The Study On The Effect And Mechanisms Of FOXM1 Pathway Promoting The Malignant Behavior Of Serous Ovarian Carcinoma

BackgroundOvarian cancer is the most lethal gynecologic malignancy.Due to the lack of obvious symptoms at the early stage and no effective screening strategy,the majority of the patients have been identified at the advanced stages of the disease.About 70%patients with ovarian cancer will recur after the standard therapy and the overall five-year survival probability is about 47%.Serous ovarian carcinoma is the most common and malignant form of ovarian cancer and accounts for up to 70%of all ovarian cancer cases.Serous ovarian carcinoma is further divided into low-grade serous ovarian carcinoma and high-grade serous ovarian carcinoma respectively.High-grade serous ovarian carcinoma is the most common type and it accounts for 70-80%of deaths caused by serous ovarian carcinoma.Thus,it is still a global problem for its early diagnosis and efficient therapy,which depends on not fully understand the molecular pathogenesis of serous ovarian carcinoma.The Cancer Genome Atlas(TCGA)researchers published the integrated genomic analysis of ovarian carcinoma,and they found that FOXM1 signaling pathway is over-activated in almost 87%high-grade serous ovarian carcinoma patients,just lower than mutant TP53(96%).FOXM1 and its proliferation-related target genes,AURKB,CCNB1,BIRC5,CDC25 and PLK1 were found to be overexpressed in high-grade serous ovarian carcinoma,which implies that FOXM1 is involved in the initiation and development of serous ovarian carcinoma.FOXM1 is a typical cell cycle-related transcription factor.It facilitates cellular proliferation by regulating the G1/S and G2/M transition and it is essential for the normal mitosis.FOXM1 is found to be overexpressed in many human malignancies.FOXM1 contributes to malignant transformation and it is widely involved in the process of initiation and progression of tumors,including angiogenesis,migration,invasion,Epithelial-mesenchymal transition(EMT),self-renew of tumor stem cells and drug resistance.As a famous transcription factor in cancer research,the reasons for the high expression of FOXM1 in serous ovarian carcinoma and related regulatory mechanisms remain unclear.The molecules that FOXM1 interacts with and the downstream target genes involved in tumorigenesis and development are not fully elucidated in serous ovarian carcinoma.Therefore,it plays a crucial role to elucidate the reasons for the overexpression,functional roles and related molecular mechanisms of the crucial downstream target genes of FOXM1 in the process of development and progression for the early diagnosis,treatment and clinical prognosis of serous ovarian carcinoma.This study focused systematicly on the effect and mechanisms of FOXM1 pathway promoting the malignant behavior of serous ovarian carcinoma,which includes the following three parts:Part Ⅰ:Study on the aberrant expression,clinical significances and biological functions of FOXM1 in serous ovarian carcinomaObjectFOXM1 is overexpressed in breast cancer,lung cancer,pancreatic cancer and other human malignancies.FOXM1 participates in the process of proliferation,metastasis,malignant transformation,DNA damage.The expression of FOXM1 was found to be increased by 5.2 folds in the expression profile microarray between serous ovarian carcinoma and normal fallopian tube in our previous study.This study intended to further investigate the expression and the biological functions of FOXM1 in vitro assay and determine the clinical application value through immunohistological staining assay.Contents and methodsBioinformatics analysis was used to detect the expression of FOXM1 in serous ovarian carcinoma,normal ovary or peritoneal tissues through Oncomine and TCGA.qRT-PCR and western blot were used to detect the mRNA and protein expression of FOXM1 in serous ovarian carcinoma and normal fallopian tube fimbria tissues or cell lines.The copy number variation of FOXM1 was analyzed by using TCGA,Oncomine and Cancer Cell Line Encyclopedia(CCLE).The effect of copy number variation on the FOXM1 mRNA and protein expression and clinical prognosis was also investigated.The effect of FOXM1 on the clinical prognosis was analyzed with the use of TCGA and Kaplan-Meier Plotter.Immunohistochemistry staining based on our tissue microarray was employed to measure the expression and clinical application value of FOXM1 in serous ovarian carcinoma.The stable ovarian cancer cell lines with FOXM1 overexpression or knockdown were constructed through lentivirus packaging system.The functional roles of FOXM1 on the