The Antifungal Drug Itraconazole Inhibits Vascular Endothelial Growth Factor Receptor 2

13 Pages·2011·2.14 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRYVOL.286,NO.51,pp.44045–44056,December23,2011
©2011byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
The Antifungal Drug Itraconazole Inhibits Vascular
Endothelial Growth Factor Receptor 2 (VEGFR2)
Glycosylation, Trafficking, and Signaling in Endothelial Cells*□S
Receivedforpublication,July1,2011,andinrevisedform,October19,2011Published,JBCPapersinPress,October24,2011,DOI10.1074/jbc.M111.278754
BenjaminA.Nacev‡§,PaolaGrassi¶,AnneDell¶,StuartM.Haslam¶,andJunO.Liu‡(cid:1)1
Fromthe‡DepartmentofPharmacologyandMolecularSciences,§MedicalScientistTrainingProgram,and(cid:1)Departmentof
Oncology,JohnsHopkinsUniversitySchoolofMedicine,Baltimore,Maryland21205andthe¶DivisionofMolecularBiosciences,
FacultyofNaturalSciences,ImperialCollegeLondon,LondonSW72AZ,UnitedKingdom
Background:Itraconazoleisawidelyusedantifungaldrugthatwasrecentlyfoundtopossesspotentantiangiogenicactivity.
Results:WereportherethatitraconazolecausedaccumulationofimmatureN-glycansonVEGFR2andinhibitedVEGFR2
traffickingandsignaling.
Conclusion:TheseresultssuggestthatitraconazoleinterfereswithVEGFR2trafficking,glycosylation,andsignalingactivity.
Significance:Itraconazolepossessesuniqueantiangiogenicpotentialthroughtargetingmultiplepathwaysthatareessentialfor
angiogenesis.
Itraconazoleisasafeandwidelyusedantifungaldrugthatwas tetra-antennary complex N-glycans in endothelial cells and
recently found to possess potent antiangiogenic activity. Cur- induced hypoglycosylation of the epidermal growth factor
rently,therearefouractiveclinicaltrialsevaluatingitracona- receptorinarenalcellcarcinomaline,suggestingthatitracona-
zole as a cancer therapeutic. Tumor growth is dependent on zole’seffectsextendbeyondVEGFR2.
angiogenesis,whichisdrivenbythesecretionofgrowthfactors
fromthetumoritself.Wereportherethatitraconazolesignifi-
cantlyinhibitedthebindingofvascularendothelialgrowthfac- ItraconazoleisaFoodandDrugAdministration-approvedanti-
tor (VEGF) to VEGF receptor 2 (VEGFR2) and that both fungaldrug,whichwepreviouslyidentifiedashavingantiangio-
VEGFR2 and an immediate downstream substrate, phospho- genicactivitybothinvitroandinmousemodels(1).Itraconazole’s
lipaseC(cid:1)1,failedtobecomeactivatedafterVEGFstimulation. longhistoryofsafeuseinhumansasanantifungalagentandits
TheseeffectswereduetoadefectinVEGFR2trafficking,lead- relativelyhighplasmaconcentrationshavespurreditsadvanceasa
ingtoadecreaseincellsurfaceexpression,andwereassociated viablecandidateforrepurposingasanantiangiogenicdrug.Itis
with the accumulation of immature N-glycans on VEGFR2. currentlyundergoingfourclinicaltrialsasananticancerthera-
Smallmoleculeinducersoflysosomalcholesterolaccumulation peutic (NCT00769600, NCT00887458, NCT00798135, and
and mammalian target of rapamycin (mTOR) inhibition, two NCT01108094)andhasrecentlybeenshowntohaveefficacyin
previouslyreporteditraconazoleactivities,failedtorecapitulate preclinicalmodelsfornon-smallcelllungcancer(2).
itraconazole’s effects on VEGFR2 glycosylation and signaling. Angiogenesis,theelaborationofnewbloodvesselsfromexist-
Likewise,glycosylationinhibitorsdidnotaltercholesteroltraf- ingvasculature,isanessentialprocessinanumberofpathological
fickingorinhibitmTOR.Repletionofcellularcholesterollevels, states,notablycancerandmaculardegeneration.Sincetheangio-
whichwasknowntorescuetheeffectsofitraconazoleonmTOR genichypothesiswasfirstputforwardbyJudahFolkmaninthe
andcholesteroltrafficking,wasalsoabletorestoreVEGFR2gly- 1970s,anumberofantiangiogenicdrugshaveenteredtheclinic
cosylationandsignaling.Thissuggeststhattheneweffectsof (3).However,thesedrugshavenotbeenassuccessfulasantici-
itraconazoleoccurinparalleltothosepreviouslyreportedbut pated. For instance, bevacizumab, an anti-vascular endothelial
aredownstreamofacommontarget.Wealsodemonstratedthat growthfactor(VEGF)antibodyandthefirsttobeapproved,only
itraconazole globally reduced poly-N-acetyllactosamine and marginallyextendssurvivalandcarriessignificantriskofadverse
events(4–7).Forthisreason,itraconazoleandotherdrugcandi-
datesarebeingactivelypursuedasnewandpotentiallymoresafe
*Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealth andmoreeffectiveantiangiogenicleads.
(NIH)MedicalScientistTrainingProgramGrantT32GM07309(toB.A.N.);
Itraconazole, like other members of the azole antifungal
by NIH, NCI Grant CA122814 (to J.O.L.); National Center for Research
Resources(NCRR),NIH,GrantUL1RR025005;andtheNIHRoadmapforMed- class,inhibitsthefungalenzymelanosterol14-(cid:1)-demethylase
icalResearch.ThisworkwasalsosupportedbytheFlightAttendantMedical (14DM),2whichgeneratesakeyintermediateinergosterolsyn-
ResearchInstituteandtheCommonwealthFoundation(toJ.O.L.).Additional
supportwasprovidedbyBiotechnologyandBiologicalSciencesResearch
CouncilGrantBBF0083091andtheMarieCurieInitialTrainingNetwork,Euro- 2Theabbreviationsusedare:14DM,Ianosterol14-(cid:1)-demethylase;HUVEC,
glycoArraysProject,partoftheFP7PeopleProgramme. humanumbilicalveinendothelialcell(s);VEGFR2,VEGFreceptor2;PLC(cid:2)1,
□S Theon-lineversionofthisarticle(availableathttp://www.jbc.org)contains phospholipaseC(cid:2)1;EGFR,epidermalgrowthfactorreceptor;mTOR,mam-
supplementalFig.1. malian target of rapamycin; RTK, receptor tyrosine kinase; endo H,
1Towhomcorrespondenceshouldbeaddressed:JunO.Liu,725N.WolfeSt., endoglycosidaseH;PNGaseF,peptide:N-glycosidaseF;dMM,deoxyman-
Hunterian 516, Baltimore, MD 21205. Tel.: [phone]; Fax: 410-955- nojirimycin;LacNAc,N-acetyllactosamine;ER,endoplasmicreticulum;Sws,
4620;E-mail:[email protected]. swainsonine;PDI,proteindisulfideisomerase.
This is an Open Access article under the CC BY license.
