P5-02-02: Divergent Effects of Zoledronate and Denosumab on Cellular Interactions between Osteoblasts and Breast Cancer Cells.
2011-12-01

Abstract Background: The development of bone metastases is a multistep process that involves interactions between tumor cells and normal host components of the bone marrow microenvironment. Emerging evidence suggests that the interplay of breast cancer cells with both RANKL-expressing osteoblasts, key mediators of osteoclastogenesis, and osteoblast-derived extracellular matrix structures is critical to the metastatic process. This relationship may contribute to the tumor cells’ colonization of bone, invasive behavior, and eventual tumor progression. Zoledronate, a nitrogen-containing bisphosphonate has demonstrated inhibitory effects on tumor cell adhesion and migration. Denosumab, a fully humanized monoclonal antibody against receptor activator of nuclear factor-κB ligand (RANKL), might also exhibit inhibitory effects on breast cancer cell adhesion and migration by neutralizing RANKL. However, despite their clinical importance, the mechanisms of zoledronate and denosumab in crosstalk between breast cancer cells and osteoblasts still remain poorly characterized. Material and Methods: To investigate the influence of zoledronate and denosumab on osteoblast-breast cancer cell interactions, cell adhesion and migration assays using different breast cancer cell lines (MCF7, MDA-MB-231, and SKBR3) were performed. Results: Cell-matrix adhesion assays revealed a strong attachment of breast cancer cells to several extracellular matrix components expressed by osteoblasts. The subsequent incubation of attached cells with 100 μM zoledronate for 72 h resulted in a prominent reduction of tumor cells’ binding to collagen type I, fibronectin, defined laminin isoforms and tenascin-C, whereas incubation with 10 μg/ml denosumab did not affect the adhesive behavior of breast cancer cells. In addition, cell-cell adhesion analyses indicated that zoledronate was able to decrease the attachment of breast cancer cells to osteoblasts. Furthermore, migration studies using the scratch assay and the transwell migration technique clearly showed that the conditioned media of osteoblasts (primary osteoblasts as well as the osteoblastic cell line CAL72) strongly enhanced the migration of the breast cancer cell line MDA-MB-231 compared to the conditioned media of osteoclast-like cells, stromal cells and control medium, suggesting that exclusively osteoblasts release factors that significantly increase tumor cell migration. Interestingly, exposure to zoledronate inhibited osteoblast-induced tumor cell migration in a concentration-dependent manner. In contrast, denosumab (0.1 — 50 μg/ml) had no inhibitory effect on the migration of MDA-MB-231 cells in scratch analysis. However, this agent was found to cause a reduction in the number of migrated cells when 10 μg/ml were used in transwell assays. Discussion: Our data clearly demonstrate that breast cancer cells interact with osteoblasts and osteoblast-secreted extracellular matrix proteins. The osteoblast-induced breast cancer cell abilities, including tumor cell adhesion and migration, were strongly affected by zoledronate, but not by denosumab. The influence by zoledronate on these interactions provides further insights into the antiresorptive effect of this substance on bone metastasis. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-02-02.