Co-Targeting IGF-1R and Autophagy Enhances the Effects of Cell Growth Suppression and

16 Pages·2016·5.35 MB·English

RESEARCHARTICLE
Co-Targeting IGF-1R and Autophagy Enhances
the Effects of Cell Growth Suppression and
Apoptosis Induced by the IGF-1R Inhibitor
NVP-AEW541 in Triple-Negative Breast Cancer
Cells
WeibinWu1,2☯,JieyiMa3☯,NanShao1,YaweiShi1,RuimingLiu3,WenLi2,YinLin1*,
a1111111111
ShenmingWang1,2*
a1111111111
a1111111111
1 DepartmentofBreastSurgery,FirstAffiliatedHospital,SunYat-senUniversity,Guangzhou,China,
a1111111111 2 DepartmentofVascularSurgery,FirstAffiliatedHospital,SunYat-senUniversity,Guangzhou,China,
a1111111111 3 LaboratoryofGeneralSurgery,FirstAffiliatedHospital,SunYat-senUniversity,Guangzhou,China
☯Theseauthorscontributedequallytothiswork.
*[email protected](SW);[email protected](YL)
OPENACCESS
Abstract
Citation:WuW,MaJ,ShaoN,ShiY,LiuR,LiW,
etal.(2017)Co-TargetingIGF-1RandAutophagy
EnhancestheEffectsofCellGrowthSuppression
andApoptosisInducedbytheIGF-1RInhibitor
Background
NVP-AEW541inTriple-NegativeBreastCancer
Cells.PLoSONE12(1):e0169229.doi:10.1371/
Triple-negativebreastcancer(TNBC)isthemostintractabletypeofbreastcancer,and
journal.pone.0169229
thereisalackofeffectivetargetedtherapy.Insulin-likegrowthfactor-1receptor(IGF-1R)is
Editor:AamirAhmad,UniversityofSouthAlabama
reportedlyapotentialtargetforTNBCtreatment.However,satisfyingtreatmentoutcomesin
MitchellCancerInstitute,UNITEDSTATES
breastcancerpatientshaveyettobeachievedwithIGF-1R-targetedagents.
Received:September19,2016
Accepted:December13,2016
Methods
Published:January3,2017
Copyright:©2017Wuetal.Thisisanopenaccess ToconfirmwhetherinhibitingIGF-1Rcouldinduceautophagy,wedetectedautophagy-
articledistributedunderthetermsoftheCreative relatedproteinsbywesternblottingandimmunofluorescencestainingofLC3-II.TheIGF-
CommonsAttributionLicense,whichpermits
1RinhibitorNVP-AEW541,autophagyinhibitor3-methyladenine(3-MA)andAtg7small
unrestricteduse,distribution,andreproductionin
interferingRNA(siRNA)wereusedtofurtherinvestigatetheeffectsofautophagyinduced
anymedium,providedtheoriginalauthorand
sourcearecredited. byIGF-1RinhibitioninTNBCcells.TheCCK8assay,EdUassay,apoptosisandcellcycle
analyseswereappliedtotestcellfunctionaftertreatment.
DataAvailabilityStatement:Allrelevantdataare
withinthepaper.
Funding:ThisworkwassupportedbyGuangzhou
Results
ScienceandTechnologyPlanProjects
(2013B021800260;http://apply.gzkj.gov.cn/).The
NVP-AEW541markedlyinducedautophagyinTNBCcellsbyincreasingthelevelsofthe
funderhadnoroleinstudydesign,datacollection
autophagy-relatedproteinBeclin-1andtheLC3-II/LC-Iratioandreducingtheselective
andanalysis,decisiontopublish,orpreparationof
themanuscript. autophagysubstratep62.Jointapplicationof3-MAorAtg7siRNAenhancedthecellgrowth
inhibitionandapoptosiseffectsofNVP-AEW541byarrestingcellsatG1/G0phaseand
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. increasingBaxexpressionanddecreasingthatofBcl-2.
