Myeloma

Hypoxia-inducible factor (HIF)-1α suppression in myeloma cells blocks tumoral growth in vivo inhibiting angiogenesis and bone destruction

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Abstract

Hypoxia-inducible transcription factor-1 (HIF-1α) is overexpressed in multiple myeloma (MM) cells within the hypoxic microenvironment. Herein, we explored the effect of persistent HIF-1α 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 profiles of MM cells. HIF-1α suppression did not have a significant impact on MM cell proliferation and survival in vitro although, increased the antiproliferative effect of lenalidomide. On the other hand, we found that HIF-1α 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-1α. The effect of HIF-1α inhibition was assessed in vivo in nonobese diabetic/severe combined immunodeficiency mice both in a subcutaneous and an intratibial MM model. HIF-1α inhibition caused a dramatic reduction in the weight and volume of the tumor burden in both mouse models. Moreover, a significant reduction of the number of vessels and vascular endothelial growth factors (VEGFs) immunostaining was observed. Finally, in the intratibial experiments, HIF-1α inhibition significantly blocked bone destruction. Overall, our data indicate that HIF-1α suppression in MM cells significantly blocks MM-induced angiogenesis and reduces MM tumor burden and bone destruction in vivo, supporting HIF-1α as a potential therapeutic target in MM.

Introduction

Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells in the bone marrow (BM) microenvironment that critically supports MM cell growth and survival.1 Particularly, an increase of BM angiogenesis and osteoclastogenesis occurs with MM cell growth and proliferation and plays a critical role in the pathophysiology and progression of MM.2, 3, 4, 5, 6, 7, 8

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 fibroblast 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.16, 17, 18 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.19, 20, 21, 22, 23 HIF-1 is a heterodimeric DNA binding complex highly inducible by hypoxia and is composed of two basic helix-loop-helix proteins, including the constitutive HIF-1β subunit and the hypoxia-inducible α-subunit.19, 20, 21 Under normoxic conditions HIF-1α has a very short half-life undergoing proteosomal degradation by oxygen-dependent hydroxylation. In contrast, under hypoxic condition, hydroxylation is suppressed and HIF-1α protein escapes proteasomal destruction and can accumulate and translocate to the nucleus.19, 20, 21 HIF-1α is a critical trigger and regulator of tumor associated angiogenesis.22, 23 Interestingly a critical role of HIF-1α 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-1α is overexpressed by MM cells, and modulates their transcriptional and pro-angiogenic profiles.27, 28, 29 Moreover, the role of hypoxia in tumor progression and dissemination has been demonstrated in MM mouse models.30, 31 These findings suggest that hypoxia could be a target in MM. The role of HIF-1α as a therapeutic target in MM is under investigation. In this study, we determined the effect of stable HIF-1α inhibition in MM cells on cell proliferation and survival in vitro and 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.32, 33, 34

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-1α knockdown

Lentivirus short hairpin RNA (shRNA) pool anti-HIF-1α (Sigma-Aldrich, Milan, Italy) was used for HIF-1α 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-1α, HIF-1β, HIF-2α and HIF-3α 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-1α 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 μM (Sigma-Aldrich, St Louis, MO, USA) or vehicle for 12–24 h. In selected experiments HMCLs stably transfected with pLKO.1 or anti-HIF-1α 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 1–50 nM of Lenalidomide (supplied by Celgene Italy srl, MiIan, Italy) (0.2–10 mM) or vehicle (dimethyl sulfoxide) for 24–72 h.

Cell proliferation and viability assays

HMCLs transfected with pLKO.1 or anti-HIF-1α were cultured in 96-well microtiter plates for 48–96 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-1α was evaluated under both normoxic and hypoxic conditions after 24–72 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 verified by FACScan, (BD Biosciences Italy, Milan, Italy).

In vivo studies

Four to six-week-old males and females severe combined immunodeficiency/nonobese diabetic (SCID-NOD) mice (Harlan Laboratories, Udine, Italy) were housed under specific 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-HIF1α containing plasmid vectors (JJN3-anti-HIF-1α), 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 μl of 5 × 104 cells of JJN3-anti-HIF-1α 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, Brüttisellen, Switzerland) at resolution of 21 μm isotropic, reconstructed, and segmented for 3-dimensional display using the instrument's analysis algorithm software. Tissue samples and cell extracts were obtained for immunohistochemical staining.

