The Role Of ZNF496 In The Development And Function Of Breast Cancer

ERα,a member of the nuclear receptor superfamily,participates in many physiological and pathological processes.Estrogen activated ERα is able to form a homodimer,binds to the palindromic estrogen response element(ERE)and plays a role of activated transcription factor.ERα contains three important domains,which are the AF1 domain,AF2 domain and DBD domain.AF1 and AF2 are transcriptional activation domains,but only AF2 is estrogen dependent,while DBD domain mainly mediates the interaction between ERα and DNA motif.As one of the most common malignant tumors in female,breast cancer possesses extremely complex pathogenesis.It is widely accepted that the abnormal endocrine level is a vital factor in inducing breast cancer.Nearly 3/4 of breast cancers are ERα positive,and the overactive ERα stimulates the normal breast epithelial cells to transform to the malignant ones and is also a prerequisite for maintaining the proliferation of breast cancer cells.The disorder of ERα binding to ligand and cofactors and the changes of its structure and posttranslational modification could affect the estrogen signaling pathway in different extent and participate in the development of breast cancer.So ERα is a crucial biomarker for breast cancer and is always used to guide the treatment of breast cancer.Targeting ERα is one of the most common strategies in breast cancer adjuvant therapy.Tamoxifen is a typical antagonist of ERα,which could inhibit the growth and proliferation of breast cancer cells by competing with estradiol(E2)for the combination to ERα.The abundance and/or activity anomaly of ERα cofactors is usually associated with the incidence and mortality rates of breast cancer and also could be the potential target in diagnosing and treating breast cancer.So the study on ERα cofactor is of great importance for revealing the regulatory mechanism of ERα and identifying the diagnostic and therapeutic targets of breast cancer.C2H2 zinc finger protein family is the biggest transcription factor and/or transcriptional regulator family,and KRAB-containing zinc fingers proteins only exist in the tetrapod,which have the similar structures and function mechanisms,and expanded rapidly along with species evolution,the number of the subfamily is increasing up to 423 members in human genome and occupies 60% of total C2H2 zinc finger proteins.The KRAB domain in the N terminal of KRAB-containing zinc finger protein recruits transcriptional repressors via interacting with KAP1,with which forms a transcriptional silencing complex,and play a role in transcriptional repression.The C2H2 domain in the C terminal mainly mediates the binding of KZNF to the specific DNA sequences or other transcription factors in order to anchor the transcriptional silencing complex to the specific chromatin sites.A growing number of evidences suggest,the emergence and quantity expansion of KRAB-containing proteins are closely associated with the establishment and perfection of transcriptional regulatory network along with the evolution in vertebrates.KRAB-containing zinc finger proteins participate in various normal physiological processes such as genome structural stabilization,embryonic development,cell proliferation and differentiation,and are also associated with the occurrence and development of tumors.Generally,the transcriptional network in tumor tends to be simplify and the abundance of KRABcontaining zinc finger proteins in the carcinoma tissues are often lower than those in the tissues adjacent to carcinoma.ZNF496 belonging to KRAB-containing zinc finger proteins is able to bind to NSD1(nuclear receptor binding SET domain protein 1)by its C2 HR domain and mediates the KAP1-independent transcriptional repression function.So far there has no explicit reports about the functions and mechanisms of ZNF496.We find that ZNF496 is highly expressing in the female reproductive system and the mammary gland,but little or not expressing in the corresponding cancer tissues by analyzing its expression profile in website www.proteinatlas.org,which hint that ZNF496 might be associated with the development of breast cancer,but the underlying mechanism is not clear.Our research finds:1.ZNF496 is highly expressing in the mammary gland and reproductive system of female and is low-expressing in the corresponding tumor tissues.We found that the expression levels of ZNF496 in breast cancer,ovary cancer and cervical cancer were lower than that in the adjacent normal tissues tumor by the human reproductive system tumor tissue microarrays we purchased.We also detected the ZNF496 protein abundance in the primary tissues of adult female and male mice by WB,and the result showed that ZNF496 was specifically expressed in lung,ovary,uterus and oviduct.The tissue specific expression of ZNF496 may be associated with the maintain of normal physiological functions in these tissues.2.ZNF496 is able to interact with ERα.In order to explore the functional mechanism of ZNF496,firstly,we screened the potential interactors of ZNF496 by IP-MS in MCF-7 nucleus and the result showed that ERα might be a potential interactor of ZNF496.And then the following Co-IP verified that ZNF496 was able to interact with ERα with the presence of E2 or not.3.ZNF496 selectively represses the transcriptional activity of ERα and functions as a selective regulator of ERα.The results of qPCR suggested the overexpression of ZNF496 could suppress and the knockdown of ZNF496 up regulated the transcriptional level of ERα target genes Greb1,pS2,Xbp1,Sgk1 and Wisp2.However,ZNF496 had no effect on the expression of Serpina3,another ERα target gene.At the same time,ZNF496 could not change the mRNA and protein levels of ERα.All these suggested that ZNF496 was able to selectively regulate the transcriptional activity of ERα.4.ZNF496 selectively represses the binding of ERα to the promoters of some of its targets.Other research in our laboratory had confirmed that the C2H2 zinc finger domain in the C terminal of ZNF496 and the DBD domain located in the middle of ERα mediated the two proteins’ interaction.The fluorescence localization experiment indicated the overexpression of ZNF496 weakened the condensation of activated ERα.In order to probe into the mechanism of ZNF496 selectively regulating ERα activity,we also conducted the EMSA experiment,which showed under the stimulation of E2,the ability of ERα binding to ERE sequence gradually descended along with the increase of ZNF496 expression.It further indicated that the overexpression of ZNF496 suppressed the binding of ERα to ERE sequence with the treatment of E2.At last we detected ability of endogenous ERα binding to the promoter of its targets via ChIPqPCR in MCF-7.The result revealed the ability of ERα binding to the promoter of pS2,Greb1 and Wisp2 showed a tendency of weakening,while the binding to Serpina3 promoter had no change.5.ZNF496 can suppress the proliferation of ERα positive breast cancer cell.we established ZNF496 stably expressing cell lines MCF-7,T-47 D and MDA-MB-231 by using lentivirus affection.CCK-8 cell proliferation assay showed that the stable overexpression of ZNF496 could inhibit the proliferation of MCF-7 and T-47 D,but had no effect on MDA-MB-231.The clonally formation assay also confirmed that ZNF496 overexpression could suppress the clonality of MCF-7 and T-47 D,but not that of MDAMB-231,which implied ZNF496 could repress cell proliferation by selectively suppressing ERα.6.The differential expression of ZNF496 in breast cancer depends on the state of ERα: The immunohistochemistry results of 29 cases of breast cancer samples indicated that the expression of ZNF496 in tumor tissues was significantly lower than that in the adjacent tumor tissues.When we divided these samples into ERα+ and ERα-,the expression of ZNF496 was high significantly lower in ERα+ tumor tissues than that in the adjacent tumor tissues,while ZNF496 in ERα-tumor tissues and adjacent tissues had no significant difference.Therefore,the differential expression of ZNF496 in breast cancer relys depends on the positive expression of ZNF496.In conclusion,this study shows ZNF496 selectively represses the binding of ERα to ERE site by binding to the DBD domain of ERα competitively and down regulates the transcriptional activity of ERα.ZNF496 inhibits the proliferation of ERα positive breast cancer cells in a ERα dependent manner.Our work provides an insight into the molecular mechanism of the development of ERα positive breast cancer and provides a potential target for the diagnosis and treatment of breast cancer and perfects the recognition of functions and mechanisms of action of KRAB-containing zinc finger proteins.