The Regulatory Molecular Mechanism Of CHES1 On The Breast Cancer Cells Proliferation Via Modulating Activity Of ER?

Breast cancer is always a threatened factor to women's health worldwide.In China,the estimated new cases of breast cancer increased sharply every year,and the average age of onset was getting younger.Therefore,revealing the molecular mechanism involved in initiation and progress of breast cancer and identifying a series of valuable clinical biomarkers will greatly contribute to clinical diagnosis and chemotherapy for breast cancer.The predisposing factors for breast caner are various,and it is well established that estrogen exposure has a tight association with malignant proliferation and metastasis of breast cancer cells.Estrogen receptor a(ERa)is the key factor that responses to estrogen stimulation and mediates signal transduction in cells.What's more,ERa is also a vital target of drugs in clinical therapy.However,the molecule and signaling pathways involved in regulatory network of estrogen-ERa signal are complex,and signals transduction regulation is a dynamic process dependent on different cellular environment.Therefore,it is always a worldwide hotpot to get a better understand to comprehensive network of estrogen signal and regulatory mechanism of ERa function.Checkpoint suppressor 1(CHES1)is identified firstly to be involved in DNA damage response and cell cycle arrest in saccharomyces cerevisiae,it is also known as FOXN3 because it contains a conserved Forkhead box(FOX)DNA binding domain.Subsequent studies have found that CHES1 could participate in various physiological process such as protein synthesis,glucose metabolism and embryo development,and it also functioned as a transcriptional repressor involved in transcriptional regulation.Increasing number of research suggest that CHES1 has a close association with occurrence,development and prognosis of many types carcinoma.And,the public dataset analysis indicates that the expression of CHES1 is downregulated in breast cancer and it may be an important tumor-suppressor.So,our work began with protein interaction analysis and investigated the the correlation and regulatory molecular mechanism between CHES1 and estrogen signaling pathway.And the main research contents are as follows:(1)Co-Immunoprecipitation(CoIP),immunofluorescence(IF)and GST-pulldown assays were performed to explore the existence of physical interaction between CHES1 and ERa in breast cancer cells,and further data confirmed the domains of two proteins responsible for the interaction.(2)Luciferase reporter gene analysis and reverse transcription polymerase chain reaction(RT-PCR)assays were conducted to establish that CHES1 functioned as a co-repressor and inhibited the estrogen-dependent transactivation of ERa,and CHES1 also repressed the mRNA and protein levels of ERa-target gene pS2,CCND1 and c-Myc.(3)Furthermore,the detailed molecular mechanism that CHES1 repressed the transcriptional activity of ERa was further to be explored.Western blot,IF and CoIP assays showed that CHES1 had little effect on the stability,dimerization and subcellular location of ERa,and it also didn't affect the interaction between ERa and HD AC 1/2.Further data suggested that CHES1 promoted the recruitment of SIRT1 to ERa and facilitated the SIRT1 mediated deacetylation of ERa,further inhibited the enrichment of ERa on its target gene promoter and its transacticvation.(4)Western blot and immunohistochemistry(IHC)assays showed that CHES1 had a lower expression level in ERa-positive breast cancer when compared with ERa-negative breast cancer.Further data indicated that a conserved Estrogen response element(ERE)motif was found in CHES1 promoter region,chromatin immunoprecipitation(ChIP)and realtime PCR results showed that ERa could recognized this ERE and bound on CHES1 promoter respond to E2 stimulus,further repressed the transcription expression of CHES1.Analysis of the protein levels in breast cancer cell lines and tissues indicated that CHES1 expression had a negative association with ERa in breast cancer.(5)Cell proliferation and flow cytometry assays confirmed that CHES1 specifically inhibited the growth and cell cycle of ERa-positive breast cancer cells in vitro,and the null mouse model showed that CHES 1 had a repressive effect on tumorigenesis of ERa-positive breast cancer cells in vivo.Bioinformatics analysis indicated that the CHES1 expression had tight association with overall survival of breast cancer patients.In summary,this work revealed a negative regulatory loop between CHES1 and estrogen-ER? signal,this model regulated the ERa-mediated proliferation and tumorigenesis of breast cancer cells.Furthermore,this study also investigated the biological function of CHES1 in breast cancer,which will provide a foundation for the clinical diagnosis and drug design of breast cancer.