Hypoxia and hypoxia inducible factor (HIF)-1α in Multiple Myeloma patients: role in the angiogenic switch

An increase of bone marrow (BM) angiogenesis occurs in multiple myeloma (MM) patients due to the overexpression of several pro-angiogenic factors by MM cells, however the molecular mechanisms involved are not jet completely investigated. Hypoxia is a common feature of solid tumors associated to angiogenesis. Tumor adaptation to hypoxia is mainly due to the hypoxia-inducible factor (HIF)-1alpha. The effect of hypoxia on MM cells and the role of HIF-1α in MM-induced angiogenesis actually are not known. In this study, first we checked the level of BM oxygen tension in a cohort of MM patients (n°=25) at the diagnosis as compared to healthy donors and MGUS subjects. The mean pO2 ± SD was 52.3±9 mmHg in MM patients similar to that observed in the controls, confirming that MM cells are exposed in vivo to hypoxic microenvironment. Thereafter HIF-1α protein expression by MM cells was checked by immunohistochemistry on bone biopsies of MM patients showing the presence of HIF-1alpha stabilization at nuclear level in malignant plasmacells into the BM. Interestingly, HIF-1alpha protein stabilization and activity was observed at nuclear level in purified CD138+ MM cells in about of 28% of MM patients evaluated suggesting that a hypoxia independent stabilization of HIF-1alpha may occur in MM cells. Consequently the effect of hypoxia and HIF-1alpha in MM cells was checked silencing HIF-1alpha by a pool of siRNA. A gene expression profiling evaluation was performed by microarray analysis using Gene Chips U133plus 2.0 (Affymetrix). Data were then validated by real time PCR. We found that hypoxia significantly upregulated the expression of the pro-angiogenic molecules in MM and cells including VEGF, OPN, IL-8 blunted by HIF-1alpha know-out. Genes belonging to glycolysis and HIF-1α regulating signal pathways were also regulated by HIF-1alpha in MM cells in hypoxic condition. These observations were confirmed in purified CD138+ MM cells exposed to hypoxia that induced a significant up-regulation of the pro-angiogenic molecules and the modulation of glycolysis and ubiquitin mediated proteolysis signal pathways. In normoxic condition, HIF-1alpha knock out significantly affected in MM cells either pro-angiogenic molecules as VEGF or several genes belonging to cell cycle regulation. In conclusion our data underline the role of hypoxia in the regulation of the angiogenic signature of MM cells and suggest that HIF-1alpha could be a potential target in MM.