PLOSONE|DOI:10.1371/journal.pone.0169229 January3,2017 1/16
Co-TargetingIGF-1RandAutophagyinTripleNegativeBreastCancerCells
Conclusion
TargetingIGF-1RinTNBCinducescell-protectiveautophagy,therebyweakeningthethera-
peuticeffectofagentsdirectedtowardIGF-1R.Ourfindingsrevealthatcombineduse
autophagy-disruptingagentscanenhancethetherapeuticefficacyofIGF-1Rinhibitorsin
TNBCcellsandmayprovideavaluabletreatmentstrategyforIGF-1Rinhibitor-basedthera-
piesforTNBCandotherIGF-1signaling-associatedtumors.
Introduction
Breastcanceristhesecondmostprevalentcancerworldwideandaccordingtoaninvestigation
bytheWorldHealthOrganization,representsoneoftheleadingcausesofdeathinwomen
cancerpatients[1,2].
Breastcancercanbedividedintofivemajorsubtypes:luminalA,luminalB,Her-2-over-
expressing,normalbreast-likeandbasal-likesubtypes.Themajorityofbasal-likesubtype
tumorsaretriple-negativebreastcancer(TNBC),whicharehighlymalignanttumors.In
thiscase,‘triplenegative’indicatesthatnoexpressionofestrogen-receptor(ER),progester-
one-receptor(PR),andhumanepidermalgrowthfactorreceptor2(HER-2)isfoundinthis
typeofbreastcancer[3].TNBCaccountsforapproximately15%to20%ofallbreastcancer
casesandisusuallyassociatedwitharelativelypoorprognosisduetoitsaggressivebehavior
andthelackofeffectivetargetingtherapiescomparedwithothersubtypes[3].Chemother-
apyiscurrentlythemostcommonadjuvanttreatmentforTNBC.However,outcomes
remaindisappointingbecauseofthehighrecurrencerateandthefactthatonlyaminority
ofTNBCcasesareactuallychemosensitive[4].Moreover,intrinsicoracquiredresistanceto
chemotherapyalsolimitsitsefficacyandapplication[5,6].Anumberofgeneshavean
importantroleintheestablishmentofdrugtolerance,includingBRCA1,TP53,PTEN,
TGFBI,ING1,Bax,PinX1,APC,CDKNandBCRP/ABCG2[7–10].Autophagyhasrecently
beenfoundtobeinvolvedinthedevelopmentofresistancetobreastcancertherapies[11].
Althoughautophagyexhibitsanti-tumoreffectsduringtumorigenesis,itmaycontributeto
thelaterdevelopmentofcancerbypromotingcancercellsurvivalandhelpingcancercells
toovercomestressduringprogressionandmetastasisaswellastreatment[12].Thus,using
autophagyinhibitorsaloneorincombinationwithothercancertherapiesmaybeapoten-
tialstrategyforbreastcancertreatment.
Insulin-likegrowthfactor-1(IGF-1)signalingisassociatedwithvarioustypesofcancers,
includingpancreatic,lungandbreastcancers[13–15].ActivationofIGF-1receptor(IGF-1R)
byIGF-1bindingresultsincellproliferation,metastasisanddrugresistance,anditisreported
thatIGF-1RpromotessurvivalandproliferationofTNBCcelllines[16].Infact,targeting
IGF-1RinhibitedmigrationandinvasionoftheTNCBcelllineMDA-MB-231[15].Further-
more,invivoexperimentshaveshownthatIGF-1Rknockdownreducedthepotentialof
MDA-MB-231cellstoestablishbrainmetastases[17].Inviewofthesefindings,inhibitorstar-
getingIGF-1Rmayserveasantitumoragents,andseveralofthemarecurrentlyundergoing
clinicaltrialsforvarioustypesofcancer[18].Regardless,IGF-1Rinhibitorshaveyettobesuc-
cessfullytranslatedintoclinicalmedicine,possiblyduetothecomplexityofIGF-1signaling.
