Hypoxia-Inducible Factor (HIF)-1α Is a Therapeutic Target in Myeloma-Induced Angiogenesis

Abstract 3927

Hypoxia-inducible factor (HIF)-1α is a critical trigger and regulator of tumor associated angiogenesis. It has been previously reported that bone marrow (BM) microenvironment is hypoxic in multiple myeloma (MM) patients and that HIF-1α is overexpressed by CD138⁺ MM cells and modulates the transcriptional and pro-angiogenic profiles of MM cells. The potential role of HIF-1α as a therapeutic target in MM is under investigation. To deepen this issue, in this study we explored the effect of a stable HIF-1α inhibition in MM cells on cell proliferation, survival and on MM-induced angiogenesis either in vitro or in vivo using a plasmocytoma model. Human myeloma cell lines (HMCL)s were screened for HIF-1α protein expression in normoxia condition finding that 2 out 5 HMCLs were positive (JJN3 and RPMI-8226). Anti-HIF-1α Lentivirus sh RNA pool was used for HIF-1α stable knock-out in human myeloma cell lines (HMCL)s whereas the pKLO.1 lentiviral vector was used as the empty control vector. HMCLs have been infected and selected with 0.4 ug/ml puromycin for 21 days and the selected clones screened for HIF-1α, HIF-1β, HIF-2α and HIF-3α. A selective inhibition on HIF-1α mRNA and protein expression was observed both in normoxic and hypoxic conditions. HMCLs proliferation and apoptosis were not affected by HIF-1α inhibition neither in hypoxia nor normoxia.

Thereafter the transcriptional profiles on the HMCL JJN3 transduced with shRNA anti-HIF-1α (JJN3-anti-HIF-1α), as compared to those infected with the control vector pKLO.1 (JJN3-pKLO.1) have been analyzed by U133 Plus2.0 Arrays (Affymetrix ®) either in hypoxic or normoxic conditions. The raw intensity signals were extracted from CEL files and normalized using the RMA package for Bioconductor and custom GeneAnnot-based Chip Definition Files in R software. Absolute fold change higher than 1.5 was used to select differentially expressed genes. Among the genes significantly modulated (327 and 361 genes in hypoxic and normoxic condition, respectively) we found that the pro-angiogenic molecules VEGF, IL8, IL10, CCL2, CCL5, MMP9 were down-regulated in JJN3-anti-HIF-1α as compared to JJN3-pKLO.1 either in hypoxic or normoxic conditions. Microarray data were further validated by quantitative real time PCR. In line with this observation, we found that the pro-angiogenic properties of JJN3, evaluated by an in vitro angiogenesis assay (Angio-Kit, TCS), were significantly inhibited by the HIF-1α knock-out. Finally the potential involvement of HIF-1α in MM-induced angiogenesis was assessed in vivo in NOD/SCID mice in a subcutaneous model. We found that the mice which were injected subcutaneously with JJN3-anti-HIF-1α, showed a dramatic reduction of the weight and volume of the plasmocytoma mass than mice inoculated with the JJN3-pKLO.1 (P=0.0007 and P=0.012; respectively). Moreover we observed a significant reduction of the number of vessels X field stained by CD31 antigen (anti-HIF-1α vs. pKLO.1: −76%; P=0.003) and VEGF immunostaining, whereas the Ki67 index of the plasmocytoma mass only showed a slight reduction (anti-HIF-1α vs. pKLO.1: −14%, P=NS).

Overall our data indicate that HIF-1α suppression in myeloma cells significantly blocks MM-induced angiogenic switch both in vitro and in vivo and consequently reduces the MM tumoral burden suggesting that HIF-1α is a potential therapeutic target in MM.