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Human Cancer Biology

HIF-1aofBoneMarrowEndothelial Cells ImpliesRelapseandDrug Resistance in Patients with Multiple Myeloma and MayAct as a Therapeutic Target

Roberto Ria1, IvanaCatacchio1, Simona Berardi1, Annunziata De Luisi1, Antonella Caivano5, Claudia Piccoli5,6,Vitalba Ruggieri5, Maria Antonia Frassanito1, Domenico Ribatti2, Beatrice Nico3, Tiziana Annese3,Simona Ruggieri3, Attilio Guarini4, Carla Minoia4, Paolo Ditonno7, Emanuele Angelucci8, Daniele Derudas8,Michele Moschetta1, Franco Dammacco1, and Angelo Vacca1

AbstractPurpose: To investigate the role of hypoxia-inducible factor-1a (HIF-1a) in angiogenesis and drug

resistance of bone marrow endothelial cells of patients with multiple myeloma.

Experimental Design: HIF-1a mRNA and protein were evaluated in patients with multiple myeloma

endothelial cells (MMEC) at diagnosis, at relapse after bortezomib- or lenalidomide-based therapies or

on refractory phase to these drugs, at remission; in endothelial cells of patients with monoclonal

gammapathies of undetermined significance (MGUS; MGECs), and of those with benign anemia (con-

trols). The effects of HIF-1a inhibition by siRNA or panobinostat (an indirect HIF-1a inhibitor) on the

expression of HIF-1a proangiogenic targets, on MMEC angiogenic activities in vitro and in vivo, and on

overcoming MMEC resistance to bortezomib and lenalidomide were studied. The overall survival of the

patients was also observed.

Results: Compared with the other endothelial cell types, only MMECs from 45% of relapsed/refractory

patients showed a normoxic HIF-1a protein stabilization and activation that were induced by reactive

oxygen species (ROS). The HIF-1a protein correlated with the expression of its proangiogenic targets. The

HIF-1a inhibition by either siRNA or panobinostat impaired the MMECs angiogenesis–related functions

both in vitro and in vivo and restored MMEC sensitivity to bortezomib and lenalidomide. Patients with

MMECs expressing the HIF-1a protein had shorter overall survival.

Conclusions: The HIF-1a protein in MMECs may induce angiogenesis and resistance to bortezomib

and lenalidomide and may be a plausible target for the antiangiogenic management of patients with

well-defined relapsed/refractory multiple myeloma. It may also have prognostic significance. Clin Cancer

Res; 20(4); 847–58. �2013 AACR.

IntroductionAngiogenesis plays a critical role in the pathophysiology

and progression of multiple myeloma because it supportsthe growth and survival of plasma cells (1). Hypoxia is amajor angiogenic stimulus (2), and hypoxia-inducible fac-tor-1 (HIF-1) is themaster regulator of the cellular responseto hypoxia (3). HIF-1 is a heterodimeric transcription factorcomposed of a constitutively expressed subunit b (HIF-1b)and an oxygen-regulated subunit a (HIF-1a; ref. 3). Undernormoxia, HIF-1a is unstable and rapidly degraded via theVon Hippel–Lindau (VHL)-mediated ubiquitin–protea-some pathway. Under hypoxia, it escapes the VHL bindingand proteasomal degradation, translocates to the nucleus,heterodimerizes with HIF-1b, and induces transcriptionof numerous target genes, whose products are involvedin cell migration, vascular remodeling, and angiogenesis(3). Under normoxia, HIF-1a may also be activated inresponse to growth factors, cytokines, and peptide media-tors whose binding to their receptor tyrosine kinasesactivates the phosphatidylinositol 3-kinase/protein kinase

Authors' Affiliations: 1Section of Internal Medicine, Department ofBiomedical Sciences and Human Oncology; 2Department of BasicMedical Sciences, Neurosciences and Sensory Organs, University ofBari Medical School, Bari, Italy, National Cancer Institute "GiovanniPaolo II", Bari, Italy; 3Department of Human Anatomy, Histology andEmbryology, and Pathological Anatomy, University of Bari MedicalSchool; 4Hematology Unit, Istituto di Ricerca e Cura a Carattere Scien-tifico (IRCCS) Oncologic Hospital; 5Laboratory of Preclinical and Trans-lational Research, IRCCS Basilicata Cancer Reference Centre, Potenza;6Department of Clinical and Experimental Medicine, University of FoggiaMedical School, Foggia; 7Hematology Unit, Ospedale Di Venere, Car-bonara di Bari, Bari; and 8Department of Haematology, Businco Hos-pital, Cagliari, Italy

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

R. Ria and I. Catacchio contributed equally to this work.

Corresponding Author: Angelo Vacca, Department of BiomedicalSciences andHumanOncology, Section of Internal Medicine "G. Baccelli,"University of Bari "Aldo Moro" Medical School, Policlinico, Piazza GiulioCesare, 11, I-70124 Bari, Italy. Phone: 39-080-5478057; Fax: 39-080-5478895; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-1950

�2013 American Association for Cancer Research.

ClinicalCancer

Research

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on August 30, 2021. © 2014 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst December 2, 2013; DOI: 10.1158/1078-0432.CCR-13-1950

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B/mammalian target of the rapamycin (PI3K/AKT/mTOR)pathway, which stimulates the HIF-1a expression (4). Alsounder normoxia, reactive oxygen species (ROS) can activateHIF-1a, thus stimulating its transcriptional activity (5).HIF-1a overexpression has been detected in several humantumors (6) as positively related to growth, angiogenesis (7),chemoresistance (8), and poor prognosis (9).

