| Ovarian cancer is the eighth most common malignant cancers affecting women worldwide. It is estimated to be diagnosed in more than225,000women per year worldwide. With a mortality rate of more than140,000deaths per year, it is the most lethal gynaecological malignancy. Though the prognosis of ovarian cancer is quite good when women diagnosed at early stage (stage Ⅰ and Ⅱ), the majority of women present at advanced stages (stage Ⅲ and Ⅳ) and the5-year survival rate is about40%. The mechanism on how ovarian cancers happen is not very clear. There are no sensitive and specific screening methods available for the early detection of ovarian cancer patients. The first response rate of ovarian cancer patients who received surgery combining chemotheorapy could reach80%. However, most of them will relapse in the near future. Though there are many target therapies for ovarian cancer treatment, there still not find any methods that could act better than the platinum based chemotherapy.During the past years, great changes have been made about the typing of ovarian cancer and its origin. More and more evidence suggested that the ovarian cancer is a heterogeneous disease and the epithelial ovarian cancer can be divided into two types. Type Ⅰ tumors are classified as low-grade serous carcinomas, endometrioid carcinomas, clear cell carcinomas, mucinous carcinomas and Brenner tumor. They usually presented at early stages and took a stepwise progression, from benign tumor to boardline tumor and finally to malignant tumor. The genome of this kind of tumor is stable. Frequent gene mutations are KRAS、BRAF、ERBB2、CTNNB1、 PTEN、PIK3CA、ARID1A and PPP2R1A. Type Ⅱ tumors are mainly high-grade serous carcinomas. It usually progresses quickly and is of high aggressiveness. P53gene mutation and chromatin instability happen a lot in this type of tumor. Recent studies have shown that both low grade and high grade serous ovarian carcinomas may arise from the fallopian tube rather than the ovary, which may shed light on the research of early detection and prevention of ovarian cancers.Previously the ovarian cancer had been treated as one entity. The identifications of distinct molecular pathways characterizing of individual subtypes have fuelled enthusiasm for the development of targeted therapiesthat direct at specific subtypes of ovarian cancers. Understanding more about how best to use our knowledge of the molecular abnormalities involved in ovarian cancer will be important in improving clinical outcomes in ovarian cancers. NAC1has been suggested as one of the most critical molecules related to ovarian cancer progression. But how it regulates its down stream genes is still largely unknown. Based on microarray transcriptome analysis, we finally focused on FOXQ1as one of the NAC1regulating genes. By revealing how the NAC1/FOXQ1pathway participating in the tumorigenesis and progression of epithelial ovarian cancers, we hope our study could have some contribution to the translational research of ovarian cancers.Part Ⅰ. The Regulation Mechanism of NAC1to FOXQ1in Epithelial Ovarian CarcinomasBackgrounds and Purpose:Recently, more and more reports have been published about the genes of Bric-a-Brac Tramtrack Broad (BTB) family involving in tumorigenesis and progression. The BTB domain is a very important domain that mediating protein protein interaction. Previously our group using SAGE method to analysis130BTB family genes and found that nucleus accumbens-associated protein1(NAC1) functions as a transcriptional co-regulator and is a new tumor assocated gene. Although NAC1lacks a DNA binding domain (unlike other BTB family proteins), the BTB domain of NAC1mediates protein homo-or hetero-dimerization to assemble a higher order transcription complex. NACC1gene is localized in chromatin19p13.2. Based on the analysis of The Cancer Genome Atlas (TCGA) ovarian cancer database, we identified NACC1, the gene encoding NAC1, as one of the top genes showing a significant positive correlation between DNA and RNA copy number in human cancers, indicating that NAC1plays a potential "driving" role in promoting cancer development.IHC analysis showed that NAC1did not express in normal ovarian epithelial cells, but it became to express more and more when the grade of ovarian cancer became higher and higher. In fact, increased expression of NAC1is associated with disease aggressiveness, development of resistance to chemotherapeutic agents, and tumor recurrence in several types of human cancers including ovarian, endometrial, and cervical carcinomas. NAC1performs multiple roles in various pathobiological processes including the maintenance of pluripotency in embryonic stem cells, participation in acute psychomotor stimulant responses in mice, tumor pathogenesis as well as the development of chemoresistance. Given the significant and diverse roles of NAC1in cancer biology, it is important to define NAC1-regulated transcriptomes and identify tumor-associated genes that are controlled by NAC1. Such information would have important biological and clinical implications. In this study, we compared the transcriptomes of NAC1-overexpressing SKOV3cells and the SKOV3cells with NACC1gene knockdown. As a result, we identified several new NAC1-regulated genes with known or potential involvement