The Role And Mechanism Of Estrogen Receptor ? 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 ?(ER?)is expressed in about 70% of breast cancer tissues.ER? 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 ER? positive breast cancer cells,ER? 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 ER? negative metastases in ER? positive breast cancer patients after the treatment of tamoxifen.It is also reported that the expression of ER? decreases in invasive breast cancer.These suggest that ER? may suppress breast cacer metastasis.However,it is still not quite clear about the relationship between ER? expression and breast cancer metastasis.?Aims?This study intends to reveal the relationship between ER? 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 ER? in breast cancer,but also to provide a theoretical basis for the discovery of potential therapeutic targets for treating breast cancer metastasis.?Methods?1.ER?-positive breast cancer primary tumor specimens and corresponding lymph node metastases specimens were collected,and the expression of ER? was detected by immunohistochemistry;2.ER? plasmid/siRNA was transfected transiently to observe the effect of ER? on the invasive ability of breast cancer cells by transwell assay.We also constructed breast cancer cell lines with ER? stable expression or ER? 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 ER? on breast cancer metastasis;3.We constructed a 3D cell culture system and observed the movement patterns of ER? knockout cells and control cells with phase holographic imaging assays.The expression of F-actin and pMLC in ER? knockout cells and control cells were detected by confocal assay;4.To screen the target molecules,ER? overexpressing cells and control cells were collected for transcriptomic sequencing;5.We transfected ER? plasmid/siRNA transiently in breast cancer cells and detected the effect of ER? 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 ER? 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 ER? 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 ER? mediated inhibition of breast cancer metastasis;10.Through immunohistochemistry experiments,we investigated the expression of ER? and vinculin in breast cancer tissues,and analyzed the relationship of vinculin expression and breast cancer metastasis.?Results?1.The expression level of ER? 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 ER? in the invasive front of breast cancer invasion was significantly lower than that in the non-invasive front;2.When ER? was over-expressed in breast cancer cells,it can inhibit the invasion and metastasis of breast cancer cells.Conversely,when ER? was silenced,it can promote the invasion and metastasis of breast cancer cells;3.In 3D Matrigel,the invasive ability of ER? knockout cells was significantly higher than that of control cells.More importantly,this process was not affected by MMP inhibitors.The knockout of ER? 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 ER?-positive breast cancer cells,the interference of ER? expression led to a significant decrease in the expression of vinculin;while in ER?-negative breast cancer cells,the overexpression of ER? led to a significant increase in the expression of vinculin;6.ER? 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 ER? mediated inhibition of breast cancer metastasis;10.ER? 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 ER? can inhibit breast cancer metastasis;2.Loss of ER? can promote amoeboid-like migration of breast cancer cells;3.ER? 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 ER? could inhibit breast cancer metastasis by promoting the expression of vinculin.