Carcinogenesis Of Ovarian Cancer Research Articles

Abstract 4891: GULP1, a potential tumor suppressor gene in ovarian tumors and its utility as a biomarker

Abstract Identification of tumor suppressor genes (TSGs) silenced by CpG methylation uncovers the molecular mechanism of tumorigenesis and potential tumor biomarkers. GULP1, a molecule not previously related to cancer, is a cytoplasmic adaptor protein with a phosphotyrosine binding domain that plays a role in one of two partially redundant pathways that lead to the engulfment and clearance of apoptotic cells, according to several genetic studies performed in Caenorhabditis elegans. Performing a pharmacologic unmasking technique we observed that this molecule is downregulated in ovarian tumor tissues when compared to normal ovary. Ovarian cancer is the leading cause of death among gynaecological cancers worldwide, this due to the fact that women are diagnosed with advanced stage disease and because of the lack of truly sensitive/specific screening techniques and unavailability of individualized therapy. We performed an expression microarray on 15 ovarian tumor samples, 10 ovarian normal surface epithelium, 3 ovarian cancer cell lines and 3 normal cell lines. All cells have been treated with a demethylating agent which comprehensively uncovers genes silenced by promoter hypermethylation. The selection of the best candidates was based upon the differential expression between normal and tumor samples (or cell lines), them being downregulated in cancer when compared to normal, and finally the re-activation after the treatment with the demethylating agent. One of these promising genes was GULP1, which expression pattern was then assessed by Reverse Transcriptase PCR and Western Blot and compared to its methylation status in the same six ovarian cell lines and showed correlation between absence of expression and presence of methylation (or the opposite situation). We developed quantitative fluorogenic methylation specific PCR (QMSP) for GULP1 and profiled 437 ovarian tumor samples, 17 borderline tumors, 19 cystadenoma samples and 13 normal ovarian samples, finding 34.7% (151/437), 11.7% (2/17), 10.5% (2/19) and 0% (0/13) of methylation frequency, respectively, (establishing an empiric cutoff). Using fisher's exact test, we observe a significant increase in methylation when comparing tumors with cystadenomas and normals (p=0.0439 and p=0.0131, respectively). Late stage tumors also showed a higher frequency of methylation versus early stage (p=0.004). So far, we have demonstrated reduced log growth rates after transiently overexpressing GULP1 in one ovarian cell line (IGROVCP). To assess its potential as a TSG, we have ongoing experiments to assess its biological function as ectopic overexpression of this gene in ovarian cancer cell lines as well as creating knock downs using shRNAs. These preliminary data indicate that GULP1 may be an ovarian cancer biomarker. Further studies on the biology of this gene are needed to evaluate its potential as a TSG and its role in ovarian cancer carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4891.

Frequent Inactivation of a Putative Tumor Suppressor, Angiopoietin-Like Protein 2, in Ovarian Cancer

Angiopoietin-like protein 2 (ANGPTL2) is a secreted protein belonging to the angiopoietin family, the members of which are implicated in various biological processes, although its receptor remains unknown. We identified a homozygous loss of ANGPTL2 (9q33.3) in the course of screening a panel of ovarian cancer (OC) cell lines for genomic copy-number aberrations using in-house array-based comparative genomic hybridization. ANGPTL2 mRNA expression was observed in normal ovarian tissue and immortalized normal ovarian epithelial cells, but was reduced in some OC lines without its homozygous deletion (18 of 23 lines) and restored after treatment with 5-aza 2'-deoxycytidine. The methylation status of sequences around the ANGPTL2 CpG-island with clear promoter activity inversely correlated with expression. ANGPTL2 methylation was frequently observed in primary OC tissues as well. In an immunohistochemical analysis of primary OCs, ANGPTL2 expression was frequently reduced (51 of 100 cases), and inversely correlated with methylation status. Patients with OC showing reduced ANGPTL2 immunoreactivity had significantly worse survival in the earlier stages (stages I and II), but better survival in advanced stages (stages III and IV). The restoration of ANGPTL2 expression or treatment with conditioned medium containing ANGPTL2 inhibited the growth of OC cells originally lacking the expression of this gene, whereas the knockdown of endogenous ANGPTL2 accelerated the growth of OC cells with the expression of ANGPTL2. These results suggest that, at least partly, epigenetic silencing by hypermethylation of the ANGPTL2 promoter leads to a loss of ANGPTL2 function, which may be a factor in the carcinogenesis of OC in a stage-dependent manner.