proliferation and metastasis were determined through cellular proliferation and transwell assay in vitro.Western blot was used to detect the changes of Epithelial-mesenchymal transition markers and cell cycle proteins after FOXM1 knockdown.ResultsThe mRNA expression of FOXM1 in serous ovarian carcinoma was significantly higher than that in normal ovarian or peritoneal tissues through Oncomine and TCGA.The mRNA expression of FOXM1 in serous ovarian carcinoma tissues or ovarian cancer cell lines was found to be obviously increased than that in normal fallopian tube fimbria through qRT-PCR.The protein expression of FOXM1 in serous ovarian carcinoma tissues or ovarian cancer cell lines was found to be apparently increased than that in normal fallopian tube fimbria through western blot.The mRNA expression of FOXM1C was the highest,followed by FOXM1B,and FOXM1A was the lowest through qRT-PCR.The genomic amplication of FOXM1 was found in 11%sequenced cases and the patients of serous ovarian carcinoma containing genomic amplication of FOXM1 showed a higher mRNA and protein expression in TCGA cohort.The copy number variation of FOXM1 in serous ovarian carcinoma samples was dramatically increased than normal ovary and blood samples from Oncomine.FOXM1 copy number was also found to be positively associated with its mRNA and protein expression in 45 kinds of ovarian cancer cell lines through CCLE.Kaplan-Meier survival analysis for serous ovarian carcinoma showed that the genetic amplication of FOXM1 led to shortening the overall survival and progression free survival of the patients in TCGA cohort.High expression of FOXM1 was significantly correlated with progression-free survival by using Kaplan-Meier plotter websites(p<0.05).The immunohistochemistry staining intensity of FOXM1 in our serous ovarian carcinoma was commonly higher than that in normal fallopian tube fimbria.Most positive staining of FOXM1 was observed in the nucleus,cytoplasm and high expression of FOXM1 was found to account for 82.09%(110/134).Kaplan-Meier analysis showed that overexpression of FOXM1 was significantly correlated with overall survival(p<0.01).The FOXM1 overexpression and knockdown cell lines were established according to the differential expression of FOXM1 in ovarian cancer cell lines.The clonogenic capacity and proliferation speed was found to be increased with overexpression of FOXM1 and decreased after FOXM1 knockdown.The ability of migration and invasion was significantly enhanced after overexpression of FOXM in A2780.Conversely,when we decreased the expression of FOXM1 in HO8910 and SKOV3 cells,the aggressive capacity of cancer cells was suppressed effectively.Overexpression of FOXM1B promoted the growth and migration of FTE187.These results suggested that FOXM1 could significantly promote the proliferation and metastasis of ovarian cancer cells.We evaluated the effect of FOXM 1 on Epithelial-mesenchymal transition markers and cell cycle proteins by western blot.The expression of E-cadherin was up-regulated and N-cadherin,Vimentin,Snail and Slug were significantly decreased in HO8910 cells after FOXM1 knockdown.In addition,FOXM1 could affect the proliferation of ovarian cancer cells by regulating the expression of CCNB1.ConclusionFOXM1 is significantly overexpressed in serous ovarian carcinoma and high expression of FOXM1 suggests poor prognosis.FOXM1 promotes the malignant behavior of serous ovarian carcinoma.FOXM1 could be used as a potential therapeutic target.Part Ⅱ:Study on the identification and biological functions of the downstream target genes of FOXM1 in serous ovarian carcinomaObjectFOXM1 is overexpressed in the majority of human cancers.The abnormal overexpression of FOXM1 facilitates the initiation and progression of malignant tumors and is intimately associated with the tumorigenesis.Although some downstream targets of FOXM1 have been studied,most of downstream targets driven by FOXM1 remain unclear.Due to the high heterogeneity of malignancies,the individual signaling pathway and downstream target genes are insufficient to elucidate the molecular mechanisms of the oncogene functions of FOXM1.The downstream target genes regulated by FOXM1 involved in tumorigenesis and development are not fully understood in serous ovarian carcinoma.Therefore,it plays a vital role to illustrate the functional roles and molecular mechanisms of the downstream target genes in the process of initiation and progression for the early diagnosis and individualized treatment of serous ovarian carcinoma.This study focused on the screening and identification of the crucial downstream target genes