Targeting the calcium sensing receptor in breast cancer cells diminishes colonization and migratory response in a trabecular bone explant model, and reduces osteolytic lesions in vivo
Résumé
Breast cancer is the most common form of cancer affecting women worldwide with bones being the prevalent site of metastasis. The calcium sensing receptor (CaSR), whose primary physiological role is calcium homeostasis, has emerged as a new target in the “vicious cycle” that amplifies the metastatic cascade. Our team had already shown that overexpression of the CaSR dramatically increased the osteolytic potential of MDA-MB-231 cells in vivo. Our objective here was to study the involvement of CaSR in the metastasis of breast cancer cells to the bone using human femur tissue explants and to abate it with calcilytics (CaSR antagonists). Methods: To investigate the role of CaSR in migration of breast cancer cells, MDA-MB-231 cells were made to overexpress either a full-length wild-type CaSR (CaSR-WT) or a dominant negative mutant (CaSR-DN). For control, cells were transfected with empty pcDNA™3.1/Zeo(+) plasmid (CaSR-EV). NPS 2143 was used as a calcilytic in relevant assays. ERK phosphorylation was checked across all cell types in response to calcium. Trabecular bone fragments, extracted from human femoral heads, were used as explants to study the osteoinvasion in vitro. Transwell inserts seeded with the transfected cells were exposed to the bone fragment supernatant before quantifying migration. Treated/untreated cells were seeded directly onto the bone fragments, and zeocin-selective colony formation assay was performed with the extracted marrow population. Mice were inoculated intra-tibially with MDA-B02 cells and received NPS-2143 (IP) for 7 days. Results: The cultured supernatant elicited a strong migratory response across all transfected cell types. In these culture conditions, the CaSR-WT and CaSR-EV cells had a 50% increase in migration levels over the CaSR-DN (p<0.01). The difference between CaSR-WT and CaSR-EV was not statistically significant. Interestingly, similar results were obtained when cells were directly added to the bone fragment and allowed to colonize. The CaSR-WT and CaSR-EV had a 3-fold increase in colony formation over CaSR-DN. This led us to select the CaSR-EV in further experiments, where treating the cells with NPS 2143 decreased their colony formation by around 42%, diminished migration toward bone supernatants (p< 0.01), and blunted proliferation sensitized by the conditioned media (p<0.01). In vivo, administration of calcilytics reduced incidence (4/5 vs. 1/6) and extent (by 86%, p<0.05) of osteolytic lesions. Perspectives: In a co-culture model benefitting from an architecturally intact tissue micro-environment, we demonstrate that the CaSR in breast cancer cells is involved in engraftment and chemoattraction to the bone. Further experiments are underway in vivo to see if calcilytics can be repurposed as a therapeutic intervention for bone metastases from breast cancer.