SMURF1 Facilitates Estrogen Receptor α Signaling In Breast Cancer Cells

Background Breast cancer is one of the most common cancers in women.Among them,60%-70% percent are ER(estrogen receptor)positive.Tamoxifen is a classical treatment for ER positive breast cancer.However,tamoxifen resistance will develop during the long time treatment.The post-translational modification of ERα protein has been linked with tamoxifen resistance.Smurf1 has different functions in cell cycle,proliferation,differentiation,metabolism,genomic stability,aging and so on.However,the Smurf1 function in ERα pathway is still unclear.Our primary data shows that Smurf1 modulates ERα signaling.In this project,we use different methods(such as q RT-PCR,western blot,immunoprecipitation,and ubiquitination related immunoprecipitation)to investigate the mechanism of SMURF1 in ERα pathway,which will contribute to the understanding of breast cancer and tamoxifen resistance.Objective To explore the mechanism of SMURF1 and ERα protein in breast cancer cell lines,Targeting SMURF1 could be one promising strategy for ERα positive breast cancer treatment.Methods 1.Query the TCGA、Kamplot、Oncomine databases to clarify the expression of SMURF1 in human breast cancer tissue.2.Using genome-wide RNA sequencing to explore whether SMURF1 affects ERα target genes in ERα positive breast cancer cells.3.The effect of SMURF1 on the proliferation of breast cancer cell lines was studied by cell proliferation assay,real-time q RT-PCR and Western blotting to detect ERα protein level after depleting SMURF1 in ERα positive breast cancer cells.4.Using Western Blot,q RT-PCR and Luciferase to validate that SMURF1 depletion decreases ERα target genes in the presence and absence of estrogen.5.Clarity SMURF1 regulating mechanism of ERα with exogenous and endogenous Co-IP experiment and further explore whether SMURF1 and ERα combined with each other,and build different domain of plasmid of SMURF1 and ERα,furthering to explore the specific binding site of SMURF1 and ERα.6.The mechanism of SMUFR1 on ERα protein was elucidated by the method of MG132,CHX,Co-IP and Ub assays.7.The the xeno-graft tumor model were used for in vivo study.Results 1.By querying the TCGA、Kamplot、Oncomine database,we found that SMURF1 protein expression is higher in breast cancer patients than normal breast tissues,and With higer SMURF1 expression correlates with a certain relationship between the poor prognosis.2.Using RNA-Sequencing found that depleting SMURF1 could increase the expression of ERα target genes in ERα positive breast cancer cells.3.After depleting SMURF1,WST-1 cell proliferation assay showed that the proliferation ability of breast cancer cells was lower than that of normal breast cancer cells.Furthermore real-time q RT-PCR and Western blotting detect ERα protein level much lower after depleting SMURF1 in ERα positive breast cancer cells.4.SMURF1 depletion decreases ERα target genes in the presence and absence of estrogen which was confirmed by Western Blot,q RT-PCR and Luciferase.5.SMURF1 HECT domain interact ERα AF1 domain. 6.SMURF1 increases ERα stability,possibly by inhibiting K48-specific poly-ubiquitination process on ERα.7.The the xeno-graft tumor model were used for in vivo study,Our data showed that SMURF1 depletion by lent-virus based sh RNA decelerated breast tumor growth.Conclusions Our study reveals a novel mechanism between SMURF1 and ERα signaling in supporting breast cancer growth.Furthermore SMURF1 HECT domain interact ERα AF1 domain.SMURF1 increases ERα stability,possibly by inhibiting K48-specific poly-ubiquitination process on ERα.Targeting SMURF1 could be one promising strategy for ERα positive breast cancer treatment.