cholesterol regulates vegfr-1 (flt-1) expression …...signaling and regulation cholesterol...

Signaling and Regulation

Cholesterol Regulates VEGFR-1 (FLT-1) Expression andSignaling in Acute Leukemia Cells

Cristina Casalou1,2,3, Ana Costa1,2,3, Tania Carvalho1,2,3, Ana L. Gomes1,2,3, Zhenping Zhu4, Yan Wu4, andS�ergio Dias1,2,3

AbstractVEGF receptors 1 (FLT-1) and 2 (KDR) are expressed on subsets of acute myeloid leukemia (AML) and acute

lymphoid leukemia cells, in which they induce cell survival, proliferation, and migration. However, little is knownabout possible cofactors that regulate VEGF receptor expression and activation on leukemia cells. Here we showthat cholesterol accumulates in leukemia-rich sites within bone marrow of xenotransplanted severe combinedimmunodeficient (SCID) mice. Therefore, we hypothesized that cholesterol-rich domains might regulate FLT-1signaling and chemotaxis of acute leukemias. We then showed that FLT-1 accumulates in discrete cholesterol-richmembrane domains where it associates with caveolin-1 and that placenta growth factor (PlGF)/VEGF stimulationpromotes FLT-1 localization in such cholesterol-rich domains. Accordingly, FLT-1 localization and its phos-phorylation are abrogated bymethyl-b-cyclodextrin (MbCD), which removes cellular cholesterol, and by nystatin,an inhibitor of lipid-raft endocytosis. Mechanistically, cholesterol increases FLT-1 expression and promotes PlGF/VEGF-induced leukemia cells viability and also induces VEGF production by the leukemia cells in vitro. Takentogether, we conclude that cholesterol regulates VEGF:VEGFR-1 signaling on subsets of acute leukemias,modulating cell migration, and viability, which may be crucial for disease progression. Finally, we provideevidence obtained from human AML samples that primary leukemia cells accumulate significantly morecholesterol than do normal cells and that cholesterol accumulation correlates with disease aggressiveness.Mol Cancer Res; 9(2); 215–24. �2011 AACR.

Introduction

Acute leukemia cells have been previously shown toexpress VEGF receptors (VEGFR), which can be stimulatedin a paracrine or autocrine manner, resulting in increasedcell survival, proliferation, and migration (1). VEGF signal-ing through VEGFR-1 (FLT-1) on acute leukemias wasshown to involve p38 and Erk1/2 activation, resulting incaveolae formation (2). Others have shown that VEGFstimulation of subsets of leukemias results in the activationof a downstream signaling pathway which mainly involvesthe activation of the phosphoinositide-3 kinase pathway (3).

Concerning the function of the different VEGF receptorson leukemia cells, we have recently shown that FLT-1mediates leukemia migration within the bone marrowmicroenvironment, promoting leukemia expansion andultimate exit to colonize extramedullary sites (4). Thesefindings led us to hypothesize that other signals within thebone marrow microenvironment might cooperate or pro-mote VEGF signaling on leukemia cells, which, in turn,would contribute toward favoring leukemia migration andinvasion, worsening disease outcome.Plasma membrane lipid-raft domains, which contain high

concentrations of cholesterol and sphingolipids, are knownto function as centers of signaling complexes. The ability oflipid rafts to enhance receptor signaling has led to theconcept of a signalosome, a region where proteins arelocalized together to facilitate receptor signaling. A vastbody of literature is available about the localization ofepidermal growth factor (EGF) receptors in lipid raftsand the subsequent regulatory pathway involving the intra-cellular transport of EGF receptor (5). Much less is knownabout the involvement of membrane-rich lipid domains andVEGF signaling (6). Our recent data showed that inhibitorsof lipid-raft assembly, including nystatin, blocked VEGF-induced leukemia migration (4), which strongly suggestedthat cholesterol-rich domains might in fact regulate VEGFsignaling on malignant cells such as acute leukemias.

Authors' Affiliations: 1Angiogenesis Group, Instituto Portugues de Onco-logia Franscisco Gentil de Lisboa, EPE (CIPM/IPOLFG), Lisbon; 2Neoan-giogenesis Group, Instituto Gulbenkian de Ciencia, Oeiras; 3CEDOC,Faculdade de Ciencias M�edicas, Universidade Nova de Lisboa, Lisbon,Portugal; and 4Imclone Systems, New York.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: S�ergio Dias, Angiogenesis Group, InstitutoPortugues de Oncologia Franscisco Gentil de Lisboa, EPE (CIPM/IPOLFG), Lisbon 1099-023, Portugal. Phone: 351-217229818; Fax: 351-217229895.E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-10-0155

�2011 American Association for Cancer Research.

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Published OnlineFirst January 5, 2011; DOI: 10.1158/1541-7786.MCR-10-0155

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In the present report, we exploit the biochemical path-ways involved in VEGF signaling in acute myeloid leukemia(AML) cells and show that FLT-1 is modulated by cellularcholesterol. We show that FLT-1 colocalizes with mem-brane-rich cholesterol domains, whose assembly is essentialfor FLT-1 expression and activation on leukemia cells.Moreover, cholesterol content of acute leukemia patientsamples correlates with disease aggressiveness. As such, ourdata reveal novel possibilities of therapeutic intervention onsubsets of acute leukemias.

