Molecular and cellular mechanisms of increased angiogenesis in multiple myeloma : a role for CXCL12.

Abstract

Multiple myeloma (MM) is an incurable haematological malignancy characterised by the clonal proliferation of plasma cells (PCs) within the bone marrow (BM). MM PC survival and expansion is dependent upon an adequate supply of oxygen and nutrients, and increased BM angiogenesis is a critical feature of MM progression. While MM PCs express and secrete a number of angiogenic factors, our current understanding of the precise mechanisms by which MM-induced angiogenesis occurs is incomplete. In this study, we collected specimens from patients with MM and the benign precursor condition MGUS, and demonstrated for the first time that circulating levels of the CXCL12 chemokine positively correlate with the degree of BM angiogenesis. Using conditioned media from a MM PC line, the contribution of MM PC-derived CXCL12 to angiogenesis was also examined and found to strongly induce vascular tube formation in vitro. In several other cell types, hypoxia has been shown to up-regulate CXCL12 expression. Studies investigating the hypoxic regulation of CXCL12 in MM PCs revealed that, while acute hypoxia is unable to stimulate CXCL12 expression, prolonged hypoxia significantly up-regulates CXCL12 mRNA and protein expression. To determine the mechanism(s) responsible for this, over-expression and RNA interference technology was employed to create genetically modified MM cells in which either HIF-1α or HIF-2α were overexpressed or knocked down. These studies showed that HIF-2α is the predominant mediator of the hypoxic induction of CXCL12 in MM PCs. The ability of HIF-2α to bind to the CXCL12 promoter was confirmed using EMSA and ChIP analyses. The role of CXCL12 in in vivo angiogenesis and the contribution of HIF-1α and HIF-2α were also examined. In these studies, transduced MM cells, in which HIF-1α, HIF-2α and CXCL12 were over-expressed or knocked down, were implanted into a vessel-poor, subcutaneous environment in immunocompromised mice. Tumour-induced angiogenesis was assessed after two weeks by measuring the haemoglobin content of excised implants. These studies confirmed that over-expression of CXCL12, HIF-1α and HIF-2α each stimulates a strong angiogenic response. Using the well-characterised CXCR4 antagonist, T140, CXCL12 was found to play a key role in the increased angiogenesis observed in response to HIF-1α and HIF-2α over-expression. These novel studies have shown that CXCL12 is an important mediator of angiogenesis in MM patients, and that aberrant CXCL12 expression by MM PCs is due, in part, to its hypoxic up-regulation mediated predominantly by HIF-2.