In multiple myeloma, the expression of HIF-1a has beenfound in both cell lines and primary plasma cells in nor-moxic conditions (10, 11), in which it induces the expres-sion of VEGF (12) and other angiogenic cytokines (13).Because of the central role ofHIF-1a in tumor aggressivenessand progression, several strategies have been adopted toinhibit its expression and transcriptional activity (4). His-tone deacetylase inhibitors (HDACI) are attractive anti-mul-tiple myeloma agents (14) that suppress the growth andsurvival of plasma cells in vitro (15), and are currently beingused in clinical trials (16). HDACIs induce degradationof HIF-1a independently of the VHL function (17), andrepress the transactivation potential of HIFs (18).

Although HIF-1a has already been studied in multiplemyeloma plasma cells as an angiogenic factor (12, 13), nostudies on its expression and function in bone marrowmultiple myeloma endothelial cells (MMECs) of patientsat different disease phases and in endothelial cells ofpatients with monoclonal gammapathies of undeterminedsignificance (MGUS; MGECs) have been carried out. Here,we show that HIF-1a protein is constitutively expressed andactivated in MMECs from a well-defined proportion ofrelapsed/refractory patients, and that it confers resistanceto antiangiogenesis mediated by bortezomib or lenalido-mide. Inhibition of HIF-1a has antiangiogenic power andovercomes the drug resistance. HIF-1a may thus be envis-aged as a new target for antiangiogenic management of afraction of patients with relapsed/refractory multiple mye-

loma. Because MMECs expressing the HIF-1a proteinimplied shorter overall survival (OS), these cells may serveas a prognostic marker.

Materials and MethodsPatients and endothelial cells

Patients fulfilling the International Myeloma WorkingGroup diagnostic criteria (19) for multiple myeloma (n ¼76) and MGUS (n ¼ 35) were studied. The patients withmultiple myeloma (52 male and 24 female), ages 45 to 82(median 63.5) years, were at diagnosis (n¼18), at completeremission (n ¼ 16), at relapse after bortezomib- or lenali-domide-based chemotherapies (n ¼ 20), or on refractoryphase to these drugs (n ¼ 22). The M component was IgG

(n ¼ 46), IgA (n ¼ 20), and k or l (n ¼ 10). The MGUSpatients (23 male and 12 female), ages 42 to 79 (median

60.5) years, were IgG (n¼ 22), IgA (n¼ 8), and k or l (n¼5). Normal (control) endothelial cells were derived from12subjects with anemia due to iron or vitamin B12 deficiency(20). The study was approved by the Ethics Committee ofthe University of Bari Medical School (Bari, Italy), and allpatients provided their informed consent in accordancewith the Declaration of Helsinki. Bone marrow primaryMMECs, MGECs, and normal endothelial cells wereobtained and cultured as described previously (21). Bonemarrow derived primary macrophages from MGUS andmultiple myeloma patients at different disease phases wereobtained as previously described (22).

Reverse transcriptase PCR, real-time RT-PCR, andWestern blot analysis

Reverse transcriptase PCR (RT-PCR) and real-time RT-PCR were performed with the primers shown in Supple-mentary Table S1 (Invitrogen) and the Applied Biosystemsmethodology (23). The PCR products were separated byelectrophoresis on 1.5% agarose gels and stained withethidium bromide. The mRNA level was measured with thecomparative threshold cycle (Ct) method using b-actin as

the reference and the 2�DDCt formula (24). Total proteinlysates (50 mg) from MMECs, MGECs, and normal endo-thelial cells were immunoblotted with anti-HIF-1a (BDBiosciences), anti-b-actin (Sigma-Aldrich), anti-VEGF recep-tor 2 (VEGFR-2; Cell Signaling Technology), anti-fibroblastgrowth factor receptor-2 (FGFR-2), anti-mesenchymal–epi-thelial transition (c-MET), and anti-VHL (both fromAbcam), anti-AKTandanti-phospho(p)AKT(both fromCellSignaling Technology), as described previously (25). Immu-noreactive bands were detected with enhanced chemilumi-nescence (LiteAblot; EuroClone), andGel-Logic1500system(Eastman Kodak Co.), and quantified as optical densityunits by the Kodak imaging software.

Conditioned media and ELISAMMECs and MGECs (1� 106 cells/mL) were cultured in

serum-free Dulbecco’s Modified Eagle Medium (DMEM)for 24 hours, then supernatants centrifuged (380� g for 10minutes), and stored at �80�C as conditioned media.VEGF, FGF-2, and hepatocyte growth factor (HGF) were

Translational RelevanceBonemarrowangiogenesis is anattractive target for the

treatment of multiple myeloma. Here we demonstratethat constitutive and normoxic expression of the hypox-ia-inducible factor-1a (HIF-1a) protein in BM endothe-lial cells (ECs) of patients with relapsed/refractory MM(MMECs) is a key inducer of angiogenesis in vitro andin vivo, and mediates resistance to antiangiogenesisexertedbybortezomiband lenalidomide. The expressionof the HIF-1a protein by MMECs was also associatedwith shorter overall survival. The HIF-1a inhibitionby using siRNA or the histone deacetylase inhibitor(HDACI) panobinostat impaired the angiogenesis-relat-ed functions of the HIF-1a protein–expressing MMECsand overcame their resistance to bortezomib and lena-lidomide. HIF-1amay thus be envisaged as an attractivetarget for the antiangiogenic management of patientswith relapsed/refractory multiple myeloma, and as apossible prognostic factor.

