The Mechanism Of ZNF213 In Regulating Estrogen Signaling In Breast Cancer

BackgroundAccording to the World Health Organization’s International Agency for Research on Cancer in 2020,breast cancer is the most prevalent malignancy among Chinese women.There are approximately more than 1 million new cases worldwide each year.According to the latest global epidemiological data,breast cancer accounts for 24.2%of female tumor incidence and 15.1% of tumor-related deaths.In breast cancer,estrogen receptor alpha(ER alpha)positivity accounts for 60%-70% of cases,and the ER alpha signaling pathway plays a very important role in the proliferation and metastasis of ER-positive breast cancer cancer cells.Thus,ER alpha is an important therapeutic target for the treatment of ER-positive breast cancer.Tamoxifen is a classic drug for ER alpha positive breast cancer treatment,but more than half of the patients develop endocrine resistance,making it an important clinical challenge for breast cancer treatment.ZNF213 is a zinc finger structural protein,which has been found to be involved in post-translational modifications of the protein.However,the function of this protein in breast cancer is not clear.Therefore,it is important to further clarify the function of ZNF213 in breast cancer and its correlation with tamoxifen resistance,which has an important role and clinical significance.ObjectiveOur aim is to investigate the mechanism of ZNF213 protein inregulating estrogen signaling pathway and tamoxifen resistance.To provide a new theoretical basis for the endocrine therapy of breast cancer,and to provide a new theoretical basis and target for the subsequent development of a new generation of clinical breast cancer endocrine therapy drugs.MethodsWe explored the expression of ZNF213 in the tissues of breast cancer patients and its correlation with endocrine therapy by consulting the public open databases TCGA,Oncomine,and Kmplot databases.Then CCK8 assay,Edu assay,scratch assay and mouse transplantation tumor assay were used to investigate the effect of ZNF213 on cell proliferation and migration,and the effect of ZNF213 on tamoxifen resistance.Then we used whole gene transcriptome sequencing technology(RNA-Seq)to explore the effect of ZNF213 on estrogen signaling pathway and other transcriptome.The effect of ZNF213 on estrogen signaling pathway activity was then examined by q RT-PCR,Western blot,and luciferase reporter gene techniques.Subsequently,immunofluorescence assay was applied to investigate whether ZNF213 and ER alpha bind to each other and the specific binding site using exogenous protein immunoprecipitation Co-IP assay.Finally,MG132 assay,CHX assay,Co-IP and Western Blot were used to explore the regulatory mechanism of ZNF213 on ER alpha protein.Results1,By querying TCGA and Oncomine databases,we found that ZNF213 protein was highly expressed in breast cancer patient tissues compared to normal breast tissues.we also found that breast cancer patients with high ZNF213 expression had low survival after endocrine therapy compared with breast cancer patients with low ZNF213 expression by querying the Kmplot database.2,We found that ZNF213 promoted cell proliferation and migration by CCK8 assay,Edu assay,mouse transplantation tumor assay and scratch assay.3,We found that silencing ZNF213 could significantly inhibit the expression level of genes downstream of estrogen signaling pathway by whole gene transcriptome sequencing technology(RNA-Seq),such as PKIB,PDZK1,GREB1,IL20,etc.4,It was demonstrated that silencing ZNF213 significantly down-regulated the activity of estrogen signaling pathwayby q RT-PCR,Western blot and luciferase reporter gene technique(Luciferase).5,CCK8 assay demonstrates that silencing ZNF213 increases the sensitivity of estrogen receptor-positive breast cells to tamoxifen.6,Immunofluorescence assay and Co-IP assay demonstrated that ZNF213 and ER alpha bind to each other,and the ZF domain of ZNF213 could bind to the AF1 domain of ER alpha.7,MG132 assay,CHX assay,Co-IP and Western Blot experiments clearly demonstrated that ZNF213 could prolong the half-life of ER alpha and inhibit the ubiquitinated degradation of ER alpha possibly via inducing ER alpha K48-linked poly-ubiquitination.ConclusionsOur study reveals a novel mechanism between ZNF213 and ER alpha signaling.ZNF213 ZF domain interact ER alpha AF1 domain.ZNF213 increases ER alpha stability,possibly by inhibiting K48-specific poly-ubiquitination process on ER alpha.Furthermore,ZNF213 could inhibit cellular sensitivity to tamoxifen.Targeting ZNF213 could be one promising strategy for ER alpha positive breast cancer treatment.