Functional And Molecular Mechanism Of FOXM1 In Endometrial Carcinoma

Aim:Endometrial cancer is one of the three most important gynecological cancers,but its pathogenesis is not clearly understood and also lacking the effective treatment.The nuclear transcriptional protein Forkhead box protein M1(FOXM1)has crucial functions on development and progression of cancer and is treated as prognostic biomarker and therapeutic target in many types of cancers.However,the situation and underlying mechanism of FOXM1 involved in Endometrial cancer(EC)is largely underestimated.In our present study,by analyzing the clinical significance of FOXM1 in EC,the biological functions using FOXM1 knock down in EC cells lines and the possible molecular mechanism,we aim to identify whether FOXM1 could act as a biomarker or therapeutic target in EC and to provide a new molecular mechanism of endometrial cancer.Methods:(1)Analysis of TCGA,GEO endometrial cancer data to understand the expression level of FOXM1 and its expression level on the prognosis of patients with endometrial cancer;(2)Using immunohistochemistry on the tissue microarray of adenocarcinoma patients to identify the protein level of FOXM1 and verify the results of database analysis;(3)Screening of FOXM1 expression level on five common endometrial cancer cell lines,the proliferation and migration ability of two endometrial cancer cells were detected by CCK8 and Transwell assays after interfering by FOXM1 siRNAs;(4)Two endometrial cancers cell lines AN3CA and Ishikawa were used for RNA-seq analysis to look for potential downstream pathways and regulatory genes for FOXM1;(5)Bio-and biochemical methods were used to validate genes that were down-regulated after disruption of FOXM1 in both cell lines to determine FOXM1 possible functions and signaling pathways in endometrial adenocarcinoma to explore its diagnostic and prognostic significance for the endometrium.Results:(1)According to the statistics of TCGA database,the expression of FOXM1 mRNA in 524 endometrial carcinomas was significantly different from that in 35 normal endometrial tissues(P<0.005).The FOXM1 mRNA level in 115 of SEC was significantly different from that of 409 EEC patients(P<0.005).The relationship between the expression of 517 FOXM1 mRNA levels and the clinical stage of endometrial cancer was analyzed,the expression of FOXM1 mRNA in all fourth stages of endometrial samples was significantly different from that of normal endometrium(P<0.005).There was no significant difference between stage I and stage II(P>0.05),and the difference between stage I and stage III and stage IV was statistically significant(P<0.05).In addition to 137 cases of endometrioid adenocarcinoma,the expression of FOXM1 mRNA level between G1 and G3 was statistically significant(P<0.01),and the difference in expression of FOXM1 mRNA between G2 and G3 was also statistical difference(P<0.05).According to the analysis of GEO database(GSE17025),the mRNA expression level of FOXM1 in 79 cases of endometrioid adenocarcinoma and 12 cases of serous endometrial carcinoma was significantly up-regulated compared with 12 normal endometria(P<0.005).540 endometrial cancer samples from the TCGA database were classified according to the median FOXM1 mRNA expression level.The expression group(n=270)and the low expression group(n=270)were followed by a one-way disease-free survival prognosis analysis.The overall survival rate(OS)of endometrial cancer patients in the endometrial cancer was found statistically different(P=0.0281).(2)Verification of FOXM1 protein levels in tissue microarray of endometrial adenocarcinoma patients showed that FOXM1 positive expression was detected in 48%(58/120)of EC clinical samples,only one of 17 cases(0.06)%)the normal endometrial tissues showed positive FOXM1 staining,indicating that FOXM1 expression was significantly different between EC and normal endometrial tissue(P<0.001);we also analyzed FOXM1 expression and EC clinical grade and the relationship between pathological staging.Statistics show that 38%(33/87)G1-G2 EC samples showed high expression of FOXM1,and in 76%(25/33)of G3 EC samples FOXM1 were highly expressed,and the difference between them was statistically significant(P<0.001);in addition,44%(44/100)of patients with stage I-II had high expression of FOXM1,while at 70%(14/20)of patients with stage III EC,FOXM1 was highly expressed,(P<0.05).(3)Compared with the control cells,the proliferation ability of AN3CA cells after interfering with FOXM1 was significantly decreased at 72 and 96 hours,P value was 0.0125 and less than 0.0001,respectively;the proliferation ability of Ishikawa cells interfering with FOXM1 was also significantly inhibited at 96 hours,the P value was 0.0067.Transwell results showed that compared with the control group,the number of AN3CA cells transfered into the transwell chamber 24 hours after FOXM1 knock down was significantly lower than that of the control group,P value was less than 0.0001;The number of cells in the transwell chamber after 24 hours of Ishkawa cells interfering with FOXM1 was also significantly lower than that in the control group,and the P value was less than 0.0001.(4)Our RNA-seq results showed that 8 co-upregulated genes were found in endometrial cancer cell lines AN3CA and Ishikawa after interference with FOXM1(TPM4,BUB1B-PAK6,AC098614.2,CXCL8,HABP4,S1-44D20.1.RP1-80N2.3,PRR5-ARHGAP)and 6 common down-regulated genes(SEPHS2,VMA21,SLC27A2,GSDMD,ALDH2,FOXM1).(5)After literature review,we selected the western blot analysis of the co-downregulated genes SLC27A2 and ALDH2 in the cell line after FOXM1 interference,and showed that only the protein level of SLC27A2 was down-regulated in both AN3CA and Ishikawa cells,instead of ALDH2(6)The enrichment analysis of GO and KEGG on these 700 common differential genes revealed that the signal pathway was enriched into the cell cycle,and part of the pathway was also enriched to metabolic pathways,which consistent with the results of GSEA and KEGG enrichment analysis of differential genes in patients with endometrial cancer with high expression and low expression of FOXM1 gene in TCGA,we speculate that FOXM1 mainly passes through cell cycle and metabolism in endometrial cancer,which play important roles in promoting the proliferation and migration of endometrial cancer.In the future research direction,we decided to continue to conduct more in-depth research from these two aspects.Conclusion:The high expression of FOXM1 is closely associated with the prognosis,the pathological stages and clinical grades.FOXM1 can promote the proliferation and cell migration of EC cells.Through SLC27A2,FOXM1 may influence the metabolic activity of EC cells.In addition,through GESA and KEGG enrichment analysis,we speculate that it may also regulate the development of endometrial cancer by affecting the cycle activity.Taken together,FOXM1 itself and FOXM1-SLC27A signaling could treated as potential cellular target for therapeutic strategy of EC.