Role Of Decorin In Multiple Myeloma (MM) Bone Marrow Microenvironment

Abstract

Decorin is a small leucine rich proteoglycan found in the extracellular matrix of various connective tissues with potential effective tumor suppressor properties. Recently, it has been shown that decorin decreases in multiple myeloma (MM) patients compared to healthy volunteers, and also in patients with osteolytic bone lesions than non-osteolytic lesions. By using a cytokine array analysis we showed that decorin is down regulated in patients withdrawn from aminobisphosphanates for six months. These results led us to focus on decorin and its role in the bone marrow micro environment.We looked at the expression of decorin in MM cell lines. The mRNA expression of decorin in MM cells (OPM1, OPM2, RPMI, INA6, U266, KMS18 and KMS20) was not detectable when compared to MG63, an osteosarcoma cell line that constitutively produces decorin. Consistent with the mRNA expression, decorin protein secretion was also very low in the MM cell lines as demonstrated by ELISA. A 48 hour MTT assay on OPM1, OPM2, RPMI, MM1.S and CD138+ patient myeloma cells with increasing concentrations (0, 5, 10, 20 and 40μg/ml) of exogenous decorin did not show a significant decrease in cell viability. These results indicate that decorin is not produced by MM cells and does not have a direct effect on MM cell viability.Having looked at decorin in the tumor, we then observed its expression in the stromal compartment. We investigated decorin mRNA and protein expression in bone marrow stromal cells (BMSC), osteoclasts (OC) and osteoblats (OB) differentiated from MM patients. BMSC and OB, differentiated for 4 and 7 days, showed a significantly higher expression of decorin compared to the OC. Decorin secretion was more pronounced by differentiating OB than BMSCs. Consistent with previously reported data, we also found that decorin protein levels were higher in BMSCs and OB differentiated for 4 and 7 days from healthy volunteers than MM patients. Moreover, addition of exogenous decorin (at 5 and 10 μg/ml) to OB increased OB differentiation as seen by increased mRNA expression for markers of OB differentiation (alkaline phosphatase and RUNX2) suggesting that decorin plays a role in OB differentiation.Given the significance of the interaction between the MM tumor and its micro-environment we next evaluated the effect of decorin in the stroma when co-cultured with MM cells. Decorin mRNA levels were down regulated when OPM2 cells were co-cultured without direct cell contact with BMSC and OB differentiated for 4 and 7 days suggesting that MM cells decreased decorin and the effect is probably cytokine mediated.Having studied the effect of decorin on OB in the stromal compartment, we then focused on the OCs. When exogenous decorin was added at 5 and 10mg/ml to peripheral blood mononuclear cells from MM patients in OC differentiation media for 7, 14 and 21 days of differentiation, there was a significant decrease in OC number as observed by TRAP staining (80% decrease in OC number in 5mg/ml and 95% decrease in 10mg/ml compared to control). Decorin at the same concentrations greatly decreased the area of pits as compared to the control (43% decrease in area of pit in 5mg/ml and 67% decrease in 10mg/ml compared to control) suggesting that exogenous decorin inhibited OC differentiation and function. TRAP staining and MTT assays on 14 day mature OCs with decorin treated for 48 hours after maturation, did not show a decrease in cell number and viability of OC indicating that decorin does not affect mature OCs.In conclusion, our data suggests that decorin secreted by stromal cells and in particular differentiating OBs, does not directly affect the viability of MM cells. It inhibits OC differentiation and function and promotes OB differentiation. Importantly, decorin levels decreased significantly in the BMSC and OB when co-cultured with MM cells. We speculate that this decrease is a result of decreased OB differentiation by MM cells. Since, all the above lines of evidence point to decorin modulating the myeloma microenvironment with potential indirect anti-tumor effects without acting as a direct antagonist to the tumor, ongoing studies that understand mechanisms to stimulate decorin secretion can prove to be useful with respect to MM microenvironment interactions. Disclosures:Raje:Celgene: Consultancy, Research Funding; Millenium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding; Amgen: Consultancy; Acetylon: Consultancy, Research Funding; Eli Lilly: Research Funding.