Histone Acetyltransferase MOF Is Involved In Suppression Of Endometrial Cancer And Maintenance Of ER? Stability

Objective:Histone acetyltransferase MOF is a member of the MYST family of histone acetyltransferases(HATs).MOF is responsible for acetylating histone H4 at lysine 16(H4K16),which is required for maintaining active chromatin state.MOF participates in various critical cellular functions such as cellular stress response,cell cycle progression,response to DNA repair and autophagy.It has also been demonstrated that MOF exerts suppression or promotion function in different human tumors.MOF is lowly expressed in hepatocellular carcinoma and breast cancer,and involved in suppressing hepatocellular carcinoma and breast cancer process.MOF is highly expressed in lung cancer and involved in promoting lung cancer process.However,the functional specificity of MOF action in endometrial cancer still need to be further defined.ECa are common gyaecological malignancy,which are classified into two types on the basis of biological and histopathological variables.Clinically,approximately 80% of ECa are type I,which is generally well-differentiated,endometrioid histology and associated with a history of unopposed estrogen exposure and other hyperestrogenic risk factors.The estrogen receptor ?(ER?)is a ligand-dependent transcription factor that regulates cell proliferation and homeostasis in many hormone-related tissues,including endometrium.ER? expression is decreased in grade III ECa samples and high expression of ER? is associated with long disease-free survival in ECa.These studies indicate that ER? action is crucial and complicated in ECa development and progression.The mechanisms underlying the reduced expression of ER? in higher grade ECa remains to be clarified.Here in this study,we aim to identify the biological function of MOF in endometrial cancer.In addition,this study is designed to investigate the correlation between MOF and characteristic expression and complicated function of ER? in endometrial cancer to explore a new target for endometrial cancer treatment.Methods:1.We firstly examined the biological role of MOF in ECa cells and mice.2.We then examined expression difference MOF in 26 benign endometrial tissues and 200 endometrioid cancer tissues,and correlation between MOF and clinicopathological features.Based on these studies,we confirmed correlation between MOF and ER?,and further investigated the molecular mechanism.4.First,Co-immunoprecipitation(Co-IP)experiments tested interaction between MOF and ER?.Second,western blot experiments examined the influence of MOF depletion and ectopic expression on ER? protein expression in ECa cell lines(Ishikawa or HEC-1A cells).We further treated the cells with protein synthesis inhibitor,cycloheximide(CHX)to examine the influence of MOF and a catalytically inactive MOF mutant(K274R)on ER? stability.In addition,ECa cell lines carrying shMOF were individually treated with a proteasome inhibitor MG132 to examine whether ER? reduction by MOF depletion was reversed by addition of MG132.At last IP experiments were performed to examine whether ER? was aectylated by MOF and a catalytically inactive MOF mutant and the possible acetylation sites of ER? mediated by MOF.4.RNA microarrays were performed to investigate the impact of MOF on genome-wide ER?-regulated genes in Ishikawa cells carrying shMOF with or without E2.We then examined cellular processes in which these differentially expressed genes regulated by MOF are mainly involved by KEGG pathway analysis.We performed real-time quantitative PCR(qPCR)and ChIP experiments to further confirm the analysis results of RNA microarray.Results: 1.Knockdown of MOF promotes growth and proliferation of endometrial cancer cells in vivo and in vitro.Growth curves analysis showed that MOF depletion promoted cell proliferation.Consistently,colony formation experiments demonstrated that knockdown of MOF formed higher number of colonies than that of control.Flow cytometry analysis results revealed that knockdown of MOF induced a decrease in the percentage of G1 phase cells as well as an increase in percentage of G2/M phase cells.Xenograft experiments were conducted in female NOD/SCID mice with Ishikawa cells infected with lentivirus carrying shMOF and shCtrl.The size of xenograft tumors from shMOF cells was larger than tumors from shCtrl cells.The tumor volume from shMOF cells demonstrated higher growth rate than tumors from the shCtrl cells.The average tumor weight from shMOF cells was higher than tumors from the control cells.2.MOF is lower expressed and positively correlated with ER? expression in endometrial cancer tissues.MOF is more lightly stained in