Leukemia (2013) 27, 16971706

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ORIGINAL ARTICLE
Hypoxia-inducible factor (HIF)-1a suppression in myeloma
cells blocks tumoral growth in vivo inhibiting angiogenesis
and bone destruction
P Storti1,11, M Bolzoni1,11, G Donofrio2, I Airoldi3, D Guasco1, D Toscani1, E Martella4, M Lazzaretti5, C Mancini4, L Agnelli6, K Patrene7,
S Maga8, V Franceschi2, S Colla9, J Anderson10, A Neri6, M Amiot8, F Aversa1, G David Roodman10 and N Giuliani1

Hypoxia-inducible transcription factor-1 (HIF-1a) is overexpressed in multiple myeloma (MM) cells within the hypoxic
microenvironment. Herein, we explored the effect of persistent HIF-1a inhibition by a lentivirus short hairpin RNA pool on MM cell
growth either in vitro or in vivo and on the transcriptional and pro-angiogenic proles of MM cells. HIF-1a suppression did not have
a signicant impact on MM cell proliferation and survival in vitro although, increased the antiproliferative effect of lenalidomide. On
the other hand, we found that HIF-1a inhibition in MM cells downregulates the pro-angiogenic genes VEGF, IL8, IL10, CCL2, CCL5
and MMP9. Pro-osteoclastogenic cytokines were also inhibited, such as IL-7 and CCL3/MIP-1a. The effect of HIF-1a inhibition was
assessed in vivo in nonobese diabetic/severe combined immunodeciency mice both in a subcutaneous and an intratibial MM
model. HIF-1a inhibition caused a dramatic reduction in the weight and volume of the tumor burden in both mouse models.
Moreover, a signicant reduction of the number of vessels and vascular endothelial growth factors (VEGFs) immunostaining was
observed. Finally, in the intratibial experiments, HIF-1a inhibition signicantly blocked bone destruction. Overall, our data indicate
that HIF-1a suppression in MM cells signicantly blocks MM-induced angiogenesis and reduces MM tumor burden and bone
destruction in vivo, supporting HIF-1a as a potential therapeutic target in MM.

Leukemia (2013) 27, 16971706; doi:10.1038/leu.2013.24

Keywords: myeloma; angiogenesis; bone disease; hypoxia; mice

INTRODUCTION hypoxia and is composed of two basic helix-loop-helix proteins,

Multiple myeloma (MM) is characterized by the accumulation of
malignant plasma cells in the bone marrow (BM) microenviron-
ment that critically supports MM cell growth and survival.1
Particularly, an increase of BM angiogenesis and osteoclasto-
genesis occurs with MM cell growth and proliferation and plays a
critical role in the pathophysiology and progression of MM.28
The angiogenic switch in MM is mainly sustained by the
overproduction of vascular endothelial growth factors (VEGFs)
by MM cells and the BM microenvironment.2,9,10 Other pro-
angiogenic molecules, such as basic broblast growth factor,11,12
interleukin-8 (IL-8),13 angiopoietin-1 14 and osteopontin15 are
involved in the pro-angiogenic process. Hypoxia is a common
feature of solid tumors and is associated with angiogenesis
and malignant phenotype. It is critical to the regulation of the
angiogenic switch in those cancers, by regulating the production
of the pro-angiogenic molecules.1618 Tumor adaptation to
hypoxia is mainly due to the hypoxia-inducible factor (HIF)-1, a
key transcription factor that regulates pro-angiogenic factors,
angiogenesis and tumor progression in solid tumors.1923 HIF-1 is
a heterodimeric DNA binding complex highly inducible by
including the constitutive HIF-1b subunit and the hypoxia-
inducible a-subunit.1921 Under normoxic conditions HIF-1a has
a very short half-life undergoing proteosomal degradation by
oxygen-dependent hydroxylation. In contrast, under hypoxic
condition, hydroxylation is suppressed and HIF-1a protein
escapes proteasomal destruction and can accumulate and
translocate to the nucleus.1921 HIF-1a is a critical trigger and
regulator of tumor associated angiogenesis.22,23 Interestingly
a critical role of HIF-1a in osteoclast formation and regulation
has been shown24,25 as well as its role in the development of
osteolytic metastasis in breast cancer model.26
Recently it has been demonstrated that the BM microenvironment
is hypoxic in MM patients27 and that HIF-1a is overexpressed by MM
cells, and modulates their transcriptional and pro-angiogenic
proles.2729 Moreover, the role of hypoxia in tumor progression
and dissemination has been demonstrated in MM mouse
models.30,31 These ndings suggest that hypoxia could be a target
in MM. The role of HIF-1a as a therapeutic target in MM is under
investigation. In this study, we determined the effect of stable HIF-1a
inhibition in MM cells on cell proliferation and survival in vitro and

