Establishment Of Fulvestrant-resistant Breast Cancer Cell Strains And Preliminary Study On The Mechanism Of Drug Resistance

Background Breast cancer is one of the most common malignant tumors in Chinese females.Endocrine therapy is currently one of the most effective treatments for estrogen receptor-positive breast cancer,which mainly includes selective estrogen receptor modulators(SERM),aromatase inhibitors(AI),and selective estrogen receptor downregulators(SERD).Although endocrine therapy increases the survival rate of patients,the development of drug resistance greatly affects the prognosis of breast cancer patients.Objective Establishment of fulvestrant ER~+breast cancer resistant cell lines.To study the sensitivity and phenotypic changes of fulvestrant resistance to ER~+breast cancer and to explore the changes of the ectodomain protein lysine specific demethylase 1(LSD1)in fulvestrant ER~+breast cancer resistant cell lines.To elucidate the possibility of LSD1 inhibitors reversing drug-resistance in fulvestrant ER~+breast cancer,thus providing new therapeutic ideas and approaches for clinically targeted treatment of fulvestrant ER~+breast cancer resistance.Methods(1)Long-term addition of fulvestrant at 100 n M and 1μM drug concentrations treated three ERα-positive breast cancer MCF7,T47D and SSM2 cells to establish fulvestrant-resistant cell lines.(2)Cell morphology of parental and drug-resistant cell lines was observed by fluorescence microscopy.(3)Crystalline violet staining was used to observe proliferation and monoclonal formation in parental and drug-resistant cell lines.(4)Cell cycle changes in parental and drug-resistant cell lines were detected by flow cytometry.(5)Western blotting was performed to detect the expression of ERα,LSD1 protein and related cyclin in parental and drug-resistant cell lines,ERαprotein expression in drug-resistant cell lines after withdrawal of fulvestrant,and LSD1 protein expression in cell lines after addition of CHX,MG132 and Baf A1.(6)Cytotoxicity assay to detect the resistance index of drug-resistant cell lines,to detect whether the drug-resistant cell lines will be re-susceptible to fulvestrant after withdrawal of fulvestrant,and to evaluate whether the LSD1-specific inhibitor reverses the drug resistance of fulvestrant-resistant cell lines.(7)RT-q PCR assay to explore the changes in LSD1 m RNA levels in drug-resistant cell lines.Results(1)Six ER~+breast cancer fulvestrant-resistant cell lines(Ful RL-MCF7,Ful RH-MCF7,Ful RL-T47D,Ful RH-T47D,Ful RL-SSM2,Ful RL-SSM2)were successfully established.(2)Drug-resistant cell lines proliferate in a non-estrogen-dependent manner while developing varying degrees of cross-resistance to tamoxifen drugs.(3)Parental cells were blocked in G1 phase after fulvestrant administration,and changes in cell cycle pathway regulation in drug-resistant strains largely converged,while there was heterogeneity in cyclin changes across drug-resistant strains.(4)The recovery of ERαprotein was inconsistent among the different resistant cell lines after withdrawal of fulvestrant,and no significant increase in fulvestrant sensitivity was observed in the Ful R-SSM2 cell line.(5)LSD1 levels in drug-resistant cell lines are reduced through the lysosomal pathway,not down-regulated through the m RNA pathway,and targeted inhibition of LSD1 may reverse resistance in fulvestrant-resistant cells.Conclusion Fulvestrant-resistant breast cancer cells from different origins have a heterogeneous presentation.Targeting LSD1 may reverse the resistance of fulvestrant-resistant cell lines by decreasing LSD1 through the lysosomal pathway,and this study provides a new idea for the treatment of fulvestrant-resistant breast cancer.