Ithasbeenrevealedthatdown-regulationofIGF-1RstimulatesthePI3K-Aktpathway,
whichisinvolvedincellautophagy.However,itremainsunknownwhetherautophagyis
responsiblefortheunsatisfactoryoutcomesofIGF-1Rinhibitorsinclinicaltrials.Inthepres-
entstudy,wesoughttoinvestigatetheeffectofautophagyonTNBCcelllinesinwhichIGF-1R
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Co-TargetingIGF-1RandAutophagyinTripleNegativeBreastCancerCells
hasbeeninhibitedandtoclarifywhethercombiningautophagy-disruptingagentscanenhance
thetherapeuticefficacyofinhibitorsthattargetIGF-1RinTNBC.
MaterialsandMethods
Celllinesandreagents
Thehumantriple-negativebreastcancercelllinesMDA-MB-231andBT-549werepurchased
fromAmericanTypeCultureCollection(ATCC,Rockville,MD,USA)andculturedinDul-
becco’smodifiedEaglemedium(DMEM;Gibco,Karlsruhe,Germany)supplementedwith
10%fetalbovineserum(FBS;Gibco,Karlsruhe,Germany)and1%antibiotics(penicillin/
streptomycin,Invitrogen,Carlsbad,CA,USA).Thecellsweremaintainedat37˚Cinahumidi-
fiedatmospherecontaining5%carbondioxide.
NVP-AEW541(IGF-1Rinhibitor)waspurchasedfromSelleckChemicals(SelleckChemi-
cals,Houston,TX,USA).Rapamycin(mTORinhibitor)wasobtainedfromCellSignaling
Technology(CST;Beverly,MA,USA).3-Methyladenine(3-MA;AutophagyInhibitor)was
obtainedfromSigma-AldrichCorporation(StLouis,MO,USA).Alldrugsweresolubilized
toproduceastocksolution,andworkingdilutionswerefreshlypreparedinculturemedium
beforeuse.
Antibodiesagainstthefollowingwereusedforwesternblottingandimmunofluorescence
staining:totalIGF-1R(#3027,Rabbit),p-IGF-1RTyr1316(#6113,Rabbit),totalAkt(#4691,Rab-
bit),p-AktSer473(#9271,Rabbit),Atg7(#2631,Rabbit),Beclin-1(#3495,Rabbit),SQSTM1/p62
(#5114,Rabbit),LC3B(#3868,Rabbit),GAPDH(#5174,Rabbit),Bax(#5023,Rabbit)andBcl-
2(#2870,Rabbit);allwerepurchasedfromCellSignalingTechnology(Beverly,MA,USA).
AppropriatesecondaryantibodieswereobtainedfromThermoFisherScientific(Rockford,IL,
USA).
Westernblottinganalysis
Westernblottingwasconductedaspreviouslydescribed[19].Proteinswereextractedfrom
lysatesofMDA-MB-231andBT-549cellstreatedwiththeindicatedconcentrationofdrugs
for24h.Proteinconcentrationsweredetermined,andequalamountsofprotein(30μg)were
loadedandseparatedby10%-15%sodiumdodecylsulfatepolyacrylamidegelelectrophoresis
(SDS-PAGE)underdenaturingconditions.Theproteinswerethentransferredontopolyviny-
lidenefluoride(PVDF)membranes(Millipore,Bedford,MA,USA).Afterblockingwith5%
non-fatmilkinTris-bufferedsaline(TBS)/Tween20,themembraneswereincubatedwithpri-
maryantibodiesovernightat4˚C.Themembraneswerewashedandincubatedwithsecondary
horseradishperoxidase(HRP)-conjugatedantibodiesfor1hatroomtemperature.Immunor-
eactionwasvisualizedbyenhancedchemiluminescence(ECL,Millipore,Bedford,MA,USA);
imageswereobtainedusingaGEImageQuantLas4000mini(GE,Fairfield,CT,USA).
ImmunofluorescencestainingforLC3-II
MDA-MB-231andBT-549cellsweregrownovernightoncoverslipsin24-wellplatesand
treatedwithdimethylsulfoxide(DMSO),NVP-AEW541(1μmol/L)orrapamycin(10nmol/
L)for24h.Thecellswerefixedwithice-coldparaformaldehydefor15minfollowedbymem-
branepermeabilizationusing0.1%TritonX-100inphosphate-bufferedsaline(PBS)for5min.