DECEMBER23,2011•VOLUME286•NUMBER51 JOURNALOFBIOLOGICALCHEMISTRY 44045
ItraconazoleInhibitsVEGFR2GlycosylationandSignaling
thesis. Conflicting reports on the potency of itraconazole CellCulture—HUVEC(Lonza)weregrowninEGM-2bullet
againsthuman14DM,whichisinvolvedincholesterolsynthe- kitmedium(Lonza)andusedforexperimentsatpassageeight
sis,callintoquestionwhether14DMinhibitionistherelevant or lower. ACHN cells (a gift of Prof. Hans Hammers, Johns
mechanism for itraconazole’s antiangiogenic effects. In addi- Hopkins) were grown in minimum Eagle’s medium (Invitro-
tion,wehaveshownthatotherazoleantifungalsarenotpotent gen)supplementedwith10%FBS(Invitrogen)and1%penicil-
inhibitorsofendothelialcellproliferation,aninvitroassayfor lin/streptomycin(Invitrogen).Bothcelllinesweremaintained
antiangiogenicpotential,andthatinacompleteseriesofitra- inahumidifiedincubatorat37°Cwith5%CO .
2
conazole stereoisomers, antifungal activity did not track with Drug Treatment and Western Blotting—Unless otherwise
activity against endothelial cell proliferation (1, 8). This evi- noted,HUVECwereseededin6-welldishesatadensityof5(cid:1)
dence strongly suggests that itraconazole’s antiangiogenic 104cells/wellin3mlofmediumandallowedtorecoverover-
propertiesareelaboratedthroughanasyetunidentifiedmech- night.Themediumwasthenchangedto2mloffreshmedium,
anism. Previous attempts to explore itraconazole’s cellular anddrugswereimmediatelyaddedfrom200(cid:1)DMSO(Fisher)
effectsrevealedthatinhumanumbilicalveinendothelialcells stocksalongwithvehiclecontrols.InexperimentswithACHN
(HUVEC),itraconazoleinducesanaccumulationofcholesterol cells,cellswereseededatadensityof2(cid:1)105cells/well.Aftera
inlateendsomes/lysosomesandinhibitsthemammaliantarget
24-h treatment, the cells were lysed by the addition of SDS
ofrapamycin(mTOR)(9).Itraconazolealsoinhibitshedgehog
samplebufferandincubatedonicefor10minfollowedbyboil-
signalinginNIH-3T3cellsandinmedulloblastomaxenografts
ingfor10min.Forphospho-VEGFR2(CellSignaling,catalog
andhasbeenshowntoinhibitN-glycosylationinmacrophages
no.2478),VEGFR2(CellSignaling,catalogno.2479),VEGFR1
(10,11).Howcloselytheseeffectsaretiedtothedirectmecha- (CellSignaling,catalogno.2893),PLC(cid:2)1(CellSignaling,cata-
nismofitraconazoleandtowhatdegreetheydrivetheinvivo logno.2822),andphospho-PLC(cid:2)1(CellSignaling,catalogno.
antiangiogeniceffectsofthedrugisnotknown.
2821) blotting, lysates were subjected to 6 or 8% SDS-PAGE.
Angiogenesisispromotedbytumor-elaboratedgrowthfac-
For pS6K (Cell Signaling, catalog no. 9205), S6K (Santa Cruz
tors,suchasVEGFandfibroblastgrowthfactor(FGF).These
Biotechnology, Inc., catalog no. sc-8418), phospho-ERK1/2
ligandsbindtoreceptortyrosinekinases(RTKs)expressedon
(CellSignaling,catalogno.9101),ERK1/2(SantaCruzBiotech-
endothelialcellsandinduceactivationofdownstreamsignaling
nologies,Inc.,catalogno.sc-94),andepidermalgrowthfactor
after receptor autophosphorylation. Phospholipase C, phos-
receptor(EGFR)(CellSignaling,catalogno.2232)blotting,pro-
phatidylinositol-3kinase,andproteinkinaseCareamongthe
teinswereseparatedby10%SDS-PAGE.Proteinsweretrans-
keysignalingintermediatesthatrelaytheVEGFbindingsignal
ferredtoPVDF(Bio-Rad)ornitrocellulose(Bio-Rad)inthecase
toeffectorsthatultimatelydriveendothelialcellproliferation,
of phospho-S6K blots. Membranes were blocked with 5%
migration,andsurvival(12,13).ForbothFGFandVEGF,there
bovineserumalbumin(BSA)(Sigma)inTBS-T(0.5%Tween20
aremultipleRTKsthatbindeachligand,andtherearemultiple
(Sigma))andthenincubatedwithprimaryantibodyin1%BSA
isoformsandsplicevariantsofeachgrowthfactor.Inthecaseof
in TBS-T, followed by incubation with HRP-conjugated anti-
VEGF-A,thepredominantVEGFfamilymembertoparticipate
rabbit or anti-mouse IgG (GE Healthcare) also in 1% BSA in
inpathologicalangiogenesis,thereareseveralRTKsthatactas
bindingpartners.TheseincludeVEGFR1,VEGFR2,PDGFR(cid:1), TBS-T.Inthecaseofphospho-S6Kblots,blockingandallincu-
andPDGF(cid:3)aswellasthenon-RTKco-receptors,neuropilin1 bationstepswerecarriedoutin5%blotto(SantaCruzBiotech-
nology, Inc.) in TBS-T. Membranes were washed three times
and2(13–15).OfthefamilyofVEGFreceptors,VEGFR2plays
with TBS-T before and after the secondary antibody incuba-
theprimaryroleinpathologicalangiogenesis(12,16–18).
tions. HRP substrate (Immobilon Western, Milipore) was
Inourcontinuingeffortstobetterunderstandhowitracona-
addedfor1–5min,andbandswerevisualizedwithanEastman
zole influences endothelial cell function, we serendipitously
KodakCo.ImageStation440CF.Quantitationofbandinten-
observed that treatment of HUVEC with itraconazole signifi-
sitywascarriedoutwithImageJusingthebackgroundsubtrac-
cantly altered the migration pattern of VEGFR2 during SDS-
tionmodule(version1.43u,NationalInstitutesofHealth).All
PAGE.Inthiswork,wehaveidentifiedthecauseofthemigra-
tion shift, explored the functional consequences of the Westernblotsshownarerepresentativeofthreeindependent
itraconazole-induced VEGFR2 changes, and sought to deter- experiments with the exception of the 125I-VEGF165 experi-
minetherelationshipbetweenthepreviouslyknownactivities ments,whichwereperformedinduplicate.
of itraconazole and the effects we observed on VEGFR2. We VEGFStimulationExperiments—1.5(cid:1)105HUVECin4ml
havealsoassessedthedegreetowhichotherRTKfamilymem- ofmediumwereseededina6-cmdishandallowedtorecover
bersandothercelltypesweresimilarlyaffectedbyitraconazole. overnight. The medium was then changed to 2 ml of basal
EGM-2 medium with 0.1% FBS, and cells were treated with
EXPERIMENTALPROCEDURES drugs from 200(cid:1) DMSO stocks or vehicle alone for 24 h.