Materials and Methods

Cell cultureHEL, HL60, and 697 cells were obtained from Amer-

ican Type Culture Collection. Cells were maintained inRPMI 1640 medium supplemented with 10% FBS, 100mmol/L L-glutamine, and 1% penicillin-streptomycin(Invitrogen Life Technologies). HUVECs were main-tained in endothelial cell growth medium containing5% FBS (EBM-2; Cambrex, Inc.) at 37�C with humidi-fied 95% air/5% CO2.

Human samplesSixteen patient samples (bone marrow or peripheral

blood) were used and grouped in 4 pediatric AML sam-ples, 2 of them in clinical bone marrow remission; 4 adultsamples I, between 30 and 35 years of age; 7 adult samplesII, between 74 and 79 years of age; 1 in clinical bonemarrow remission. Also, 1 MDS sample was used in theadult sample II group. Mononuclear cells were collectedfrom bone marrow/peripheral blood and isolated usingFicoll/Histopaque gradient. Dry pellets were processed forcholesterol quantification according to Amplex Red kit.

RNA extraction and quantitative real-time PCRLeukemia cells were analyzed for VEGF-1 (FLT-1) by

quantitative real-time PCR (qRT-PCR; ABI PRISM 7700Sequence Detection System and SYBR Green Master MixKit; Applied Biosystems). Total cellular RNA was extractedusing Trizol protocol (Sigma-Aldrich), and cDNA wassynthesized following conventional protocols. The 18S genewas used as a standard reference. The relative expression ofFLT-1 and KDR was obtained using comparative thresholdcycle (CT) method. Primer sequences used were as follows:FLT-1 sense; 50-CCTCGCCGGAAGTTGTAT-30; FLT-1antisense; 50-GTCAAATAGCGAGCAGATTTCTCA-30;KDR sense; 50-ATTCCTCCCCCGCATCA-30; KDRantisense; 50-GCTCGTTGGCGACTCTT-30.

Whole-cell lysate preparation and Western blottingAcute leukemia cells were stimulated for 30 minutes

with recombinant VEGF165 (50 ng/mL), placenta growthfactor (PlGF; 100 ng/mL) and/or treated with for 2 to4 hours with MbCD (10 or 20 mmol/L), cholesterol þMbCD (0.2 or 0.4 mmol/L), and nystatin (50 mg/mL).After stimulation/treatment, total protein extracts wereobtained by suspending cell pellets in cold buffer A

[50 mmol/L Tris-HCl, pH 7.4; 1% (v/v) TritonX-100; 150 mmol/L NaCl; 1 mmol/L EDTA; 0.1%(v/v) SDS], in the presence of protease and phosphataseinhibitors, for 30 minutes on ice followed by centri-fugation at 12,000 � g, for 15 minutes at 4�C. Proteinconcentrations were determined using the Bio-RadLaboratories DC protein assay kit and equal amountswere separated by SDS-PAGE gels, transferred ontonitrocellulose membranes, and processed for Westernblotting. Primary antibodies used were as follows: anti-FLT-1 (0.5 mg/mL; Santa Cruz Biotechnology, Inc.), anti-caveolin-1 (1:100; BD-Biosciences), anti-phosphotyrosine(1:50, 0.5 mg/mL; Santa Cruz Biotechnology, Inc.), anti-b-actin (1:2,500; Sigma-Aldrich). Horseradish peroxi-dase–conjugated secondary antibodies were used at adilution of 1:2,500, and the enhanced chemiluminescencedetection system and Kodak films (Amersham PharmaciaBiotech) were used to visualize the presence of proteins onthe nitrocellulose blots. Bands were quantified usingImage J software (rsb.info.nih.gov/ij/).

Immunoprecipitation assayHEL lysates (900 mg) were precleared (1 hour) with

Protein G-Sepharose beads (Sigma Aldrich) and then incu-bated overnight at 4�C with anti-FLT-1 (1 mg/mL; SantaCruz) or with rabbit IgG as a negative control of thecoimmunoprecipitation procedure. Protein G-Sepharosebeads were then added and mixed for 2 hours at 4�C.Beads were recovered by centrifugation, washed with coldbuffer A or with buffer A supplemented with high saltconcentration (500 mmol/L NaCl), and resuspended in(20 mL) Laemmli buffer. After boiling at 95�C for 5 min-utes, the immunoprecipitates were analyzed by 8% SDS-PAGE gels followed by Western blotting with anti-phosphotyrosine and anti-FLT-1 antibodies (1 mg/mL;Santa Cruz).