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quantifiedby ELISA (SearchLight humanangiogenesis array2; TEMA RICERCA SRL).

Immunofluorescence and dual immunofluorescence–confocal laser-scanning microscopyFor immunofluorescence, 5 � 103 MMECs, MGECs, and

normal endothelial cells (these treated or not with defer-oxamine 380 mmol/L for 6 hours as positive and negativecontrol, respectively) were cultured on fibronectin-coatedchamber slides (LabTek; Nalge Nunc International), fixedwith paraformaldehyde, permeabilized with Triton X-100,and incubated with an anti-HIF-1a antibody (BD Trans-duction Laboratories) then with a secondary rabbit anti-mouse IgG-TRITC and phalloidin–fluorescein isothiocya-nate (both fromSigma-Aldrich); nucleiwere counterstainedwith 40,6-diamidino-2-phenylindole (VectashieldHard_Setmounting medium; Vector).

HIF-1a DNA–binding assayHIF-1a activation was measured in MMECs, MGECs, and

normal endothelial cells with the TransAM HIF-1a assay(ActiveMotif). ActivatedHIF-1a contained in nuclear extracts(NE-PER Nuclear and Cytoplasmic Extraction Reagents;Thermo Scientific) specifically binds to an erythropoietin30 hypoxia-response element–derived oligonucleotide probeimmobilized on a 96-well plates, and it is identified by ananti-HIF-1a antibody. Wild-type consensus oligonucleotidewas used as a competitor for HIF-1a binding to monitor theassay specificity. Amutated consensus oligonucleotide servedas an additional negative control; nuclear extracts of CoCl2-stimulated HeLa cells as the positive control.

Treatment of MMECs with siRNA and anti-multiplemyeloma drugsThe HIF-1a protein–positive MMECs (5 � 105) of

relapsed or refractory patients were transiently transfectedwith HIF-1a�siRNA 50 nmol/L for 3 to 5 days, with controlsiRNAs (SMART-pool; Dharmacon RNA Technologies) orwith the transfection reagent alone (Lipofectamine, RNAi-MAX siRNA transfection reagent; Invitrogen; ref. 20). Inseparate experiments, MMECs (5 � 105) were treated withbortezomib (Velcade; Millennium Pharmaceuticals Inc.)10 nmol/L for 24 hours (26), lenalidomide (Revlimid;Celgene Co.) 1.75 mmol/L for 72 hours (25), or pan-obinostat (LBH589; Novartis Oncology) 100 nmol/L for72 hours (27). The HIF-1a�siRNA transfected MMECswere then incubated with/without bortezomib or lenalido-mide. All MMECs groups were evaluated in functionalstudies. Isobologram analysis was performed with the Cal-cuSyn software (Biosoft; Ferguson), and a combinationindex (CI) < 1.0 indicated synergism.

Functional studiesViability and apoptosis. Viability was assessed by trypan

blue staining, and apoptotic rate by phycoerythrin–Annexin V and 7-amino-actinomycin D (Apoptosis detec-tion kit; BD Biosciences) followed by cytofluorimetry onFACScantoII (BD Biosciences).

Adhesion and spreading. MMECs (1 � 104) were platedinDMEM(EuroClone) onfibronectin-coated 96-well platesin triplicate for 30 minutes (adhesion) or 90 minutes(spreading), fixed with 4% paraformaldehyde, and quan-tified by the crystal violet assay at 595 nm in a MicroplateReader (Molecular Devices Corp.; ref. 20).

Chemotaxis. MMECs (5� 104) were tested in a Boydenmicrochamber assay toward DMEM with 1.5% fetal calfserum alone (negative control) or added with VEGF (10ng/mL; SigmaChemical Co.), and FGF-2 (10 ng/mL; Pepro-tech Inc.) as chemoattractants (20). After 8 hours at 37�C,the migrated cells were fixed, stained, and counted by theEVOS inverted microscope (EuroClone) at �400.

Angiogenesis on Matrigel. MMECs were plated on 48-well plates coated withMatrigel (BD Biosciences) in serum-free medium (SFM): after 12 hours, the skeletonization ofthe mesh was followed by measurement of mesh areas andvessel length in three randomly chosen fieldswith the EVOSmicroscope at �200.

Chorioallantoic membrane assayFertilized white Leghorn chicken eggs were incubated at

37�Cat constant humidity (25).Onday3, the shellwasopen-ed and 2-to-3 mL of albumen removed to detach the chorio-allantoic membrane (CAM). On day 8, the CAMs wereimplanted with 1 mm3 sterilized gelatin sponges (GelfoamUpjohn Co.) loaded with SFM alone (negative control) orwith conditioned media from untreated (positive control) orHIF-1a�siRNA- or panobinostat-treated MMECs. On day 12,the angiogenic response was evaluated as the number ofvessels converging toward the sponge at �50 and photo-graphed in ovo by a stereomicroscope (Olympus Italia S.R.L).

Measurement of ROS and treatment of MMECs withantioxidants

MMECs and MGECs (2 � 105) treated or not with 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (tro-lox) 0.5mmol/L for 24 hours were trypsinized and incubatedwith 2,7-dichlorodihydrofluorescein-diacetate (H2DCF-DA;both from Sigma-Aldrich) 2 mmol/L at 37�C for 15 minutes,washed, and resuspended in PBS. Each sample was analyzedfor 20,000 events on the FACSCanto II cytofluorimeter.