Chemicals and Reagents—Itraconazole and ketoconazole RecombinanthumanVEGF wasthenaddedtoafinalcon-
165
werepurchasedfromSigma.Otherazoleswereobtainedfrom centrationof25ng/mlfroma100(cid:1)stockinbasalEGM-2with
theJohnsHopkinsDrugLibrary(1).Rapamycinandsunitinib 0.1%FBSfreshlypreparedfromfrozen100(cid:4)g/mlstocksin0.1%
werefromLCLaboratories.Swainsonineandcastanospermine BSAinPBS.Thecellswerestimulatedfor2–60minandtrans-
were from Tocris, and deoxymannojirimycin was from Enzo ferredtoicealongwithanunstimulatedcontrol.Themedium
Life Sciences. Recombinant human VEGF was purchased was aspirated, and the cells were immediately lysed with the
165
fromR&Dsystems. addition of 240 (cid:4)l of 2(cid:1) SDS sample buffer. After a 10-min
44046 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER51•DECEMBER23,2011
ItraconazoleInhibitsVEGFR2GlycosylationandSignaling
incubationonice,thelysateswereboiledfor10minandthen P-40inPBS.Thebeadswerethenresuspendedin100(cid:4)lofPBS
subjectedtoSDS-PAGE. andtransferredtoanewtube.Thesupernatantwasremoved,
FilipinStaining—Theseexperimentswerebasedonprevious andthebeadswereboiledin90(cid:4)lof1(cid:1)SDSsamplebuffer.
work (9). HUVEC were seeded at 3 (cid:1) 104 cells/well of an The samples were split into two equal volumes, and the pro-
8-chamberglassslide(Lab-Tek)in400(cid:4)lofmedium.Afteran teinsweresubjectedto6%SDS-PAGE.Onesetofsampleswas
overnightrecovery,drugswereaddedfrom200(cid:1)DMSOstocks transferredtoaPVDFmembrane,andVEGFR2wasanalyzed
along with vehicle controls. Following a 24-h treatment, the byWesternblot.Theotherwasfixedinthegelbyincubation
cellswerefixedin4%paraformaldehydeinPBS(pH7.4)for20 with10%aceticacidand30%MeOHfor0.5handthendriedfor
minandthenwashedthreetimeswith400(cid:4)lofPBS.200(cid:4)lof 2hat80°C.Thedriedgelwasexposedtofilmfor3–4weeksat
filipin staining solution (50 (cid:4)g/ml filipin (Sigma, catalog no. (cid:2)70°Cpriortodeveloping.
F9765)dilutedfroma100(cid:1)stockinDSMOstoredinthedark GlobalN-GlycanAnalysis—9.2(cid:1)105HUVECwereseeded
and made from powder stored under argon) and slides were in15-cmdishesandallowedtorecoverovernightin20mlof
incubated in the dark for 2 h atroom temperature. The cells medium. The medium was then changed to 36.5 ml of fresh
werethenwashedthreetimeswith400(cid:4)lofPBS,mountedwith medium, and the cells were treated with 800 nM itraconazole
Immu-mount(Thermo),andstoredinthedarkpriortoimag- (froma200(cid:1)DMSOstock)orvehiclealonefor24h.Theplates
ingwithaZeissAxioExaminerwith710NLO-Metamultipho- werewashedthreetimeswith15mlofice-coldPBSandthen
tonandconfocalmodule.Theseexperimentswereperformed scrapedinto5mlofice-coldPBS,collectedbycentrifugation
induplicate. (500(cid:1)g),andfrozeninliquidN .Approximately9(cid:1)106cells
2
Proliferation Assays—These assays were performed as from each treatment condition were processed as described
reported previously (19). ACHN cells were seeded at 8,000 previously (22, 23). Briefly, all samples were subjected to
cells/well. homogenizationusinga130-wattVibra-Cellultrasonicproces-
125I-VEGFReceptorCross-linking—Theseexperimentswere sor (VC 130 PB, Sonics & Materials) within a sound-abating
adaptedfromthemethodsofNeufeldandco-workers(20,21). enclosureinextractionbuffer(25mMTris,150mMNaCl,5mM
3(cid:1)105HUVECwereseededon6-cmdishesin4mlofmedium EDTA, and 1% CHAPS at pH 7.4) and subsequently dialyzed
andallowedtorecoverovernight.Themediumwaschangedto against4(cid:1)4.5litersof50mMammoniumbicarbonate,pH8.5,
2mlofbasalEGM-2with0.1%FBS,andeithervehicleoritra- at4°Cfor48h.
conazole (800 nM final concentration) from a 200(cid:1) DSMO Afterdialysis,thesampleswerelyophilizedandsubjectedto
stockwasadded.Aftera24-hincubation,theplatewastrans- reduction, carboxymethylation, and tryptic digestion. Reduc-
ferredto4°Candwashedtwicewith5mlofice-coldPBS.An tionwascarriedoutin1mlof50mMTris-HClbuffer,pH8.5,
aliquot of 1.4 ml of serum-free EGM-2 with 5 ng/ml 125I- containing2mg/mldithiothreitol,at37°Cinawaterbathfor
VEGF (PerkinElmer Life Sciences) (diluted from a 100(cid:1) 1h.Thesampleswerethencarboxymethylatedbytheaddition
165
stock) was added. For one of two vehicle-treated plates, 100 ofiodoaceticacid(5-foldmolarexcessoverdithiothreitol),and
ng/ml cold VEGF was also added to the medium prior to thereactionwasallowedtoproceedatroomtemperatureinthe
165
transfer to the cells (competition sample). The cold VEGF darkfor1.5h.Carboxymethylationwasterminatedbydialysis
165
wasdilutedfroma100(cid:4)g/mlstockin0.1%BSAinPBS.Aftera against4(cid:1)4.5litersof50mmammoniumbicarbonate,pH8.5,
2-hincubationwithrockingat4°C,thecellswerewashedonce at4°Cfor48h.Afterdialysis,thesampleswerelyophilized.The
with4.5mlofice-coldPBSandmovedtoroomtemperature. reduced carboxymethylated proteins were then digested with
Analiquotof2.5mlof0.15mMdisuccinimidylsuberate(Sigma) N-p-tosyl-L-phenylalanine chloromethyl ketone-pretreated
inPBS(dilutedfromafreshlymade20mMstockinDMSO)was bovinepancreastrypsin(EC3.4.21.4;Sigma)for16hat37°Cin
added,andtheincubationwascontinuedfor15minpriortothe 50 mM ammonium bicarbonate buffer, pH 8.4. The products
addition of 200 (cid:4)l of quenching buffer (10 mM Tris-HCl (pH were purified by C18 Sep-Pak(cid:2) (Waters) as described previ-
7.5),200mMglycine,2mMEDTA).Aftera1-minincubation, ously(24).
theplateswerereturnedto4°Candwashedoncewith4.5mlof Peptide N-glycosidase F digestion of the tryptic glycopep-
PBS.Analiquotof200(cid:4)lofice-coldlysisbuffer(50mMTris- tideswascarriedoutin50mMammoniumbicarbonatebuffer,
HCl(pH7.5),150mMNaCl,1%NonidetP-40,10mMEDTA, pH8.5,for20hat37°Cwith5unitsofenzyme(RocheApplied
10%glycerol,proteaseinhibitors(CompleteEDTA-free;Roche Science).ThereleasedN-glycanswerepurifiedfromO-glyco-
AppliedScience))wasthenadded,andthecellswerescraped peptides and peptides by chromatography on a Sep-Pak C18
fromtheplatesandcollectedina1.5-mltube,whichwasthen cartridge(WatersCorp.,Milford,MA)andsubsequentlymeth-
restedat4°Cfor15min.Thetubeswerethenspunat10,000(cid:1) ylatedusingthesodiumhydroxidepermethylationprocedure
gat4°Cfor10min,andthesupernatantwastransferredtoa asdescribedpreviously(22).
newtube.Analiquotof200(cid:4)lofthesupernatantwascombined MALDI-TOFdatawereacquiredonaVoyager-DESTRmass
ina0.5-mltubewith1.5(cid:4)gofanti-VEGR2antibody(CellSig- spectrometer (Applied Biosystems, Foster City, CA) in the
naling,catalogno.2479)froma1mg/mlstockinPBSandincu- reflectronmodewithdelayedextraction.Permethylatedsam-
batedfor2hat4°Conarotatingwheel.Themixturewasthen plesweredissolvedin10(cid:4)lof70%(v/v)aqueousmethanol,and
addedto30(cid:4)lofproteinADynabeads(Invitrogen),whichwere 1(cid:4)lofthedissolvedsamplewaspremixedwith1(cid:4)lofmatrix
prewashedwith1mlofPBSina0.5-mltubeandrotatedfor20 (20 mg/ml 2,5-dihydroxybenzoic acid in 70% (v/v) aqueous
minat4°C,afterwhichtimethemixturewastransferredtoa methanol), spotted onto a target plate, and dried under
1.5-mltubeandwashedthreetimeswith500(cid:4)lof0.1%Nonidet vacuum.