1
Hematology, Department of Clinical and Experimental Medicine, University of Parma, Parma, Italy; 2Dipartimento di Scienze Medico-Veterinarie, University of Parma, Parma, Italy;
3
A.I.R.C. Laboratory of Immunology and Tumors, Department of Experimental and Laboratory Medicine, Istituto Giannina Gaslini, Genova, Italy; 4Anatomia e Istologia patologica,
Azienda Ospedaliero-Universitaria di Parma, Parma, Italy; 5Department of Biosciences, University of Parma, Parma, Italy; 6Department of Clinical Sciences and Community Health,
University of Milan, and Hematology 1, Fondazione IRCCS Ca Granda Ospedale Maggiore Policlinico, Milan, Italy; 7Department of Medicine, Division of Hematology/Oncology and
the Center for Bone Biology of UPMC, University of Pittsburgh, Pittsburgh, PA, USA; 8INSERM, U892, University of Nantes, CNRS, UMR 6299, Nantes, France; 9MD Anderson Cancer
Center, University of Texas, Houston, TX, USA and 10Division of Hematology Oncology, Indiana University School of Medicine Department of Medicine, Indianapolis, IN, USA.
Correspondence: Professor N Giuliani, Hematology, Department of Clinical and Experimental Medicine, University of Parma, Via Gramsci 14, Parma 43126, Italy.
E-mail: nicola.giuliani@unipr.it
11
These authors contributed equally to this work.
Received 5 December 2012; revised 14 January 2013; accepted 17 January 2013; accepted article preview online 24 January 2013; advance online publication, 12 February 2013
 Targeting HIF-1a in multiple myeloma in vivo
P Storti et al
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growth, angiogenesis and osteolysis in vivo using plasmocytoma
xenograft or intratibial mouse models previously used to evaluate
the effect on myeloma cell growth and bone destruction.3234
MATERIALS AND METHODS
Cells and cell culture conditions
Cell lines. Human myeloma cell lines (HMCLs) JJN3, Roswell Park Memorial
Institute medium (RPMI)-8226 and OPM-2 were purchased from DSMZ
(Braunschweig, Germany), while the U266 was obtained from the ATCC
(LGC Standards S.r.l., Venezia, Italy). HMCLs were cultured in RPMI media at
10% fetal bovine serum with 2 nM of glutamine and antibiotics (Invitrogen
Life Technologies, Milan, Italy).
HIF-1a knockdown. Lentivirus short hairpin RNA (shRNA) pool anti-HIF-1a
(Sigma-Aldrich, Milan, Italy) was used for HIF-1a stable knockdown in
HMCLs, whereas the pLKO.1 lentiviral vector was the empty control.
Recombinant lentivirus was produced by transient transfection of 293T
cells following a standard protocol. HMCLs were infected as previously
described32, and selected in culture by the presence of 4 ug per ml
puromycin for 21 days. Selected clones of HMCLs were then screened for
HIF-1a, HIF-1b, HIF-2a and HIF-3a mRNA and/or protein expression. Stably
transfected HMCLs were maintained in RPMI medium containing 10% of
fetal bovine serum with 4 ug per ml puromycin until use.
Hypoxic and drugs treatments. HMCLs stably transfected with pLKO.1 or
anti-HIF-1a shRNA were incubated in the presence or absence of hypoxic
conditions (1%O2, 5%CO2) or treated with the hypoxic mimetic drug cobalt
chloride at 100 mM (Sigma-Aldrich, St Louis, MO, USA) or vehicle for 1224 h. In
selected experiments HMCLs stably transfected with pLKO.1 or anti-
HIF-1a were treated either in normoxic or in hypoxic conditions in the presence
of the absence of Bortezomib (supplied from Janssen-Cilag; Milan, Italy) at
concentrations ranging from 150 nM of Lenalidomide (supplied by Celgene Italy
srl, MiIan, Italy) (0.210 mM) or vehicle (dimethyl sulfoxide) for 2472 h.
Cell proliferation and viability assays. HMCLs transfected with pLKO.1 or
anti-HIF-1a were cultured in 96-well microtiter plates for 4896 h in the
presence of 3H-thymidine (3H-TdR) (Biocompare South San Francisco, CA,
USA) and thymidine incorporation was detected by liquid scintillation
spectroscopy (1205 Betaplate; Wallac; Markham, ON, Canada). Viability
of HMCLs stably transfected with pLKO.1 or anti-HIF-1a was evaluated under
both normoxic and hypoxic conditions after 2472 h of culture by adapted
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test assay
(Cell Counting Kit-8; Alexis, Vinci-Biochem srl, Italy). Cell apoptosis was
determined by Apo 2.7 mAb staining (Immunotech, Fullerton, CA, USA) and
veried by FACScan, (BD Biosciences Italy, Milan, Italy).
In vivo studies
Four to six-week-old males and females severe combined immunodeciency/
nonobese diabetic (SCID-NOD) mice (Harlan Laboratories, Udine, Italy) were
housed under specic pathogen-free conditions. All procedures involving
animals were performed in accordance with the National
and International current regulations (D.l.vo 27/01/1992, n.116, European
Economic Community Council Directive 86/609, OJL 358, Dec. 1, 1987). Three
groups of six animals each were injected subcutaneously with 5  106 JJN3
cells stably transfected with anti-HIF1a containing plasmid vectors (JJN3-anti-
HIF-1a), or with JJN3 stably transfected with empty vector (JJN3-pLKO.1), or
JJN3 wild type (JJN3). Twenty days after tumor cell inoculation, mice were
killed and autopsies were performed. Tumor mass was measured as
previously described.32 Maximum length and width of the tumor masses
were measured with a caliper, and tumor volume (mm3) was calculated
according to the following formula: 0.523  length  width2. Tumors were
removed and subjected to immunohistochemical staining.
In a separate set of experiments SCID and NIH-III nude mice (4 weeks of
age) were injected intratibially with 20 ml of 5  104 cells of JJN3-anti-HIF-1a
or JJN3-pLKO.1 or JJN3 or saline alone. All research protocols were approved
by the Pittsburgh VA Healthcare System Institutional Animal Care and Use
Committee. Four weeks after injection, the animals were killed and the tibias
were dissected out. Images of dissected tibias were acquired on a vivaCT 40
scanner (Scanco Medical, Bruttisellen, Switzerland) at resolution of 21 mm
isotropic, reconstructed, and segmented for 3-dimensional display using the
instruments analysis algorithm software. Tissue samples and cell extracts
were obtained for immunohistochemical staining.
Leukemia (2013) 1697 1706
Gene expression proling and microarray analysis
The transcriptional proles of JJN3-anti-HIF-1a and JJN3-pLKO.1
cells exposed to hypoxic or normoxic conditions were analyzed.
To perform gene expression proles, total RNA was puried using the
RNeasy total RNA Isolation Kit (Qiagen, Valencia, CA, USA). Preparation of
biotin-labeled complementary RNA, hybridization to GeneChip Human
Genome U133 Plus2.0 Arrays and scanning (GeneChip Scanner 3000 7G,
Affymetrix Inc., Santa Clara, CA, USA) were performed according to
manufacturers protocols. The raw intensity signals were extracted from
cell intensity (CELL) les and normalized using the robust multi-chip
average (RMA) package for Bioconductor and custom GeneAnnot-based
Chip Denition Files version 2.2.0 in R software. The most differentially
expressed genes between two experimental conditions were dened as
follows: for each gene, the ratio between the difference and the average
value in the expression signals under the two conditions was calculated.
Those genes with a minimum of 1.5 absolute fold change between hypoxic
and normoxic conditions were selected for further analysis. Then, those
genes with ratios exceeding two s.d. from the mean were considered
differentially expressed. NetAffx (https://www.affymetrix.com/analysis/
netaffx/) and Database for Annotation, Visualization and Integrated
Discovery (DAVID) (http://david.abcc.ncifcrf.gov) tools were used for the
functional annotation studies of the selected lists. The data have been
deposited at NCBI Gene Expression Omnibus repository (http://
www.ncbi.nlm.mih.gov/geo) and are accessible through Gene Expression
Omnibus series accession number GSE40326.
RNA isolation and reverse-transcriptase polymerase chain
reaction amplication
Total RNA was extracted from the cells using the RNeasy total RNA
isolation kit (Qiagen, Valencia, CA, USA), and then quantied using a
Nanodrop ND-100 (Celbio S.p.A., Milan, Italy). 1 mg of RNA was reverse-
transcribed with 400 U Moloney murine leukemia reverse transcriptase
(Applied Biosciences, Life Sciences, Carlsbad, CA, USA) in accordance with
the manufacturers protocol. The complementary DNAs were amplied by
means of PCR using the following specic primer pairs:
 HIF1A: F: 50 -CTCAAAGTCGGACAGCCTCA-30 ; R: 50 -CCCTGCAGTAGGTTTCTG
CT-30
 VEGFA: F:50 -CGAAGTGGTGAAGTTCATGGATG-30 ; R: 50 -TTCTGTTCAGTCTTT
CCTGGTGAG-30
 IL8: F: 50 -TACTCCAAACCTTTCCACCC-30 ; R: 50 -AACTTCTCCACAACCCTCT
G-30
 MMP9: F: 50 -CGCAGACATCGTCATCCAGT-30 ; R: 50 -GGATTGGCCTTGGAAG
ATGA-30
 CCL2: F: 50 -ACTGAAGCTCGTACTCTC-30 ; R: 50 -CTTGGGTTGTGGAGTGAG-30
 GAPDH: F 5-CAACGGATTTGGTCGTATTG-3; R: 5-GGAAGATGGTGATGGG
ATTT-3
Annealing temperature: HIF1A: 59 1C; VEGFA: 66 1C; IL8: 64 1C; CCL2: 60 1C
MMP9: 58 1C; GAPDH: 58 1C. Product size: HIF1A: 454 bp; VEGFA: 375 bp; IL8:
158 bp; CCL2: 354 bp MMP9: 369 bp GAPDH: 209 bp. Pictures of the
electrophoresed complementary DNAs were recorded with a digital DC
120 Kodak camera and quantied.
Real-time quantitative PCR and angiogenesis PCR array
Real-time PCR was performed using the TaqMan Gene Expression Assay
(Applied Biosystems Applera, Milan, Italy) for the following genes:
 HIF1A: HS00936379_M1
 HIF-1B (ARNT): HS00231048_M1
 HIF2A (EPAS1): HS01026149_M1
 HIF2B (ARNT2): HS00208298_M1
 HIF3A: HS00223818_M1
 BNIP3: HS00969291_M1
 VEGFA: HS99999034_M1
 IL8: Hs99999034_M1
 CCL2: HS00234140_M1
 MIP1A: Hs00234142_M1
 IL7: Hs00174202_M1
 DKK1: Hs00183740_M1
The PCR amplications were performed in duplicate using the iCycler iQ
Real-Time Detection System (Bio-Rad, Milan, Italy). The comparative
& 2013 Macmillan Publishers Limited