endometrioid ECa,compared with that in benign endometrial tissue samples.In addition,lower expression of MOF is associated with higher grade.Moreover,it is unexpected that MOF expression is positively correlated with ER? status in endometrioid ECa tissues.We then examined the expression level of MOF or ER? in 20 freshly frozen endometrioid ECa tissues.Consistent with the IHC results,the expression of MOF is correlated with that of ER? in endometrioid ECa samples.3.MOF interacts with ER? and modulates ER? stability.Co-immunoprecipitation(Co-IP)experiments showed that the endogenous MOF associated with ER? in mammalian cells.In addition,Ishikawa cells were co-transfected with ER? and FLAG-tagged MOF expression plasmids and perfomed Co-IP experiments.Precipitated proteins were detected by western blotting using the antibodies as indicated.The results showed that exogenous MOF was precipitated with ER?.Moreover,the results of immunofluoresence experiments demonstrated that the endogenous MOF was mainly located in the nucleus with or without the treatment of E2 and MOF were partially distributed together with ER? in the nucleus in Ishikawa cells with the treatment of E2.We observed MOF depletion downregulated ER? expression without affecting ER? mRNA levels in ECa cell lines(Ishikawa and HEC-1A cells),and ectopic expression of MOF increased ER? expression in a dose-dependent manner.We further treated the cells with protein synthesis inhibitor,cycloheximide(CHX)to examine the influence of MOF and MOF mutant(K274R)on ER? stability.The results demonstrated that ER? degradation was decreased in Ishikawa cells transfected with wild-type MOF,however ER? stability maintenance was abolished by a catalytically inactive MOF mutant(K274R).In addition,ECa cell lines carrying shMOF were individually treated with a proteasome inhibitor MG132,and ER? reduction by MOF depletion was reversed by addition of MG132.IP experiments were performed to show that ER? in immunoprecipitated protein complex was acetylated in ECa cells.The COS-7 cells were then cotransfected with ER? together with p300 or MOF expression plasmids.The data demonstrated that MOF obviously promoted the acetylation on ER?,which was similar with that induced by p300.However,a ctalytically inactive MOF mutant(K274R)failed to acetylate ER?.To further determine the acetylation sites on ER? mediated by MOF,we then turn to generate several ER? mutants.Five lysines on ER? have been reported to be acetylated by p300,including K266,K268,K299,K302 and K303.Based on the above publication,we substituted K266,K268,K299,K302,K303,and K302/K303 with arginine(R)to make an acetylation-resistant mimic.The acetylation assay results have shown that compared with the acetylation level of ER? wild type mediated by MOF,acetylation levels of ER? mutants(K266R,K268 R,K299R,K303 R and K302R/K303R)were reduced.4.MOF regulates a subset of endogenous ER?-induced genes in endometrial carcinomas cells.RNA microarrays were performed in Ishikawa cells carrying shMOF with or without E2.Gene array analysis showed that there were 1280 differentially expressed genes(DEGs)regulated by E2 treatment,2807 DEGs and 2054 DEGs respectively in cells carrying shMOF in presence or absence of E2.The KEGG analysis demonstrated that the DEGs influenced by MOF with E2 treatment are mainly involved in metabolic pathway,pathways in cancer,MAPK signaling pathway,cytokine-cytokine receptor interaction,as well as cell cycle pathway.Among the 1280 E2-regulated genes,18.8% were significantly downregulated in MOF knockdown cells in presence of E2.We further selected a subset of genes,which are obviously regulated by MOF in presence of E2 and involved in tumor process.In addition,we performed real-time quantitative PCR(qPCR)experiments to further confirm the analysis results of RNA microarray.Consistent with the RNA microarray analysis results,we demonstrated that DNA damage-regulated autophagy modular 1(DRAM1)and TAGLN induced by E2 were regulated by MOF.PGR and E2F1,which are ER? target genes in breast cancer,were not remarkably regulated by MOF in Ishikawa cells.ChIP assays data demonstrated that MOF or ER? was recruited to DRAM1-EREI,but not DRAM1-EREII upon E2 induction.In addition,histone H4K16 ac,but not H3K9 ac was significantly decreased by shMOF Conclusions:1.MOF involved in suppression of endometrial cancer cell growth and proliferation.2.Expression of MOF is reduced and positively correlated with that of ER? in endometrial cancer tissues.3.MOF associates with ER? to be involved in maintenance of ER? stability via acetylation of ER? mediated by MOF.4.MOF regulates a subset of genome-wide ER?-regulated genes in endometrial cancer cells.