DECEMBER23,2011•VOLUME286•NUMBER51 JOURNALOFBIOLOGICALCHEMISTRY 44047
ItraconazoleInhibitsVEGFR2GlycosylationandSignaling
The MS data were processed using Data Explorer 4.9 soft- withrotationat4°C,thebeadswerewashedthreetimesin1ml
ware (Applied Biosystems). The mass spectra were base line- of lysis buffer (5 min of rotation per wash) and subsequently
corrected (default settings) and noise-filtered (with a correc- boiledin60(cid:4)lof2(cid:1)SDSsamplebufferfor10min.Thesuper-
tion factor of 0.7) and then converted to ASCII format. The natantwasthenrunonSDS-PAGEalongwiththeinputfrac-
processed spectra were then subjected to manual assignment tionsandanalyzedbyWesternblot.
and annotation with the aid of the glycobioinformatics tool Sterol Rescue Experiments—These experiments were based
GlycoWorkBench(25). onworkbyChristianetal.(26)thatwaspreviouslyappliedto
N-Glycan Digestions—HUVEC were seeded at 4 (cid:1) 105 the rescue of itraconazole-induced effects in HUVEC (9).
cells/10 ml of medium in a 10-cm dish. After an overnight HUVECwereseededat5(cid:1)104cells/wellina6-welldishin3ml
recovery, the medium was changed to 8 ml of fresh medium, of medium. After an overnight recovery, the medium was
andthecellsweretreatedwithitraconazolefroma200(cid:1)stock changed to 2 ml of fresh medium, and the free sterols, free
inDMSOorvehiclealone.Thecellswerewashedtwicewith10 methyl-(cid:3)-cyclodextrin,andsterol(cid:3)methyl-(cid:3)-cyclodextrincom-
mlofice-coldPBSandlysedbytheadditionof500(cid:4)loflysis plexeswereaddedfrom100(cid:1)stocksfollowedbydrugaddition
buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% from200(cid:1)stocksinDMSOorvehiclealone.Topreparethe
Triton-X-100, protease inhibitors (Complete EDTA-free, sterol(cid:3)methyl-(cid:3)-cyclodextrin complexes, cholesterol (Sigma)
RocheAppliedScience))withrockingat4°Cfor20min.The or(cid:3)-estradiol (Sigma) from 20 mg/ml stocks in ethanol were
lysateswerecollectedbyscrapingthedishandthencentrifuged combined with filter-sterilized 10% methyl-(cid:3)-cyclodextrin
for10min,10,000(cid:1)gat4°C.Theproteinconcentrationinthe (Sigma)inPBStogiveafinal0.4mg/mlsterolconcentration.
supernatant was measured using a D protein assay kit (Bio- Aliquotsof0.4mg/mlsterolinPBSonly,10%methyl-(cid:3)-cyclo-
c
Rad)andnormalizedbytheadditionoflysisbufferasneeded. dextrinwithethanolvehicle,andethanolinPBSonlywerealso
Equalamountsofprotein(19–34(cid:4)g)weredigestedat37°Cin prepared.Allsamplesweresolubilizedbythreecyclesofvor-
300-(cid:4)lreactionswithsialidase(specificityfor(cid:1)2–3,(cid:1)2–6,and texing followed by (cid:3)5-min incubations at 42°C prior to the
(cid:1)2–8N-acetyl-neuraminicacidresidues),endoH,andPNGase addition to cells to give 4 (cid:4)g/ml sterol and 0.1% methyl-(cid:3)-
FpurchasedfromNewEnglandBiolabsaccordingtotheman- cyclodextrinfinalconcentrations.Allsolutionswereprepared
ufacturer’sinstructions.Thesialidasereactionwascarriedout fresh.Attheendofa24-hincubation,thesampleswerepro-
for5min,andtheendoHandPNGaseFreactionswerecarried cessedforWesternblottingasdescribedabove.
outfor40min.Attheendofthereaction,thesamples,along Immunofluorescence—HUVECwereseededat2(cid:1)104cells/
withundigestedcontrols,wereboiledinSDSsamplebufferand wellofa12-welldishin1mlofmediumonanEtOH-sterilized
analyzedbyWesternblot.InthecaseofACHNcells,thepro- 18-mmglasscoverslip(Fisher).Afteranovernightrecovery,the
cedurewasmodifiedasfollows.1.5(cid:1)106cellswereseededon medium was changed to 1 ml of fresh medium, and the cells
10-cmdishesin10mlofmediumwith10%FBSandallowedto weretreatedwithdrugsfrom200(cid:1)stocksinDMSOorvehicle
recoverovernight.Themediumwaschangedto10mloffresh alonefor24h.Themediumwasthenaspirated,andthecells
mediumwith2%serum,andthecellsweretreatedwithdrugs werefixedin0.7mlof4%paraformaldehydeinPBSfor20min.
for 24 h. A sample of 100 (cid:4)g of protein was used for each Followingthreewasheswith0.7mlofPBS,thecellswereper-
digestion. miabilizedwith0.7mlof0.1%TritonX-100inPBSfor2min
Surface Protein Biotinylation—HUVEC were seeded and andthenblockedwith0.7mlof2%filteredgoatserum(Sigma)
treatedwithdrugsasintheVEGFbindingexperiments,except inPBSfor1hatroomtemperature.Theblockingsolutionwas
thatthedrugincubationsweredonein4mlofmedium.Fol- removed,andthecoverslipswereincubatedovernightat4°C
lowingthetreatmentperiod,thecellswerewashedtwicewith5 with 25 (cid:4)l of primary antibody solution (1:50 anti-VEGFR2
mlofice-coldPBSandthenincubatedwith2.5mlofice-cold (CellSignaling,catalogno.2479)and1:50anti-PDI(Enzo,cat-
0.5mg/mlSulfo-NHS-LC-Biotin(Thermo)inPBSfor30min alogno.ADI-SPA-891)or1:1000anti-GM130(BDBiosciences,
withrockingat4°C.Afterwashingoncewith5mlofice-cold catalogno.610823)in1.25%BSAinPBS).Thecoverslipswere
PBS,thecellswereincubatedwith2.5mlof25mMTris(pH8.0) thenwashedthreetimesin0.7mlofPBSandincubatedfor1h
for 15 min at 4°C. The cells were again washed with 5 ml of atroomtemperaturewith25(cid:4)lofsecondaryantibodysolution
ice-coldPBSandthenlysedin200(cid:4)loflysisbuffer(50mMTris (1:800goatanti-rabbitAlexafluor488(Invitrogen,catalogno.
