The Role And Molecular Mechanisms Of ELF5 Acetylation In The Occurrence And Development Of Breast Cancer

Acetylation is an important type of protein post-translational modification.Acetylation participates in the regulation of protein sTable ility,enzyme activity,subcellular localization,protein-protein interactions and protein-DNA interactions,and plays an important role in a variety of physiological,biochemical and pathological processes,such as gene transcription,DNA damage repair,cell cycle changes,protein folding and signaling pathway.Breast cancer is one of the three most common malignancies in the world,its occurrence,progression,invasion,metastasis and other processes are closely related to the acetylation of a variety of proteins.Among them,ER81,an important member of the ETS family,its acetylation is closely related the abnormality of the HER2 signaling pathway in breast cancer.In addition,ELF5,another important member of the ETS family,also plays an important role in the progression of breast cancer.In luminal breast cancer,ELF5 inhibits the transcription of ER,FOXA1,MYC,and other proliferation-related genes.A sustained increase in ELF5 expression has been demonstrated in endocrine-resistant breast cancers and basal-like subtype breast cancer,resulting in the suppression of estrogen sensitivity and lung metastasis.However,ELF5,as a member of the ETS family,is structurally similar to the members of the same family that can be acetylated,such as ER81,ETS1,PEA3,etc.,it is still unclear that whether ELF5 can also be acetylated.Protein sequence analysis showed that ELF5 has multiple highly conserved lysines,suggesting that acetylation modification may occur at these sites.Therefore,our study started with the acetylation modification of ELF5,and explored the mechanism by which the acetylation of ELF5 regulates the occurrence and development of breast cancer.And the main research contents are as follows:Determination of the interaction between ELF5 and p300.ELF5 was identified as an interatcted protein of p300 by immunoprecipitation combined with mass spectrometry experiments.The endogenous and exogenous co-immunoprecipitation experiments,GST pulldown assay confirmed that ELF5 interacts with p300.ELF5 and p300 co-localization in the nucleus was confirmed by immunofluorescence experiments.Determination of ELF5 acetylation.It is the first time to confirm that ELF5 is an acetylated protein in human breast cancer MCF7 and T47D cells.The results of immunoprecipitation experiments,siRNA interference experiments,inhibitor treatment experiments,and in vitro acetylation experiments proved that p300 catalyzes the acetylation process of ELF5.These six positions of K130,K134,K143,K197,K228,K245 in ELF5 were confirmed to be acetylated sites by mass spectrometry identification and point mutation experiments.Identification of ELF5 deacetylase.Through deacetylase inhibitor assay,it is determined that the deacetylation of ELF5 is regulated by the HD AC family and the Sirtuin family,and the Sirtuin family is the main one.The co-immunoprecipitation experiment,immunofluorescence experiment,GST pull-down experiment,etc.confirmed the interaction between SIRT6 and ELF5.SIRT6 negatively regulates the acetylation modification of ELF5 was confirmed by siRNA interference assay,inhibitor treatment assay,and in vitro deacetylation assay.Acetylation of ELF5 promotes ubiquitination degradation.The protein sTable ility of ELF5 is regulated by acetylation and proteasome pathways through inhibitor treatment assays.Acetylation of ELF5 promotes its ubiquitination modification and shortens its half-life was proved by half-life experiments and ubiquitination experiments.Immunohistochemical staining proved that the expression of ELF5 and SIRT6 in breast cancer tissue samples was positively correlated.ELF5 suppresses the proliferation of breast cancer by targeting CCND1 and relies on acetylation modification.Cell proliferation experiments,cell colony formation,soft agar clone formation experiments and xenograft model assays were conducted and proved that both wildtype and 6KQ mutant ELF5 can effectively inhibit the proliferation of breast cancer cells,while the 6KR mutant cannot effectively inhibit breast cancer cell growth.Those results indicate that the suppression of ELF5 breast cancer cell relies on its acetylation.Cell cycle assay,western blot,chromatin immunoprecipitation assay,luciferase reporter assay and immunohistochemical staining assay identified CCND1 as a new target gene of ELF5 to inhibit breast cancer cell proliferation.ELF5 acetylation can enhance the transcription repressive effect on CCND1,and decrease the expression level of cyclin D1,thereby inhibiting the proliferation of breast cancer cells.In summary,our data confirmed that the transcription factor ELF5 was acetylated,and identified CCND1 as a new target gene of ELF5.This study demonstrated that acetylation not only regulates the protein sTable ility of ELF5,but also inhibitions the proliferation of breast cancer cells by targeting CCND1.This not only broadens the understanding of ELF5’s function,but also helps to explore the mechanism of the occurrence and development of breast cancer and provides new ideas for the treatment of breast cancer.