Gene expression profiling and microarray analysis

The transcriptional profiles of JJN3-anti-HIF-1α and JJN3-pLKO.1 cells exposed to hypoxic or normoxic conditions were analyzed. To perform gene expression profiles, total RNA was purified 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 manufacturer’s protocols. The raw intensity signals were extracted from cell intensity (CELL) files and normalized using the robust multi-chip average (RMA) package for Bioconductor and custom GeneAnnot-based Chip Definition Files version 2.2.0 in R software. The most differentially expressed genes between two experimental conditions were defined 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 amplification

Total RNA was extracted from the cells using the RNeasy total RNA isolation kit (Qiagen, Valencia, CA, USA), and then quantified using a Nanodrop ND-100 (Celbio S.p.A., Milan, Italy). 1 μg of RNA was reverse- transcribed with 400 U Moloney murine leukemia reverse transcriptase (Applied Biosciences, Life Sciences, Carlsbad, CA, USA) in accordance with the manufacturer’s protocol. The complementary DNAs were amplified by means of PCR using the following specific primer pairs:

  • HIF1A: F: 5′-IndexTermCTCAAAGTCGGACAGCCTCA-3′; R: 5′-IndexTermCCCTGCAGTAGGTTTCTGCT-3′

  • VEGFA: F:5′-IndexTermCGAAGTGGTGAAGTTCATGGATG-3′; R: 5′-IndexTermTTCTGTTCAGTCTTTCCTGGTGAG-3′

  • IL8: F: 5′-IndexTermTACTCCAAACCTTTCCACCC-3′; R: 5′-IndexTermAACTTCTCCACAACCCTCTG-3′

  • MMP9: F: 5′-IndexTermCGCAGACATCGTCATCCAGT-3′; R: 5′-IndexTermGGATTGGCCTTGGAAGATGA-3′

  • CCL2: F: 5′-IndexTermACTGAAGCTCGTACTCTC-3′; R: 5′-IndexTermCTTGGGTTGTGGAGTGAG-3′

  • GAPDH: F 5'-IndexTermCAACGGATTTGGTCGTATTG-3'; R: 5'-IndexTermGGAAGATGGTGATGGGATTT-3'

Annealing temperature: HIF1A: 59 °C; VEGFA: 66 °C; IL8: 64 °C; CCL2: 60 °C MMP9: 58 °C; GAPDH: 58 °C. 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 quantified.

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 amplifications were performed in duplicate using the iCycler iQ Real-Time Detection System (Bio-Rad, Milan, Italy). The comparative Ct method was applied to normalize the differences in the quantity and quality of RNA, and mRNA was quantified using the comparative ΔCt method using the endogenous reference gene ABL (ΔCt=mean Ct gene–mean Ct ABL); ΔΔCt was evaluated as the difference between the ΔCt of a sample and the ΔCt of the control. The fold change in mRNA expression was calculated as 2−ΔΔCt.

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 Profiler PCR Array and RT2 Real-Timer SyBR green/ROX PCR Mix (PAHS-024, Superarray, SABiosciences, Frederick, MD, USA) that profiles 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 manufacturer's protocol. A total of 40 μg of nuclear extracts were tested. A polyclonal goat anti-HIF-1α Ab (1:1000; R&D system, Minneapolis, MN, USA) or anti-HIF-1β or anti-HIF-2α or anti-HIF-3α (1:500; Novus Biologicals, Littleton, CO, USA) were used to detect HIF-1α, HIF-1β, HIF-2α and HIF-3α, 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-1α, anti-mouse Immunoglobulin G (BD Biosciences) for HIF-1β, HIF-2α and p27 and anti-rabbit Immunoglobulin G (Chemicon International, Millipore, Billerica, MA, USA) for HIF-3α.

HIF-1α activity

HIF-1α 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 manufacturer’s 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 manufacturer’s 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-1α and JJN3 were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 3 μm, and stained with hematoxylin and eosin or with Toluidine blu and Gomori’s three-chromic methods. Tumors obtained from intratibial injections underwent EDTA decalcification before embedding in paraffin. 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-1α 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-1α and VEGF staining were revealed using the UltraVision LP Large Volume Detection System HRP polymer (Thermo Scientific, Erembodegem, Belgium) and quantified according to semiquantitative immunohistochemical 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 fixed 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 3-3′-diaminobenzidine tetrahydrochloride (liquid DAB substrate chromogen system, DAKO, Glostrup, DK).