Ct method was applied to normalize the differences in the quantity
and quality of RNA, and mRNA was quantied using the comparative
DCt method using the endogenous reference gene ABL (DCt mean
Ct genemean Ct ABL); DDCt was evaluated as the difference between the
DCt of a sample and the DCt of the control. The fold change in mRNA
expression was calculated as 2  DDCt.
The expression levels of the pro-angiogenic molecules were evaluated
on mRNA extracted from tumors removed from SCID-NOD mice with the
Human Angiogenesis RT2 Proler PCR Array and RT2 Real-Timer SyBR
green/ROX PCR Mix (PAHS-024, Superarray, SABiosciences, Frederick, MD,
USA) that proles the expression of 84 key genes involved in modulating
the biological processes of angiogenesis.
Western blot analysis
Western blot analysis was performed as previously described.26 Nuclear
extracts were prepared using the Nuclear extraction kit (Active Motif,
Carlsbad, CA, USA) according to the manufacturers protocol. A total of
40 mg of nuclear extracts were tested. A polyclonal goat anti-HIF-1a Ab
(1:1000; R&D system, Minneapolis, MN, USA) or anti-HIF-1b or anti-HIF-2a
or anti-HIF-3a (1:500; Novus Biologicals, Littleton, CO, USA) were used to
detect HIF-1a, HIF-1b, HIF-2a and HIF-3a, respectively, whereas anti-
Histone H1 monoclonal Ab was used as internal control (Upstate, Lake
Placid, NY, USA). A monoclonal anti-p27 (BD Pharmingen, San Jose, CA,
USA) was used as primary antibody to detect p27 on whole lysates.
Appropriate secondary antibodies that were used: anti-goat
Immunoglobulin G (Rockland Immunochemicals, Gilberrtsville, PA, USA)
for HIF-1a, anti-mouse Immunoglobulin G (BD Biosciences) for HIF-1b,
HIF-2a and p27 and anti-rabbit Immunoglobulin G (Chemicon
International, Millipore, Billerica, MA, USA) for HIF-3a.
HIF-1a activity
HIF-1a activation in nuclear extracts of HMCLs was evaluated using an
Enzyme-linked immunosorbent assay based method (TransAM HIF-1 kit,
Active Motif) according to the manufacturers procedures.
Enzyme-linked immunosorbent assays
Soluble VEGF, IL-8, IL-7 and CCL3 proteins were detected in the
conditioned media of HMCLs using Enzyme-linked immunosorbent assays
purchased from R&D system according to the manufacturers protocols.
Cytokine levels in the conditioned media were normalized to the number
of cells at the end of culture period.
Histological and Immunohistochemical analysis
Tissue samples obtained from tumors removed from mice injected either
subcutaneously or intratibially with JJN3-pLKO.1, JJN3-anti-HIF-1a and JJN3
were xed in 10% neutral buffered formalin, embedded in parafn,
sectioned at 3 mm, and stained with hematoxylin and eosin or with
Toluidine blu and Gomoris three-chromic methods. Tumors obtained from
intratibial injections underwent EDTA decalcication before embedding in
parafn. On the basis of cell location and morphology, the number of all
Osteoclasts (OCLs) and active prismatic osteoblasts was evaluated on the
bone surface of each section (3  10 mm2). Osteocyte number and vitality
were recorded on a total of 500 lacunae per histological section.
Sections were immuno-stained either with mouse monoclonal anti-HIF-
1a Ab (NOVUS Biologicals Littleton, CO; Working dilution 1:100) or mouse
anti-human VEGF (R&D; dilution 1:20) or with 1:100 diluted mouse anti-
Ki67 primary antibody (Clone MIB-1, Dako, Carpinteria, CA, USA) for 30 min.
HIF-1a and VEGF staining were revealed using the UltraVision LP Large
Volume Detection System HRP polymer (Thermo Scientic, Erembodegem,
Belgium) and quantied according to semiquantitative immunohisto-
chemical score. Detection of Ki67 was performed using a high-sensitive
detection system (Advance-HRP, Dako Carpinteria, CA, USA) and 3,3-
diaminobenzidine was used as chromogen substrate. Angiogenesis was
evaluated on frozen tissues samples obtained from both series of mice.
Tissues were xed in acetone and treated with rabbit anti-mouse CD34
(1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing
sections were incubated with a secondary antibody (1:250; Rat anti-
Immunoglobulin G horseradish peroxidase; Millipore) and reaction
revealed with a solution of 3-30 -diaminobenzidine tetrahydrochloride
(liquid DAB substrate chromogen system, DAKO, Glostrup, DK).
& 2013 Macmillan Publishers Limited
Targeting HIF-1a in multiple myeloma in vivo
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RESULTS
Permanent HIF-1a silencing in HMCLs: effects on MM cell
proliferation, survival and drug sensitivity
HMCLs were initially checked for HIF1A mRNA and HIF-1a protein
expression under normoxic conditions (Figure 1a). All HMCLs
tested expressed HIF1A mRNA, whereas HIF-1a protein was
present at low levels in JJN3 and U266, but not in RPMI-8226
and OPM-2 (Figure 1a). All HMCLs overexpressed HIF-1a protein
upon hypoxic treatment. The JJN3 and RPMI-8226 were used as
cell lines for HIF-1a inhibition studies. The selected clones were
screened both under normoxic and hypoxic conditions for HIF1A
mRNA by qualitative PCR (Figure 1b), and for HIF1A, BNIP3, HIF1B,
HIF2A, HIF2B and HIF3A mRNA levels by real-time PCR. These
studies showed a selective inhibition of HIF1A and its target gene
BNIP3 by the shRNA pool used (Figure 1c left panel). HIF1B, HIF2A,
HIF2B and HIF3A mRNA were not inhibited by the shRNA pool
(Figure 1c). Western blot for HIF-1a, HIF-1b and HIF-3a proteins
showed specic inhibition of HIF-1a protein under both normoxia
and hypoxia (Figure 1d). HIF-2a and HIF-2 b were not expressed
either in normoxia or upon treatment with hypoxic mimetic
(data not shown). Consistently HIF-1a activity was signicantly
inhibited in JJN3 anti-HIF-1a as compared with JJN3 pLKO.1 under
hypoxic conditions (Figure 1e). Similar results were obtained with
RPMI-8226 (data not shown).
The effect of HIF-1a suppression on HMCLs proliferation and
survival was next investigated. Interestingly, there was no signi-
cant inhibition of cell proliferation either with JJN3 or RPMI-8226
(Figure 2a) at different time points. Cell viability was not
signicantly changed by HIF-1a suppression when checked at
12 and 24 h either under normoxia or in hypoxia (Figure 2b).
A statistically signicant effect on cell viability was observed in
normoxia condition at 72 h for JJN3 cells (  23%; P 0.01)
(Figure 2c upper panel) and maintained at the same entity for
longer culture period, but not for RPMI-8226 cells (Figure 2c, lower
panel). Treatment with lenalidomide at a wide range of
concentrations induced a signicantly higher inhibition of cell
proliferation in JJN3 anti-HIF-1a as compared with JJN3 pLKO.1
(P 0.01) (Figure 2d) without changing their viability signicantly.
Consistently p27 expression was increased by lenalidomide in
JJN3 anti-HIF-1a as compared with JJN3 pLKO.1 (Figure 2e). On the
other hand, Bortezomib (410 nM) induced a similar rate of cell
proliferation and cell death in JJN3 anti-HIF-1a and JJN3 pLKO.1
(data not shown).
HIF-1a suppression affects the transcriptional prole of HMCLs:
inhibitory effect on the pro-angiogenic and pro-osteoclastogenic
genes
The transcriptional proles of JJN3 cells transduced with shRNA
anti-HIF-1a (JJN3-anti-HIF-1a) were compared with those infected
with the control vector pLKO.1 (JJN3-pLKO.1) either under hypoxic
or normoxic conditions. Among the signicantly modulated genes
(326 and 361 genes under hypoxic and normoxic condition,
respectively) (Supplementary Table S1), we found downregulation
of the pro-angiogenic molecules VEGFA, IL8, CCL2, MMP9 in JJN3-
anti-HIF-1a cells under both hypoxic and normoxic conditions.
Microarray data were further validated by both qualitative PCR
(Figure 3a) and real-time quantitative PCR, which showed that
VEGFA and IL8 were induced by hypoxia and inhibited by HIF-1a
suppression (Figure 3b), whereas CCL2 mRNA was not induced by
hypoxia but was inhibited by HIF-1a suppression either under
normoxia or hypoxia. Finally, other genes with pro-angiogenic
properties, such as IL10 and CCL5, were inhibited by HIF-1a
suppression in JJN3 under both normoxic and hypoxic conditions
(Supplementary Table S1). Consistent with our previous observa-
tion on small interfering RNA-mediated HIF-1a inhibition,27 stable
HIF-1a suppression by shRNA signicantly blunted vessels
formation induced by the conditioned media of HMCLs as
Leukemia (2013) 1697 1706
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Figure 1. HIF-1a expression by HMCLs and validation of permanent HIF-1a knockdown by shRNA. HIF1A mRNA and HIF-1a protein expressions
were evaluated in RPMI-8226, U266, OPM-2 and JJN3 by qualitative PCR and western blot, respectively. JJN3 treated under hypoxic conditions
for 12 h was used as positive control for HIF-1a expression (a). Anti-HIF-1a Lentivirus shRNA pool was used for HIF-1a stable knockdown in
JJN3, whereas the pLKO.1 lentiviral vector was used as the empty control vector. Cells were infected and maintained in RPMI medium at 10%
of fetal bovine serum in the presence of puromycin at 4 ug/ml for 21 days. Cells were then incubated with the hypoxic mimetic drug cobalt
chloride or the vehicle for 12 h. HIF1A mRNA expression was evaluated by qualitative PCR using Glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) mRNA as internal control (b). mRNA levels of HIF1A and BNIP3 as well as those of HIF1B, HIF2A, HIF2B and HIF3A were evaluated by real-
time PCR. Graphs represent the median fold change in the mRNA levels for three independent experiments performed in triplicate (c). HIF-1a,
HIF-1b and HIF-3a protein expression at the nuclear level were evaluated by western blot. Histone H1 was used as internal control (d). HIF-1a
activity in the nucleus was checked as described in the methods. Graphs represent the median optical density related to HIF-1a activity for
10 ug of proteins (e).