ResultsNormoxic activation of HIF-1a in MMECs correlateswith relapse and drug resistance

HIF-1a mRNA and protein were measured in normoxicconditions in MMECs from patients at different diseasephases as well as in MGECs and normal endothelial cells.Although mRNA levels overlapped between all the endo-thelial cells types (Fig. 1A), theproteinwasonly expressed inMMECs of 19 of 42 (45%) patients at relapse after borte-zomib- or lenalidomide-based therapies or on refractoryphase to these drugs (Fig. 1B). HIF-1awas active as assessedby aDNA-binding assay (Fig. 1C), and found in the nucleus(Fig. 1D, top). In contrast, it was always absent (or irrele-vant) in patients at diagnosis (Fig. 1D, middle left) or inremission (data not shown), or in those with MGUS (Fig.

Endothelial HIF-1a and Relapsed/Refractory Multiple Myeloma

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1D, middle right), as well as in endothelial cells of thecontrol subjects (Fig. 1D, bottom right). The normoxicexpression of HIF-1a in MMECs from relapsed/refractory

patients was not confirmed in another bone marrowhematopoietic cell population, such as macrophages (Sup-plementary Fig. S1).

Figure 1. HIF-1a is expressed and activated in MMECs from patients with relapsed/refractory disease cultured in normoxia. A, HIF-1a mRNA levels wereanalyzed by RT-PCR and real-time RT-PCR and normalized to endogenous b-actin mRNA. Gene expression fold changes in normal endothelial cells werearbitrarily set as 1. B, HIF-1a protein was examined by Western blot analysis (b-actin ¼ loading control), and data shown as optical density (OD). C, HIF-1atranscription activity was quantified by spectrophotometry at 450 nm. ��, P < 0.01 by the Wilcoxon signed-rank test. D, immunofluorescence for HIF-1a (redsignal), actin (green signal), and nuclei (blue signal) in endothelial cells from representative patients with multiple myeloma and MGUS, and control subjects.Merge (pink signal) shows colocalization of HIF-1a, nuclei, and actin. Top, merge for HIF-1a and nuclei in MMECs from relapsed/refractory patients. Middle,merge for HIF-1a and nuclei in MMECs at diagnosis and MGECs. Bottom, merge for HIF-1a and nuclei in normal endothelial cells treated or not withdeferoxamine (DFO). DFOmimics hypoxia and served as the positive control. Pictures by anOlympus photomicroscope (Olympus,Milan, Italy) equippedwiththe DP20-5E digital camera. Magnification, �600; scale bar, 16 mm.

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HIF-1a induces overexpression of its proangiogenictargetsWe wondered whether HIF-1a protein expression may

result in theupregulationof its proangiogenic targets: VEGF,FGF-2, and c-MET. Thesewere significantly overexpressed in

conjunction with the HIF-1a protein, as both mRNA(Fig. 2A) and protein (Fig. 2B). A similar correlation wasalso found with VEGFR-2, FGFR-2, and HGF (the c-METligand; Supplementary Fig. S2). To determine whetherVEGF, FGF-2, and c-MET overexpression was induced by

Figure 2. HIF-1 pathway promotes the transcription of VEGF, FGF-2, and c-MET inMMECs. A, real-time RT-PCR (normalized to b-actin) for VEGF, FGF-2, andc-MET. B, protein levels of VEGF, FGF-2 by ELISA (left graphs), and of HIF-1a and c-MET by Western blot analysis measured by OD (right). Data areexpressed as mean � SD of MMECs from patients at diagnosis (n ¼ 12), in remission (n ¼ 14), relapsed after bortezomib- or lenalidomide-basedtherapies (n ¼ 9) or on refractory phase to these drugs (n ¼ 10), and of MGECs (n ¼ 20); C, MMECs treated with HIF-1a�siRNA compared with nontargetingsiRNA (control) or untreated MMECs showed significant reduction (�90% as average) of HIF-1a mRNA at 72 hours after transfection (on real-timeRT-PCR normalized to b-actin, left graph), and of HIF-1a protein (�70% as average) at 5 days (onWestern blot analysis with b-actin as loading control, right);D, downregulation of HIF-1a target genes: VEGF, FGF-2, and c-MET following the HIF-1a�siRNA (on real-time RT-PCR, left graph) and cell viabilityupon siRNA after 5 days (right graph). �, P < 0.05; and ��, P < 0.01 by the Wilcoxon signed-rank test.






the HIF-1a activation, we measured each mRNA followingHIF-1a knockdownby siRNA. Interestingly, this produced a90% reduction (as average) of HIF-1a mRNA at 72 hours(Fig. 2C, left graph), whereas the protein was reduced by70% (as average) only at day 5 (Fig. 2C, right) suggestingthat it was stabilized substantially in the MMECs. TheHIF-1a silencing sizeably suppressed the VEGF (�60% asaverage), FGF-2 (�54%), and c-MET (�50%) mRNAs (Fig.2D, left graph). No effect on cell viability was observedupon siRNA after 5 days (Fig. 2D, right graph).