RESULTS

Permanent HIF-1α silencing in HMCLs: effects on MM cell proliferation, survival and drug sensitivity

HMCLs were initially checked for HIF1A mRNA and HIF-1α protein expression under normoxic conditions (Figure 1a). All HMCLs tested expressed HIF1A mRNA, whereas HIF-1α protein was present at low levels in JJN3 and U266, but not in RPMI-8226 and OPM-2 (Figure 1a). All HMCLs overexpressed HIF-1α protein upon hypoxic treatment. The JJN3 and RPMI-8226 were used as cell lines for HIF-1α 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-1α, HIF-1β and HIF-3α proteins showed specific inhibition of HIF-1α protein under both normoxia and hypoxia (Figure 1d). HIF-2α and HIF-2 β were not expressed either in normoxia or upon treatment with hypoxic mimetic (data not shown). Consistently HIF-1α activity was significantly inhibited in JJN3 anti-HIF-1α as compared with JJN3 pLKO.1 under hypoxic conditions (Figure 1e). Similar results were obtained with RPMI-8226 (data not shown).

Figure 1
figure1

HIF-1α expression by HMCLs and validation of permanent HIF-1α knockdown by shRNA. HIF1A mRNA and HIF-1α 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-1α expression (a). Anti-HIF-1α Lentivirus shRNA pool was used for HIF-1α 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-1α, HIF-1β and HIF-3α protein expression at the nuclear level were evaluated by western blot. Histone H1 was used as internal control (d). HIF-1α activity in the nucleus was checked as described in the methods. Graphs represent the median optical density related to HIF-1α activity for 10 ug of proteins (e).

The effect of HIF-1α suppression on HMCLs proliferation and survival was next investigated. Interestingly, there was no significant inhibition of cell proliferation either with JJN3 or RPMI-8226 (Figure 2a) at different time points. Cell viability was not significantly changed by HIF-1α suppression when checked at 12 and 24 h either under normoxia or in hypoxia (Figure 2b). A statistically significant 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 significantly higher inhibition of cell proliferation in JJN3 anti-HIF-1α as compared with JJN3 pLKO.1 (P=0.01) (Figure 2d) without changing their viability significantly. Consistently p27 expression was increased by lenalidomide in JJN3 anti-HIF-1α as compared with JJN3 pLKO.1 (Figure 2e). On the other hand, Bortezomib (4–10 nM) induced a similar rate of cell proliferation and cell death in JJN3 anti-HIF-1α and JJN3 pLKO.1 (data not shown).

Figure 2
figure2

Effect of HIF-1α inhibition on the proliferation, viability and drug sensitivity of HMCLs. Cell proliferation was evaluated in JJN3-pLKO.1, JJN3-anti-HIF-1α, RPMI-8226-pLKO.1 and RPMI-8226-anti-HIF-1α after 48–96 h of culture by 3H-thymidine uptake as described in the methods. Graphs and bars represent the mean±s.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-1α were incubated under normoxic or hypoxic conditions for 12–24 h and viability evaluated by adapted MTT test. Graphs and bars represent the mean±s.d. for cell viability in four independent experiments performed in duplicate (b). Cell viability was evaluated in JJN3-pLKO.1, JJN3-anti-HIF-1α, RPMI-8226-pLKO.1 and RPMI-8226-anti-HIF-1α after 12–72 h of culture. Graphs and bars represent the mean±s.d. percentage change in the cell viability vs control (c). JJN3-pLKO.1 and JJN3-anti-HIF-1α were incubated with Lenalidomide (0.2–10 μM) 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-1α treated for 24 h with Lenalidomide (Len) (2 and 10 μM) or vehicle (DMSO); actin was used as internal control (e).

HIF-1α suppression affects the transcriptional profile of HMCLs: inhibitory effect on the pro-angiogenic and pro-osteoclastogenic genes

The transcriptional profiles of JJN3 cells transduced with shRNA anti-HIF-1α (JJN3-anti-HIF-1α) were compared with those infected with the control vector pLKO.1 (JJN3-pLKO.1) either under hypoxic or normoxic conditions. Among the significantly 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-1α 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-1α suppression (Figure 3b), whereas CCL2 mRNA was not induced by hypoxia but was inhibited by HIF-1α suppression either under normoxia or hypoxia. Finally, other genes with pro-angiogenic properties, such as IL10 and CCL5, were inhibited by HIF-1α suppression in JJN3 under both normoxic and hypoxic conditions (Supplementary Table S1). Consistent with our previous observation on small interfering RNA-mediated HIF-1α inhibition,27 stable HIF-1α suppression by shRNA significantly blunted vessels formation induced by the conditioned media of HMCLs as determined by AngioKit (TCS Biologies, London, UK) (data not shown).

