Receptor activator of NFκB (RANK) plays an important role in mammary gland development and breast cancer metastasis. Here, we examined the cancer cell-autonomous role of RANK in regulating breast cancer induced osteolysis. First, we observed that RANK is highly expressed in various bone-tropic (BT) clones of parenteral human MDA-MB-231 (MDA-231-P) and mouse 4T1 (4T1-P) breast cancer cells. Epigenetic analysis revealed that enhanced RANK expression in these cells correlates with increased promoter methylation, and further analysis of genetic variations showed that bone-tropic cells predominantly expressed two altered isoforms of the RANK receptor, namely Δ9- and Δ8,9-RANK. Moreover, both isoforms were found to express the regulatory motif for p38 MAPK (559PVQEET564), but lacked TRAF6-binding motif, which is essential for NFκB activation. Mechanistic studies in MDA-231-BT and 4T1-BT cells showed that RANK ligand stimulated p38 phosphorylation (MDA-231-P, 39% and 4T1-P, 20% p<0.05), while it had no effects on the phosphorylation of IKKβ and I?B, degradation of I?B and NFκB DNA binding at concentrations up to 300 ng/ml. Functional studies in breast cancer and bone marrow cells demonstrated that exposure of MDA-231-P and 4T1-P or their bone-tropic clones to RANK ligand (100-300 ng/ml) stimulated cell spreading by 2 folds (p<0.05), increased 2D random (MDA-231-P, 37%, p<0.05), directed (MDA-231-P, 160% and MDA-231-BT, 189%, p<0.05) and chemotactic migration (MDA-231-P, 367%, p<0.05), and enhanced the ability of bone-tropic breast cancer cells to stimulate osteoclastogenesis (MDA-231-BT, 196% and 4T1-BT, 93%, p<0.01), whereas stable knockdown of RANK was inhibitory (p<0.05). Collectively, these findings suggest that genetic variations in RANK in highly metastatic bone-tropic breast cancer cells contribute to phenotypic modifications that alter the host tissue environment to foster formation of osteolytic bone lesions. Studies to further examine the role of different variants of RANK in the regulation of breast cancer induced osteolysis are currently in progress.
Disclosure: The authors declared no competing interests. This work was supported by the Ministry of Health of Saudi Arabia.