HIF-1a knockdown affects key MMEC angiogenesis–related functions

To investigate whether HIF-1a plays a role in multiplemyeloma angiogenesis, the effects of HIF-1a�siRNA on thefunctions of MMECs expressing the HIF-1a protein werestudied. TheHIF-1a silencing impacted chemotaxis (�60%as average), cell adhesion (�50%), and spreading (�47%)but not cell viability (Fig. 3A) nor apoptosis (data notshown). The HIF-1a�siRNAMMECs seeded onto the Matri-gel surface lacked angiogenesis as assessed by substantialreduction of vessel length (�62% as average) and emptyareas (�70%; Fig. 3B). On the in vivo CAM assay, whenCAMs were implanted with a gelatine sponge soaked withthe conditioned media of MMECs expressing the HIF-1aprotein, many newly-formed capillaries converging radiallytoward the sponge in a "spoked-wheel" pattern were seen(vessel count¼ 31� 6, positive control; Fig. 3C,middle). Incontrast, the conditioned media of the HIF-1a�siRNAMMECs gave poor angiogenesis (12 � 5; Fig. 3C, right),

which was similar to physiologic angiogenesis obtainedwith SFM (9 � 3, negative control; Fig. 3C, left).

HIF-1a knockdown restores MMECs sensitivity tobortezomib and lenalidomide

Interestingly, the HIF-1a protein mediated drug resis-tance of MMECs. Indeed, HIF-1a protein expressingMMECs of patients at relapse or on refractory phase tobortezomib- or lenalidomide-based therapieswere resistantto the previously shown (26, 25) antiangiogenic effect ofthese drugs: neither bortezomib nor lenalidomide impairedMMECs chemotaxis, adhesion, spreading (Fig. 4A) and thewhole angiogenesis (Fig. 4B). In contrast, HIF-1a protein–negative MMECs of the same patients’ series were sensitiveto the antiangiogenic effect of the drugs (SupplementaryFig. S3). When HIF-1a was knocked down by siRNA,resistance to bortezomib or lenalidomide was overcome.Specifically, the combination of HIF-1a�siRNA þ bortezo-mib showed a synergistic inhibition of chemotaxis (�60%as average), adhesion (�38%), spreading (�45%; Fig. 4A),andwhole angiogenesis (�80% vessel length;�78% emptyareas; Fig. 4B) compared with HIF-1a�siRNA alone (CI < 1;isobologram analysis). The combination of HIF-1a�siRNAþ lenalidomide also showed a synergistic inhibition ofchemotaxis (�54%; Fig. 4A) and whole angiogenesis(�63% vessel length and �60% empty areas; Fig. 4B). Nochanges in cell viability (Fig. 4A) nor apoptosis (data notshown) were seen in HIF-1a protein expressing MMECstreated with bortezomib or lenalidomide singularly or inassociation with HIF-1a�siRNA.

Figure 3. HIF-1a knockdownimpairs angiogenesis-relatedfunctions of MMECs fromrelapsed/refractory patients.HIF-1a�siRNA transfected MMECswere tested for: A, chemotaxis,adhesion, spreading, and viability;B, angiogenesis on Matrigel(measurement of vessel length andempty areas by the EVOS imagesoftware). Matrigel magnification,�200; scale bar, 50 mm. C, CAMassay: poor angiogenesis byconditioned media of HIF-1a�siRNA (right) versus untreatedMMECs (positive control, middle)from a representative relapsedpatient; physiologic angiogenesiswith SFM (left). Pictures with astereomicroscope: originalmagnification�50. Data aremeans� SD of MMECs from relapsed(n ¼ 9) or refractory (n ¼ 10)patients. �,P<0.05; and ��,P<0.01by the Wilcoxon signed-rank test.

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Panobinostat inhibits MMEC angiogenesis bydownregulating the HIF-1a transcriptional activityWe investigated whether panobinostat may exert an

antiangiogenic effect on the HIF-1a protein expressingMMECs because it is a HDACi, i.e., an indirect HIF-1ainhibitor (18). Similarly to what was seen in HIF-1a�siRNA MMECs, panobinostat impacted chemotaxis(�61% as average), cell adhesion (�50%), and wholeangiogenesis (�64% vessel length; �64% empty areas;Fig. 5A and B). The combination of panobinostat þbortezomib induced a synergistic inhibition of chemo-taxis, cell adhesion, and spreading (�64%,�50%, �33%,respectively; Fig. 5A), and of whole angiogenesis (�77%vessel length and �47% empty areas; Fig. 5B, CI < 1)compared with panobinostat alone. Also, panobinostatþlenalidomide showed a synergistic inhibition of chemo-taxis (�64%; Fig. 5A) and of whole angiogenesis (�80%vessel length; �54% empty areas; Fig. 5B). No changes incell viability (Fig. 5A) were seen in HIF-1a protein expres-sing MMECs treated with panobinostat singularly or inassociation with bortezomib or lenalidomide. Interest-

ingly, we also demonstrated that panobinostat inhibitsMMEC angiogenesis either directly by acting on MMECsand indirectly by acting on total bone marrow cells. Weobserved that the conditioned media of the total bonemarrow cells (BMCM) increased MMEC angiogenesisbecause of the proangiogenic factors and cytokinesreleased by bone marrow cells (Supplementary Fig.S4A, right). Indeed, the direct inhibitory effect of pano-binostat, on the isolated MMEC angiogenesis (Supple-mentary Fig. S4B, left), was partially subverted by theBMCM (Supplementary Fig. S4B, middle). However,when the total bone marrow cells were pretreated withpanobinostat, their BMCM inhibited MMEC angiogenesis(Supplementary Fig. S4B, right). In the CAM assay, dif-ferent from the conditioned media of untreated MMECs(vessel count ¼ 37 � 7; Fig. 5C, middle) the conditionedmedia of panobinostat-treated MMECs gave irrelevantangiogenesis (10 � 5, P < 0.01; Fig. 5C, right), reequili-brating the vessel counts to physiologic angiogenesis (8 �3; Fig. 5C, left). Next, we wondered whether treatmentswith panobinostat impacted on the HIF-1a protein

