Heparanase Promotes Osteolysis by Upregulating RANKL– A Novel Role for Heparanase in Multiple Myeloma.

Abstract 2821

Poster Board II-797

Heparanase, an enzyme that cleaves the heparan sulfate chains of proteoglycans, is upregulated in a variety of human tumors including multiple myeloma. We have previously demonstrated that heparanase promotes robust myeloma tumor growth and supports spontaneous metastasis to bone. In addition we showed that expression of heparanase is a major determinant of the osteolytic phenotype in myeloma and that heparanase expression in a distal tumor can promote systemic osteolysis due to enhanced osteoclastogenesis within distal bones prior to metastasis. Because it has been shown that myeloma cells can produce and secrete RANKL, we hypothesized that heparanase upregulates the expression of RANKL thereby leading to osteolysis. To test this idea, CAG myeloma cells transfected with human heparanase cDNA (heparanase-high) or empty vector (heparanase-low) were injected into SCID mice and the levels of human RANKL expression were evaluated by immunohistolochemical staining and western blotting. A significant elevation of RANKL was observed in heparanase-high tumors compared to heparanase-low tumors formed in human bones implanted in SCID mice (SCID-hu), as well as in subcutaneous myeloma tumors. In addition, RANKL levels were significantly elevated in both in vitro cell extracts and conditioned medium of myeloma cells when heparanase expression was enhanced. The elevation of RANKL expression was also found in other myeloma cell lines (MM.1S and U266) when cells were cultured in the presence of recombinant human heparanase (50 ng/ml and 100 ng/ml each cell line, added every 12 hours) for 48 hours. In addition, in vitro osteoclastogenesis is significantly enhanced by the medium conditioned by heparanase-high cells as compared to heparanase-low cells and this enhanced osteoclastogenesis is completely abolished by addition of OPG (a RANKL inhibitor) to the medium. From these data, we conclude that elevated heparanase in myeloma cells increases RANKL expression and secretion, resulting in the increased osteolysis in myeloma.