Figure 3
figure3

Effect of HIF-1α 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-1α 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 -ΔCt 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-1α by real-time PCR. Graphs represent the median fold change in mRNA levels for two independent experiments performed in triplicate (*P<0.05) (c). CCL3/MIP1α and IL-7 protein levels were evaluated in the conditioned media of JJN3-pLKO.1 and JJN3-anti-HIF-1α. Graphs and bars represent the mean±s.d. protein levels for two independent experiments measured in duplicate. (*P<0.05) (d).

The effect of HIF-1α suppression on the production of pro-osteoclastogenic cytokines by HMCLs was then investigated. A significant 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 confirmed at translational level under normoxia as shown for JJN3 (Figure 3d). In contrast, no significant effect was observed on the expression of the osteoblast inhibitor gene DKK1 by JJN3 with HIF-1α suppression (data not shown).

HIF-1α suppression in JJN3 cells blocks the growth of subcutaneous MM in SCID-NOD mice and inhibits angiogenesis

We next investigated whether inhibition of HIF1α in JJN3 may influence tumor growth in vivo. Therefore, we analyzed the tumorigenicity of JJN3 wild type, JJN3 transfected with a plasmid with silenced HIF1α (JJN3-anti-HIF-1α) 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-1α cells developed significantly smaller tumors than mice inoculated with the JJN3-pLKO.1 (P=0.00018) or with JJN3 (P=0.032) (Figure 4a and b). Both weight and volume of the tumors in JJN3-anti-HIF-1α mice were significantly reduced as compared with JJN3-pLKO.1 (Figure 4c and d). The median weight of tumors formed by JJN3-anti-HIF-1α 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-1α was 72.6 mm3 (range 3–221 mm3) and that of JJN3-pLKO.1 was 369.8 mm3 (range 221.8–671 mm3) (P=0.0012) (Figure 4d). Interestingly in the tumoral mass removed from JJN3-anti-HIF-1α mice the microvascular density (number of vessels positive for CD34/mm3) was significantly reduced as compared with that obtained from JJN3-pLKO.1 (JJN3-anti-HIF-1α vs. JJN3-pLKO.1: −76%; P=0.003). Similarly VEGF immunostaining was reduced in JJN3-anti-HIF-1α derived tumors as compared with JJN3 pLKO.1 injected mice (Figure 4e and f).

Figure 4
figure4

HIF-1α 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 HIF1α (JJN3-anti-HIF-1α) 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-1α and JJN3-pLKO.1) after 20 days (a) or tumor masses removed from JJN3-anti-HIF-1α 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-1α 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-1α and JJN3-pLKO.1. Original magnification × 400 (e) and × 1000 (f).

HIF-1α suppression in JJN3 cells blunts angiogenesis and bone destruction in an intratibial mouse model

The effect of HIF-1α suppression in MM cells was further investigated in vivo in intratibial mouse model. The SCID mice were injected intratibially with saline or JJN3-pLKO.1 or JJN3-anti-HIF-1α cells, and lytic lesions were allowed to develop for 2–4 weeks before the mice were analyzed. Histological analysis showed a significant reduction in the tumor mass with an increase in the bone thickness in JJN3-anti-HIF-1α 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-1α as compared with JJN3 pLKO.1 mice although the difference did not reach statistical significance (Figure 5b). However, immunostaining for CD34 showed a significant reduction in the number of CD34-positive vessels in JJN3-anti-HIF-1α 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-1α cells showed a dramatic reduction of osteolytic lesions (Figure 5d). Finally, histological analysis showed a significant reduction in the number of OCLs in JJN3-anti-HIF-1α as compared with JJN3-pLKO.1 bone sections (P<0.05), whereas the number of active prismatic osteoblasts arranged in laminae did not significantly changed as well as that of viable osteocytes for 500 lacunae (P=0.4; NS, data not shown).

Figure 5
figure5

HIF-1α inhibition in JJN3 blunts myeloma-induced neo-angiogenesis and bone destruction after intratibial inoculation in mice. SCID mice were injected intratibially with 20 μl containing 5 × 104 cells of JJN3-anti-HIF-1α 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-1α 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-1α 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, *P<0.001). Images show the dissected tibias of representative mice (saline, JJN3-anti-HIF-1α and JJN3-pLKO.1) acquired on a vivaCT 40 at resolution of 21 μm isotropic, reconstructed and segmented for 3-dimensional display using the instrument's analysis algorithm software (d).

