Hypoxia and Hypoxia Inducible Factor (HIF)-1α in Multiple Myeloma: Effect on the Pro-Angiogenic Signature of Myeloma Cells and the Bone Marrow Microenvironment.
Authors: Colla Simona , Storti Paola , Donofrio Gaetano , Lazzaretti Mirca , Bonomini Sabrina , Lunghi Paolo , Crugnola Monica , Bolzoni Marina , Galla Luisa , Ippolito Luigi , Martella Eugenia , Sammarelli Gabriella , Craviotto Luisa , Caramatti Cecilia , Mangoni Marcellina , Bonati Antonio , Rizzoli Vittorio , Giuliani Nicola 
Published: April 2020

Abstract
Hypoxia is a common feature of solid tumors associated to angiogenesis and malignant phenotype. Tumor adaptation to hypoxia is mainly due to the hypoxia-inducible factor (HIF)-1α, a key transcription factor that regulates angiogenesis and tumor progression. As known multiple myeloma (MM) is a hematological malignancy characterized by the accumulation of malignant plasmacells into a hypoxic microenvironment as the bone marrow (BM) that critically supports their growth and survival. However the effect of hypoxia on MM cells and the role of HIF-1α in MM-induced angiogenesis actually are not known. Recently we have demonstrated that the tumor suppressor gene ING4 may exert an anti-angiogenic effect through the inhibition of HIF-1α activity in MM cells in hypoxic condition suggesting a role of HIF-1α in MM-induced angiogenic switch. To go further insight this issue, 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 (p=NS) 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 showing the presence of HIF-1α stabilization at nuclear level in malignant plasmacells as well as in BM stromal cells (BMSC) into the BM. Consequently the effect of hypoxia and HIF-1α in both MM and BMSC cells was checked. Human myeloma cell lines (JJN3 and RPMI-8226) and BMSC were transfected with a pool of siRNA anti-HIF-1α to knockout HIF-1α and then exposed to low oxygen tension. 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 both MM and BMSC cells including Vascular Endothelial Growth Factor (VEGF), Osteopontin (OPN) and Interleukin-8 (IL-8) blunted by siRNA anti-HIF-1α. Genes belonging to glycolysis and HIF-1α regulating signal pathways were found to be also regulated by HIF-1α in MM cells in hypoxic condition. These observations were confirmed in purified CD138+ MM cells (n°=11) 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. Finally, the potential expression and role of HIF-1α in MM cells was also investigated in normoxic condition. Whereas the presence of HIF-1α mRNA was observed in all HMCLs and primary MM cells tested, HIF-1a protein stabilization and activity was observed at nuclear level in 2 out of 6 HMCLs and in about 38% of MM patients evaluated suggesting that a hypoxia independent stabilization of HIF-1α may occur in MM cells. Consistently, in normoxic condition, HIF-1α knock out by siRNA significantly affected in HMCLs 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 the BM microenvironment and suggest that HIF-1α could be a potential target in MM.