Sucrose density centrifugation and isolation of lipid-raft fractionsAcute leukemia cells (HEL; 1 � 108) stimulated for

30 minutes with PlGF (100 ng/mL) or treated for 1 hourwith IR1 inhibitor (2 mmol/L; Calbiochem), MbCD(10 mmol/L), and cholesterol þ MbCD (0.2 mmol/L)were washed with PBS and cell pellets were suspended in1.0 mL of 1% (v/v) Triton X-100 in TNEV buffer[100 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl,5 mmol/L EDTA, 1 mmol/L Na2VO3, 1 mmol/L PMSF,1� protease inhibitors (Roche)] on ice for 60 minutes. Cellswere homogenized with 10 passages through a 22-gaugeneedle and nuclei were removed by centrifugation at 800�g for 8 minutes at 4�C. The supernatants were mixed 1:1 in85% sucrose (v/v)/TNEV buffer. The mixture (2 mL) wastransferred to the bottom of the ultracentrifuge tube, and2 solutions with different sucrose concentrations in TNEVbuffer were added sequentially [6 mL of 35% (v/v) sucroseand 3.5 mL of 5% (v/v) sucrose]. The discontinuousgradients were separated by centrifugation in a swing-outrotor (SW41TI) at 38,000 � g during 18 hours at 4�C in a

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Mol Cancer Res; 9(2) February 2011 Molecular Cancer Research216




Beckman XL-80 Ultracentrifuge. One-milliliter fractionswere collected sequentially from the top to the bottom ofthe tube, and Western blot analyses were done with anti-bodies against FLT-1, caveolin-1. After identification byWestern blot fractions from sucrose gradients (lipid raft, 4and 5; cytosolic fractions, 10 and 11) were concentrated bycentrifugation (4,000 � g for 20 minutes at 4�C), usingAmicon Ultra-4 devices with 10-kDa cutoff membranes(Millipore).

Cell viability and migration assaysCells (1 � 105/mL) were cultured in serum-free RPMI

medium for 48 hours in the presence of PlGF (100 ng/mL),MbCD (10 mmol/L), and cholesterolþMbCD complexes(0.4 mmol/L). When applicable, cells were previouslytreated for 2 hours with 2 mmol/L IR1 (tyrosine kinaseinhibitor of FLT-1; Calbiochem.) or with neutralizingantibodies directed to FLT-1 (1 mg/mL 6.12 antibody;Imclone). Cell viability was determined at 24 and 48 hoursby Trypan Blue exclusion and cell counts (with the aid of ahemocytometer). Each experiment was done in triplicate.Cell migration was assayed using a modified version of aTranswell migration technique described previously (7).Serum-starved cells (1 � 106 cell/mL) were treated for 4or 18 hours with cholesterol þ MbCD. Cell aliquots (100mL) were added to 8-mmpore Transwell inserts of migrationsystem (6.5 mm in diameter; Costar) and allowed to migratefor 4 to 6 hours toward PlGF in the absence/presence ofneutralizing antibodies directed to FLT-1. Cell counts weredone in 7 distinct power fields (20�magnification) with anOlympus CK2 microscope. Experiments were done intriplicate, and results are shown as the number of migratingcells per milliliter.

Human VEGF ELISA and cholesterol measurementVEGF production by leukemia cell lines used was deter-

mined by ELISA. VEGF in serum-free medium was quan-tified using the human VEGF ELISA kit (Calbiochem.).Cellular cholesterol was detected using the Amplex RedCholesterol Assay Kit according to manufacturer's instruc-tions (Molecular Probes).

ImmunofluorescenceLeukemia cells (5� 106) were serum starved for 16 hours

and further attached to poly-L-lysine–coated coverslips for10 minutes at 37�C. After a brief wash in PBS, cells werestimulated with PlGF (100 ng/mL) for 30 minutes and/ortreated with MbCD (10 mmol/L) or cholesterol þ MbCDcomplex (0.2 mmol/L cholesterol) for 2 to 4 hours. Thecells were fixed in 2% (v/v) paraformaldehyde for 10minutes at room temperature, washed twice with PBS,and permeabilized in 0.1% (v/v) Triton X-100 for 30seconds. After blocking in PBS (Invitrogen Life Technol-ogies) supplemented with 0.1% (w/v) BSA, 5% (v/v)complete goat serum, rabbit anti-human FLT-1 (1.5 mg/mL; Santa Cruz Biotechnology, Inc.), and mouse anti-caveolin-1 (1:100; BD biosciences) were used overnightat 4�C. The cells were washed and incubated with Alexa

Fluor 594 or 488 secondary antibodies at 1:500 (MolecularProbes) for 90 minutes and washed with PBS. Samples weremounted in Vectashield and analyzed by confocal micro-scopy in a True Confocal Scanner Leica TCS SP2 (LeicaMicrosystems; objectives HCX PL APOCS 63 � 1.4 oil).Sets of optical sections with 0.5-mm intervals along thez-axis were obtained from the top to the bottom of cells. Z-projections of the acquired images were obtained usingImageJ software (rsb.info.nih.gov/ij/).

Bone marrow smears and Nile Red stainingTen to 12 days after 697 and HL60 cells xenotrans-

plantation, mice were sacrificed and bone marrow [Balb/SCID (severe combined immunodeficient) mice] wasremoved in toto by flushing a femoral cavity. Bone marrowsmears were prepared by streaking the exposed bonemarrow onto a glass slide. Pressure while executing thesmears was adjusted to disperse cells in a monolayerwithout disrupting cells and vascular structure integrity.Bone marrow smears were air dried, fixed in cold acetonefor 10 minutes, and stained with Nile Red for 15 minutes.Images were acquired on a Zeiss Axioplan microscope witha Zeiss Axioxcam MRm (amplification � 200, 630). NileRed solution [1:6 diluted in 75% (v/v) glycerol] wasprepared from a stock Nile Red solution (100 mg/mLin ethanol; N3013; Sigma). Nile Red stained neutral lipids(yellow-gold emission) with an excitation wavelength of450 to 500 nm and polar lipids (red emission) with anexcitation wavelength of 515 to 560 nm (8).