Afterblockingin1%bovineserumalbumin(BSA)for1h,thecellswereincubatedovernight
withananti-LC3antibodyat4˚C.ThecellswerethenwashedandincubatedwithAlexaFluor
488(Invitrogen,Carlsbad,CA,USA)for1hatroomtemperature,protectedfromlight.
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Co-TargetingIGF-1RandAutophagyinTripleNegativeBreastCancerCells
ImageswereobtainedbyfluorescencemicroscopyandanalyzedusingImage-ProPlus6.0
software.
TransfectionofAtg7smallinterferingRNA(siRNA)
ScrambledsiRNAsandspecificvalidatedsiRNAsforAtg7werepurchasedfromAppliedBio-
systems(FosterCity,CA).Cellswereseededinthewellsofculturedishes.After24h,thecells
weretransfectedwith50nmol/LscrambledsiRNAsor50nmol/LAtg7siRNAusingtheLipo-
fectamineRNAiMAXtransfectionreagent(Invitrogen,Carlsbad,CA,USA)accordingtothe
manufacturer’sprotocol.
Cellproliferationassays
Cellproliferationwasmeasuredusingcellcountingkit-8(CCK-8;Dojindo,Kumamoto,Japan)
andEdU(RiboBio,Guangzhou,China)assays.Cellswereseededin96-wellplates(5×103cells/
well)andculturedovernightat37˚C,followedbytreatmentwithDMSO,NVP-AEW541(1μmol/
L)and/or3-MA(5mmol/L)for24hortransfectionofAtg7siRNA.FortheCCK-8assay,10μlof
CCK-8solutionwasaddedtoeachwell,andthecellswereincubatedat37˚Cfor2h.Theabsor-
bancewasmeasuredat450nm,aspreviouslydescribed[20].FortheEdUassay,cellswere
exposedtoEdU(50μmol/L)for2hat37˚Candthenfixedin4%formaldehydefor30min.The
cellsweresubsequentlytreatedwithApollococktailfor30min,followedbynuclearstainingwith
Hoechst33342for30min.Imageswerevisualizedbyinvertedfluorescencemicroscopy(CarlZeiss
AxioObserverZ1,Jena,Germany)andanalyzedusingImage-ProPlus6.0software.
Apoptosisandcellcycleanalysis
Apoptosisandthecellcycledistributionweremeasuredbycytometryanalysis.Fortheapopto-
sisassay,cellswereseededin6-wellplatesandincubatedovernightat37˚C.Afterdrugtreat-
mentorAtg7transfection,thecellswereharvestedandstainedusinganAnnexinV-FITC
apoptosiskit(Dojindo,Kumamoto,Japan)accordingtothemanufacturer’sinstructionsand
thenanalyzedbyflowcytometry(BDBioscience,USA).Forcellcycleanalysis,cellssubjected
totheindicatedtreatmentswereharvestedandfixedovernightat-20˚Cwith70%ethanol.The
cellswerethenstainedwithpropidiumiodide(50μg/mL)withRnaseI(100μg/mL)and0.1%
TritonX-100inPBSat37˚Cfor30min.Cellcycleanalysiswasperformedbyflowcytometry
(BDBioscience,USA).
Statistics
Dataareexpressedasthemeans±SD.DifferencesbetweengroupswereassessedbyStudent’s
t-testandone-wayANOVAfollowedbyDunnett’smultiplecomparisontest.GraphPadPrism
version5.0(GraphPad,SanDiego,CA,USA)andSPSSversion17.0(SPSSInc.,Chicago,IL,
USA)wereusedforallofthestatisticalcalculations.Resultswithpvalues<0.05wereconsid-
eredstatisticallysignificant.