Figure 4. HIF-1a knockdownsensitizes MMECs to bortezomiband lenalidomide. MMECs treatedwith bortezomib or lenalidomidesingularly, or in conjunction withHIF-1a�siRNA were tested for: A,viability, chemotaxis, adhesion,and spreading; B, angiogenesis onMatrigel (measurement of vessellength and empty areas by theEVOS image software). Originalmagnification, �200; scale bar,50 mm. Histograms are means �SDofMMECs from relapsed (n¼ 9)or refractory (n ¼ 10) patients.�, P < 0.05 or ��, P < 0.01 by theWilcoxon signed-rank test. TheCI < 1.0 indicates synergism(isobologram analysis).






Figure 5. Angiogenesis inhibition by panobinostat in MMECs of relapsed/refractory patients is mediated by downregulation of the HIF-1a transcriptionalactivity. MMECs treated with panobinostat, bortezomib, or lenalidomide singularly or in combination were tested for: A, viability, chemotaxis, adhesion,and spreading; B, angiogenesis on Matrigel (measurement of vessel length and empty areas by the EVOS image software). Matrigel magnification, �200;scale bar, 50 mm. (Continued on the following page.)

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expression and/or activation: the drug did not reduce theprotein levels (Fig. 5D, left),whereas it reduced its activationwhen given singularly (�50%) and in combination withbortezomib (�79%) or lenalidomide (�53%; Fig. 5D,middle graph). These treatments, respectively, inhibitedVEGF (�50%; �80%; and �68%), FGF-2 (�40%; �70%;and �50%), and c-MET (�45%; �70%; and �50%) tran-scription (Fig. 5D, right graph).

HIF-1a�siRNA and panobinostat induce similarchanges in the MMEC proteomeTo further examine the inhibitory effects of HIF-

1a�siRNA and panobinostat at the molecular level, wecompared the proteome of HIF-1a protein expressingMMECs following HIF-1a�siRNA or panobinostat treat-ment. At least three two-dimensional electrophoresis(2-DE) gels were run per sample followed by comput-er-assisted spot matching to enable the identification ofspots variations. Nine proteins were identified as differ-entially expressed by peptide sequencing and tandemmass spectrometry followed by database searching (Sup-plementary Fig. S4C and S4D). Eight proteins weredownregulated by both HIF-1a�siRNA and panobinostat(2-fold changes vs. control): glutathione S-transferase P1(GSTP1), HSPB1, Annexin A4 (ANXA4), protein disul-fide-isomerase A3, prolyl 4-hydroxylase subunit a-2, gas-trin-releasing peptide (GRP), LIM and SH3 domain pro-tein 1 (LASP1), and ANXA1; the polymerase I and tran-script release factor was instead upregulated. These pro-teins govern cell shape, cell metabolism, chemotaxis, andangiogenesis (Supplementary Table S2).

The HIF-1a protein is stabilized in MMECs by ROS andhas a prognostic valueTo search the mechanism that stabilizes the HIF-1a

protein in MMECs, we studied the pathways governing itsexpression in normoxia. The expression was not associatedwith neither the loss of pVHL nor the activation of the AKTpathway (Supplementary Fig. S5). Interestingly, the ROSproduction was highly correlated with the HIF-1a proteinexpression (Fig. 6A), as assessed in HIF-1a protein–positiveversus -negative MMECs of relapsed/refractory patients andof the other groups of the patients, or versus MGECs.Accordingly, the antioxidant trolox reduced both the ROS(Fig. 6B, left)—without affecting cell viability (Fig. 6B,right)—and the HIF-1a protein levels (Fig. 6C), suggestinga key role of ROS in mediating the HIF-1a protein stabili-zation in normoxia.Worth of note is that a 12-month follow-up of patients

with HIF-1a protein–positive and -negative MMECsshowed a significantly shorter OS in the former (Fig. 6D).

DiscussionAmong hematologic malignancies, chronic lymphocytic

leukemia (28), diffuse large B cell and follicular non-Hodg-kin lymphomas (29), Hodgkin lymphoma (30), and mul-tiple myeloma (10, 11) express HIF-1a in tumor cells. HIF-1a has been found in plasma cells cultured in normoxia of28% of patients with multiple myeloma (10), and it wasenhanced by plasma cell growth factors such as insulin-likegrowth factor-1 and interleukin-6 (11).Here, the role ofHIF-1a in multiple myeloma angiogenesis and drug resistancewas investigated in MMECs harvested from patients at dif-ferent disease phases and cultured in normoxia. HIF-1amRNA overlapped between MMECs, MGECs, and normalendothelial cells, whereas the protein was expressed, stabi-lized, and activated only in MMECs of 45% of patients whowere relapsed after bortezomib- or lenalidomide-based ther-apies orwere refractory to these drugs. Because theseMMECshad the protein innormoxic conditions one canhypothesizethat it was regulated at posttranslational level. Accordingly,hypoxia-independent mechanisms may govern HIF-1a inMMECs from patients’ relapsed/refractory to bortezomib orlenalidomide as already found in plasma cells (10). HIF-1ainducers in normoxic cells include reduced expression of thetumor suppressor proteinVHL(31), activationof PI3K/AKT/mTOR pathway (32) and ROS (5). In MMECs, the HIF-1aprotein did not correlate either with expression of VHL orwith activation of the AKT pathway, but it didwith increasedROS production; and the ROS inhibition by trolox reducedthe HIF-1a protein stabilization. Data support a key role ofROS in mediating the HIF-1a protein stabilization inMMECs similarly to what observed in human prostate(33) and gastric carcinoma cells (5).

