The Mechanisms Of RANKL/RANK Pathway In Cell Migration Of Breast Cancer And Its Regulation

BackgroundBreast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among females in worldwide, and mortality is related to metastatic spread of the primary tumor.The migration of breast cancer cells is well-orchestrated and not a random process. Bone is the most preferential metastatic target site for breast cancer metastasis, and the frequency of metastasis to bone is much higher than to the lung and liver. The exact mechanism for this preferential metastasis requires further investigation.The Receptor Activator of NF-κB (RANK) and its ligand, known as RANKL, was originally shown to be crucial for osteoclastogenesis and lymph node development. Osteoprotegerin (OPG) acts as a soluble decoy receptor and competes with RANK for binding to RANKL. Accumulating studies have shown that the expression of both RANKL and RANK have been observed in primary and secondary bone tumors, it has been proposed that the RANKL/RANK system can regulate bone metastases of epithelial tumours. So we speculated that RANKL might act as a‘soil’factor facilitating cancer cell metastasis to bone. Although the mechanism of RANKL/RANK pathway in regulation of bone was well established, the role of RANKL/RANK signal transduction pathways in inducing the migration of human breast cancer cells is still not well understood. Moreover, less is known about the mechanisms that what stimulies controls the expression of RANKL and RANK in breast cancer cells. In this study, we confirmed that RANKL could trigger the migration of human breast cancer cells in vitro, and explored the downstream signaling pathways of RANKL/RANK in MDA-MB-231 cells to induce cell migration. We also determined for the first time that the regulation of RANKL/RANK by hypoxia led to increased migration of breast cancer cells in vitro. MethodsPartⅠ. Effects of RANKL/RANK pathway on the migration of MDA-MB-231 cellsThe migration of MDA-MB-231 human breast cancer cells induced by RANKL was confirmed using wound-healing and transwell chemotaxis assays. In order to inhibit RANKL-induced migration, RANKL was incubated with OPG before adding into the plate. MDA-MB-231 cells were also co-cultured with osteoblastic cells (hFOB 1.19) in the transwell assay, and then the migration of MDA-MB-231 from the upper insert towards the osteoblastic cells in lower insert were examined. To silence RANK gene expression, short hairpin RNAs (shRNAs) targeting mRNA regions of RANK were designed. The sequences were inserted into the plasmid, and lentiviral vectors expressing shRNA were constructed. Another HIV-derived lentiviral vector was packaged using the pLenti-TNFRSF11A-Neo which contained the full length cDNA sequence of the human RANK gene. The RT-PCR and Western blot assays were performed to test the expression of RANK in the transfected cells. The transwell assays to test the migration of the transfected cells.PartⅡ. The related signal transduction pathways in RANKL-induced cell migrationAfter the MDA-MB-231cells were treated with RANKL at different times, the levels of phosphorylated Src were tested by Western blot, and after the cells were treated with different doses of RANKL or co-incubated with OPG/RANKL the levels of phosphorylated Src by Western blot were tested. The change of phosphorylated Src in cells transduced with RANK-targeted shRNA was also tested. To confirm the involvement of Src kinases in RANKL-induced MDA-MB-231 cell migration, MDA-MB-231 cells were pretreated with the specific Src inhibitor or transfected with Src small interfering RNA (siRNA). After the MDA-MB-231cells were treated with RANKL at different times or in different doses, the levels of phosphorylated MAPKs (p-ERK1/2, p-JNK, and p-p38) were tested by Western blot. The phosphorylation levels of ERK1/2, JNK, and p38 were tested after the cells were pretreated with specific Src inhibitor or transfected with Src siRNA. The cell migration was examined via treatment with PD98059 (30μM), SB203580 (20μM), SP600125 (20μM)PartⅢ. RANK and RANKL expressions regulated by hypoxia and its mechanism.The human breast cancer MDA-MB-231 cells and MCF-7 cells were cultured in normoxia or hypoxic conditions. Hypoxia-dependent expressions of RANK and RANKL in different time were investigated by RT-PCR. Western blot assay tests was used to test the protein level of RANK, RANKL and hypoxia inducing factor 1α. (HIF-1α). To determine the effect of HIF-1αon hypoxia-induced expression of RANK and RANKL of MDA-MB-231 cells, siRNA against HIF-1αwas used to downregulate the expression of HIF-1αin MDA-MB-231 cells. We further investigated the role of PI3K/Akt signaling on HIF-1α-mediated expressions of RANK and RANKL in MDA-MB-231 cells by Western blot. Finally, the wound-healing assay was used to test the effect of hypoxia on RANKL-induced cell migration.Results(1)RANKL could induce MDA-MB-231 cell migration in a dose-dependent manner(P<0.05). OPG could inhibit RANKL-induced cell migration, especially when the ratio between OPG and RANKL was 5: 1.(2)After co-culturing MDA-MB-231 cells with hFOB 1.19 cells, the migreation of MDA-MB-231 cells was increased(P<0.05). OPG could suppress the migration of MDA-MB-231cells in this assay.(3)The lentiviral vectors containing RANK-targeted shRNA were constructed and packaged successfully and the silencing effect of lentivirus-mediated RANK-targeted shRNA was confirmed. Another lentiviral vector was packaged using the pLenti- TNFRSF11A-Neo which containing the full length cDNA sequence of the human RANK gene. With to the changes of the expression of RANK, the ability of cell migration toward RANKL was changed too.(4)The levels of phosphorylated Src was increased after treated with RANKL in a concentration-dependent and time-dependent manner. The RANKL-induced expression of phosphorylated Src was significantly lower in the MDA-MB-231-I002 cells transduced with lentivirus-mediated RANK-targeted shRNA than in the MDA-MB-231 cells. The cells transfected with Src siRNA and treated with Src inhibitor antagonized RANKL-induced migration(P<0.05)(5)The levels of phosphorylated ERK1/2, p-38 and JNK were increased after treatment with RANKL in a concentration-dependent and time-dependent manner. The cells transfected with Src siRNA and treated with Src inhibitor suppressed the expression of phosphorylated ERK1/2, p-38 and JNK induced by RANKL. Cell migration induced by RANKL was also effectively decreased by treatment with inhibitors of ERK1/2, p-38 and JNK.(6)Hypoxia upregulated the expressions of RANK and RANKL in human breast cancer cells(P<0.05),which was similar to the findings of the upregulation of HIF-1αprotein induced by hypoxia. The treatment with HIF-1αsiRNA not only blocked the upregulation of HIF-1α, but also suppressed the upregulation of RANK and RANKL protein levels after the cells were exposed to hypoxic conditions at 4 h, 8 h, and 12 h. Exposure of MDA-MB-231 cells to hypoxia led to increased phosphorylation of Akt. The PI3K/Akt inhibitor abolished hypoxia- mediated upregulation of RANK and RANKL protein expressions.(7)In wound healing assay, exposure of MDA-MB-231 cells to hypoxic conditions resulted in significantly increased RANKL-induced migration compared with normoxia(P<0.05). These effects were blocked by additional treatment of OPG. Moreover, the rate of wound recovery was decreased following downregulation of HIF-1αby siRNA against HIF-1α. Pretreatment with PI3K/Akt inhibitor also blocked the RANKL-induced migration of MDA-MB-231cells under hypoxia(P<0.05).Conclusion(1) RANKL can induce MDA-MB-231 cell migration and the RANKL/RANK pathway mediates the process of MDA-MB-231 cells migrated towards osteoblasts.(2) Src-MAPKs (ERK1/2, p38, and JNK) signaling pathway is involved in RANKL-induced cell migration of MDA-MB-231 cells.(3) The expression of RANK and RANKL in breast cancer cells can be upregulated by hypoxia. The hypoxia-PI3K/Akt -HIF-1α-RANKL-RANK pathway is likely to be involved in the migration of breast cancer cells in hypoxic environments.