(pH 8.0), 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.5% A11008), 1:800 goat anti-mouse Alexafluor 594 (Invitrogen,
sodiumdeoxycholate,10mMpyrophosphate,10mMglycero- catalog no. A11005), and DAPI (0.1 (cid:4)g/ml) in 1.25% BSA in
phosphate, 50 mM NaF, 0.5 mM NaVO , protease inhibitors PBS).Followingthreewasheswith0.7mlofPBS,thecoverslips
4
(Complete EDTA-free, Roche)) by incubation for 20 min at weremountedwith15(cid:4)lofImmumount(Fisher)andsealed
4°C.Thelysateswerecollectedbyscrapingthedishandthen withclearnailpolish.Confocalimagingwasperformedusinga
centrifugedfor10min,10,000(cid:1)gat4°C.Theproteinconcen- Zeiss710NLOMetamultiphotonconfocalmicroscopeandZen
trationinthesupernatantwasdeterminedusingaD protein software(CarlZeiss).Representativemicrographsweretaken
c
assay kit (Bio-Rad) and normalized by the addition of lysis of2fields/condition/independentexperiment.
bufferasneeded.Analiquotof200(cid:4)lofsupernatantwasthen
RESULTS
applied to high capacity steptavidin-agarose beads (Thermo),
whichhadbeenequilibratedwithtwo5-minincubationswith1 Itraconazole Induces the Accumulation of a Low Apparent
mloflysisbuffer.Asampleoflysatewasalsoboiledfor10min MolecularWeightVEGFR2Species—VEGFR2ordinarilyexists
inSDSsamplebufferasaninputcontrol.Aftera1-hincubation as a major high molecular weight species and a minor lower
44048 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER51•DECEMBER23,2011
ItraconazoleInhibitsVEGFR2GlycosylationandSignaling
(30–32)(Fig.2A).Invehicle-treatedcells,VEGFR2andPLC(cid:2)1
phosphorylationpeakedby2minafterVEGFaddition,andthe
signaldecayedby30minafterstimulation.InHUVECtreated
with400nMitraconazole,VEGFR2phosphorylationwasinhib-
itedby80%(p(cid:4)0.05),andPLC(cid:2)1phosphorylationwasinhib-
itedby60%(p(cid:4)0.05)at2minafterVEGFstimulation(Fig.2,A
and B). There was no increase in phosphorylation with time,
suggestingthatthedecreaseinphosphorylationat2minwas
not simply a result of itraconazole delaying the kinetics of
receptor activation. Treatment with 800 nM itraconazole
slightly increased the inhibition of PLC(cid:2)1 phosphorylation
compared with the 400 nM dose but did not further suppress
VEGFR2phosphorylation,whichwasalreadynearthelimitof
detection by Western blot. Notably, the degree of VEGFR2
phosphorylationinhibitionby400nMitraconazolewassimilar
tothatobservedbytreatmentwithsunitinib,anantiangiogen-
esisdrugthathasefficacyagainstrenalcellcarcinoma,gastro-
FIGURE 1. Itraconazole induces a change in the migration pattern of intestinal stromal tumors, and pancreatic neuroendocrine
VEGFR2.A,HUVECweretreatedfor24hwiththeindicateddosesofitracona-
tumors and directly inhibits VEGFR2 kinase activity (33, 34).
zoleorvehicle(DMSO),andVEGFR2wasanalyzedbyWesternblot.B,HUVEC
weretreatedasinAwiththreeinhibitorsoffungal14DM(terconazole,flu- Interestingly, although sunitinib strongly inhibited the phos-
conazole,andketoconazole)andaninhibitorofhuman14DM(azalanstat). phorylationofERK1/2,adownstreameffectoroftheVEGFR2
signaling, itraconazole at doses up to 800 nM did not block
molecularweightspeciesasseenonSDS-PAGE.Weobserved ERK1/2activationafterVEGFaddition.
thatinHUVECtreatedwithitraconazole,themigrationpattern InorderforVEGFR2tobecomeactivated,thereceptormust
of VEGFR2 was altered such that only the lower molecular firstbindVEGF,dimerize,andthenundergoautophosphoryl-
weightspecieswaspresent(Fig.1A).Theshiftfromthehighto ation in trans (18, 35). Given the strong inhibition of VEGF-
lowmolecularweightspecieswasapparentwhenHUVECwere stimulatedVEGFR2signalingbyitraconazole,wenextsought
treated with 200 nM itraconazole and was nearly complete at todeterminewhichstepofVEGFR2activationwasperturbed.
400 nM itraconazole, which is in line with the IC we have FollowingthetechniquesusedbyNeufeldandco-workers(20,
50
previouslyreportedfortheitraconazole-inducedinhibitionof 21) to initially identify the VEGFR2 receptor on the basis of
HUVEC proliferation, cholesterol accumulation, and mTOR VEGFbinding,weevaluatedtheabilityofVEGFR2initracona-
inhibition(1,9).Thecorrelationofthesepotenciessuggestedto zole-treated cells to bind 125I-VEGF . After drug treatment
165
usthattheeffectswererelatedtoasimilarunderlyingmecha- using the same conditions as in the signaling experiments,
nismandthereforewarrantedfurtherexploration. HUVEC were stimulated with the radiolabeled ligand, which
Becauseitraconazoleinhibits14DM,wefirsttestedwhether wasthencross-linkedtothereceptorwiththebidentatecross-
thisactivitycontributedtotheVEGFR2migrationpatternshift. linking agent disuccinimidyl suberate. Because VEGF binds a
We treated HUVEC with terconazole, fluconazole, and keto- number of receptors but we were specifically interested in
conazole,whicharethreeothermembersoftheazoleantifun- VEGFR2,weimmunoprecipitatedVEGFR2andevaluatedthe
galclass,andazalanstat,anon-azolesmallmoleculeinhibitorof resultant fraction by Western blot and autoradiography (Fig.
human14DMwithanapparentK of800pM(27).Nonewere 2C). As expected, there was strong labeling of VEGFR2 after
i
abletoinducesimilarchangesinVEGFR2migration(Fig.1B). vehicle treatment, which was abrogated by the addition of a
Notably, ketoconazole has significant crossover inhibition of 20-foldexcessofcoldVEGF duringtheligand-bindingstep.
165
human14DMatdosesmuchlowerthanweusedhere(28,29). In contrast, itraconazole treatment resulted in a nearly com-
Thus, 14DM inhibition could not explain the effects of itra- plete loss of labeling, suggesting that itraconazole prevented
conazoleonVEGFR2. VEGFbindingtoVEGFR2.
