SMURF1 Facilitates Estrogen Receptor ? 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 ER? 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 ER? pathway is still unclear.Our primary data shows that Smurf1 modulates ER? 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 ER? pathway,which will contribute to the understanding of breast cancer and tamoxifen resistance.Objective To explore the mechanism of SMURF1 and ER? protein in breast cancer cell lines,Targeting SMURF1 could be one promising strategy for ER? 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 ER? target genes in ER? 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 ER? protein level after depleting SMURF1 in ER? positive breast cancer cells.4.Using Western Blot,q RT-PCR and Luciferase to validate that SMURF1 depletion decreases ER? target genes in the presence and absence of estrogen.5.Clarity SMURF1 regulating mechanism of ER? with exogenous and endogenous Co-IP experiment and further explore whether SMURF1 and ER? combined with each other,and build different domain of plasmid of SMURF1 and ER?,furthering to explore the specific binding site of SMURF1 and ER?.6.The mechanism of SMUFR1 on ER? 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 ER? target genes in ER? 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 ER? protein level much lower after depleting SMURF1 in ER? positive breast cancer cells.4.SMURF1 depletion decreases ER? 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 ER? AF1 domain. 6.SMURF1 increases ER? stability,possibly by inhibiting K48-specific poly-ubiquitination process on ER?.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 ER? signaling in supporting breast cancer growth.Furthermore SMURF1 HECT domain interact ER? AF1 domain.SMURF1 increases ER? stability,possibly by inhibiting K48-specific poly-ubiquitination process on ER?.Targeting SMURF1 could be one promising strategy for ER? positive breast cancer treatment.