Discussion

It is well established that intratumoral hypoxia and consequently HIF-1α activation in solid tumors critically trigger the angiogenic switch, and induce modifications of cancer cell metabolism leading to tumor progression and metastasis.16, 17, 18, 22 HIF-1α is overexpressed in many tumors22, 23 including some hematological malignancies such as MM.27, 28, 29, 35 We and others27, 35 have recently shown that BM microenvironment is hypoxic both in MM patients27 and in mice.35 In addition HIF-1α 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-1α 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-1α could be a potential therapeutic target for MM. To test this hypothesis, we performed HIF-1α knockdown in HMCLs using a pool of shRNA anti-HIF-1α and evaluated the effect permanent suppression of HIF-1α on MM cell proliferation, survival, the pro-angiogenic profile and tumor growth in vivo. Firstly, we showed that the inhibition of HIF-1α by shRNA was specific because no significant inhibitory effect was demonstrated on HIF1B, HIF2A, HIF2B and HIF3A expression. Interestingly, we did not find a significant inhibitory in vitro effect on MM cell proliferation by HIF-1α suppression only showing a significant inhibitory effect on cell survival after 72 h in JJN3, but not in RPMI-8226. These results suggest that HIF-1α inhibition did not directly induce a significant effect on MM cell proliferation and survival. Similarly, it has been previously reported that small interfering RNA anti-HIF-1α 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-1α stable suppression on the sensitivity of HMCLs to IMiDs and Bortezomib in vitro. Interestingly, we found that shRNA anti-HIF-1α increased the inhibitory effect of IMiDs on HMCL proliferation. This result was also supported by the previous finding that the response to lenalidomide treatment involves oxidative stress response in MM cells.37 In contrast, we did not find 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-1α upregulation38 rather than by HIF-1α inhibition.

Although, we did not find a significant effect on cell proliferation and survival by knockdown of HIF-1α, stable HIF-1α suppression by shRNA modified the pro-angiogenic and osteoclastogenic profiles of MM cells either under hypoxic or normoxic conditions. Microarray analysis and real-time PCR showed that HIF-1α suppression significantly inhibited some pro-angiogenic factor genes such as VEGFA, IL-8, CCL2, MMP9. The capacity of HIF-1α to regulate VEGFA and IL8 gene expression by MM cells confirmed our previous data,27, 39 and supports HIF-1α 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-1α, because both cytokines are produced by MM cells and contribute to angiogenesis and BM spreading.41, 42, 43

Interestingly, our data suggest that other than angiogenic cytokines, HIF-1α 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/MIP1α44 and IL-7.45 To the best of our knowledge, this is the first time that the capacity of HIF-1α has been reported to regulate pro-osteoclastogenic cytokine in MM cells, although the evidence that CCL3/MIP1α and IL-7 are potential target genes of HIF-1α has been shown in other cell types such as acute myeloid leukemia cells for CCL3/MIP1α46 and mesenchymal/osteoblastic cells for IL-7.47

To evaluate the role of HIF-1α 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-1α suppression showing a significant reduction of tumor mass. Because of the lack of a significant effect of HIF-1α 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-1α to regulate the pro-angiogenic profiles of HMCLs in vitro and by our previous results showing that HIF-1α suppression blocks MM-induced in vitro angiogenesis.27 The significant inhibition of angiogenesis (evidenced by reduction in the number and length of vessels in mice injected with JJN3-anti-HIF-1α) confirmed that the antitumor effect of HIF-1α suppression was mainly due to the inhibition of angiogenesis. In support of these data, the inhibitory effect on angiogenesis by HIF-1α 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 significance. Finally, in line with our data, it has been previously reported that HIF-1α 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-1α 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 significant reduction of the number of OCLs in JJN3 anti-HIF-1α as compared with JJN3-pLKO.1.

The capacity of HIF-1α 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-1α 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-1α 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-1α inhibition results in a potent anti-MM effect by blocking angiogenesis and the development of osteolytic bone lesions. These results suggest that HIF-1α is a potential therapeutic target in MM. In this context, it has been recently reported that adaphostin inhibits HIF-1α expression and induces a significant 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-1α 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-1α mRNA, such as EZN-296850 in MM patients.

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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, Università 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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Correspondence to N Giuliani.

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Keywords

  • myeloma
  • angiogenesis
  • bone disease
  • hypoxia
  • mice

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