determined by AngioKit (TCS Biologies, London, UK) (data not
shown).
The effect of HIF-1a suppression on the production of
pro-osteoclastogenic cytokines by HMCLs was then investigated.
A signicant inhibitory effect was observed for both MIP1A/CCL3
and IL7 mRNA levels either under normoxia or in hypoxia as
shown for JJN3 (Figure 3c). This inhibitory effect was also
conrmed at translational level under normoxia as shown for
JJN3 (Figure 3d). In contrast, no signicant effect was observed on
the expression of the osteoblast inhibitor gene DKK1 by JJN3 with
HIF-1a suppression (data not shown).
HIF-1a suppression in JJN3 cells blocks the growth of
subcutaneous MM in SCID-NOD mice and inhibits angiogenesis
We next investigated whether inhibition of HIF1a in JJN3 may
inuence tumor growth in vivo. Therefore, we analyzed the
tumorigenicity of JJN3 wild type, JJN3 transfected with a plasmid
with silenced HIF1a (JJN3-anti-HIF-1a) and JJN3 with empty vector
(JJN3-pLKO.1) cells injected subcutaneously into SCID-NOD
animals. Twenty days after cell inoculation, mice were killed,
tumors removed and measured. At this time point, all animals
developed tumors that grew at the site of injection in the absence
of metastases to distant sites. Mice injected with the JJN3-anti-
HIF-1a cells developed signicantly smaller tumors than mice
inoculated with the JJN3-pLKO.1 (P 0.00018) or with JJN3
(P 0.032) (Figure 4a-b). Both weight and volume of the tumors
in JJN3-anti-HIF-1a mice were signicantly reduced as compared
with JJN3-pLKO.1 (Figure 4c-d). The median weight of tumors
formed by JJN3-anti-HIF-1a was 0.05 g vs 0.3 g for JJN3-pLKO
(P 0.0007) (Figure 4c). The median volume of tumors formed by
JJN3-anti-HIF-1a was 72.6 mm3 (range 3221 mm3) and that of
JJN3-pLKO.1 was 369.8 mm3 (range 221.8671 mm3) (P 0.0012)
(Figure 4d). Interestingly in the tumoral mass removed from
JJN3-anti-HIF-1a mice the microvascular density (number of
vessels positive for CD34/mm3) was signicantly reduced as
compared with that obtained from JJN3-pLKO.1 (JJN3-anti-HIF-1a
vs. JJN3-pLKO.1:  76%; P 0.003). Similarly VEGF immunostain-
ing was reduced in JJN3-anti-HIF-1a derived tumors as compared
with JJN3 pLKO.1 injected mice (Figure 4e-f).
HIF-1a suppression in JJN3 cells blunts angiogenesis and bone
destruction in an intratibial mouse model
The effect of HIF-1a suppression in MM cells was further
investigated in vivo in intratibial mouse model. The SCID mice