Itraconazole Prevents VEGF-stimulated Signaling by Complete N-Glycosylation of VEGFR2 Is Inhibited by
VEGFR2—Havingobservedtheitraconazole-inducedchanges Itraconazole—GiventheclearinhibitionofVEGFR2signaling
inVEGFR2migration,wehypothesizedthatVEGFR2function and ligand binding after itraconazole treatment, we next
may be perturbed. To test this, HUVEC were incubated in focused on understanding the nature of the itraconazole-in-
reducedserummediuminthepresenceofvaryingdoesofitra- duced VEGFR2 molecular weight changes. Because itracona-
conazoleorvehicleandthenstimulatedwithVEGF for2–60 zolewasrecentlyshowntointerferewiththeglycosylationof
165
min alongside unstimulated controls. After stimulation, the theCD14receptorandotherproteinsinmacrophages,wehad
cells were immediately lysed, and the ligand-induced auto- a strong suspicion that itraconazole was also modulating
transphosphorylation of the receptor at Tyr-1175, a require- VEGFR2glycosylation,therebyleadingtothealteredmigration
mentforVEGFR2signaling,wasdeterminedbyWesternblot- oftheprotein(11).Thismodelisconsistentwithearlyworkon
ting (30). Phosphorylation of phospholipase C (cid:2)1 (PLC(cid:2)1), a VEGFR2, which revealed that the protein was N-glycosylated
directbindingpartnerofVEGFR2thatsignalsdownstreamina and that in [35S]methionine pulse-chase experiments, the
phospho-Tyr-1175-dependent manner, was also examined newly synthesized protein transits through a low molecular
DECEMBER23,2011•VOLUME286•NUMBER51 JOURNALOFBIOLOGICALCHEMISTRY 44049
ItraconazoleInhibitsVEGFR2GlycosylationandSignaling
FIGURE2.ItraconazoleblocksVEGF bindingtoVEGFR2andinhibitsVEGFR2signaling.A,HUVECweregrowninlowserummediumfor24hinthe
165
presenceofvehicle(DMSO),itraconazole(Ita),ortheVEGFR2inhibitorsunitinib.ThecellswerethenstimulatedfortheindicatedtimeswithVEGF ,lysed,and
analyzedbyWesternblotforanactivatingphosphorylationonVEGFR2(Tyr-1175),itsimmediatedownstreambindingpartnerPLC(cid:2)1(Tyr-783)1,6a5ndadown-
streameffectorERK1/2(Thr-202/Tyr-204).Totalproteinlevelsareshownascontrols.B,forthe400nMitraconazolesamples,thephosphorylatedsignalswere
quantitated,normalizedtothelevelsoftotalproteinandthentovehiclecontrol.n(cid:5)3independentexperiments;errorbars,S.E.;*,p(cid:4)0.05.C,HUVECwere
treatedwith800nMitraconazoleorvehicleunderthesameconditionsasinAandthenwereincubatedwith125I-VEGF165withorwithoutcompetitionwithcold
VEGF (20-foldexcess).Theligandwascross-linkedtoVEGFR2,whichwasthenimmunoprecipitatedandsubjecttobothautoradiographyandWestern
165
blotting(IB)forVEGFR2.
weightintermediatesimilarinsizetothespeciesthataccumu- wassensitivetobothendoHandPNGaseFbutwasinsensitive
lates after itraconazole treatment (31). The same study also to sialidase. This indicated that the VEGFR2 migration shift
showedthatonlythematureproteincontainedphosphorylated observedafteritraconazoletreatmentwasduetotheaccumu-
tyrosinesafterVEGFstimulation,whichissimilartoourobser- lationofahypoglycosylatedVEGFR2species.
vationsontheitraconazole-inducedchangesinVEGFR2. Because the ER contains the calnexin/calreticulin quality
Wethusexaminedtheeffectsofvariousknowninhibitorsof control chaperones that retain improperly glycosylated pro-
N-linked glycosylation on VEGFR2 and compared them with teins, we performed cell surface biotinylation experiments to
that of itraconazole. Altering glycosylation in HUVEC using determine if trafficking of the hypoglycosylated VEGFR2 was
tunicamycin, swainsonine, deoxymannojirimycin (dMM), or disturbed (36). After treatment under the same conditions in
castanospermine,inhibitorsofdifferentenzymesintheN-gly- whichweassessedVEGFR2signalingandVEGFbinding,cell
cosylation pathway, clearly affected the migration pattern of surfaceproteinswerereactedwithNHS-biotinandthencap-
VEGFR2(Fig.3,AandC).Thissuggestedthatperturbedglyco- turedonstreptavidinbeadsafterlysis(pull-downfraction)and
sylationinHUVECmightunderliethemobilityshiftsseenafter analyzed by Western blot. In contrast to tubulin, which is an
itraconazoletreatment.Interestingly,timecourseexperiments intracellular protein and therefore not biotinylated, VEGFR2
with tunicamycin and dMM indicated that they induced fromvehicle-treatedcellswaspresentinthepull-downfraction
VEGFR2shiftsmuchmorerapidlythanitraconazole(Fig.3,B (Fig.3F).However,itraconazoletreatmentstronglyreducedthe
andC).Wenextdigestedlysatesfromitraconazoleorvehicle- levelsofVEGFR2onthecellsurface.
treatedHUVECwithsialidase,whichcleaves(cid:1)2–3,(cid:1)2–6,and TodeterminethefateofthehypoglycosylatedVEGFR2,its
(cid:1)2–8N-acetyl-neuraminicacidresiduesfromcomplexN-gly- subcellular localization was determined by immunofluores-
cans; endo H, which releases either hybrid or oligomannose cence(Fig.4A).Initraconazole-treatedcells,VEGFR2accumu-
N-linkedsugars;andPNGaseF,whichreleasesallN-linkedsug- latedinaunipolar,perinuclear,Golgi-likestructure,whichpar-
ars, respectively (Fig. 3D). Western blots of vehicle-treated tially colocalized with the cis-Golgi marker, GM130. In
lysates showed that the upper band was sensitive to sialidase contrast,VEGFR2invehicletreatedcellswasuniformlydistrib-
andPNGaseF,whereasthelowerbandwassensitivetoendoH utedinsmallpunctithroughoutthecytoplasm.TheVEGFR2
andPNGaseF.ThisconfirmsthatbothspeciesareN-glycosy- signal was clearly not associated with the ER marker PDI in
lated and indicates that the upper band contains the more eithervehicle-oritraconazole-treatedsamples(Fig.4B).Treat-
highly processed complex N-glycans. In contrast, all of the mentwitheithercastanospermine,dMM,orswainsoninefailed
VEGFR2presentinthelysatesfromitraconazole-treatedcells toinduceasimilarVEGFR2accumulation(Fig.4C),suggesting
44050 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER51•DECEMBER23,2011
ItraconazoleInhibitsVEGFR2GlycosylationandSignaling
FIGURE3.ItraconazolealtersN-linkedglycosylationinHUVEC.A,WesternblotofVEGFR2fromHUVECtreatedfor24hwithitraconazole(Ita)orN-linked
glycosylationinhibitorsSws,dMM,orcastanospermine(Cast).HUVECweretreatedfortheindicatedtimeswith800nMitraconazole(B),tunicamycin,ordMM
(C)priortoanalysisofVEGFR2byWesternblot.D,lysatesfromitraconazole-orvehicle-treatedHUVECweredigestedwithsialidase,endoH,orPNGaseF,and
themigrationpatternofVEGFR2wasanalyzedbyWesternblot.E,partialMALDI-TOFmassspectraofthehighmolecularmassregionoftheglobalpopulation
ofN-glycansfromHUVECtreatedwith800nMitraconazoleorvehiclealone.TheinsetshowsaWesternblotforVEGFR2fromsamplesofthecellpelletsusedfor
theglobalprofiling.SeesupplementalFig.1forcompletespectraandamoredetaileddescriptionoftheannotations.F,cellsurfaceproteinsfromitraconazole-
treatedHUVECorvehiclecontrolswerebiotinylated,capturedonstreptavidinbeads,andsubjectedtoWesternblotting.Tubulin,acontrolintracellular
protein,waspresentonlyintheinputfraction.Yellowcircles,galactose;greencircles,mannose;bluesquares,N-acetylglucosamine;yellowsquares,N-acetyl-
galactosamine;redtriangles,fucose;purplediamonds,N-acetylneuraminicacid.
DECEMBER23,2011•VOLUME286•NUMBER51 JOURNALOFBIOLOGICALCHEMISTRY 44051
ItraconazoleInhibitsVEGFR2GlycosylationandSignaling
tionpatternwithaneffectiveconcentrationstartingfrom200
nM(Fig.5A).Thiswassimilartotheconcentrationthatinduced
hypoglycosylation of VEGFR2 and the IC for proliferation
50
andtheotheritraconazole-inducedphenotypesinHUVEC.