HIF-1awas closely involved in theMMECsoverangiogenicphenotype.Bonemarrowangiogenesis is a constanthallmarkof multiple myeloma progression (21), and enhanced by anautocrine VEGF loop of MMECs (34). The HIF-1a stabiliza-tion may plausibly lead, in turn, to enhanced angiogenesisbecause it increases the expression of the angiogenic factorsVEGF, FGF-2, and c-MET. In plasma cells too the HIF-1aactivation leads to the production of angiogenic factors (13).

To further elucidate the role of HIF-1a in multiple mye-loma angiogenesis, we evaluated the effects of its knock-down in theMMECs.HIF-1amRNAwas reduced by 90% at72hours after transfection,whereas the proteinwas reducedby 70% 5 days after transfection, which implied its strongstabilization. HIF-1a knockdown affected MMECs adhe-sion, spreading, andmigration, as others have found in cellsof malignant glioma (35) and renal carcinoma (36). It alsoinhibited MMEC angiogenesis in vitro (Matrigel) and in vivo(CAM) assays. Recently, Calvani and colleagues reportedthat human umbilical vein endothelial cells cultured in

(Continued.) C, CAMs were incubated with gelatine sponges loaded with SFM (physiologic angiogenesis, left), with the conditioned media of MMECs nottreated (positive control, middle) or treated with panobinostat (right) showing insufficient angiogenesis. Pictures with a stereomicroscope: originalmagnification�50. D, evaluation of: HIF-1a protein (onWestern blot analysis with b-actin as loading control, left); HIF-1a activation (by a DNA-binding assay,middle); expression of the HIF-1a target genes VEGF-A, FGF-2, c-MET (by real-time RT-PCR, right) of MMECs treated with panobinostat, bortezomib,and lenalidomide singularly or in combination. All histograms are means � SD of MMECs from relapsed (n ¼ 9) or refractory (n ¼ 10) patients. �, P < 0.05 or��, P < 0.01 by the Wilcoxon signed-rank test. The CI < 1.0 indicates synergism (isobologram analysis).






growth factors enriched medium form tube-like structuresunder both normoxia and hypoxia, whereas they formvessels only under hypoxia when cultured in a growthfactors–reduced medium (37). Here, the enhanced angio-genesis shown byMMECs in normoxic conditions could beexplained with constitutive HIF-1a protein stabilizationand consequent production of its angiogenic targets.

The stabilization of HIF-1a protein in MMECs of a well-defined percentage of relapsed/refractory patients suggestsits involvement inmultiplemyelomadrug resistance. In factMMECs expressing theHIF-1a protein showed resistance tobortezomib and lenalidomide compared withMMECs neg-

ative for expression as shown previously (26, 25). Interest-ingly, the HIF-1a knockdown restored the sensitivity tobortezomib or lenalidomide showing a synergistic effect incombination with these drugs. All these evidences suggestthat HIF-1a may be an antiangiogenic target in relapsed/refractory patients having MMECs with the HIF-1a protein.

We investigated the pharmacologic inhibition of HIF-1aby panobinostat, an HDACI (i.e., an indirect HIF-1a inhib-itor; ref. 18),whichhas alreadybeen shown todeliver potentin vitro (38, 39) and in vivo (38) anti-multiple myelomaactivity. The deacetylase activity is needed for the transacti-vation potential of HIF-1a (18). We show, in fact, that

Figure 6. HIF-1a of MMECs from relapsed/refractory patients is stabilized by ROS. A, intracellular ROS production by flow cytometry analysis in MMECs andMGECs treated with the oxidation sensitive dye H2DCF-DA. Data are expressed as mean fluorescence intensity � SD of MMECs of patients at diagnosis(n ¼ 12), in remission (n ¼ 14), or relapsed after bortezomib- or lenalidomide-based therapies (n ¼ 15), or on refractory phase to these drugs (n ¼ 14),and of MGECs (n ¼ 20). B, ROS levels measured by flow cytometry analysis in MMECs treated with the antioxidant trolox and then incubated withH2DCF-DA (left graph); evaluation of cell viability after trolox treatment (right graph). C, HIF-1a protein levels examined by Western blot analysis (b-actin asloading control) and shown as OD in MMECs treated with Trolox. Data are expressed as mean fluorescence intensity � SD of HIF-1a–positiveMMECs of relapsed (n ¼ 9) or refractory (n ¼ 8) patients. �, P < 0.05 and ��, P < 0.01 by the Wilcoxon signed-rank test. D, Kaplan–Meier functions ofOS of patients relapsed after bortezomib- or lenalidomide-based therapies or refractory to these drugs, and calculated from the day of bonemarrow samplingat relapse/refractory phase to the date of final follow-up (12 months). Patients with MMECs expressing the HIF-1a protein were 19 (continuous line);those with MMECs negative for the HIF-1a protein were 23 (sketched line). P < 0.001 ¼ significance by the log-rank test.