of FOXM1 in serous ovarian carcinoma through ChIP-seq assay.Contents and methodsThe downstream target genes of FOXM1 in serous ovarian carcinoma were screened through analyzing the results of ChIP-seq in MCF7 cell lines disposed with FOXM1 inhibitor thiostrepton or DMSO control and expression profile microarray of serous ovarian carcinoma.The expressions of downstream target genes were further analyzed by using TCGA.The mRNA and protein expressions of candidate downstream target genes were further validated after FOXM1 knockdown in ovarian cancer cell lines through qRT-PCR and western blot.The expression relationship between FOXM1 and target genes was analyzed through TCGA and CCLE.qRT-PCR was also used to study the coexpression between FOXM1 and target genes in serous ovarian carcinoma tissues.The potential binding sites at the promoter of target genes that FOXM1 could bind to were predicted by using gene-regulation website.Dual-luciferase report assay was used to validate the exact binding sites at the promoter which FOXM1 binds to.ChIP-PCR was employed to determine that FOXM1 could bind to the promoters of target genes.The differential expression of target genes between serous ovarian carcinoma and normal ovary tissues was analyzed by using TCGA data.qRT-PCR and western blot were used to analyze the differential expressions of target genes between serous ovarian carcinoma and normal fallopian tube tissues.The effect of target genes on the clinical prognosis was analyzed through Kaplan-Meier Plotter website.Immunohistochemistry staining based on our tissue microarray was employed to detect the expression and clinical application value of target genes in serous ovarian carcinoma.The stable ovarian cancer cell lines with target genes overexpression or knockdown were constructed through lentivirus packaging system according to the differential expression in ovarian cancer cell lines.Functional experiments were used to verify the effect of the target genes on proliferation and metastasis of ovarian cancer cells.ResultsChIP-seq and differential expression microarray were analyzed to identify novel targets of FOXM1 in serous ovarian carcinoma:TOP2A,TROAP,CCNA2,KIF20A,ASPM,HMGB2,CCNB1,TTF2,CCNF.CCNF and KIF20A were chosen to serve as the novel potential downstream target genes driven by FOXM1 in serous ovarian carcinoma after validation through qRT-PCR and western blot.The expression of CCNF was positively associated with the expression of FOXM1 in TCGA(r=0.68,p<0.01)and CCLE(r=0.4096,p<0.01).qRT-PCR was used to determine the coexpression between FOXM1 and CCNF in serous ovarian carcinoma tissues and the results showed that FOXM1 expression was positively correlated to CCNF(r=0.5235,P<0.01).The potential binding sites at the promoter of CCNF that FOXM1 could bind to were predicted by using gene-regulation website.The results of dual-luciferase report assay demonstrated that FOXM1 could trans-active the promoter activity of CCNF through TFBS2(Transcription factor binding site 2).Additionally,a ChIP-PCR was performed to further determine FOXM1 could bind to the promoter of CCNF through TFBS2.CCNF was found to be overexpressed in serous ovarian carcinoma in TCGA cohort.qRT-PCR was used to validate the mRNA expression of CCNF in serous ovarian carcinoma tissues and the results showed that it was obviously increased than that in normal normal fallopian tube fimbria.CCNF protein in serous ovarian carcinoma tissues was apparently upregulated than that in normal normal fallopian tube fimbria through western blot.High expression of CCNF was significantly correlated with overall survival by using Kaplan-Meier plotter websites.The positive immunoreactivity for CCNF was observed in the nucleus of serous ovarian carcinoma based on our immunohistochemistry staining.The proportion of high expression of CCNF was about 44.95%and patients with higher expression of CCNF had shorter overall survival than those with lower expression(p<0.05).Clonogenic assay followed by showed that upregulation of CCNF accelerated the clonogenic and proliferation capacity and repression of CCNF impaired the clonogenic and proliferation ability of ovarian cancer cells.The results of transwell assay showed that the ovarian cancer cells with CCNF knockdown exhibited weaker migration and invasion ability and the invasion ability of FOXM1 was abrogated through downregulation of CCNF.KIF20A was overexpressed in serous ovarian carcinoma and the expression of KIF20A was positively associated with the expression of FOXM1 in TCGA(r=0.66,P<0.01)and CCLE data(r=0.3855,P<0.01).The proportion of high expression of