Bone Abstracts

Bone is the primary site of metastasis for breast cancer, which leads mainly to osteolytic lesions, Cancer cells can expand into the bone for their ability to ‘dialogue’ with resident cells, interfering with the physiological processes of bone turnover. In this study, a large-scale analysis comparing gene expression of biopsies of bone and visceral metastases from human breast cancer patients showed that the receptor (G protein-coupled) activity modifying protein-2 (RAMP2) gene, encoding for a co-receptor calcitonin-receptor-like receptor, was overexpressed 2.7-fold in bone metastases. Gene expression also showed a significant increase of components of the RAMP2-pathway, both receptors (calcitonin-receptor-like receptor, +2.08-fold) and ligands (amylin +1.22-fold). To elucidate the potential role of RAMP2 in osteolytic lesions, we stably transfected the human osteotropic breast cancer cell line MDA-MB-231 with RAMP2 (MDA-RAMP2) and found an increased ability of in vitro migration and proliferation, compared to empty vector transfected (MDA-empty) cells. Moreover, osteoclast precursors treated with conditioned medium (CM) from MDA-RAMP2 cells showed a significant increase of osteoclast differentiation (+2.1-fold, P=0.01) and function (pit index: +6.1-fold, P=0.0001) compared to MDA-empty-CM treated preosteoclasts. Semi-quantitative RT-PCR revealed an increase in RankL/Opg ratio in primary osteoblasts treated with MDA-RAMP2-CM, indicating further pro-osteoclastogenic action of tumour cells mediated by osteoblasts. We also observed that MDA-RAMP2 cells formed oncospheres larger (+2.61-fold, P=0.04) but less numerous (−2.87-fold, P=0.02) than MDA-empty cells, indicating a reduced stemness in favour of proliferation and differentiation. Finally, in vivo experiments of intratibial injection of MDA-RAMP2 cells in Balb-c nu/nu mice showed an increased osteolytic area (+1.6-fold, P=0.048) compared to MDA-empty cell injected tibias. In conclusion, our data suggest that RAMP2 plays a role in tumour aggressiveness and promotes the growth of cancer cells in bone through their ability to communicate with the resident cells, thus contributing to the osteotropism of breast cancer cells.