Eletrophoretic mobility shift assayNuclear extraction and electrophoretic gel mobility

shift assays were conducted following standard methodo-logy as described elsewhere (9). Briefly, oligonucleotideprobe (sequence: ACCCCTTGAGTCACCAGAAGG)was labeled with [g32-P]ATP, using T4 polynucleotidekinase (Promega), and purified in Micro-spin G-50 col-umns (Bio-Rad). For the eletrophoretic mobility shift assay(EMSA) analysis, 10 mg of nuclear proteins was preincu-bated with EMSA binding buffer (Promega) and 15 ng/mLpoly(dI)-poly(dC) at room temperature 10 minutes beforethe addition of the radiolabeled oligonucleotide for anadditional 25 minutes. For Supershift studies, before theaddition of the radiolabeled probe, samples were incubatedfor 30 minutes with 4 mg of CREB-1 antibody (H-74; SantaCruz).

Statistical analysisResults are expressed as mean � SD. Data were analyzed

using the unpaired 2-tailed Student t test. The values ofP < 0.05 were considered significant.

Results

Acute leukemia cells localize in cholesterol-rich nichesin the bone marrow microenvironment in vivoWe have previously observed that acute leukemia cells

migrate in an FLT-1 dependent manner within the bone

FLT-1 Signaling Is Modulated by Cellular Cholesterol

www.aacrjournals.org Mol Cancer Res; 9(2) February 2011 217




marrow microenvironment toward the epiphysis, en route tocolonize other organs (4). In the present study, we detectedspecific cholesterol accumulations in leukemia-rich bonemarrow sites in vivo. For this, we stained smears of bonemarrow collected on days 10 to 12 after mice inoculationwith leukemia cells, together with Nile Red, to detect intra-cellular lipids (8, 10). We observed major accumulations oflipids (neutral lipids, i.e., cholesterol esters and polar lipids)around bone marrow sinusoids, where leukemia cells alsotend to accumulate (Fig. 1). These data suggest that leukemiacell migration within the bone marrow microenvironmentresults in their accumulation in cholesterol-rich areas.

Avidity for cholesterol of leukemia cellsWe compared cholesterol content of mononucleated cells

(MNC) isolated from healthy donors with MNCs obtainedfrom AML patients (2A). Patient samples were grouped intopediatric samples, adult sample I (AML patients between 30and 35 years of age), and adult sample II (AML patients

between 64 and 79 years of age). In peripheral blood AMLpatient samples (AML8 and 15), the levels of intracellularcholesterol were increased by 2-, 4- to 3-, and 4-fold inrelation to healthy donor samples. AML patients displayincreased intracellular cholesterol, in particular olderpatients with 4- to 6-fold increase (adult samples II). Afterclinical bone marrow remission, intracellular cholesterollevels decreased to levels compared with that obtained fromhealthy donors (AML3, 4, and 16-BM R). Also, MDSsample (patient with myelodysplasic syndrome, MDS) haslower levels of cholesterol. Furthermore, pediatric AMLsamples present 3- to 4-fold increased levels of cellularcholesterol. In Figure 2B, AML cells (HEL and HL60 celllines) showed 2- to 4-fold significant increase of cellularcholesterol (controls 1 and 2, respectively) when comparedwith cells isolated from healthy donors. Next, we disturbedcholesterol homeostasis of leukemia cells by exposure for 4hours to 10 mmol/L methyl-b-cyclodextrin (MbCD),which depletes cholesterol from cellular membranes, orby increasing its cellular cholesterol levels with the use ofcholesterol þMbCD complexes (0.4 mmol/L). CholesterolþMbCD complex treatment increased by 1-, 5- to 2-, and3-fold cellular cholesterol of leukemia cells when comparedwith untreated cells. This increase was abolished by bothMbCD and nystatin treatments, which inhibit lipid-raftendocytosis. In addition, PlGF treatment also increasedintracellular cholesterol of AML cells, an effect that wasreverted to control levels (control 2; Fig. 2B) by the use of aneutralizing antibody (Ab) against FLT-1 (6.12 Ab; P <0,02; Imclone). Acute leukemia cells possess more intracel-lular cholesterol than normal cells. Furthermore, FLT-1activation by PlGF further potentiates this effect. In addi-tion, cellular cholesterol is highly increased in AMLpatients, an effect that was reverted after clinical bonemarrow remission.

PlGF induces FLT-1 accumulation and colocalizationwith caveolin-1 in lipid raftsWehave previously reported that FLT-1 associates in vitro

with caveolin-1, the main component of a subtype of lipidrafts (caveolae), in AML cells (2). This suggested that FLT-1–mediated signaling in acute leukemia cells might dependon cholesterol-raft membrane domains. Therefore, in thepresent study, we determined a more precise membranelocation of FLT-1 on leukemia cells and examinedwhether itwas affected by PlGF stimulation and/or cholesterol distur-bance. We isolated lipid rafts by sucrose density gradients,and this analysis revealed that FLT-1 and caveolin-1 colo-calize in 2 distinct regions of the sucrose gradients: a caveolin-enriched membrane region (fractions 4 and 5; 6%–30%sucrose) and a cytosolic region (fractions 10–11; 35%sucrose), as assessed by caveolin-1 (a component of lipidrafts) co-sedimentation (Fig. 3A). On PlGF/VEGF stimula-tion, FLT-1 was localized preferentially into lipid-raft frac-tions (fractions 4 and 5), an effect that was reverted in thepresence of the FLT-1 inhibitor IR1 (Fig. 3A). Also, inhibi-tion of FLT-1 by IR1 treatment, removes caveolin-1 fromthe lipid-raft fractions. Using confocal microscopy, we

