Functional And Mechanistic Study Of RAD21 In Promoting Human Breast Cancer Proliferation And Metastasis

Background:Breast cancer is one of the most common female malignancies in the world.About 90%of breast cancer deaths are due to metastasis.It is important to understand the key molecular mechanism and regulation networks of breast cancer growth and metastasis.Cohesin,a multi-subunit protein complex,can maintain sister chromatid cohesion and ensure accurate homologous recombination,DNA repair and genome stability.As the core subunit of the cohesin complex,RAD21 can regulate the binding betwen cohesin and chromatin.Recent studies have indicated that genes encoding cohesin subunits are somatic mutated or abnormally expressed in many types of human cancer.S.F.Mahmood et al.identified RAD21 as one of the driver genes in breast cancer.As a transcription cofactor,Rad21 can not only mediate the formation of advanced chromatin structure,but also participate in the regulation of tumor-related genes expression such as MYC,CD44,HOXA7 and TGFB1.Transcription coactivator YAP(Yes-associated protein)plays an important role in regulating tumor cell proliferation,metastasis and chemotherapy resistance.It is an important downstream effector of Hippo signaling pathway.However,the function of YAP in breast cancer is controversial.Emerging evidence has indicated that YAP can promote breast cancer progression,but some studies show that YAP acts as a tumor suppressor gene.However,the mechanisms of Rad21 and YAP in metastasis of breast cancer remains unclear.Methods:Western Blotting and Real-time quantitative PCR(RT-qPCR)were used to detect the expression of RAD21 in different breast cancer cells.Immunohistochemistry was used to detect the expression level of RAD21 in tumor tissues and paracancerous tissue of breast cancer patients.Colony formation assay,EdU proliferation assay,and Cell counting kit-8(CCK8)proliferation assay were performed to examine the effect on cell proliferation in the RAD21 depleting breast cancer cells.Migration assay and invasion assay were used to investigate the pro-metastasis effect of RAD21 in breast cancer cells.RT-qPCR,immunofluorescence and ChIP assay were used to examine whether YAP expression and activity were regulated by RAD21.The activators and inhibitors of autophagy were used to analyze the effect of RAD21 on autophagy.Electron microscopy was performed to detect the autophagosomes in the cells of RAD21 low-expressing.Nude mice xenografts were performed to determine whether RAD21 affects breast cancer growth and metastasis.Results:1.RAD21 is highly expressed in breast cancer tissues and breast cancer cell lines,and high RAD21 level is associated with patients' poor prognosis.By analyzing the TCGA breast cancer dataset,we found that RAD21 shows gene amplification and mRNA high expression in 37%invasive breast cancer patients.High level of RAD21 expression is correlated with poor outcome.It was shown that RAD21 had higher intensity of immunostaining in breast cancer tissues than paracancerous tissue by immunohistochemistry.Compared with normal breast cell line MCF-10A,the expression level of RAD21 in breast cancer cells is higher in breast cancer cell lines,especially in triple negative breast cancer cells.2.Depletion of RAD21 reduces the cell proliferation,invasion and tumorigenic ability of breast cancer cell.Knocking down RAD21 can significantly inhibit proliferation and colony formation in the triple-negative breast cancer cell lines MDA-MB-231 and BT549.RAD21 depleted breast cancer cells exhibit lower migration and invasion ability,and higher levels of epithelial markers and lower levels of mesenchymal markers.The average tumor volume of RAD21 depletion groups was significantly smaller than that of the control group.However,the average tumor volume of RAD21 overexpression group did not change significantly,while the proportion of Ki-67 positive cells increased significantly compared with the control group.3.RAD21 negatively regulates the expression of YAP in breast cancer.After analyzing RNA-seq data of MDA-MB-231 cells with or without RAD21 depletion and comparing with YAP/TAZ binding sites,we found that about 28%up-regulated genes in RAD21 depletion cells may be regulated by YAP.We also found that RAD21 can bind to the promoter region of the YAP by ChIP assay.RAD21 knockdown increased YAP protein level significantly and the nuclear distribution in MDA-MB-231 and BT549 cells.In addition,YAP downstream target genes were also upregulated significantly.In MDA-MB-231 cells with RAD21 overexpression,the level of YAP protein was significantly decreased.Low level of YAP protein was detected in the tumor tissue of breast cancer patients.4.RAD21 depletion inhibits metastasis through YAP activation.Depletion of YAP can rescue the reduction of the migration and invasion ability in RAD21-knockdown MDA-MB-231 cells.5.RAD21 Knockdown promotes autophagy in breast cancer cells.In RAD21-knockdown MDA-MB-231 cells,the autophagosomes were significantly increased compared with the control cells by electron microscopy,and autophagy-related protein LC3-? was significantly reduced.However,depletion of both RAD21 and YAP caused LC3-? accumulation,and inhibited autophagy.In MDA-MB-231 cells with RAD21 overexpression,the reduction of YAP and the accumulation of LC3-? indicates that the process of autophagy was blocked.Conclusions:1.RAD21 is highly expressed in breast cancer cell lines and breast cancer tissues.High RAD21 level is also related to the poor prognosis of breast cancer patients.2.Depletion of RAD21 can reduce the proliferation,migration and invasion of breast cancer cells in vitro and tumorigenic ability in vivo;3.RAD21 negatively regulates the expression of YAP gene in breast cancer,and the biological function of RAD21 depends on YAP repression.