KIF20A was about 58.33%(7/12)and the positive immunoreactivity for KIF20A was focused in the nucleus through the human protein atlas website.The patients with higher expression of KIF20A had shorter overall survival and proeression-free survival than those with lower expression(p<0.01).The proliferation and invasion ability of ovarian cancer cell lines SKOV3 and OVCAR3 was decreased after KIF20A knockdown through CCK8 proliferation assay and transwell assay.ConclusionFOXM1 could promote malignant behavior by regulating CCNF and KIF20A in serous ovarian carcinoma.CCNF and KIF20A are overexpressed in serous ovarian carcinoma and both of them could indicate poor prognosis.Part Ⅲ:Study on the upstream regulation mechanisms of FOXM1 in serous ovarian carcinomaObjectAlternative splicing is a crucial regulation mechanism of proteomic diversity.Recent evidence indicates that almost 95%of multi-exon genes involves alternative splicing.Alternative splicing has also been found to be associated with various diseases including human cancers.Aberrant splicing can lead to loss-of-function in tumor suppressors or activation of oncogenes and cancer pathways.Spliceosome-associated factor CTNNBL1 was found to be overexpressed in serous ovarian carcinoma and associated with poor clinical prognosis in our previous work.In addition,our transcriptome analysis showed that CTNNBL1 regulated many alternative splicing events including FOXM1.This study intended to investigate the expression and biological functions of CTNNBL1 in vitro assay and determine the clinical application value through immunohistological staining.The mechanisms that CTNNBL1 regulated alternative splicing of FOXM1 was further studied.The tumor suppressor TP53 is mutant in about 50%of human cancers.The proportion of mutant TP53 is about 96%and FOXM1 is activated in nearly 87%samples in TCGA cohort.It is reported that p53 could bind to the promoter and suppress the mRNA and protein expression of FOXM1.It is unknown whether high FOXM1 expression is related to TP53 mutation.Inhibition effect of p53 on the expression of FOXM1 is decreased after wild type p53 inactivated.It remains unclear whether mutant p53 promotes FOXM1 expression through suppressing the activity of wild type p53 and mutant p53 with gain of function(GOF)could enhance the expression of FOXM1.This study intended to further explain the reasons for the high expression of FOXM1 in serous ovarian carcinoma from two aspects including alternative splicing and TP53 regulation.On one hand,we studied the expression,biological functions and clinical prognosis of CTNNBL1 in serous ovarian carcinoma and we further clarified the regulatory mechanisms of CTNNBL1 on alternative splicing of FOXM1.On the other hand,the relationship between TP53 mutation and FOXM1 expression was studied by bioinformatics analysis and in vitro experiments.Contents and methodsWestern blot was used to detect the differential expression of CTNNBL1 in normal fallopian tube fimbria and serous ovarian carcinoma tissues or cell lines.Bioinformatics analysis was used to determine the expression,copy number variation and the impact on clinical prognosis of CTNNBL1 by using Oncomine and TCGA.Immunohistological staining based on our tissue microarray was used to detect the protein expression of CTNNBL1 and evaluate its clinical prognosis value in serous ovarian carcinoma.The impact of CTNNBL1 expression and gene alteration on clinical prognosis was analyzed in TCGA cohort.The CTNNBL1 overexpression and knockdown cell lines were established according to the differential expression of CTNNBL1 in ovarian cancer cell lines.The functional roles of CTNNBL1 in ovarian cancer cell lines were determined through clonogenic,proliferation and transwell assay.Affymetrix Human HTA 2.0 arrays were used to identify alternative splicing events regulated by CTNNBL1 in serous ovarian carcinoma.The effect of CTNNBL1 on FOXM1 alternative splicing was analyzed through in vitro splicing experiments.Bioinformatics analysis was employed to determine the correlation between TP53 and FOXM1 from Oncomine and TCGA.Immunohistological staining was used to evaluate the correlation between p53 and FOXM1 expression.The effect of mutant or wild type TP53 overexpression on the FOXM1 expression was determined by cell experiments in vitro.ResultsThe protein expression of CTNNBL1 was found to be significantly increased in serous ovarian carcinoma compared with normal fallopian tube fimbria through western blot.The protein expression of CTNNBL1 in ovarian cancer cell lines was also found to be much higher than that in normal cell line