsreleasedfromtumorcells(e.g.,TGF-b)flow
intosentinellymphnodesandaffectthesurvivalandlongevity
ofDCs(8,24,25).DCnumbersinthesentinellymphnodesof
tumorshavebeenfoundtoberemarkablyreducedinmultiple
cancers(4).However,themechanismoftumor-mediatedapo-
ptosisofDCsisunclear.Ourresultsdemonstratethattumorcells
inducetheupregulationofmultiplemiRNAs,includingmiR-22,
miR-503,miR-16-1,andmiR-155.ArecentstudybyCubillos-
Ruizetal.(46)reportedthattumor-associatedDCshadreduced
miR-155levels.Thismaybecausedfromdifferenttimepoints
and/ordifferentmodels.Indeed,miR-155andalsomiR-503are
immediatelyupregulated,buttheirexpressionisremarkablydown-
regulatedwithtimeextensionuponexposuretotumors.However,
thesetumor-associatedmiRNAs,particularlymiR-22andmiR-
503,reducethesurvivalandlongevityofDCsbypromotingtheir
apoptosis.OtherstudieshavealsoshownthatmiR-16andmiR-
155areinvolvedintheapoptosisoftumorcells(15,18)and
DCs(16)bytargetingYWHAZandBcl2.
DC-basedimmunotherapieshavebeenextensivelyexploredfor
cancertreatments,buttheyhavehadlimitedsuccess.Amajor
problemisthatDCsundergoapoptosisafterinjection.Thus,de-
liveryofapoptosis-resistantDCsmayimprovetheefficacyofDC
immunotherapies.ThedecreasedviabilityoftheDCscouldbe
explainedbythereducedexpressionoftheantiapoptoticproteins
Bcl2andYWHAZ,whichhavepreviouslybeenshowntocontrol
thelongevityofmurineDCs(30,40–42).Theadministrationof
siRNAstargetingtheseproapoptoticmoleculesinDCsincreases
DCsurvival.TreatmentofDCswithTRANCE,aprosurvival
factor,resultsinbetteradjuvantproperties.Ourdatashowthat
DCstransfectedwithmiR-22andmiR-503inhibitorsmayim-
proveantitumorresponse.However,theroleofoverexpressed
miRNAsonDCsurvival/apoptosismightonlybeapartoftheDC
conditionedbytumorcells.Otherssuchassolubleandmembrane-
boundfactorsarelikelyinvolved,andthedeleteriouseffectson
DCsisnotlimitedtotheirsurvival.
Disclosures
Theauthorshavenofinancialconflictsofinterest.
FIGURE7.miR-22andmiR-503impair
DC-basedimmunotherapyagainsttumors.
(AandB)Tumorsgrewslowerinmice
injectedwithDCstransfectedwithmiR-
22andmiR-503inhibitors.BMDCswere
transfectedwithmiR-22inhibitors(miR-
22I),miR-503inhibitors(miR-503I),or
vectorscontainingBcl2orYWHAZ.Con-
trolBMDCsweretransfectedusingamix-
tureofmocksiRNAswithemptyvectors.
(CandD)Tumorsgrewfasterinmiceim-
munizedwithDCstransfectedwithtumor-
associatedmiRNAs.BMDCsweretrans-
fectedwithmiR-22mimics(miR-22M),
miR-503mimics(miR-503M),Bcl2siRNAs
(Bcl2siR),orYWHAZsiRNAs(YWHAZ
siR).ThemurineB16tumormodelwas
establishedaccordingtotheprotocolde-
scribedinMaterialsandMethods.Trans-
fectedBMDCswereinjectedintotumor
tissueatdays3,9,and15(2310
6
cells/
mouse).Tumorsizesweremeasuredatthe
indicatedtimesandstatisticallyanalyzedby
ANOVAfollowedbytheposthocTukey–
Kramermultiple-comparisonTest.Tumor
weightsweremeasuredatday28afterin-
jectingtumorcellsandstatisticallyanalyzed
usingaStudentttest.
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