Results
TargetingIGF-1RinhibitedTNBCcellproliferationbyattenuatingtheAkt
pathway
ToinvestigatetheeffectofIGF-1RonTNBCcells,wechoseaselectiveinhibitorofIGF-1R,
NVP-AEW541[21],todown-regulatethelevelofphosphorylatedIGF-1RinMDA-MB-231
andBT-549cells.WesternblottingconfirmedtheeffectofNVP-AEW541onphospho-IGF-
1R(Fig1A).EdUandCCK-8assayswereperformedtoexaminetheeffectofNVP-AEW541
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Co-TargetingIGF-1RandAutophagyinTripleNegativeBreastCancerCells
Fig1.EffectofNVP-AEW541onproliferationandlevelsofIGF-1RandAktinMDA-MB-231andBT-549
cells.(A)WesternblottingvalidatedthatNVP-AEW541significantlyreducedexpressionofphosphorylated
IGF-1RandAkt.Onerepresentativefromthreeindependentexperimentsisshown.(B-D)NVP-AEW541
markedlydecreasedtheproliferationrateofMDA-MB-231andBT-549cells,asassessedbyEdU(BandC)
andcellcountingkit-8assays(D).Resultswereobtainedfromatleastfourindependentexperiments,and
dataarepresentedasthemean±SD,*p<0.05,**p<0.01,***p<0.001.
doi:10.1371/journal.pone.0169229.g001
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Co-TargetingIGF-1RandAutophagyinTripleNegativeBreastCancerCells
oncellproliferation,andtheresultsshowedthattreatmentwith1μmol/LNVP-AEW541for
24hsignificantlyreducedTNBCcellproliferation(Fig1B–1D).ThePI3K-Aktpathwayis
downstreamofIGFsignalingandrelatedtoautophagy[11].Ourresultsshowedthatthelevel
ofactivatedAkt,anautophagyinhibitiongene[22],wasalsoreducedbyNVP-AEW541(Fig
1A),suggestingthatIGF-1RinhibitionmightinduceautophagyinTNBCcells.
NVP-AEW541inducedautophagyinMDA-MB-231andBT-549cells
ToconfirmourhypothesisthatIGF-1Rinhibitionmightinduceautophagy,rapamycin,an
autophagy-promotingagent,waschosenasapositivecontrol.AsshowninFig2A,both
NVP-AEW541(1μM)andrapamycin(10nM)notablyincreasedtheexpressionlevelofthe
autophagy-relatedproteinBeclin-1andtheratioofLC3-II/LC-Iandreducedthelevelofthe
selectiveautophagysubstratep62.TheeffectofNVP-AEW541onLC3-IIpunctaformation
wasalsotested.AftertreatmentofcellswithDMSO,NVP-AEW541orrapamycinfor24h,
immunofluorescenceanalysisshowedtheremarkableaccumulationofLC3-IIpuncta,achar-
acteristicmammalianautophagyphenotype,inNVP-AEW541orrapamycintreatmentgroups
(Fig2Band2C).TheseresultsindicatedthatNVP-AEW541couldsignificantlyinduceautop-
hagyinTNBCcells.
InhibitionofautophagyenhancedNVP-AEW541-inducedcellgrowth
suppressioninTNBCcells
ToexplorewhetherNVP-AEW541-inducedautophagyisbeneficialforTNBCcells,wecom-
binedtheautophagyinhibitor3-MAorsiRNAtargetingtheautophagy-promotinggeneAtg7
andexaminedtheeffectsofNVP-AEW541onTNBCcells.Westernblottinganalysisverified
theautophagy-suppressiveeffectof3-MAandAtg7siRNA:Beclin-1andtheLC3-II/LC3-I
ratiowerereduced,andthelevelofp62wasenhanced(Fig3AandFig4A).Comparedwith
theNVP-AEW541group,cellstreatedwithNVP-AEW541togetherwith3-MAorAtg7
siRNAshowedlowerlevelsofautophagy(higherBeclin-1andLC3-II/LC3-Iratioandlower
p62level)(Fig3AandFig4A).CCK-8andEdUassaysshowedthatNVP-AEW541combined
with3-MAorAtg7siRNAtreatmentsignificantlysuppressedcellproliferationcomparedto
NVP-AEW541alone(Fig3B–3DandFig4B–4D).ThesefindingssuggestthatNVP-AE-
W541-inducedautophagymightprotectTNBCcellsfromgrowthsuppression.