in ovarian cancers development and further investigated the role of one of these genes, FOXQ1, in the biological functions mediating by NAC1.Methods and Results:We compared the global gene expression profiles of NAC1-overexpressing SKOV3ovarian cancer cells and SKOV3cells with silenced NACC1. We found that NAC1knockdown was associated with the upregulation of apoptotic genes and downregulation of the genes involved in cellular growth, proliferation, movement, NOTCH signaling, as well as epithelial mesenchymal transition. Representative NAC1downstream candidate genes including FOXQ1, FOXA2, JAGGED1, NOTCH1, IGFBP6, DBNL, DVL1, SNF2H, and KBTBD8were validated by quantitative real-time polymerase chain reaction. Among these, FOXQ1, a gene involved in cellular motility and epithelial-mesenchymal transition, was further characterized. NACC1knockdown decreased expression and promoter activity of FOXQ1. Similarly, inactivation of NAC1by expression of the BTB domain of truncated NAC1, which inhibits NAC1dimerization, also suppressed FOXQ1expression. Ectopic expression of NAC1in NACC1null cells increased FOXQ1expression. NACC1knockdown resulted in decreased cellular motility and invasion, while constitutive expression of FOXQ1significantly increased cellular motility in NACC1knockdown cells compared with control cells without FOXQ1expression. Finally, there was significant co-occurrence of upregulation of NACC1and FOXQ1in high-grade serous carcinomas based on bioinformatics analysis.Conclusions and Significance:In conclusion, we have defined the NAC1-regulated transcriptome in ovarian cancer cells and identified several NAC1-controlled genes that participate in multiple cancer-related pathways. We further demonstrate that NAC1is essential and sufficient for FOXQ1transcription activation and that the role of NAC1in cellular motility is mediated at least in part by FOXQ1. Our results should have some translational significance for developing target therapies of NAC1overexpressing epithelial ovarian cancers. Part Ⅱ. The Role of Forkhead Box Q1Transcription Factor in Ovarian Epithelial CarcinomasBackgrounds and Purpose:While studying the transcriptome regulated by NACC1, a gene that encodes the NAC1protein and participates in the pathogenesis of ovarian cancer recurrence and chemoresistance, we found that FOXQ1was significantly upregulated by NAC1in ovarian high-grade serous carcinomas. Ectopic expression of FOXQ1reversed the NAC1knockdown-mediated decrease in cell invasion. FOXQ1belongs to the Forkhead box (FOX) transcription factor superfamily, which is characterized by a conserved stretch of110amino acids that is responsible for DNA binding. There are a total of17FOX subfamilies (FOXA-FOXR) containing at least43unique proteins. Deregulation of FOX proteins such as FOXO, FOXM, FOXP, FOXC, FOXA, and FOXQ has been implicated in tumorigenesis, given that they are involved in a wide spectrum of biological activities including metabolism, development, differentiation, proliferation, apoptosis, migration, and invasion.Specifically, the FOXQ1gene, which encodes a protein of403amino acids, was first identified in2001, and its biological importance was highlighted by its involvement in embryonic stem cell biology and vertebrate development by acting as a downstream mediator of HOXA1and HOXC13. Moreover, FOXQ1is also known as a critical transcription factor that regulates epithelial-mesenchymal transition (EMT) a developmental process that is often activated during cancer invasion and metastasis. Given the potential role of FOXQ1in cancer biology, the present study was undertaken to characterize its functional role in ovarian carcinomas.Methods and Results:We found that FOXQ1was significantly overexpressed in both type Ⅰ and type Ⅱ epithelial ovarian cancers. The ovarian cancer subtypes including clear cell carcinoma, endometrioid carcinoma, mucinous carcinoma, and high-grade serous carcinoma showed a higher level of FOXQ1(p<0.01,p<0.0001, p<0.01, and p<0.0001, respectively) compared with normal ovarian surface epithelium. When compared to normal fallopian tube epithelium, FOXQ1was overexpressed in endometrioid and high-grade serous types of ovarian carcinomas (p<0.001and p<0.0001, respectively). We compared the relative transcript levels of FOXQ1in a panel of nine ovarian cancer cell lines and revealed that the expression of FOXQ1was upregulated in all ovarian cancer cell lines when compared with OSE10cell. We then used a human ovarian cancer cell line, SKOV3, which expressed a higher level of FOXQ1, as a cell model to investigate the biological effects of FOXQ1by using RNA interference. Silencing of FOXQ1expression using a shRNA knockdown approach affected the expression of several cell cycle regulators, leading to suppressed cell proliferation, reduced cell motility/invasion, and upregulation of epithelial cell markers and the downregulation of mesenchymal cell markersConclusions and Significance: Taken together, the results suggest that FOXQ1expression is essential to maintain cell proliferation, motility/invasion, and epithelial-mesenchymal transition phenotypes in epithelial ovarian carcinoma cells. It may serve as an useful target molecule for the future ovarian cancer therapies. |
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