The Role Of Progesterone And Estrogen In Breast Cancer Bone Metastasis Mediated By RANKL And Its Receptors

Skeletal metastases are common in patients with soild tumors, affecting approximately 70% of patients with advanced breast cancer. Histological evidences have demonstrated that most of skeletal metastases are osteolytic which is due to the increased number and activity of bone-resorbing oseoclasts. Morbidity caused by skeletal complications includes pain, hypercalcemia, pathologic fracture and compression of the spinal cord. The median survival after the diagnosis of bone metastases from breast cancer is only 2 to 3 years. However, the molecular mechanisms of breast cancer metastasis to the skeleton are still undetermined.Recently, a novel cytokine triad consisting of receptor activator of NF-κB ligand (RANKL), its receptor (RANKL) and the endogenous decoy receptor osteoprotegerin (OPG) was identified for their roles in bone remodeling. It is well known that RANKL/RANK/OPG axis controls osteoclastogenesis and bone resorption. The TNF ligand superfamily member RANKL, which is expressed on the surface of osteoblasts, is important for the formation, function and survival of osteoclasts. RANK is activated when it is bind by RANKL. OPG is a soluble member of TNF receptor super family secreted by osteoblasts .OPG competes with RANK for binding to RANKL.RANKL/RANK/OPG was believed to be involved in the formation of solid tumor bone metastasis. Recently, several researches have demonstrated that RANKL mediated the breast tissues proliferation and breast tumorogenesis induced by progesterone. Additionally, RANKL was observed as a regulator of stem cell subpopulation in breast epithelial cells. Lifetime exposure to reproductive hormones, in particular estrogens and progesterone, affects the risk of breast cancer, a complex disease that is under hormonal control. The same hormones also control the development of the mammary gland.To investigate the role of progesterone and estrogen in the regulation of RANKL and the receptors expression in Breast Cancer cell lines, we treat T47D, MCF-7 and MDA-MB-231 breast cancer cell lines with 1×10~6M progesterone or 1×10~7M estrogen for 24h. The mRNA level of RNAKL, RANK OPG and PR were detected by Real-time PCR with the RNA isolated from the treated and untreated cell lines. The RANKL and OPG expression were significantly increased by progesterone in T-47D and MCF7 cell lines. Both progesterone and estrogen down-regulated the expression of RANK in T47D and MCF7 cell lines. Mammosphere formation assay was performed with 4T1 cells and MDA-MB-231 cells when these cells were treated with 1μg/ml sRANKL. Compared with the contrl cells, a significantly increased number of mammospheres were observed in the cells treated with sRNAKL in both 4T1 cells and MDA-MB-231 cells. Interestingly, when we did the migration assay with MDA-MB-231 cells, the result suggested that sRANKL in the bottom chamber could increase the mobility of MDA-MB-231 cells and attract these cell to migrate to the bottom chamber. Furthermore, 0.1×10~6 of MDA-MB-231 cells was injected intracardiacally into the 3-4 weeks old SCID mouse (BALB/cJHan, Hsd-Prkdcscid). 5 mice were injected with 100ul of striped corn oil as a control group. 5 mice were injected with 100ul of 10mg/ml (1mg/mouse) progesterone as P group. 5 mice were injected with 200ul of 1mg/ml RU486 (Progesterone antagonist) as RU group. Low density of bone metastasis signal in the P group detected by IVIS indicates the protective role of RU in the formation of bone metastasis. However, we did not find significant differences of metastasis incidence between the P group and RU group.In conclusion, progesterone and estrogen play an important role in the RANKL and the receptors expression in T47D and MCF-7 cell lines. Furthermore, RANKL has the ability to stimulate more stem cells subpopulation and to attract the breast cancer cells in migration assay. Finally, RU486 attenuate the breast cancer leg metastasis which could be induced by RANKL pathway. Our results suggest that progesterone and estrogen could regulate breast cancer bone metastasis through the RANKL pathway.