CCL3 Impairs Osteoblast Function Via Downregulation of Osteocalcin.

Abstract 739

A common feature of bone disease in cancer is hyperactivity of osteoclasts (OC). However, osteoblast (OB) inhibition is critical to the development of osteolytic lesions. Tumor-OC interactions have been extensively studied, the mechanism of OB inhibition however still remains elusive. Several chemokines are upregulated in the multiple myeloma (MM)/bone microenvironment. CCL3 (MIP-1αa), in particular, mediates MM cell migration and stimulates OC differentiation, directly, by stimulating precursor cell fusion, and indirectly, by inducing OB secretion of RANKL. Here, we investigate whether CCL3 interferes with OB differentiation and activity. In vitro osteoblastogenesis consists of differentiation of alkaline phosphatase (ALP) positive cells, followed by secretion and mineralization of the extracellular matrix. We observed that ALP-positive cells express both CCL3 receptors, CCR1 and CCR5, late in differentiation (40% and 32%, respectively, isotype control 9%). No significant CCL3 secretion was detected in OB culture supernatant, suggesting that paracrine CCL3 may primarily affect matrix mineralization. Indeed, exogenous CCL3 (25 to 100 ng/ml) did not modify OB number, instead it decreased calcium deposition (10% decrease at 25ng/ml, 33% decrease at 50 and 100 ng/ml, p<0.05). We next assessed the expression levels of proteins critical to matrix formation and mineralization including osteopontin, bone-sialoprotein and osteocalcin. Osteopontin expression was not affected by CCL3, while both bone-sialoprotein and osteocalcin were downregulated. Importantly, either continuous exposure to CCL3 or 24h stimulation of mature OB impaired RNA expression of osteocalcin (30% to 80%) and bone-sialoprotein (26% to 60%) but not ALP, confirming that CCL3 interferes with OB function rather than formation. We also observed a correlation between osteocalcin expression by IHC on BM biopsies and CCL3 levels in BM serum of MM patients. These data suggest that MM-derived CCL3 interferes with bone mineralization by inhibiting osteocalcin expression. Using neutralizing antibodies against CCL3 we restored both osteocalcin and bone sialoprotein RNA expression levels in the presence of CCL3. Importantly, pretreatment with a small molecule CCR1 inhibitor, MLN3897 (Millennium Pharmaceuticals) completely abrogated the inhibition on osteocalcin and partially reversed bone-sialoprotein expression, suggesting CCR1 as the main mediator of CCL3 effects. We further verified these results in an in-vivo setting of MM bone disease, using the SCID-hu model. CB17 SCID mice bearing a human fetal bone implant were engrafted with CCL3-expressing INA6 MM cells and treated orally with MLN3897 for a total of 49 doses. After 4 weeks of treatment the bones were harvested and stained for TRAP activity, hematoxylin-eosin and osteocalcin. The number of OC/400x field was significantly reduced in the treated group (2.7 vs 1.9, p<0.05), thus confirming in vivo the anti-osteoclastogenic effect of CCR1 inhibition. Moreover, in the presence of INA6 MM cells osteocalcin levels were downregulated compared to non-injected bones and treatment with the CCR1 inhibitor partially restored osteocalcin expression. These results suggest a new role for the CCL3/CCR1 pathway in the development of osteolytic lesions in MM, as inhibitor of OB function other than OC growth factor. Targeting this pathway represents a promising strategy for the treatment of bone disease.