The Role Function And Underlying Mechanism Of A Novel Variant Of Estrogen Receptor ER-α25 In Breast Cancer Cells

Objective: In this study,we aim to elucidate the function and underlying mechanisms of a recently cloned ER variant ER-α25 in breast cancer cells.We also aim to study ER-α25 in the exosomes secreted from LPS-induced macrophages,and its role in regulation of the stemmness of breast cancer cells.Method:(1).We established the cell with forced expression of ER-α25from breast cancer T47 D and MDA-MB-231 cells.Western blot analysis was used to assess the stemmness gene expression in these cells,tumorsphere formation assay to assess the ability of tumorsphere formation,and colony formation assay to evaluate the ability of colony formation.Cell scratch and Transwell assays were used to test examine the migration and invasion ability of these cells.(2).We identified and purified Purification and identification of the exosomes secreted from LPS-treated THP-1 cells,and examination ofexamined ER-α25 expression in the exosomes with Western blot analysis.We evaluated the expression levels of Western blot analysis of the Wnt/β-catenin signaling pathwayproteins with Western blot analysis,and also studied the effects of examination of the effects of the Wnt signaling inhibitor XAV939 on the expression of the stemmness genes,tumorsphere formation,colony formation,as well as migration and invasion in breast cancer cells.(3).T47 D and MDAMB-231 cells co-cultured with the exosomes from the LPS-induced macrophage were examined for the stemmness gene expression with Western blot analysis,for the ability of tumorsphere formation,and for the colony formation.Cell scratch and Transwell assays were used to examine the migration and invasion ability of these cells.Results:(1).The breast cancer T47 D and MDA-MB-231 cells with forced ER-α25 expression increased the expression of stemmness genes,enhanced tumorsphere formation and colony formation,augmented migration and invasion.(2).ER-α25 is highly expressed in the exosomes secreted from the LPS-treated macrophages.(3).The breast cancer T47 D and MDA-MB-231 cells co-cultured with the exosomes from the LPS-treated macrophages increased the expression of stemmness genes,enhanced tumorsphere formation and colony formation,augmented migration,and invasion.(4).The breast cancer T47 D and MDA-MB-231 cells with forced ER-α25 expression or co-cultured with the exosomes from the LPS-treated macrophages exhibit the EMT molecular phenotype.(5).The breast cancer T47 D and MDA-MB-231 cells with forced ER-α25 expression show high level of ?-catenin expression,and the Wnt signaling inhibitor XAV939 reduced the expression of stemmness genes,and attenuated tumorsphere formation and colony formation as well as reduced migration and invasion.Conclusion: The novel variant of estrogen receptor ER-α25 enhances the stemmness and promotes EMT in breast cancer cells presumably through the Wnt/β-catenin pathway.The exosomes from the macrophages may deliver ER-α25 to neighboring breast cancer cells to enhance their stemmness.