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Figure 2. Effect of HIF-1a inhibition on the proliferation, viability and drug sensitivity of HMCLs. Cell proliferation was evaluated in JJN3-
pLKO.1, JJN3-anti-HIF-1a, RPMI-8226-pLKO.1 and RPMI-8226-anti-HIF-1a after 4896 h of culture by 3H-thymidine uptake as described in the
methods. Graphs and bars represent the means.d. of counts per minute of incorporated 3H-thymidine for three independent experiments
performed in six replicates (a). JJN3-pLKO.1 and JJN3-anti-HIF-1a were incubated under normoxic or hypoxic conditions for 1224 h and
viability evaluated by adapted MTT test. Graphs and bars represent the means.d. for cell viability in four independent experiments
performed in duplicate (b). Cell viability was evaluated in JJN3-pLKO.1, JJN3-anti-HIF-1a, RPMI-8226-pLKO.1 and RPMI-8226-anti-HIF-1a after
1272 h of culture. Graphs and bars represent the means.d. percentage change in the cell viability vs control (c). JJN3-pLKO.1 and JJN3-anti-
HIF-1a were incubated with Lenalidomide (0.210 mM) or vehicle (dimethyl sulfoxide, DMSO) under normoxic conditions for 48 h and cell
proliferation checked by 3H-thymidine uptake. Graph represents the mean % inhibition of cell proliferation (d). p27 protein expression was
evaluated by western blot analysis in cell lysates of JJN3-pLKO.1 and JJN3-anti-HIF-1a treated for 24 h with Lenalidomide (Len) (2 and 10 mM) or
vehicle (DMSO); actin was used as internal control (e).