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Figure 1. Evidence for colocalization of acute leukemia cells incholesterol-rich bone marrow niches in vivo. Leukemia cells tend to gatheraround bone marrow sinusoids, where major lipid accumulation wasdetected (limited by the intermittent line). Nile Red fluorescence arisingfrom the dye–lipid interaction was selectively measured using anexcitation wavelength of 450 to 500 nm for neutral lipids (yellow-goldemission; B and F) and 515 to 560 nm for polar lipids (red emission; C andG). These results were obtained from 3 independent experiments and arerepresentative of 4 recipients. Images were processed with AdobePhotoshop 7.0 Software. DAPI, 40,6-diamidino-2-phenylindole.

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observed that FLT-1 (in red) and caveolin-1 (in green)colocalized in lipid-raft/caveolin-1–rich structures(Fig. 3B; see FLT-1 antibody specificity in SupplementaryFig. 1). To determine whether FLT-1 distribution in lipidrafts was affected by cholesterol disturbance, sucrose gradi-ent–generated lipid-raft fractions 4 and 5 and cytosolicfractions 10 and 11 were concentrated and analyzed by

Western blot as before. As shown in Figure 3C, cholesterolenrichment accumulated FLT-1 in lipid-raft fractions,whereas MbCD extracted FLT-1 from lipid-raft fractions(see quantification in Fig. 3D). b-Actin was used as a loadingcontrol (Fig. 3C). As determined by confocal microscopy,after cholesterol enrichment, FLT-1 colocalized with caveo-lin-1 whereas cholesterol extraction with MbCD reduced

Figure 2. Acute leukemia cellspossess high avidity forcholesterol. A, AML primary cellsdisplay high levels of intracellularcholesterol as compared withcholesterol content in cellsobtained from healthy donors, asmeasured by the Amplex RedCholesterol Assay Kit.Furthermore, intracellularcholesterol content correlateswith the aggressiveness of thedisease. HD1/2, healthy donorsample; AML, BM AML samples;AML-PB, PB AML samples; AML-BM R, BM AML in clinicalremission samples; MDS,myelodysplasic syndrome. B,acute leukemia cells possesshigher levels of intracellularcholesterol, an effect potentiatedby FLT-1 activation by PlGF.Leukemia cell lines (HEL andHL60) were exposed to 0.2 mmol/Lcholesterol complex or to MbCDalone (10 mmol/L) for 4 hours andcellular cholesterol was quantifiedusing the Amplex Red Kit. Errorbars, standard errors of 2independent experiments.

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this colocalization (Fig. 3E). These results indicating thepreferential localization of FLT-1 in lipid rafts after intra-cellular cholesterol enrichment suggest that FLT-1–raftinteractions may regulate PlGF/VEGF-mediated signaling.

Cholesterol enrichment of acute leukemia cells inducesFLT-1 activation in lipid raftsIn AML cells, FLT-1 is phosphorylated by PlGF/VEGF

treatment for 30 minutes (Fig. 4A), as assessed by coim-munoprecipitation of FLT-1 with pan-phosphotyrosineantibody. We determined the consequences of disturbingcholesterol homeostasis for FLT-1 activation on AMLcells. We observed that cholesterol extraction abolishesFLT-1 phosphorylation induced by PlGF in a dosedependent manner. In contrast, cholesterol enrichment

increased FLT-1 phosphorylation (activation) in theabsence or presence of PlGF (Fig. 4B). FLT-1 activationwas inhibited by the use of nystatin, an inhibitor of lipid-raft formation and endocytosis, which impeded PlGF-induced FLT-1 phosphorylation (Fig. 4C). b-Actin wasalways used as loading control. Taken together, these datasuggest that FLT-1 activation (phosphorylation) isaffected by cellular cholesterol levels and, in particular,by agents that perturb the formation of lipid-rafts/cho-lesterol-rich membrane domains.

Cholesterol enrichment modulates PlGF-induced AMLcells propertiesTo evaluate the role of cholesterol in acute leukemia cells

properties, we assessed cell viability, migration, and VEGF

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Figure 3.Cholesterol enrichment of acute leukemia cells recruits FLT-1 to lipid-raft/caveolin-1–rich domains. A, extracts from AML cells stimulated with PlGF,control cells, or cells treated with FLT-1 tyrosine kinase inhibitor (IR1) were separated by sucrose gradient fractionation and analyzed byWestern blot for FLT-1and caveolin-1 distribution. FLT-1 was mainly localized in caveolin-1–enriched fractions (4 and 5) on PlGF stimulation, an effect reverted by FLT-1 tyrosinekinase inhibition. B, colocalization of FLT-1 with caveolin-1 was also observed by confocal microscopy. C, after AML exposure to cholesterol-disturbingagents, cell extracts were fractionated by sucrose gradients and fractions enriched for lipid raft/caveolin-1 (fractions 4 and 5) and cytosolic (fractions 10 and 11)were concentrated using Amicon ultradevices and analyzed by SDS-PAGE. Results are representative of 3 independent experiments. D, the bands obtainedvia Western blot for FLT-1 distribution on cholesterol disturbance (C) were quantified with ImageJ software-based analysis (http://rsb.info.nih.gov/ij/). E,extraction of FLT-1 from caveolin-1–enriched rafts was observed after MbCD treatment, whereas the reverse effect was observed with cholesterol þ MbCDcell treatment (see confocal microscopic analysis). Scale bars, 10 mm. WB, Western blotting.

