Oxygen Tension in the Bone Marrow (BM) of Patients with Malignant and Indolent Monoclonal Gammopathy: Role of Hypoxia and Hypoxia-Inducible Factor (HIF)-1α in the Regulation of Gene Expression and Pro-Angiogenic Profiles of CD138⁺ Cells.

Abstract 422

Multiple myeloma (MM) is characterized by the accumulation of malignant plasmacells into the bone marrow (BM) microenvironment that supports their growth and survival. Particularly, an increase of BM angiogenesis occurs in relationship with plasmacells infiltration playing a critical role in the progression on monoclonal gammopathy. Hypoxia is known to be associated to angiogenesis in solid tumors as well as the hypoxia-inducible factor (HIF)-1α is a critical trigger and regulator of the angiogenic switch. Actually, the oxygen levels in the BM of patients with monoclonal gammopathy as well the effects of hypoxia and HIF-1α on the gene expression and pro-angiogenic profile of CD138⁺ cells are not known.

In this study, first, we investigated the level of BM oxygen saturation (sO₂) and partial pressure (pO₂) in a cohort of 44 patients with monoclonal gammopathy at the diagnosis including active MM (n°=25), smoldering MM (n° = 8) and MGUS (n°=11) showing that BM of MM patients was hypoxic (mean ± SD of pO₂: 52.5 ± 8.69 mmHg; sO2 of 83.7 ± 11%) even if any significant difference in both pO₂ and sO₂ was not observed in comparison with smoldering MM patients or MGUS as well as in relationship with the stage of disease (p=0.7). Next, we evaluated how hypoxia exposition could modify the gene expression profile of CD138+cells isolated form MM patients by U133 Plus2.0 Arrays. By supervised analysis performed on 11 paired samples we found that hypoxia significantly modulated 714 genes in CD138+ cells isolated from MM patients. Interestingly, genes belonging either to oxidative stress and hypoxia signaling, including heme oxygenase 1 (HMOX1) and the heat shock proteins 90kDa alpha (HSP90AA1; HSP90AB1), or to protein ubiquitination pathway, such as XIAP, were significantly up-regulated by hypoxia. Among the pro-angiogenic genes, we found that both VEGFA and IL8 were induced by hypoxia. Following, given that HIF-1α accumulates only in hypoxic condition, we checked HIF-1α protein expression in the BM of MM patients by immunohistochemistry on bone biopsies immediately after fixation. A strong HIF-1α immunostaining was demonstrated in MM cells at nuclear level in all patients analyzed. Interestingly, the presence of HIF-1α protein was also observed in isolated CD138⁺ MM cells of about 28% of MM patients in normoxic condition.

In order to investigate the potential role of HIF-1α on MM cell gene expression and angiogenic profile we performed HIF-1α silencing in MM cells by siRNA anti-HIF-1α and that exposed cells to normoxic or hypoxic conditions. HIF-1α suppression was associated to the modulation of pro-angiogenic molecules (VEGFA, VEGFB, IL-8 and PGF) and oxidative stress (SOD2, PTGS2, TXT) and glycogenolysis regulating (ENO2, ALDOC, PFKFB3, HK2, PDK1, PFKFB4) genes. Data obtained were than validated by real time PCR and at protein level by western blot and ELISA assay. Consistently we found that HIF-1α suppression significantly inhibited the pro-angiogenic properties, evaluated in an in vitro angiogenesis model as capillary junctions and tubules formation and tubule length.

In conclusion, we demonstrate that the MM-BM environment is hypoxic and that HIF-1α protein expressed by MM cells. Consistently we show that hypoxia and HIF-1α significantly modulate the gene expression profiles of MM cells regulating the expression of the pro-angiogenic factors by MM cells and their pro-angiogenic properties.