Study Of Mechanism On Methyltransferase SETDB1 Cooperating With PELP1 To Promote Breast Cancer Progression

Background: Breast cancer(BC)is the most common type of female malignancy.70% of BC patients present with positive estrogen receptor(ER)expression and endocrine therapy is the main treatment for these patients.Resistance to endocrine therapy leads to poor prognosis and is a major clinical problem in the treatment of ER positive BC.It is of great clinical value to elucidate the mechanisms of BC progression and to explore effective therapeutic targets.The methyltransferase SETDB1(SET domain bifurcated 1)affects tumor progression-related gene expression and signaling by methylating histone and non-histone substrates.PELP1(proline glutamic acid leucine rich protein 1)is overexpressed in BC and involved in the functional regulation of various nuclear receptors and transcription factors as a scaffold protein.SETDB1 and PELP1 are involved in multiple tumorigenesis,however,the mechanism of SETDB1 in BC endocrine resistance remains elusive.Objective:This study investigates the biological functions and signaling of SETDB1 in BC,and the molecular mechanisms by which SETDB1 cooperates with PELP1 to promote BC progression.Methods:1.To investigate the biological function of SETDB1 in BC.Analyze the SETDB1 expression in BC by TCGA databases.Construct SETDB1 knockdown models in BC cell lines and endocrine resistant cell lines using lentiviral transduction.Mithramycin A was used as a non-specific inhibitor of SETDB1,and the efficacy of the drug was investigated by Western Blotting(WB).Using SETDB1 knockdown cells and drug inhibition models,the effects of SETDB1 low expression on BC proliferation and endocrine therapy resistance were investigated by cell viability assay and colony formation assays.2.To investigate the signaling pathway of SETDB1 that regulates BC progression.RNA-seq and bioinformatics analysis were performed on SETDB1 knockdown cells to investigate signalling pathways regulated by SETDB1.The effect of SETDB1 on the transcriptional activity of estrogen receptor was explored by luciferase reporter assay.The gene expression and signaling pathways regulated by SETDB1 were verified by RT-q PCR(real time-quantitative PCR)and WB in BC cell lines with knockdown or overexpression of SETDB1.3.To investigate the mechanism by which SETDB1 cooperates with PELP1 to regulate AKT signaling.Yeast two-hybrid assay was used to clarify the binding and region of SETDB1 with PELP1.Interaction and cellular localization of SETDB1 with PELP1 were explored by immunoprecipitation and GST-pull down assay in BC models.The association of SETDB1,PELP1 and AKT was clarified by bioinformatics analysis.Constructed cell models with SETDB1,PELP1 and AKT overexpression and confirmed the interaction of three proteins using immunoprecipitation assay.The effect of PELP1 on SETDB1 mediated AKT methylation was investigated by in vitro methylation assay.BC cell models with overexpression of SETDB1 and PELP1 knockdown were constructed,and regulation of SETDB1-mediated AKT phosphorylation by PELP1 was investigated using WB.4.To investigate the biological functions of PELP1-SETDB1 axis in BC.Using BC cell models with SETDB1 overexpression and PELP1 knockdown,the roles of PELP1-SETDB1 axis on BC proliferation and endocrine resistance were investigated by colony formation assays,3D cell culture and function rescue assay.In vivo xenograft assays were constructed using MCF7 cell models to investigate the role of PELP1 in SETDB1-mediated BC tumorigenesis.The correlation between SETDB1 and PELP1 expression in BC was verified using the TCGA dataset.Results:1.SETDB1 is overexpressed in BC.In BC and endocrine resistant BC cell lines,knockdown or pharmacological attenuation of SETDB1 inhibited BC proliferation and endocrine therapy resistance.2.RNA-seq enrichment showed that SETDB1 promoted estrogen signaling pathway and AKT signaling.SETDB1 promoted transcriptional activity of estrogen receptor,knockdown of SETDB1 downregulated estrogen-responsive gene expression.SETDB1 induced AKT phosphorylation activation.3.PELP1 was found to be a novel interacting protein of SETDB1 in the cytoplasm.PELP1 regulated SETDB1-mediated AKT methylation and AKT phosphorylation.4.In BC cell lines,PELP1 knockdown attenuated SETDB1-mediated cell proliferation and endocrine resistance.PELP1 regulated SETDB1-induced AKT activation and BC tumorigenesis in xenograft models.SETDB1 expression was positively correlated with PELP1 expression in BC.Conclusions:SETDB1 promotes breast cancer proliferation and endocrine therapy resistance via AKT activation and estrogen signaling.SETDB1 interacts with PELP1 and synergistically regulates AKT-mediated breast cancer progression.