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production on cholesterol-enriched or -depleted cells,alone or in the presence of PlGF. Viability was assessedafter 24 to 48 hours by counting cell viability withTrypan Blue exclusion dye. As previously reported,PlGF induced a modest increase in AML cells viability.However, cholesterol enrichment by leukemia cellsincreased cell viability after 24 hours; interestingly,cotreatment with PlGF induced a further increase inleukemia cell viability (Fig. 5A). In contrast, PlGF-induced cell viability was significantly reduced byMbCD treatment. Cholesterol enrichment of AML cellsfor 4 to 16 hours increased their chemotactic (migra-tory) response toward PlGF (Fig. 5B), an effect that wasreverted in the presence of FLT-1 neutralizing agents. Inaddition, cholesterol enrichment also increased VEGFproduction by AML cells in vitro (see SupplementaryFig. 2). Taken together, these data suggest that choles-terol cellular levels affect FLT-1–mediated increase incell viability, chemotaxis, and VEGF production.

FLT-1 expression on AML cells increases on cholesterolexposureBesides its effects in the regulation of FLT-1 activation

and subcellular localization, next we examined whethercholesterol levels also affected FLT-1 expression. In fact,cholesterol enrichment upregulates FLT-1 mRNA expres-sion in leukemia cells (Fig. 6A). PlGF further increasedFLT-1 expression in cholesterol-enriched cells, an effectthat was reverted by the FLT-1 inhibitors. This effect onFLT-1 mRNA expression is characteristic of leukemiacells, as FLT-1 mRNA expression remains unaltered bycholesterol disturbances in human umbilical veinendothelial cells (HUVEC; Fig. 6B). It was previouslyreported that CREB (cAMP responsive element bindingprotein)/ATF (activating transcription factor) elementregulates the basal transcription of FLT-1 expression(11). Moreover, we observed that increasing intracellularcholesterol levels in AML cells further potentiate thebinding of CREB complexes to the FLT-1 promoter

Figure 4. Cholesterol enrichmentpromotes FLT-1 signaling in lipidrafts. FLT-1 receptor activationwas tested by the addition ofPlGF, MbCD, cholesterol þMbCD, and nystatin as indicated.Total cell extracts were analyzedusing anti-phosphotyrosine,anti-FLT-1, and anti-b-actinantibodies. A, immunoprecipitation(IP) of FLT-1 in AML lysatesshowed that PlGF/VEGFtreatment for 30 minutesactivates FLT-1 receptor. B,leukemia cell treatment withMbCD abolishes FLT-1phosphorylation mediated byVEGF/PlGF. In contrast, AMLintracellular cholesterol increaseactivates FLT-1. C, inhibition oflipid-raft formation/endocytosisby nystatin impedes FLT-1activation. Autoradiographs arerepresentative of similar resultsobtained from 3 independentexperiments. WB, Westernblotting.

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region, thereby regulating FLT-1 expression (see Supple-mentary Fig. 3). In contrast, KDR mRNA expression wasnot altered by increasing cellular cholesterol in leukemiacells (Fig. 6C).

Discussion

In this report, we reveal novel molecular evidence bywhich cellular cholesterol regulates the function of areceptor tyrosine kinase on malignant leukemia cells.In detail, we show that cholesterol affects FLT-1 expres-sion, localization, and activation, thereby modulatingcellular phenotypes including viability, chemotaxis/

migration, and VEGF production. The mechanisms bywhich cholesterol interferes with FLT-1 signaling andcellular localization are still not completely understoodbut may involve the intracellular transport machineryand the regulation/assembly of specific membranedomains, including vesicles and lipid rafts. Other recep-tor tyrosine kinases, most notoriously EGF receptor,have been shown to be transported intracellularly invesicles, in and out of the cell onto the cell nucleuswhere EGF receptor is shown to activate transcription(5). Whether FLT-1 recycling involves a similar mechan-ism of intracellular transport is still undisclosed. Never-theless, the present report reveals that FLT-1 localizespreferentially in specific lipid-enriched membrane

10

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0Mig

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umbe

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L x

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Via

ble

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(x

104

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hpf

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P = 0.002P = 0.01

P = 0.08P = 0.01

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B

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Control Cholesterol+MβCD

Cholesterol + MβCD

6.12

IR1

MβCD MβCD+PIGFCholesterol+MβCD+PIGF

PIGF

PIGF

Figure 5. Cholesterol enrichmentpromotes PLGF/VEGF-inducedleukemia cell viability andmigration. A, viability of AML cellswas assessed for 48 hours byTrypan Blue exclusion dye.Cholesterol þ MbCD actedsynergistically with PlGF inducingsignificantly (P ¼ 0.002) cellviability, whereas cholesterolextraction by MbCD cell treatmenthad the opposite effect (P ¼ 0.01).The chemotactic capacity ofleukemia cells toward PlGF wasevaluated using a Transwellmigration system after theirtreatment with cholesterol þMbCD complexes (4–16 hours).B, A significant increase in cellmigration was obtained, an effectreverted by FLT-1 signalingblockage (6.12 neutralizingantibody, P < 0.01; tyrosine kinaseinhibitor IR1,P¼ 0.08). Results arerepresentative of 3 independentexperiments. Error bars, SEM.

