The Role And Mechanism Of Estrogen Receptor α In Inhibiting Breast Cancer Metastasis

【Background】Breast cancer is one of the most common malignant tumors in women,and its morbidity rate ranks first in female tumors.The development of breast cancer is closely related to estrogen.Estrogen receptor α(ERα)is expressed in about 70% of breast cancer tissues.ERα is activated by sex hormones and contributes to the aberrant proliferation of breast cancer cells.The classical mechanism of ERa action involves regulating the transcription of oestrogen-responsive genes by binding to the oestrogen-responsive element(ERE)within the promoters of the target genes.As the main driving factor for the growth of ERα positive breast cancer cells,ERα is widely used as the drug target of endocrine therapy,such as tamoxifen.While the clinical epidemiological investigation show that there is a relatively greater chance of detecting ERα negative metastases in ERα positive breast cancer patients after the treatment of tamoxifen.It is also reported that the expression of ERα decreases in invasive breast cancer.These suggest that ERα may suppress breast cacer metastasis.However,it is still not quite clear about the relationship between ERα expression and breast cancer metastasis.【Aims】This study intends to reveal the relationship between ERα and breast cancer metastasis through the analysis of clinical breast cancer samples and experiments at the cellular and animal level.This study intends to not only elucidate the new role of ERα in breast cancer,but also to provide a theoretical basis for the discovery of potential therapeutic targets for treating breast cancer metastasis.【Methods】1.ERα-positive breast cancer primary tumor specimens and corresponding lymph node metastases specimens were collected,and the expression of ERα was detected by immunohistochemistry;2.ERα plasmid/siRNA was transfected transiently to observe the effect of ERα on the invasive ability of breast cancer cells by transwell assay.We also constructed breast cancer cell lines with ERα stable expression or ERα knockout by lentivirus.Then the tumor metastatic model of nude mice and the small animal in vivo imaging apparatus were used to determine the effects of ERα on breast cancer metastasis;3.We constructed a 3D cell culture system and observed the movement patterns of ERα knockout cells and control cells with phase holographic imaging assays.The expression of F-actin and pMLC in ERα knockout cells and control cells were detected by confocal assay;4.To screen the target molecules,ERα overexpressing cells and control cells were collected for transcriptomic sequencing;5.We transfected ERα plasmid/siRNA transiently in breast cancer cells and detected the effect of ERα on the expression of vinculin by qPCR,WB,and cell immunofluorescence assay;6.ChIP,luciferase reporter assay and WB experiments were applied to further clarify the mechanism of ERα on vinculin expression;7.The CRISPR/Cas9 system was used to construct the vinculin knockout cell line and the tail-vein injection model was used to observe the effects of vinculin on breast cancer metastasis;8.Confocal and phase holographic imaging assays were used to observe the effect of vinculin on the regulation of cell amoeboid-like movement;orthotopic mouse model of breast cancer and lung extravasation assay experiments were used to investigate whether vinculin involved in breast cancer metastasis by regulating amoeboid-like migration of breast cancer cells;9.In ERα overexpressing cells,we constructed vinculin knockdown and control cells.Orthotopic mouse model of breast cancer and transwell assay were used to investigate whether vinculin is involved in ERα mediated inhibition of breast cancer metastasis;10.Through immunohistochemistry experiments,we investigated the expression of ERα and vinculin in breast cancer tissues,and analyzed the relationship of vinculin expression and breast cancer metastasis.【Results】1.The expression level of ERα in lymph node metastasis of breast cancer was significantly lower than that in the primary tumor,and further analysis showed that the expression level of ERα in the invasive front of breast cancer invasion was significantly lower than that in the non-invasive front;2.When ERα was over-expressed in breast cancer cells,it can inhibit the invasion and metastasis of breast cancer cells.Conversely,when ERα was silenced,it can promote the invasion and metastasis of breast cancer cells;3.In 3D Matrigel,the invasive ability of ERα knockout cells was significantly higher than that of control cells.More importantly,this process was not affected by MMP inhibitors.The knockout of ERα resulted in enhanced amoeboid-like migration of breast cancer cells,which includds: increased formation of amoeboid-like protrusions,increased expression of p-MLC,p-MLC rear distribution and cell rounding;4.Through transcriptomic sequencing,we found the enrichment of genes related to cell motility.Further study showed that the vinculin transcript was the most significantly altered;5.In ERα-positive breast cancer cells,the interference of ERα expression led to a significant decrease in the expression of vinculin;while in ERα-negative breast cancer cells,the overexpression of ERα led to a significant increase in the expression of vinculin;6.ERα could promote the transcriptional activity of vinculin by directly binding to the ERE region on vinculin promoter;7.Vinculin knock-out promoted lung metastasis of breast cancer cells;8.In 3D Matrigel,vinculin knockout resulted in increased amoeboid-like protrusions formation,increased p-MLC expression,p-MLC rear distribution and cell rounding.Further in vivo experiments suggested that vinculin could inhibit breast cancer metastasis by regulating the amoeboid-like migration of breast cancer cells;9.The results from transwell assay and orthotopic mouse model of breast cancer suggested that vinculin involved in ERα mediated inhibition of breast cancer metastasis;10.ERα expression in breast cancer tissues was positively correlated with the expression of vinculin.Furthermore,vinculin expression is inversely correlated with breast cancer metastasis.【Conclusion】1.In this study,we demonstrat that ERα can inhibit breast cancer metastasis;2.Loss of ERα can promote amoeboid-like migration of breast cancer cells;3.ERα promotes the expression of vinculin in breast cancer cells by directly binding with the promoter region of vinculin and consequently promoting its transcriptional activity;4.Loss of vinculin promotes breast cancer metastasis,and vinculin can inhibit breast cancer metastasis by regulating the amoeboid-like migration of breast cancer cells;5.From the cellular,animal,clinical sample level,it is demonstrated that ERα could inhibit breast cancer metastasis by promoting the expression of vinculin.