Human Myeloma Cells Express the Bone Regulating Gene Runx2/Cbfa1 and Produce Osteopontin That It Is Involved in Myeloma-Induced Angiogenesis.

Osteopontin (OPN) is a multifunctional bone matrix glycoprotein that it is involved in angiogenesis, cell survival and tumor progression. OPN gene expression by human cells is mainly regulated by the bone specific trascription factor Runx2 namely also CBFA1 (Runx2/Cbfa1) even if other transcription factors may be also involved in the production of OPN by cancer cells as the CCAAT/enhancer-binding protein alpha (C/EBPα) and AML-1.

In this study we show that human myeloma cell lines (HMCLs): RPMI-8226, U266, XG-1 and XG-6 but not ARH-77 and normal CD19+ cells directly produce OPN and express its major regulating gene Runx2/Cbfa1. The activity of Runx2/Cbfa1 in human myeloma cells has been also demonstrated by a gel mobility shift assay. On the other hand we found that all HMCLs tested were negative for C/EBPα mRNA, whereas AML-1A and AML-1B mRNA were expressed in all HMCLs even if any correlation has not been found between OPN expression and AML-1A/AML-1B ratio. In addition we found that OPN production by myeloma was up-regulated at both mRNA and protein level by IL-6 and IGF-I through a Runx2/Cbfa1 mediated mechanism; in turn recombinant human OPN was able to increase myeloma cells proliferation. The potential role of OPN in MM-induced angiogenesis has been also investigated in an experimental model of angiogenesis. rhOPN treatment stimulated vessel formation as compared to control and the conditioned medium (CM) of HMCLs, significantly increased vessel formation in comparison either with control or with VEGF treatment. On the contrary OPN-immunodepleted CM of HMCLs had not a stimulatory effect on vessel formation and the presence of anti OPN Ab inhibited vessel formation induced by HMCLs.

The expression of OPN by purified bone marrow (BM) CD138+ cells has been also investigated in 60 newly diagnosed multiple myeloma (MM) patients, finding that 40% of MM patients tested expressed OPN. Higher OPN levels have been detected in the BM plasma of MM patients positive for OPN as compared to control subjects.

A significant higher MVD was observed in the group of patients positive for OPN, (mean±SE: 29.1±0.7 vs. 17.55±0.37; p<0.01) and similarly, the number of microvessels per field was higher in OPN positive patients in comparison with OPN negative ones (mean±SE: 6.7±0.15 vs. 4.28±0.04; p=0.05).

In conclusion our data highlight the direct ectopic production of OPN by human myeloma cells with a Runx2/CBFA1 mediated mechanism and the capacity of myeloma-derived OPN to stimulate angiogenesis in vitro. In addition OPN has been detected in a subset of MM patients with higher BM angiogenesis suggesting its potential involvement in pathophysiology of MM-induced angiogenesis.