Next,weexaminedtheeffectsofitraconazoleontheN-gly-
cosylation pattern of another RTK family member, EGFR, in
ACHNcells,whichisarenalcarcinomaline.Inaninitialexper-
iment, we determined whether itraconazole affected ACHN
cellproliferation.Becauseitraconazoleishighlyprotein-bound
inserum,wereasonedthatinthisexperiment,thepotencyof
itraconazole would be serum concentration-dependent and
thereforetestedmediawithboth10and2%serum(Fig.5C).As
expected,ACHNcellswereconsiderablymoresensitivein2%
serum, with an EC of 373 nM. We note that even with 2%
50
serum,ACHNcellsweresignificantlylesssensitivetoitracona-
zolethanHUVEC,asseenpreviouslywithothercelltypes(1).
BecauseHUVECarenormallygrownin2%serum,wechoseto
use the 2% serum medium for the ACHN cell glycosylation
FIGURE4.SubcellularlocalizationofVEGFR2aftertreatmentwithitra-
conazoleorotherglycosylationinhibitors.HUVECweretreatedfor24h experiments. Under these conditions, itraconazole increased
withitraconazole(Ita;800nM)orvehicle(DMSO),fixed,andstainedwithanti- themotilityofEGFRstartingnear500nM,whichissimilarto
VEGFR2andanti-GM130(A),acis-Golgimarker,oranti-PDI(B),anERmarker.
C,cellsweretreatedwith500(cid:4)Mcastanospermine(Cast),500(cid:4)MdMM,50(cid:4)M the EC50 for ACHN cell proliferation, suggesting that the
Sws,800nMitraconazole,orvehicle(DMSO)andtreatedasinA.Representa- migrationshiftmaybemechanisticallylinkedtotheeffectson
tive confocal micrographs are shown. Bars, 20 (cid:4)m. Experiments were cellproliferation(Fig.5B).ItraconazolealsoconferredendoH
repeatedintriplicateforGM130stainingexperimentsandinduplicateforPDI
sensitivitytoEGFR,indicatingthat,likeVEGFR2inHUVEC,
staining.
itraconazolewasabletomodulatetheN-glycosylationofEGFR
thatthedisruptioninVEGFR2traffickingseenafteritracona- inACHNcells(Fig.5D).
zole treatment was probably not the result of impaired GlycosylationInhibitionIsIndependentofCholesterolTraf-
glycosylation. ficking and mTOR Inhibition—We have previously reported
N-Glycosylationisacomplexprocessdependentonmultiple that itraconazole has multiple effects on endothelial cells,
enzymesthatactsequentiallyonglycoproteinsastheytransit includinginhibitionofcholesteroltrafficking(NPCphenotype)
through the secretory system. To better understand at which andmTORinhibition(9).TheIC forHUVECproliferation,
50
stageitraconazolewasarrestingVEGFR2processing,wechar- cholesteroltrafficking,mTORinhibition,andVEGFR2glyco-
acterizedtheN-glycanprofileofitraconazole-treatedHUVEC sylation were all similar, suggesting a common underlining
using MALDI-TOF mass spectrometry (Fig. 3E and supple- mechanism. Thus, we sought to determine the relationship
mental Fig. 1). Itraconazole caused a clear reduction in high betweenVEGFR2hypoglycosylationandtheseotheractivities
molecular weight tetra-antennary and poly-N-acetyllac- of itraconazole. We first determined whether glycosylation
tosamine(poly-LacNAc)-containingN-glycans.Thisisdemon- inhibition in general could affect cholesterol trafficking by
strated by the fact that the most abundant species in the treatingHUVECwithcastanospermine,swainsonine,ordMM
depictedmassrangeafteritraconazoletreatmentwasatrian- andimagingcholesterollocalizationwiththecholesterol-bind-
tennaryspeciesatm/z3055.8comparedwithatetra-antennary ing dye, filipin (Fig. 6A). None of the glycosylation inhibitors
species at m/z 3504.7 without itraconazole treatment. Addi- wasabletoperturbcholesteroltrafficking.Similarly,castano-
tionally, the poly-LacNAc-containing species at m/z 3953.7, spermine,swainsonine,anddMMallfailedtoinhibitmTOR,as
4316.2,4490.0,and4766.1werenolongerobservedafteritra- determinedbythephosphorylationstateofS6kinase,acanon-
conazole treatment. These differences would be associated icalmTORsubstrate(Fig.6B).Wealsotestedwhetherdirect
withadeficiencyinlateN-glycanprocessingstepsandinthe blockade of cholesterol trafficking using U18666A or imip-
caseofthepoly-LacNAc-containingN-glycansspecificallywith ramine,knowninducersoftheNPCphenotype,ormTORinhi-
lackofprocessingbyenzymeslocalizedinthetrans-Golgicom- bition by rapamycin would lead to hypoglycosylation of
partment(37,38).Notably,thesemassspectroscopydataalso VEGFR2(Fig.6C).Noneoftheseagentscouldrecapitulatethe
indicated an increase in the relative abundance of effectscausedbyitraconazole.
Man GlcNAc species,whichisconsistentwiththeeffectsof Becauseitraconazolehaspleiotropiceffects,itwaspossible
5 2
itraconazoleinmacrophages(11). thatanactivityotherthanhypoglycosylationcouldbecontrib-
Itraconazole Inhibits the Glycosylation of EGFR in ACHN uting to the inhibition of VEGFR2 signaling. Thus, we took
CellsandVEGFR1inHUVEC—Theresultsobtainedfromthe advantageofsmallmoleculesthatrecapitulatesubsetsofitra-
global profiling of N-glycans from itraconazole-treated conazole’s activities to test their effects on VEGF-dependent
HUVECsuggestedthattheeffectsonglycosylationarenotlim- activation of VEGFR2. Neither treatment with rapamycin,
ited to VEGFR2. To validate this at the level of an individual U18666A,nordMMcouldmimictheeffectsofitraconazoleon
protein,weexaminedasecondisoformofVEGFRinHUVEC, VEGR2 signaling (Fig. 6D). Thus, of the molecular activities
VEGFR1, and found that itraconazole also affected its migra- associated with itraconazole, induction of hypoglycosylation
44052 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER51•DECEMBER23,2011
ItraconazoleInhibitsVEGFR2GlycosylationandSignaling
FIGURE5.Itraconazole’seffectsonglycosylationextendbeyondVEGFR2andHUVEC.A,HUVECweretreatedwiththeindicateddosesofitraconazole(Ita)
orDMSOvehicle(0(cid:4)Mitraconazoledose),andthemigrationpatternofVEGFR1wasanalyzedbyWesternblot.B,ACHNcellsweretreatedwiththeindicated
concentrationsofitraconazolein2%serum,andEGFRwasanalyzedbyWesternblot.C,theeffectsofitraconazoleontheproliferationoftherenalcell
carcinomaline,ACHN,inthepresenceof2or10%serum.D,lysatesfromitraconazole-treatedACHNcellsweredigestedwitheitherendoHorPNGaseFand
analyzedbyWesternblotforEGFR.Errorbars,S.E.
