LSD1 Promotes Estrogen-driven Endometrial Carcinoma Cell Proliferation

Background and purpose Endometrial cancer(EC)is the most commonly diagnosed gynecological cancer.The incidence increased every year,while the pathogenesis is not yet clear.Histone lysine methylation is a crucial regulator of transcription,and its dysregulation is associated with oncogenesis.As the first demethylase detected,histone lysine specific demethylase 1(LSD1,KDM1A)functions as a histone demethylase that specifically mono-or di-demethylates histone 3 lysine 4(H3K4)or lysine 9(H3K9)and a transcriptional co-repressor included in the REST/Co-Rest complex.A recent study reported that LSD1 overexpressed in endometrioid endometrial carcinoma(EEC)and associated with tumor progression as well as poor prognosis.However,the underlying mechanism of LSD1 in EEC remains largely unknown.This study aimed to investigate physiological function and mechanism of LSD1 in EEC.Methods The expression levels of LSD1 and cyclin D1 protein in human tissues were analyzed through immunohistochemistry(IHC)staining.Cells were transfected with two sequences of si LSD1 or si Control prior to ethanol or 17β-estradiol treatment for another 48 h.Cell proliferation was assessed by CCK-8 assay and Clonogenic assays.Cell poptosis and cell cycle were assessed with flow cytometric analysis.We used western blot to detect the protein expression of LSD1,AKT,p-AKT,ERK,p-ERK,PTEN,cyclin D1,bcl-2,P21,Cleaved Caspase-3,H3K4me2 and H3K9me2.The level of H3K9me2 at the promoter region of cyclin D1 was explored through chromatin immunoprecipitation(Ch IP)assay.Results 1.LSD1 was significantly up-regulated in human endometrial cancer tissues and cell lines.2.β-estradiol(E2)treatment increased LSD1 expression in both dose and time effects via the GPR30/PI3K/AKT pathway in endometrial cancer cells.Both si GPR30 and the PI3K/AKT inhibitor LY294002 blocked this effect.3.Estrogen addition greatly promoted cell growth,cell cycle progression and inhibited cell apoptosis.RNAi-mediated silencing of LSD1 abolished estrogen-driven endometrial cancer cell proliferation,and induced G1 cell arrest and apoptosis.4.Mechanistically,LSD1 silencing resulted in PI3K/AKT signal inactivation,but without the elevation of PTEN expression as expected.When LSD1 was silenced,we found phospho-AKT,cyclin D1 and bcl-2 were ablated.Meanwhile,P21 and Cleaved Caspase-3 were upregulated.Moreover,interfering with cyclin D1 led to PI3K/AKT signal suppression.Re-overexpression of cyclin D1 in LSD1-knockdown ECCs reversed LSD1’s inhibitory action.An increase in total H3K9me2 was observed by western blot when LSD1 was silenced in E2 treated HEC-1-A cells.We did not detect obvious H3K4me2 changes.Consistently,LSD1 level was positively correlated with cyclin D1 in EEC tissues.Conclusion LSD1 was overexpressed in human endometrial cancer tissues and cell lines.Our data firstly suggested a possible role of LSD1/cyclin D1/PI3K/AKT loop in regulating the proliferation of EEC cells and provide novel experimental support for LSD1 as a potential target for endometrial cancer therapeutics.