Small Leucine-Rich Proteoglycans (SLRPs) Are Involved in the Anti-Myeloma Response of Osteoblasts.

Multiple myeloma (MM) is closely associated with severe osteolytic bone disease caused by stimulation of osteoclastogenesis and suppression of osteoblastogenesis. We have recently demonstrated that while osteoclasts support MM growth, osteoblasts have negative impact on survival and proliferation of MM cells from a subset of patients in vitro and in vivo (Yaccoby et al., Cancer Res 2004: Haematologica 2006), indicating that MM bone disease drives tumor progression. To unravel anti-MM molecular mechanism of osteoblasts we performed global gene expression profiling on osteoblasts and their precursors, mesenchymal stem cells (MSCs). We found and validated by qRT-PCR and Western Blotting that osteoblasts produce high level of small leucine-rich proteoglycans (SLRPs). Main SLRPs such as decorin, biglycan and lumican are essential for proper bone remodeling have been shown to inhibit growth of various tumors and their expression is inversely correlated with tumor progression. Decorin is not produced by most MM cases and recombinant decorin was rapidly internalized by MM cells. Co-culture of MM cells with the supporting osteoclasts in the presence of osteoblast’s conditioned media resulted in reduced MM cell survival (n=5, p<0.002), an effect that was partially but significantly attenuated by decorin neutralizing antibody (5 μg/ml, n=5, p<0.01). In co-culture with osteoblasts, primary MM cell growth was significantly increased by 46±15% when decorin expression in osteoblasts was effectively blocked using lentiviral-containing decorin siRNA (n=4, p<0.04). In contrast overexpression of decorin in MSCs resulted in reduced primary MM cell survival by 20±3% (n=4, p<0.04). Furthermore, in co-culture with osteoclasts recombinant or purified decorin (5–10 μg/ml) inhibited growth of primary MM cells in 6 of 9 experiments by 35±8% (p<0.007). The anti-MM effect of decorin involved direct induction of apoptosis as determined by annexin V/PI flow cytometry and activation of p21 using Western Blotting. In additional experiments we demonstrated that conditioned media from culture of MM cells stimulated tube formation by HUVECs in a matrigel assay and that decorin significantly attenuated this effect (2±1 vs. 12±1 tubes in control wells, p<0.001), indicating that certain SLRPs are antiangiogenic. Decorin (5–10 μg/ml) also effectively inhibited differentiation of osteoclasts in culture of osteoclast precursors with RANKL and M-CSF (14±2 vs. 32±4 multinucleated TRAP-expressing osteoclasts in control wells, p<0.01). For in vivo study we exploited our SCID-hu model for primary MM (Yaccoby et al., Blood 1998: 1999). Immunohistochemical analysis for decorin and lumican in nonmyelomatous human bone implanted in SCID-hu mice revealed high expression level by osteogenic cells and localization along the bone surface and extracellular matrix. Decorin and lumican levels were reduced in myelomatous bones engrafted with primary MM cells and markedly increased following treatment with osteoblast activating agents such as PTH and anti-DKK1, providing a possible molecular mechanism for anti-MM efficacy of these agents in vivo (Yaccoby et al., Blood 2007). We conclude that certain SLRPs are involved in the anti-MM effect of mature osteoblasts directly and indirectly through inhibition of angiogenesis and osteoclastogenesis, and that increasing endogenous or exogenous SLRPs in myelomatous bones are safe approaches to control MM and its associated bone disease.