FIGURE6.Itraconazole’seffectsonVEGFR2glycosylationoccurinparalleltootheritraconazole-inducedeffects.A,cholesterollocalizationwasvisual-
izedbyfilipinstainingofHUVECtreatedwith800nMitraconazole(Ita),500(cid:4)Mcastanospermine(Cast),500(cid:4)MdMM,or50(cid:4)MSws.Bar,20(cid:4)m.B,mTOR
inhibitionwasdeterminedbasedonthephosphorylationstatusofitssubstrateS6kinase(S6K)aftertreatmentwithitraconazole,castanospermine,dMM,or
Sws,asdeterminedbyWesternblot.C,VEGFR2migrationpatternswereanalyzedbyWesternblotinlysatesfromHUVECtreatedfor24hwithitraconazole,the
mTORinhibitorrapamycin,ortwoinhibitorsofcholesteroltrafficking,U18666Aandimipramine.D,activationofVEGFR2phosphorylationbyVEGF after
165
treatmentwiththeindicatedcompoundswasassessedbyWesternblotting.
wasthemostcloselylinkedtotheinhibitionofVEGF-induced CholesterolRepletionRescuesItraconazole-inducedGlycosyl-
VEGFR2 autophosphorylation. Interestingly, treatment with ation and VEGFR2 Signaling Inhibition—In earlier work, we
dMMdidnotblockVEGFR2activation,althoughdMMtreat- foundthattheadditionofcholesteroltoHUVECviaacyclo-
mentalsoleadstohypoglycosylation. dextrincarriercouldrescuetheeffectsofitraconazoleonpro-
DECEMBER23,2011•VOLUME286•NUMBER51 JOURNALOFBIOLOGICALCHEMISTRY 44053
ItraconazoleInhibitsVEGFR2GlycosylationandSignaling
tration(Fig.7A).Themethyl-(cid:3)-cyclodextrin(cid:3)cholesterolrescue
ofVEGFR2wasalsospecifictoitraconazole-inducedglycosyl-
ation changes because dMM-induced hypoglycosylation was
notaffectedbycholesterolsupplementation(Fig.7C).
DISCUSSION
Because itraconazole was found to possess previously
unknownantiangiogenicactivity,ithasbeensubjecttoexten-
sivestudiestodeconvoluteitsmechanismofinhibitionofendo-
thelial cell proliferation and angiogenesis (1, 8, 9). Concur-
rently,ithasenteredmultiplePhaseIIhumanclinicalstudiesas
a novel agent for treating cancer. Although itraconazole has
beenshowntoinhibitmTORandinducecholesteroltrafficking
defects in endothelial cells, how these activities relate to the
overall effects in endothelial cells has remained unknown. In
thiswork,wehaveidentifiedanewmechanismbywhichitra-
conazole may be affecting angiogenesis. We have shown that
itraconazoleiscapableofinducingVEGFR2hypoglycosylation
andthatitstronglyinhibitsVEGFR2autophosphorylationafter
VEGF stimulation. Blockade of VEGF binding, secondary to
VEGFR2 trafficking defects, contributes to this mechanism.
TheeffectsofVEGFR2signalinginhibitionextenddownstream
toPLC(cid:2)1activation.Consistentwithstudiesinmacrophages,
the glycosylation effects we observed were not shared with
other members of the azole class of antifungals or human
14DMinhibitors,therebyinvokingauniquemechanismunre-
latedto14DMinhibition(11).Notably,theseeffectsoccurred
startingat200nM,whichisfarbelowitraconazole’ssteadystate
troughplasmalevel(2.6(cid:4)M)inpatientsonastandard200-mg
oral dosing regimen, suggesting that this mechanism may be
FIGURE7.Itraconazole-induced VEGFR2 hypoglycosylation and sig-
nalinginhibitionisrescuedbysupplementationofcellularcholes- relevant in vivo (SPORANOX(cid:2) (itraconazole) package insert,
terol.A,HUVECweretreatedwithitraconazoleorvehiclealoneinthe PriCara,Raritan,NJ).
presenceoffreecholesterolorfree(cid:3)-estradiol,freemethyl-(cid:3)-cyclodex-
trin,methyl-(cid:3)-cyclodextrin(cid:3)cholesterol,ormethyl-(cid:3)-cyclodextrin(cid:3)(cid:3)-estra- The serendipitous discovery of itraconazole’s effects on
diolcomplexes.VEGFR2migrationpatternsinlysateswereanalyzedby VEGFR2 glycosylation and function makes itraconazole a
Westernblot.B,theabilityofmethyl-(cid:3)-cyclodextrin(cid:3)cholesteroltorescueitra- uniqueantiangiogenicagent.Ithasbeenshownthatinhibition
conazole(Itra)-inducedVEGFR2signalinginhibitioninHUVECafterVEGF
stimulationwasdeterminedbyWesternblot.C,thecapacityofmethyl-1(cid:3)65- ofmTOR,cholesteroltrafficking,orVEGFRsignalingaloneis
cyclodextrin(cid:3)cholesterol to rescue dMM-induced migration changes was sufficienttoblockendothelialcellproliferationand/orangio-
determinedasinA.
genesis (9, 39). Because itraconazole is capable of simultane-
ouslyinhibitingmTORandVEGFR2signaling,itsinvivoeffi-
liferation and mTOR inhibition (9). This suggested that itra- cacyasanantiangiogenicagentmaybesignificantlyenhanced
conazole-inducedcholesterolmislocalizationwastheprimary as a result. Interestingly, the blockade of VEGFR2 signaling
driverofitraconazole’sglobaleffects.However,givenournew after VEGF addition did not extend to the level of ERK1/2,
findingthatcholesteroltraffickingdisruptionbyU18666Aand suggestingthatitraconazolemaybeaffectingonlyasubsetof
imipraminecouldnotinduceVEGFR2hypoglycosylationand VEGF-responsive receptors. Sunitinib, a direct inhibitor of
thatU18666AdidnotinhibitVEGFsignaling,furtherexplora- multiplereceptortyrosinekinases,includingVEGFR1,-2,and
tionoftheinterplaybetweenitraconazole’seffectsandcholes- -3,PDGFR(cid:1),andPDGFR(cid:3),blockedbothPLC(cid:2)1andERKacti-
terolwaswarranted.Wefoundthattheadditionofcholesterol vationinourexperiments(33).Thus,theactivationofERK1/2
via methyl-(cid:3)-cyclodextrin(cid:3)cholesterol complexes, but not byVEGFinthepresenceofitraconazolemaybeduetoother
methyl-(cid:3)-cyclodextrin or free cholesterol alone, could rescue VEGF-binding receptors or receptor complexes on HUVEC,
the itraconazole-induced hypoglycosylation of VEGFR2 and suggesting that VEGFR2 may be preferentially sensitive to
thatmethyl-(cid:3)-cyclodextrin(cid:3)cholesterolcouldalsorescueitra- hypoglycosylation. Alternatively, the residual activation of
conazole-induced VEGFR2 signaling inhibition (Fig. 7, A and VEGFR2afteritraconazoletreatmentmaybesufficienttodrive
B). signalingviaERK1/2.
Importantly, the addition of methyl-(cid:3)-cyclodextrin com- Havingdescribedanewactivityofitraconazoleinendothe-
plexedwith(cid:3)-estradiol,asterolrelatedtocholesterol,hadno lialcells,wenextdeterminedtherelationshipbetweenglyco-
effectonVEGFR2glycosylation,suggestingthatthecholesterol sylationinhibitionandtheknownmoleculareffectsofitracona-
rescuewasprobablynotageneraleffectofeitheramethyl-(cid:3)- zole. We found that the induction of cholesterol trafficking
cyclodextrin(cid:3)sterolcomplexorthetotalcellularsterolconcen- defectsandmTORinhibitionoccurinparalleltothehypogly-
44054 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER51•DECEMBER23,2011