InhibitionofautophagyenhancedNVP-AEW541-inducedapoptosisin
TNBCcells
AstheresultspresentedaboverevealedthatNVP-AEW541-inducedautophagyprotects
TNBCcellproliferation,wethenexaminedtheeffectofNVP-AEW541-inducedautophagyon
apoptosis.Theexpressionlevelsofapoptosis-relatedproteinsBcl-2andBaxwereexaminedby
westernblottinganalysis,andAnnexinV/PIdualstainingwasperformedtoevaluatetherate
ofapoptosis.AsshowninFig5A,cellsexposedtoNVP-AEW541togetherwith3-MAorAtg7
siRNApresentedhigherBaxlevelsandlowerBcl-2levelscomparedwiththeNVP-AEW541
group.AnnexinV/PIdualstainingrevealedthatNVP-AEW541combinedwith3-MAorAtg7
siRNAtreatmentincreasedtheapoptosisratefrom30.8%to45.0%andfrom30.8%to42.4%,
respectively,inMDA-MB-231cellsandfrom18.2%to37.1%andfrom18.2%to31.6%,respec-
tively,inBT-549cells(Fig5Band5C).Theseresultsindicatedthatinhibitionofautophagy
enhancesNVP-AEW541-inducedapoptosisinTNBCcells.
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Fig2.NVP-AEW541inducesautophagyinMDA-MB-231andBT-549cells.(A)NVP-AEW541induceda
significantaccumulationofLC3-IIandBeclin-1anddown-regulatedp62inMDA-MB-231andBT-549cells,as
measuredbywesternblotting.Onerepresentativefromthreeindependentexperimentsisshown.(BandC)
ImmunostainingandfluorescencemicroscopyanalysisofLC3(green)andthenucleus(blue)demonstrated
thatNVP-AEW541promotedautophagy,asindicatedbyincreasedLC3-IIpuncta.Resultswereobtainedfrom
fourindependentexperiments,anddataarepresentedasthemean±SD,*p<0.05,**p<0.01,***p<0.001.
doi:10.1371/journal.pone.0169229.g002
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Co-TargetingIGF-1RandAutophagyinTripleNegativeBreastCancerCells
Fig3.3-MAinhibitsNVP-AEW541-inducedautophagyandenhancesanti-proliferativeeffectsofNVP-
AEW541inMDA-MB-231andBT-549cells.(A)3-MAinhibitedtheautophagyinducedbyNVP-AEW541in
MDA-MB-231andBT-549cells,asshownbywesternblotting.Onerepresentativefromthreeindependent
experimentsisshown.(B-D)3-MAcombinedwithNVP-AEW541significantlysuppressedtheproliferationof
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MDA-MB-231andBT-549cellscomparedwithNVP-AEW541alone,asdetectedbyEdU(BandC)andCCK-
8assays(D).Resultswereobtainedfromatleastfourindependentexperiments,anddataarepresentedas
themean±SD,*p<0.05,**p<0.01,***p<0.001.
doi:10.1371/journal.pone.0169229.g003
InhibitionofautophagyenhancedNVP-AEW541-inducedcellcycle
arrestinTNBCcells
BecauseNVP-AEW541-inducedautophagyenhancedthecellgrowth-suppressingeffectof
NVP-AEW541inTNBCcells,wefurtherinvestigateditseffectonthecellcycle.Cellcycle
analysisrevealedthatNVP-AEW541slightlyincreasedtheproportionofcellsinG1/G0phase
andloweredthenumberofcellsinotherphasescomparedwithcontrolgroup.Inaddition,
comparedwithNVP-AEW541alone,both3-MAandAtg7siRNAmarkedlyenhancedthe
effectofNVP-AEW541onMDA-MB-231andBT-549cellcycles,withhigherG1/G0-phase
arrest,asexaminedbyflowcytometry(Fig6Aand6B).Thesefindingssuggestedthat
NVP-AEW541-inducedautophagymightplayaroleinprotectingTNBCcellsfromcellcycle
arrest.