were injected intratibially with saline or JJN3-pLKO.1 or JJN3-anti-
HIF-1a cells, and lytic lesions were allowed to develop for
24 weeks before the mice were analyzed. Histological analysis
showed a signicant reduction in the tumor mass with an
increase in the bone thickness in JJN3-anti-HIF-1a as compared
with JJN3 pLKO.1 injected animals (Figure 5a). However,
immunostaining for Ki67 showed a reduction in the number of
Ki67-positive cells in JJN3-anti-HIF-1a as compared with JJN3
pLKO.1 mice although the difference did not reach statistical
signicance (Figure 5b). However, immunostaining for CD34
showed a signicant reduction in the number of CD34-positive
vessels in JJN3-anti-HIF-1a as compared with JJN3 pLKO.1 mice
(median number of vessels positive for CD34/mm3 22 vs 64,
P 0.001) (Figure 5c) similarly to what observed in the
subcutaneous model. Finally, micro-QCT analysis demonstrated
that mice injected with JJN3-pLKO.1 cells started developing
detectable lytic lesions at 2 weeks after cell injection with
continued further bone deterioration through the 4 weeks, that
ultimately involved the entire tibia leading to animal death from
advanced disease. In contrast, the saline injected controls at 4
weeks were similar to the 0-week time point, demonstrating that
the effects detected were not the result of the injection process.
Interestingly, mice injected with JJN3-anti-HIF-1a cells showed a
dramatic reduction of osteolytic lesions (Figure 5d). Finally,
histological analysis showed a signicant reduction in the number
of OCLs in JJN3-anti-HIF-1a as compared with JJN3-pLKO.1 bone
sections (Po0.05), whereas the number of active prismatic
osteoblasts arranged in laminae did not signicantly changed as

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Figure 3. Effect of HIF-1a inhibition on the expression of the pro-angiogenic and pro-osteoclastogenic cytokines by JJN3 cells. The presence of
the mRNA transcripts for the pro-angiogenic genes VEGFA, IL8, MMP9, CCL2 was evaluated in JJN3-pLKO.1 and JJN3-anti-HIF-1a under both
normoxic and hypoxic conditions by qualitative PCR. GAPDH was used as internal control (a). mRNA levels of VEGFA, IL8 and CCL2 were
quantified by real-time PCR. Graph represents the median -DCt values of two independent experiments performed in triplicate (b). Gene
expression of the pro-osteoclastogenic genes MIP1A and IL7 was evaluated in JJN3-pLKO.1 and JJN3-anti-HIF-1a by real-time PCR. Graphs
represent the median fold change in mRNA levels for two independent experiments performed in triplicate (*Po0.05) (c). CCL3/MIP1a and
IL-7 protein levels were evaluated in the conditioned media of JJN3-pLKO.1 and JJN3-anti-HIF-1a. Graphs and bars represent the means.d.
protein levels for two independent experiments measured in duplicate. (*Po0.05) (d).