FTE187.The expression of CTNNBL1 in serous ovarian carcinoma was significantly higher than that in normal ovarian tissue in TCGA cohort.Copy number variation of CTNNBL1 in serous ovarian carcinoma samples was found to be dramatically increased than normal ovary samples and the copy number variation of CTNNBL1 was highly associated with its aberrant expression.The immunohistochemistry staining results of CTNNBL1 was found that the distribution of CTNNBL1 mainly located in the cell nucleus and the strong staining of CTNNBL1 accounted for 56.25%(72/128)cases and the weak staining was detected in 56 tumors(43.75%).CTNNBL1 expression was correlated with age and omentum metastasis,but not with other clinicopathological features.Kaplan-Meier analysis showed that high expression of CTNNBL1 was associated with reduced survival time than the low expression group(p<0.05).CTNNBL1 genomic alterations occurred in 21%of sequenced cases(65/316)including mRNA upregulation in 16.1%(51/316)and GCN increases in 1%(3/316)and we found that the genetic alterations of CTNNBL1 led to shortening the overall survival of serous ovarian carcinoma patients in TCGA cohort.Survival analysis of TCGA patients indicated that high CTNNBL1 expression had a worse overall survival compared to the low expression subgroup.Overexpression of CTNNBL1 dramatically increased the colony-forming efficiency and proliferation speed,while knockdown of CTNNBL1 reduced the clonogenicity and growth speed in ovarian cancer cells.Cell cycle analysis demonstrated CTNNBL1 knockdown led to decrease in the percentage of cells at S phase and increase in the percentage of cells at G1 phase.Accordingly,CCND1 and CCNE were decreased after CTNNBL1 knockdown.The wound-healing assay showed that the migrating ability was dramatically attenuated by CTNNBL1.Transwell assay showed that CTNNBL1 significantly enhanced the invasion potential in A2780 cells and knockdown of CTNNBL1 suppressed invasion ability in HO8910 and HEY cells.In addition,we found that CTNNBL1 knockdown upregulated epithelial marker(E-cadherin)in OVCAR3 cells and reduced the levels of mesenchymal markers(Snail,Slug,MMP2 and β-catenin)in A2780,HO8910 and OVCAR3 cells though western blot.Our transcriptome analysis showed that more than half of CTNNBL1 regulated alternative splicing events correspond to cassette exons(which results in an open reading frame shift)including FOXM1.The expression of CTNNBL1 was positively correlated with FOXM1 in serous ovarian carcinoma in TCGA cohort.FOXM1 protein was upregulated in CTNNBL1 overexpressed cells while down-regulated in CTNNBL1 knockdown cells through western blot assay.We found that knockdown of CTNNBL1 suppressed the expression of FOXM1B and FOXM1C and upregulated the expression of FOXM1A in ovarian cancer cells.The splicing assay in vitro showed that FOXM1A was increased and the expressions of FOXM1B and FOXM1C were reduced dramatically after cotransfection minigene pcDNA3.1-Ⅶa and CTNNBLI siRNA into 293T cells.Hendrix ovarian statistics showed that the expression of FOXM1 was increased in mutant TP53 samples comparing with wild type TP53 samples and the expression of FOXM1 in p53 positive staining samples was higher than that in p53 negative samples through immunohistochemistry staining analysis.The mRNA and protein expressions of FOXM1 were found to be higher in mutant TP53 samples of serous ovarian carcinoma than that in wild type TP53 samples by using TCGA data.We also found that the expression of FOXM1 was increased in mutant TP53 samples of serous ovarian carcinoma with GOF(R175H,R273H,R248Q,R273C,R282W,Y220C,etc).There was no obvious expression difference of FOXM1 between mutant TP53 samples of serous ovarian carcinoma with GOF and without GOF,but they were both higher than the wild type TP53 samples.The expression of FOXM1 was shown to be positively correlated to the expression of mutant p53 through our immunohistochemistry staining analysis(p<0.01).The expression of FOXM1 was increased after TP53 knockdown in A2780 by using siRNA,While the expression of FOXM1 was decreased after p53 activation by using nutlin3a.The mRNA expression of FOXM1 was not changed after overexpression of mutant TP53 with GOF except R248Q in SKOV3 and the mRNA expression of FOXM1 was not decreased after overexpression of wild TP53 in SKOV3.However,we found that the protein expression of FOXM1 was enhanced after TP53 mutation(R248Q or R282W)overexpression in SKOV3 cells.ConclusionCTNNBL1 regulates the expression of FOXM1 by alternative splicing.The high expression of FOXM1 is associated with TP53 mutation in serous ovarian carcinoma.