| Ovarian cancer is the leading cause of death from a gynecological malignancyworldwide, with increasing incidence recently in China. Epithelial ovarian cancer is themost common ovarian cancer and accounts for approximately70%of all ovarianmalignant diseases. It is the sixth most common cancer and the fifth leading cause ofcancer-related death in women worldwide. This lethal gynecological malignancy iscommonly diagnosed at a late stage due to the silent early stage and easily metastasis.Despite significant advances in surgery and chemotherapy over the last few decades,survival to incidence ratio is still poor and overall cure rate remains very low. Littleprogress has been made so far to improve long-term survival, owing to its late diagnosis atadvanced stages and its resistance to conventional chemotherapy. Therefore, there is an urgent requirement for new biomarkers for epithelial ovarian cancer, so as to developbetter early diagnostic and therapeutic strategies.It is now clear that the vast majority of pre-mRNAs contain exons that can bealternatively included into the mature mRNA or removed from it, which is calledalternative splicing. It is a widespread means of increasing protein diversity and regulatinggene expression in eukaryotes. Recent survey of various mammalian tissues,developmental stages and cell-lines suggests that at least90%of the protein-coding genesuse alternative transcriptional and alternative splicing events. Alternative splicing may beone of the most extensively used mechanisms that accounts for the greatermacromolecular and cellular complexity of higher eukaryotic organisms. In metazoans,alternative splicing plays an important part in generating different protein products thatfunction in diverse cellular processes, including cell growth, differentiation and death. Aswell as being associated with cancer, the nature of the alternative gene products is usuallyconsistent with an active role in cancer. Therefore, the alternative splicing process itself isa potential target for gene therapy.FoxM1belongs to a large family of evolutionary conserved transcriptional regulatorsthat were characterized by the presence of a DNA-binding domain called the forkhead boxor winged helix domain. It is a key cell-cycle regulator of both the transition from G1to Sphase and the progression to mitosis. It has been shown to play important roles inregulating the expression of genes involved in cell proliferation, differentiation, andtransformation. Increased expression of FoxM1has been found in several human tumorssuggesting a role in human carcinogenesis. Moreover, it has been shown that higherexpression of FoxM1was associated with poor prognosis of cancer patients and couldserve as an independent predictor of poor survival in cancer. Three isoforms of FoxM1,FoxM1a, FoxM1b, and FoxM1c, have been reported previously, but only FoxM1b andFoxM1c exhibit transactivation activity in reporter assays. This can be explained by thefact that the C-terminal region is required for transcriptional activation, and thatinsertion of exon A2in the C-terminus of the protein suppresses transcriptional activity.Therefore, it would be interesting to address in more detail the expression patterns of FoxM1and its isforms.In order to identify alternative splicing events in the FoxM1gene, we performed aseries of experiments in this study. We firstly examined the FoxM1protein expression innormal human tissues by immunohistochemical staining. FoxM1is not expressed or at alow level in most normal human tissues except in normal human kidney tissues. FoxM1protein expression in kidney normal tissues was usually increased compared with that inkidney tumor tissues. We used western blot analysis to characterize protein expression ofFoxM1in six epithelial ovarian cancer cell lines. Our results showed that expression ofFoxM1protein in HO-8910cell line and A2780cell line was lower than that in theirsubline cell line HO-8910PM (a highly metastatic cell line derived from HO-8910) andA2780/CP70(a cisplatin resistant cell line derived from A2780), respectively. FoxM1a,FoxM1b and FoxM1c were detectable in all these six cell lines by using RT–PCR. Wefurther used real-time PCR analysis to characterize mRNA expression of FoxM1a,FoxM1b and FoxM1c in epithelial ovarian cancer cell lines. Our results showed thatexpression of FoxM1b mRNA in HO-8910cell line and A2780cell line was lower thanthat in their subline cell line HO-8910PM and A2780/CP70, respectively. However,expression of FoxM1a mRNA in HO-8910cell line was higher than that in its subline cellline HO-8910PM. FoxM1protein expression was positively correlated with lymph nodemetastasis (P=0.012) by immunohistochemical staining in epithelial ovarian cancer. Weexamined FoxM1a, FoxM1b and FoxM1c mRNA expressions in ovarian normal andcancer tissues by real-time quantitative PCR. Expression of FoxM1b mRNA in ovariannormal tissues was lower than that in cancer tissues (P=0.002), whereas expression ofFoxM1a mRNA in ovarian normal tissues was higher than that in cancer tissues (P <0.001). Kaplan Meier analysis and Cox proportional hazards model were applied to assessthe relationship between FoxM1b and progression-free survival (PFS) and overall survival(OS). The results showed that high FoxM1b mRNA expression was associated with lymphnode metastasis (P=0.007) and poor prognosis of epithelial ovarian cancer. Multivariateanalyses indicated that elevated mRNA levels of FoxM1b was an independent prognosticfactor for PFS (P=0.002) and OS (P=0.003) in epithelial ovarian cancer, respectively. To evaluate possible contributions of FoxM1b to cell migration and invasion, theoverexpression vectors pcDNA3.1-FoxM1b and FoxM1shRNA were transfected intoHO-8910and HO-8910PM cells respectively. Our results showed that overexpression ofFoxM1b increased MMP-2, MMP-9and VEGF-A expression and enhanced migration andinvasion of ovarian cancer cells. We also found that down-regulation of FoxM1reducedthe expression of MMP-2, MMP-9and VEGF-A, resulting in the inhibition of migrationand invasion of ovarian cancer cells. To address the role of FoxM1b in cisplatin resistance,the overexpression vectors pcDNA3.1-FoxM1b and FoxM1shRNA were transfected intoA2780and A2780/CP70cells respectively. The results showed that FoxM1b was a criticalmediator of cisplatin sensitivity and resistance in ovarian cancer cells.As far as we know, this is the first report that has examined the expression patterns ofFoxM1and its isoforms in ovarian cancer tissues and cell lines. We also investigated thecorrelations between FoxM1b mRNA expression and various clinic pathologic parameters,and its prognostic value for survival of patients with epithelial ovarian cancer.Furthermore, we determined the functional roles of FoxM1b in ovarian cancer cell lines.These data suggest that FoxM1b may serve as a promising therapeutic target for epithelialovarian cancer and that down-regulating FoxM1b could potentially be an effectivetherapeutic approach for inhibition of ovarian cancer progression. |
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