Ria et al.





panobinostat does not induce HIF-1a degradation, butreduces its binding toDNA,hence its transcriptional activity.This was especially seen when panobinostat was associatedwith bortezomib, as demonstratedby the intense decrease ofVEGF, FGF-2, and c-MET transcripts. Moreover, panobino-stat impactedMMECangiogenesis–related functions such ascell adhesion and chemotaxis, as well as the whole angio-genesis in vitro and in vivo. Similarly to HIF-1a�siRNA, thepanobinostat treatment was able to overcomeMMECs resis-tance tobortezomiband lenalidomide, thuspotentiating theantiangiogenic activity of the drugs in a synergistic way.Others have shown that panobinostat was able to potentiatecytotoxic activity of bortezomib, dexamethasone, and mel-phalan in multiple myeloma cell lines resistant to thesedrugs by impairing cell growth and survival (27).We investigated more deeply into the molecular mechan-

isms involved in the inhibition of MMEC angiogenesis byHIF-1a�siRNA and panobinostat through proteomic analy-sis. The differentially expressed MMECs proteins in responseto the HIF-1a�siRNA and panobinostat treatment wereinvolved in drug resistance (GSTP1), as well as cell shape,cytoskeletal remodeling, migration, and invasiveness (GRP;LASP-1). Specifically, GSTs are enzymes that catalyze theconjugation of xenobiotics with glutathione, thereby facili-tating their subsequent efflux throughmultiresistancepumps(40). This provides tumor cells with a selective survivaladvantage over normal cells by enhancing drug efflux, andconsequent decreasing the therapeutic efficacy of the drugs.In multiple myeloma plasma cells, indeed, GSTs entail awell-characterized mechanism of drug resistance (41). Wesuggest that the downregulation of GSTP1 inHIF-1a�siRNA–andpanobinostat-treatedMMECsmaybe away to overcomedrug resistance. The restoration of drug sensitivity in theHIF-1a�siRNA– and panobinostat-treated MMECs couldalso be explained by the downregulation of HSB1 (orHSP70), a member of the "stress-associated early responsegene" family involved in a wide range of cell functions understress conditions and oncogenesis (42). Of note, HSB1expression is upregulated in multiple myeloma plasma cells(43) and in resistant multiple myeloma cell lines, and itsinhibition reduces cell adhesion and reverts drug resistance(44), much in the same way as we show in MMECs. Amongthe downregulated proteins we found LASP-1, an acting–binding cytosckeletal protein localized at focal adhesionsalong stress fibres, which regulates cell migration (45); andGRP that elicits endothelial cells migration and cord forma-tion in vitro, and enhances angiogenesis in vivo (46).

Moreover, here we emphasize that patients withMMECs expressing the HIF-1a protein had shorter OSthan those with MMECs negative for expression suggest-ing that endothelial HIF-1a may represent a poor prog-nosis factor. Further confirmatory studies in larger seriesmay be encouraged.

In conclusion, HIF-1a of MMECs of a well-defined per-centage of relapsed patients after treatment with bortezo-mib- or lenalidomide-based therapies or refractory to thesedrugs may be targeted for antiangiogenic management, andbe regarded as a new prognostic factor.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

DisclaimerThe sponsors of this study are public or nonprofit organizations that

support science in general. They had no role in gathering, analyzing, orinterpreting the data. The authors are fully responsible for the content andeditorial decisions for this article.

Authors' ContributionsConception and design: R. Ria, I. Catacchio, C. Piccoli, A. VaccaDevelopment of methodology: I. Catacchio, S. Berardi, A.D. Luisi,A. Caivano, V. RuggieriAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): R. Ria, C. Piccoli, M.A. Frassanito, D. Ribatti, B.Nico, T. Annese, S. Ruggieri, A. Guarini, C. Minoia, P. Ditonno, E. Angelucci,D. Derudas, M. Moschetta, A. VaccaAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): R. Ria, I. Catacchio, A. Caivano,V. Ruggieri, D. Ribatti, B. Nico, S. RuggieriWriting, review, and/or revision of the manuscript: R. Ria, I. Catacchio,S. Berardi, E. Angelucci, F. Dammacco, A. VaccaAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): A. Guarini, C. MinoiaStudy supervision: R. Ria, A. Vacca

AcknowledgmentsThe authors thank Prof. Marco Presta (University of Brescia Medical

School, Brescia, Italy) for contribution to the Discussion.

Grant SupportThis work was supported by Associazione Italiana per la Ricerca sul

Cancro (AIRC, Italian Association for Cancer Research), Investigator grantand the 5 per thousandMolecular Clinical Oncology Special Program (grantno. 9965), Milan (to A. Vacca), the European Commission’s Seventh Frame-work Programme (EU FPT7) under grant agreement no. 278706 (OVER-MyR; to A.Vacca), and EU FPT7 (2007–2013) under grant agreementno. 278570 (toD. Ribatti), and grants fromMIUR PRIN 2009WCNS5C_004(to R. Ria), and 2010NECHBX (to A. Vacca).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 16, 2013; revised October 24, 2013; accepted November 11,2013; published OnlineFirst December 2, 2013.

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2014;20:847-858. Published OnlineFirst December 2, 2013.Clin Cancer Res Roberto Ria, Ivana Catacchio, Simona Berardi, et al. Therapeutic TargetResistance in Patients with Multiple Myeloma and May Act as a

of Bone Marrow Endothelial Cells Implies Relapse and DrugαHIF-1

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