well as that of viable osteocytes for 500 lacunae (P 0.4; NS, data
not shown).
DISCUSSION
It is well established that intratumoral hypoxia and consequently
HIF-1a activation in solid tumors critically trigger the angiogenic
switch, and induce modications of cancer cell metabolism
leading to tumor progression and metastasis.1618,22 HIF-1a is
overexpressed in many tumors22,23 including some hematological
malignancies such as MM.2729,35 We and others27,35 have recently
shown that BM microenvironment is hypoxic both in MM
patients27 and in mice.35 In addition HIF-1a also may be
overexpressed by MM cells under normoxic conditions27,28
dependent at least in part on c-myc upregulation.27 Finally, it
has been reported that hypoxia modulates the expression of pro-
angiogenic genes such as VEGF and IL-827 by MM cells, and that
HIF-1a is involved in MM-induced angiogenesis in vitro.27 In turn,
angiogenesis is able to support MM cell growth and survival.2 All
these results suggest that HIF-1a could be a potential therapeutic
target for MM. To test this hypothesis, we performed HIF-1a
knockdown in HMCLs using a pool of shRNA anti-HIF-1a and
evaluated the effect permanent suppression of HIF-1a on MM cell
proliferation, survival, the pro-angiogenic prole and tumor
growth in vivo. Firstly, we showed that the inhibition of HIF-1a
by shRNA was specic because no signicant inhibitory effect was
demonstrated on HIF1B, HIF2A, HIF2B and HIF3A expression.
Interestingly, we did not nd a signicant inhibitory in vitro effect
on MM cell proliferation by HIF-1a suppression only showing a
signicant inhibitory effect on cell survival after 72 h in JJN3, but
not in RPMI-8226. These results suggest that HIF-1a inhibition did
not directly induce a signicant effect on MM cell proliferation and
survival. Similarly, it has been previously reported that small
interfering RNA anti-HIF-1a alone is not able to directly affect MM
cell survival, but enhanced MM cell sensitivity to melphalan.36
Therefore, we further evaluated the potential effect of HIF-1a
stable suppression on the sensitivity of HMCLs to IMiDs and
Bortezomib in vitro. Interestingly, we found that shRNA anti-HIF-1a
increased the inhibitory effect of IMiDs on HMCL proliferation.
This result was also supported by the previous nding that the
response to lenalidomide treatment involves oxidative
stress response in MM cells.37 In contrast, we did not nd any
sensitization of MM cells to Bortezomib treatment. It has been
reported that anti-MM effects of Bortezomib may be increased by
hypoxia and HIF-1a upregulation38 rather than by HIF-1a
inhibition.
Although, we did not nd a signicant effect on cell
proliferation and survival by knockdown of HIF-1a, stable
HIF-1a suppression by shRNA modied the pro-angiogenic and
osteoclastogenic proles of MM cells either under hypoxic or

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Figure 4. HIF-1a inhibition in JJN3 blocks the development of subcutaneous plasmocytomas in SCID-NOD mice and inhibits angiogenesis.
Three groups of six SCID/NOD animals each were injected subcutaneously with 5  106 of JJN3, JJN3 transfected with a plasmid with silenced
HIF1a (JJN3-anti-HIF-1a) or JJN3 with empty vector (JJN3-pLKO.1). Twenty days after cell inoculation, mice were killed, tumors removed,
weighed and measured as described in the methods. Tissue samples from the masses were obtained for immunohistochemical staining.
Photographs represent either one representative mouse of each group of animals (JJN3, JJN3-anti-HIF-1a and JJN3-pLKO.1) after 20 days (a) or
tumor masses removed from JJN3-anti-HIF-1a and JJN3-pLKO.1 of two representative mice from each group (b). Box plots represent the
median weight (c) and volume (d) of the masses removed from all the mice injected with JJN3-anti-HIF-1a and JJN3-pLKO.1, respectively.
Images represent the immunostaining of CD34 (e) and VEGF (f ) antigens performed on frozen tissue and formalin fixed paraffin-embedded
samples, respectively, obtained from the plasmocytoma masses of two representative mice injected with JJN3-anti-HIF-1a and JJN3-pLKO.1.
Original magnification  400 (e) and  1000 (f ).

normoxic conditions. Microarray analysis and real-time PCR
showed that HIF-1a suppression signicantly inhibited some
pro-angiogenic factor genes such as VEGFA, IL-8, CCL2, MMP9.
The capacity of HIF-1a to regulate VEGFA and IL8 gene expression
by MM cells conrmed our previous data,27,39 and supports HIF-1a
as a key regulator of angiogenesis also in MM cells because
VEGF9,10 and at least in part of IL-839,40 are well-known factors
involved in MM-induced angiogenesis. CCL2 and MMP9 could be
also involved in the pro-angiogenic role of HIF-1a, because
both cytokines are produced by MM cells and contribute to
angiogenesis and BM spreading.4143
Interestingly, our data suggest that other than angiogenic
cytokines, HIF-1a suppression affects expression and production
of MM-derived pro-osteoclastogenic factors that are known to be
involved in osteoclast formation and bone destruction such as
CCL3/MIP1a44 and IL-7.45 To the best of our knowledge, this is the
rst time that the capacity of HIF-1a has been reported to regulate
pro-osteoclastogenic cytokine in MM cells, although the evidence
that CCL3/MIP1a and IL-7 are potential target genes of HIF-1a has
been shown in other cell types such as acute myeloid leukemia
cells for CCL3/MIP1a46 and mesenchymal/osteoblastic cells for
IL-7.47
To evaluate the role of HIF-1a as a potential target in MM we
used two different in vivo models: the subcutaneously and
the intratibial model. A dramatic effect on tumor burden was
demonstrated in both models by HIF-1a suppression showing a