Discussion
IGF-1Risamemberoftheinsulinreceptor(IR)family,whichincludestheIR,IGF-1R,IGF-
1R/IRhybridreceptorsandIGF-2R.ActivatingIGF-1RbyIGF-1orIGF-2bindinghasan
importantfunctionintumorigenesis.Indeed,ithasbeenreportedthatphosphorylatedIGF-
1R/1Risdetectedinallbreastcancersubtypes(luminal,48.1%;TNBC,41.9%;HER2,64.3%),
withhighermortality[23].Alargeportionoftriple-negativebreastcancercellsexpressIGF-
1R,andactivatedIGFsignalingpromotescellsurvivalandproliferation[16].Thesefindings
showthatIGFsignalingisapotentialtargetforTNBCandothertypesofcancer.Therefore,
effortsarebeingmadetodevelopagentsthattargetIGF-1Rforcancertreatment,andseveral
suchdrugshavebeenassessedinclinicaltrials[24].TherearetwotypesofIGF-1R-targeted
agents:monoclonalantibodiesagainstIGF-1R,suchasDalotuzumab[25]andGanitumab
(AMG479)[26];andIGF-1Rkinaseinhibitors,includingLinsitinib,andBMS-754807[27].
Despiteearlyaccomplishmentsinpre-clinicalinvestigations,inclinicaltrials,relativelyfew
tumortypes,e.g.,Ewingsarcoma[28]andthymoma[29],exhibitedsustainedresponsesto
IGF-1Rinhibitors.Thus,IGF-1Rinhibitorshaveyettoshowsatisfactoryclinicalbenefitto
patientswithmoreprevalentlydiagnosedcancers,includingbreastcancerandnon-smallcell
lungcancer,intheoverallpatientpopulation[30,31].
ThecomplexityofIGFsignalingmaybeoneofthereasonsforthefailureofIGF-1R-tar-
getedagentsinclinicaltrialsinvolvingmanytypesofcancers.Moreover,similartoIGF-1R
interactionwithIGF-1,bindingofIGF-2toIGF-1RorIR-AcanalsostimulateIGFsignaling.
Thesituationisfurthercomplicatedwhencellscontainhybridheterodimericreceptorscon-
sistingofIGF-1Randinsulinreceptorsubunits,whichcanactasamajortransducerofIGF
signaling[32].Inaddition,HIF-1-inducedIGF-2releaseandcompensatoryactivationofIR
wasalsoreportedinTNBCcellsunderhypoxicconditionswhenIGF-1Rwasspecificallytar-
geted,thusdisplayingthefunctionofIGF-1signaling[15].
Autophagyisalysosomaldegradationprocessthatcanbestimulatedbyvariousstresses
suchasstarvation,hypoxiaorinfection.Autophagyisinvolvedinmanydiseases,including
cancers.IthasbeenreportedthatIGF-1regulatesautophagythroughc-junN-terminalkinase
andAktpathwaysinhumanatheroscleroticvascularsmoothcells[33].However,ithasnotyet
beenelucidatedwhetherIGF-1RinhibitioncaninduceautophagyinTNBCcells.Inthisstudy,
wefoundthatIGF-1RinhibitionbyNVP-AEW541couldnotonlyinhibitcellproliferation
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Fig4.Atg7siRNAenhancestheinhibitionofproliferationbyblockingNVP-AEW541-inducedautophagy
inMDA-MB-231andBT-549cells.(A)Atg7siRNAblockedNVP-AEW541-inducedautophagyinMDA-MB-231
andBT-549cells,asassessedbywesternblotting.Onerepresentativefromthreeindependentexperimentsis
shown.(B-D)Atg7siRNAcombinedwithNVP-AEW541significantlysuppressedtheproliferationofMDA-MB-
231andBT-549cellscomparedwithNVP-AEW541alone,asdetectedbyEdU(BandC)andCCK-8assays
(D).Resultswereobtainedfromatleastfourindependentexperiments,anddataarepresentedasthe
mean±SD,*p<0.05,**p<0.01,***p<0.001.
doi:10.1371/journal.pone.0169229.g004
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