Casalou et al.





domains. Detailed biochemical analysis of cellularextracts, together with the use of biochemical inhibitors,suggests that these membrane domains may be consid-ered lipid rafts. As such, our data strongly suggest thatlipid rafts are essential for VEGF receptor signaling onmalignant leukemia cells.We have previously shown that subsets of acute

leukemias respond to VEGF/PlGF gradients withinthe bone marrow microenvironment, migrating towardthe epiphysis of long bones, from where the leukemiacells leave the bone marrow onto the peripheral blood,en route to other target organs (4). From these findings,it became clear that leukemia cells migrate towardspecific "niches" within the bone marrow microenviron-ment. Recent studies have suggested that leukemia cells"create" specific niches within the bone marrow micro-environment, where the malignant cells thrive and pro-liferate, eventually replacing the normal hematopoieticelements (12). The signals/molecular cues involved inthis "invasion" of the bone marrow by malignant cellsare still largely undisclosed but are believed to involve

SDF-1:CXCR4 and possibly integrin-mediated signaling(13–15).Our present report suggests that bone marrow choles-

terol levels and the cholesterol distribution throughout thebone marrow may play an important part during leukemiaengraftment, expansion, and perhaps also during leukemiaspread. In detail, we provide evidence that PlGF/VEGFsignaling via FLT-1 is affected by cellular cholesterol levelsand that specific cholesterol accumulations are seen inleukemia-rich sites in the bone marrow, in vivo. Interest-ingly, we have recently discovered that systemic choles-terol levels also affect the levels of SDF-1 in the bonemarrow microenvironment (16). This strongly suggeststhat cholesterol may affect leukemia engraftment andexpansion by interfering with SDF-1:CXCR4 signalingand VEGF:FLT-1 signaling, respectively.A link between cholesterol avidity and acute leukemias

has been previously suggested. In our present study, AMLpatient samples show increased levels of intracellular cho-lesterol compared with healthy donor samples. Moreover,the bone marrow cholesterol content correlates with disease

Figure 6. Acute leukemia cellexposure to cholesterolupregulates FLT-1 expression. A,as determined by real-time PCR,FLT-1 expression after AML celltreatment with cholesterol þMbCD complexes (4 hours) in thepresence/absence of PlGF issignificantly upregulated (withoutPlGF, P ¼ 0.048; with PlGF,P < 0.025). Inhibition of FLT-1signaling (6.12 neutralizingantibody; IR1 chemical inhibitor)significantly abolishes theupregulated FLT-1 expressionobtained after cholesterol cellexposure (P < 0.03). In contrast,FLT-1 mRNA expression was notaffected by cellular cholesteroldisturbance in HUVECs. B, KDR(also expressed in these cells)mRNA expression remainsunaltered by enriched cellularcholesterol. C, experiments weredone in triplicate and representedas the mean (n ¼ 3).

Control Cholesterol+MβCD

MβCD

3

2.5

2

1.5

1

0.5

0

A

B C

1.4

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-1/1

8S m

RN

A (

rela

tive

units

)

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-1/1

8S m

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A o

ver

cont

rol (

rela

tive

units

)

KD

R/1

8S m

RN

A o

ver

cont

rol (

rela

tive

units

)

Cholesterol+MβCD+PIGF

Cholesterol+MβCD

+IR1

Cholesterol+MβCD+6.12

PIGF

Cholesterol+MβCD

MβCDPIGF Cholesterol+MβCD

MβCDPIGF

P < 0.025 P < 0.03

P < 0.048






aggressiveness (and stage); leukemia patients undergoingclinical remission show a corresponding decrease in intra-cellular cholesterol levels. Other studies have reported thatcholesterol uptake by leukemia cells promotes their sur-vival and resistance to chemotherapy (17, 18). Thesestudies led to the use of statins (cholesterol-loweringagents) as therapeutic targets for subsets of acute leuke-mias (19, 20), with reported clinical activity and efficacy.Nevertheless, to our knowledge, there have been noreports explaining the importance of cholesterol inVEGF-mediated signaling on acute leukemia cells. Alltogether, the findings reported in this article have impli-cations for the understanding of the regulation of the bonemarrow microenvironment during the onset/engraftmentand expansion/progression of acute leukemias. The bio-chemical mechanisms described here, showing that lipid-enriched membrane domains regulate VEGFR signaling,may be relevant also in the context of other receptortyrosine kinases and other tumor types.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank other members of the Angiogenesis Laboratory for usefuldiscussions and for critically reading the manuscript.

Grant Support

This study was supported by GlaxoSmithKline. C. Casalou, A. Costa, A.L.Gomes, and T. Carvalho are recipients of FCT (Portuguese Government)fellowships.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 13, 2010; revised November 27, 2010; accepted December 20,2010; published OnlineFirst January 5, 2011.

