Effects Of Bone Sialoprotein On The Proliferation And Invasion Of Breast Cancer And The Possible Mechanism Involved In Its Bone Metastasis

Objective:We have investigate the effects of BSP on the proliferation and invasion of breast cancer cells and the mechanism leads to its bone metastasis.Methods:1.The breast cancer cell lines with BSP knockdown or overexpression in MCF-7 and MDA-MB-231 background were established.The proliferation and migration of breast cancer cells were examined by CCK-8 proliferation assay and transwell migration assay respectively.2.The impact of BSP on the expression of migration-related proteins in MCF-7/MDA-MB-231 cell line were detected by Western Blot analysis.The physical interaction between BSP and TGF?R1,TGFPR3 or VDR were determined by Co-immunoprecipitation.3.Pre-osteoblast cell line MC3T3-E1-Sub14 was differentiated into osteoblasts and macrophage cell line RAW264.7 into osteoclast.The degree of osteoblast differentiation was examined by alkaline phosphatase(ALP)stain and the osteoclast differentiation was examined by tartrate-resistant acid phosphatase(TRAP)stain and by Western blot analyses of osteoclast differnetiation markers.4.A co-culture system of MCF-7/MDA-MB-231 breast cancer cells with osteoblasts/osteoclasts was established.ALP and cytoskeleton stains were performed to detect the degree of osteoblast differentiation.The impact of co-culture on the differentiation of osteoclast differentiation was examined by TRAP stain and by Western blot analyses of the osteoclast differentiation markers.Results:1.BSP knockdown can significantly inhibit the proliferation and migration ability of MCF-7 cells and to a lesser degree on the proliferation of MDA-MB-231 cells.In contrast,BSP overexpression in breast cancer cells can promote the proliferation and migration of breast cancer cells.BSP overexpression has a stronger effect in promoting migration of MDA-MB-231 cells than MCF-7 cells.2.BSP knockdown or overexpression differentially affect the protein level of phosphorylated SMAD3?SMAD4 and VDR in MCF-7 and MDA-MB-231 cells.BSP also interacts with TGF?R1,TGF?R3 and VDR physically.3.We have differentiated osteoblast successfully in vitro evidenced by ALP staining,Positive TRAP staining and the expression of osteoclast marker CTSK,MMP9,and OPG suggested the successful differentiation of osteoclast in vitro.4.Breast cancer cell co-culture can inhibit the differentiation of pre-osteoblast.Overexpressing BSP in breast cancer cells significantly decreased the differentiation of pre-osteoblasts in this co-culture model.When breast cancer cells were co-cultured with osteoblasts,no significant changes were observed in the cytoskeleton structure of the differentiated osteoclast.When osteoclasts were co-cultured with breast cancer cells overexpressing BSP,TRAP-positive osteoclasts are smaller or the same compared with osteoclasts co-cultured with wild-type breast cancer cells or cells with BSP knockdown.Conclusion:BSP knockdown inhibits the proliferation and migration of MCF-7 and MDA-MB-231 breast cancer cells.In contrast,BSP overexpression promotes the proliferation and migration of breast cancer cells.BSP may affect breast cancer cell proliferation and migrationg by interacting with TGFP signaling pathway or by its physical interaction with VDR.BSP differentially affected the proliferation and differentiation of osteoblasts and osteoclasts during bone metastasis of MCF-7/MDA-MB-231 breast cancer cells,suggesting that breast cancer cell BSP level may promote bone metastasis of breast cancer cells by breaking the dynamic balance between osteoblasts and osteoclasts.