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Figure 5. HIF-1a inhibition in JJN3 blunts myeloma-induced neo-angiogenesis and bone destruction after intratibial inoculation in mice. SCID
mice were injected intratibially with 20 ml containing 5  104 cells of JJN3-anti-HIF-1a or JJN3-pLKO.1 or saline alone. At 4 weeks after injection,
the animals were killed and the tibias were dissected out. Tissue samples were obtained for the histological evaluation and
immunoistochemistry. Hematoxylin and eosin staining of tissues samples obtained from two representative mice of each mice injected
with JJN3-anti-HIF-1a and JJN3-pLKO.1, respectively. Original magnification  400 (a). Immunostaining for Ki67 (b) and CD34 (c) antigens
performed on tissue samples of representative JJN3-anti-HIF-1a and JJN3-pLKO.1 mice (original magnification  630). Plots represent the
number of Ki67-positive cells (b) and the number of vessels X mm2 of tissues positive for CD34 (c), respectively (bars show the median value,
*Po0.001). Images show the dissected tibias of representative mice (saline, JJN3-anti-HIF-1a and JJN3-pLKO.1) acquired on a vivaCT 40 at
resolution of 21 mm isotropic, reconstructed and segmented for 3-dimensional display using the instruments analysis algorithm software (d).

signicant reduction of tumor mass. Because of the lack of a
signicant effect of HIF-1a suppression on cell proliferation and
survival of HMCLs in vitro, we guess that the anti-MM in vivo effect
was mainly due to the inhibitory effect on angiogenesis. This was
supported by the capacity of HIF-1a to regulate the pro-
angiogenic proles of HMCLs in vitro and by our previous results
showing that HIF-1a suppression blocks MM-induced in vitro
angiogenesis.27 The signicant inhibition of angiogenesis
(evidenced by reduction in the number and length of vessels in
mice injected with JJN3-anti-HIF-1a) conrmed that the antitumor
effect of HIF-1a suppression was mainly due to the inhibition of
angiogenesis. In support of these data, the inhibitory effect on

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angiogenesis by HIF-1a suppression was accompanied by a
reduction of VEGF immunostaining and of the proliferative
index, Ki67, that could be related to angiogenesis in MM,6
although it did not reach a statistical signicance. Finally, in line
with our data, it has been previously reported that HIF-1a
inhibition by RNA interference inhibits in vivo angiogenesis in
mice in other tumor models.48
In addition to the inhibition of angiogenesis, we showed in the
intratibial bone model that HIF-1a suppression also affects MM-
induced in vivo formation of osteolytic bone lesions. This could be
due in part to the inhibitory effect on MM growth via
the inhibition of angiogenesis, but also to a direct effect on the
production of pro-osteoclastogenic cytokines as shown in vitro.
Accordingly, histological analysis showed a signicant reduction of
the number of OCLs in JJN3 anti-HIF-1a as compared with JJN3-
pLKO.1.
The capacity of HIF-1a suppression to block the development of
osteolytic lesions in the intratibial MM model may explain in turn
its anti-MM effect, because it is well-known that osteoclast
activation supports MM cells growth in vivo.7 Similarly, it has
been previously reported that hypoxia and HIF-1a lead to the
development of osteolytic lesions and tumor growth in breast
cancer in vivo models.26
In conclusion, as in other tumoral models where it has been
shown that HIF-1a inhibition exerts a potent in vivo antitumor
effect, including the squamous cell carcinoma, liver and lung
cancer,48,49 our data indicate that selective HIF-1a inhibition
results in a potent anti-MM effect by blocking angiogenesis and
the development of osteolytic bone lesions. These results suggest
that HIF-1a is a potential therapeutic target in MM. In this context,
it has been recently reported that adaphostin inhibits HIF-1a
expression and induces a signicant antiangiogenic and anti-MM
activity in a MM xenograft mouse model.28 Furthermore, a
growing number of novel anticancer agents have been
demonstrated to inhibit HIF-1a with different molecular
mechanisms including histone deacetylases, molecules acting on
RAS-RAK-MEK-ERK pathways and PI3K-AKT, and mTOR inhibitors,
that are also currently in phase I-II clinical trials in MM.1,29 Overall,
our study provides the rationale for clinical trial with selective
inhibitors of HIF-1a mRNA, such as EZN-296850 in MM patients.
CONFLICT OF INTEREST
The authors declare no conict of interest.
ACKNOWLEDGEMENTS
This study was supported by grants from the International Myeloma Foundation;
and Associazione Italiana per la Ricerca sul Cancro A.I.R.C, IG2009 no. 8530 (N.G.),
no. IG4569 (A.N.) and no. 13018 (I.A.); Special Program Molecular Clinical Oncology 5
per mille no. 9965 (N.G.) and no. 9980 (A.N.), the Multiple Myeloma Research
Foundation and grant support R01 AR059679, R21 CA141426, R01AR057308, and
Merit Review funds from the Veterans Administration. (G.D.R.). We thanks Ministero
Italiano dell Istruzione, Universita e Ricerca (MIUR) and Italian Association against
Leukemia, Lymphoma and Myeloma (AIL). We thanks Carla Palumbo and Marzia
Ferretti for their support in the histological analysis.
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