References1. Hicklin DJ, Ellis LM. Role of vascular endothelial growth factor path-

way in tumor growth and angiogenesis. J Clin Oncol 2005;23:1011–27.

2. Casalou C, Fragoso R, Nunes JFM, Dias S. VEGF/PLGF inducesleukemia cell migration via P38/ERK1/2 kinase pathway, resulting inRho GTPases activation and caveolae formation. Leukemia2007;21:1590–4.

3. Fragoso R, Elias AP, Dias S. Autocrine VEGF loops, signaling path-ways, and acute leukemia regulation. Leuk Lymphoma2007;48:481–8.

4. Fragoso R, Pereira T, Wu Y, Zhu Z, Cabecadas J, Dias S. VEGFR-1(FLT-1) activation modulates acute lymphoblastic leukemia localiza-tion and survival within the bone marrow, determining the onset ofextramedullary disease. Blood 2006;107:1608–16.

5. Patra SK. Dissecting lipid raft facilitated cell signaling pathways incancer. Biochem Biophys Acta 2008;1785:182–206.

6. Labrecque L, Royal I, Surprenant DS, Patterson C, Gingras D,B�eliveau R. Regulation of vascular endothelial growth factor recep-tor-2 activity by caveolin-1 and plasma membrane cholesterol. MolBiol Cell 2003;14:334–47.

7. Hamada T, Mohle R, Hesselgesser J, Hoxie J, Nachman RL, MooreMA, Rafii S, et al. Transendothelial migration of megakaryocytes inresponse to stromal cell-derived factors (SDF-1) enhances plateletproduction. J EXP Med 1998:188:539–48.

8. Greenspan P, Fowler SD. Spectrofluorometric studies of the lipidprobe, Nile Red. J Lipid Res 1985;26:781–9.

9. Santos SC, Dias S. Internal and external autocrine VEGF/KDR loopsregulate survival of subsets of acute leukemia through distinct signal-ing pathways. Blood 2004;103:3883–9.

10. Diaz G, Melis M, Batetta B, Angius F, Falchi AM. Hydrophobiccharacterization of intracellular lipids in situ by Nile Red/Yellow emis-sion ratio. Micron 2008;39:819–24.

11. Morishita K, Johnson DE, Williams LT. A novel promoter for vascularendothelial growth factor receptor (flt-1) that confers endothelial-specific gene expression. J Bio Chem 1995;270:27948–53.

12. Colmone A, Amorim M, Pontier AL, Wang S, Jacblonski E, SipkinsDA. Leukemic cells create bone marrow niches that disrupt the

behavior of normal hematopoietic progenitor cells. Science2008;322:1861–5.

13. Peled A, Kollet O, Ponomaryov T, Petit I, Franitza S, Grabovsky V, et al.The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34(þ) cells: role in transendothelial/stromalmigration and engraftment of NOD/SCID mice. Blood 2000;95:3289–96.

14. Kijowski J, Baj-Krzyworzeka M, Majka M, Reca R, Marquez LA,Christofidou-Solomidou M, et al. The SDF-1-CXCR4 axis stimulatesVEGF secretion and activates integrins but does not affect prolifera-tion and survival in lymphohematopoietic cells. Stem Cells2001;19:453–66.

15. Hidalgo A, Sanz-Rodriguez F, Rodriguez-Fernandez JL, Albella B,Blaya C,Wright N, et al. Chemokine stromal cell-derived factor-1alphamodulates VLA-4 integrin-dependent adhesion to fibronectin andVCAM-1 on bone marrow hematopoietic progenitor cells. Exp Hema-tol 2001;29:345–55.

16. Gomes AL, Carvalho T, Serpa J, Torre C, Dias S. Hypercholester-olemia promotes bone marrow cell mobilization by perturbing theSDF-1:CXCR4 axis. Blood 2010. [Epub ahead of print].

17. Li HY, Appelbaum FR,Willman CL, Zager RA, Banker DE. Cholesterol-modulating agents kill acute myeloid leukemia cells and sensitizethem to therapeutics by blocking adaptive cholesterol responses.Blood 2003;101:3628–34.

18. Banker DE, Mayer SJ, Li HY, Willman CL, Appelbaum FR, Zager RA.Cholesterol synthesis and import contribute to protective choles-terol increments in acute myeloid leukemia cells. Blood2004;104:1816–24.

19. Sassano A, Katsoulidis E, Antico G, Altman JK, Redig AJ, Minucci S,et al. Suppressive effects of statins on acute promyelocytic leukemiacells. Cancer Res 2007;67:4524–32.

20. Kornblau SM, Banker DE, Stirewalt D, Shen D, Lemker E, Verstov-sek S, et al. Blockade of adaptive defensive changes in cholesteroluptake and synthesis in AML by the addition of pravastatin toidarubicin þ high-dose Ara-C: a phase 1 study. Blood 2007;109:2999–3006.

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2011;9:215-224. Published OnlineFirst January 5, 2011.Mol Cancer Res Cristina Casalou, Ana Costa, Tânia Carvalho, et al. in Acute Leukemia CellsCholesterol Regulates VEGFR-1 (FLT-1) Expression and Signaling

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