Myeloma Cells Block Runx2/Cbfa1 Activity in Human Bone Marrow Osteoblast Progenitors and Inhibit Osteoblast Formation and Differentiation.

The decreased bone formation contributes to the development of bone lesions in multiple myeloma (MM) patients. Runx2/Cbfa1 is a transcription factor highly restricted to the osteoblastic lineage that has a critical role in the osteoblast formation and differentiation. In this study we investigated the potential effect of myeloma cells on osteoblastogenesis and the role of Runx2/Cbfa1 in the decreased bone formation in MM.

In a co-culture system performed in presence or absence of a transwell insert with human myeloma cell lines (HMCLs) (RPMI-8226, U266, XG-1, XG-6, OPM-2) and human bone marrow (BM) pre-osteoblastic cells, obtained from BM stromal cells (BMSC), we observed an inhibition of osteocalcin, alkaline phosphatase and collagen I expression at both mRNA and protein level. Consistently we found that the formation of both Colony Forming Unit Fibroblasts (CFU-F) and Colony Forming Unit Osteoblasts (CFU-OB) were suppressed in long term BM culture by several HMCLs. Moreover, by a gel mobility shift assay (EMSA) we found that Runx-2/Cbfa1 activity was significantly inhibited in BMSC/pre-osteoblastic cells after 48 hours of co-cultures with OPM-2 whereas the level of Runx2/Cbfa1 protein was not affected. The inhibition of osteoblast formation, differentiation and the block of the Runx2/Cbfa1 activity in BMSC/pre-osteoblastic was more pronounced in the cell-to-cell contact conditions as compared to those without the cellular contact.

Further we evaluated Runx2/Cbfa1 expression by immunohistochemistry in BM biopsies of 16 MM patients finding a significant reduction of the number of Runx2/Cbfa1 positive cells in osteolytic patients as compared to those without bone lesions (median %: 19.93% vs. 38%; p=0.001) that supports the in vitro results.

To identify molecules that could contribute to the inhibitory effect of myeloma cells on osteoblast formation, differentiation and Runx2/Cbfa1 activity, we screened HMCLs and fresh purified CD138+ MM cells for the expression of DKK1, IL-7, noggin, gremlin and secreted frizzled-related protein (sFRP)-2, -3, -4 also evaluating the potential involvement of these osteoblast inhibitors. DKK1 mRNA was expressed by 2 out of 5 HMCLs tested and by 8 out of 12 MM patients tested as well as by BMSC/pre-osteoblastic cells and hOB, however soluble DKK-1 at a widely range of concentration had not effect on the number of CFU-F and CFU-OB in human BM cultures and it did not inhibited Runx2/Cbfa1 activity in BMSC/pre-osteoblastic cells. Moreover, blocking anti-DKK1 Ab did not blunted the effect of HMCLs on osteoblast formation in human BM cultures. All the HMCLs and fresh CD138+ MM cells tested expressed IL-7 mRNA, as we have previously reported. IL-7 reduced Runx2/Cbfa1 activity in BMSC/pre-osteoblastic cells showing an inhibitory effect on CFU-F and CFU-OB formation in BM culture, moreover blocking anti-IL-7 Ab partially reduced the effect of HMCLs in co-cultures. Finally, we found that HMCLs and fresh CD138+ MM cells did not expressed noggin, gremlin, sFRP-2 and rarely produced sFRP-3 and sFRP-4 whereas BMSC/pre-osteoblastic cells and hOB were positive for these factors even if HMCLs had not effect on their expression in co-cultures.

In conclusion our data indicate that human myeloma cells inhibit osteoblast formation and differentiation in human BM cultures blocking Runx2/Cbfa1 activity in BMSC/pre-osteoblastic cells through the cell-to-cell contact. Soluble factors produced by myeloma cells as IL-7 could contribute to the inhibitory effect on osteoblastogenesis.