Methylation of RUNX3 gene in epithelial ovarian carcinoma

Objective To access the role of methylated RUNX3 ggene in the carcinogenesis an progression of ovarian carcinoma.Methods Sample of 32 epithelial ovarian carcinoma tissues,32 para-carcinoma tissues,36 benign epithelial tumors and 10 normal ovarian tissues and 2 cell lines(3A0 and SKOV3),were collected and subject to methylation-specific PCR(MSP).Promoter methylation status of RUNX3 in two tumor cell lines were analyzed before and after 5-aza-2'deoxycytidine treatment.In addition. mRNA expression of RUNX3 were investigated by quantitative reverse transcription-PCR Results CpG island methylation of RUNX3 was observed in 53.1%(17 of 32)of epithelial ovarian cancer,and 37.5% (12 of 32)in corresponding noncancerous tissues,16.7% in benign epithelial tumors(6 of 36),and all celllines,but not in normal control tissues.The prevalence of RUNX3 gene CpG methylation in malignantwas significantly higher than those in benign and normal tissues(X2=10.060,X2=8.925,P<0.05). Nineteen percent(6 of 32)of ovarian epithelial carcinoma expressed RUNX3 mRNA,while its expression Was present in 28% (9 of 32)corresponding noncancerous tissues and 72% (26 of 36)of benign ovarian tumor and 80% (8 of 10)of normal ovarian tissues.The RUNX3 promoter methylation was found in all cell lines tested.The ratio of expression of RUNX3 mRNA in ovarian epithelial carcinoma was significantly lower than those of normal and benign tumors(X2=19.443,X2=12.862,P<0.05).After 5-aza-2'deoxycytidine treatment,methylation was partially or completely reversed,and its mRNA expression was increased.The relationship between gene expression and promoter methylation was reversely correlated.Conclusions Our results suggest that promoter hypermethylation of RUNX3 genes is common in ovarian cancer. Therefore, hypermethylation of RUNX3 genes may be involved in the carcinogenesis of ovarian cancer and may serve as an early diagnostic marker for ovarian cancer. The close correlation between RUNX3 methylation of its mRNA suggests that methylation which can be reversed.Thus,it provides a new way for therapy of ovarian Cancer. Key words: Ovarian neoplasms; DNA binding proteins; Transcription factors; Promoter regions(genetic)DNA methelation

Detection of human papillomavirus DNA and genotyping in patients with epithelial ovarian carcinoma

The purpose of this study was to investigate the possible role of human papillomavirus (HPV) in Turkish patients with epithelial ovarian cancer by using the highly sensitive technique of polymerase chain reaction (PCR) to identify all the subtypes of this unique oncogenic virus. All patients were subjected to initial surgery, and subsequently recruited for postoperative chemotherapy depending on the extent of the disease and their condition. HPV PCR screening was done from paraffin embedded samples. PCR amplifications were done using the MY09/11 primer set after digestion and phenol-chloroform extraction of the DNA. HPV PCR-positive samples were analyzed and genotyped using an OpenGene automated DNA sequencing system. Overall, 94 patients were included in this study. The mean age was 52.7 years (range, 21-76 years). As a histopathologic diagnosis, the majority of the patients had serous papillary tumors (81%). HPV was found to be positive in eight patients (8.5%). All of the positive patients had serous papillary tumors (8/76, 10.5%) and advanced stage disease. Six patients had HPV type 16, and the remaining two patients had HPV type 33. None of the patients had more than one type of HPV. HPV may have a role in the carcinogenesis of ovarian cancer. It is worth investigating this possible relation both in large case-control studies and in vitro models by using more sensitive techniques.

Promoter Hypermethylation Contributes to Frequent Inactivation of a Putative Conditional Tumor Suppressor Gene Connective Tissue Growth Factor in Ovarian Cancer

Connective tissue growth factor (CTGF) is a secreted protein belonging to the CCN family, members of which are implicated in various biological processes. We identified a homozygous loss of CTGF (6q23.2) in the course of screening a panel of ovarian cancer cell lines for genomic copy number aberrations using in-house array-based comparative genomic hybridization. CTGF mRNA expression was observed in normal ovarian tissue and immortalized ovarian epithelial cells but was reduced in many ovarian cancer cell lines without its homozygous deletion (12 of 23 lines) and restored after treatment with 5-aza 2'-deoxycytidine. The methylation status around the CTGF CpG island correlated inversely with the expression, and a putative target region for methylation showed promoter activity. CTGF methylation was frequently observed in primary ovarian cancer tissues (39 of 66, 59%) and inversely correlated with CTGF mRNA expression. In an immunohistochemical analysis of primary ovarian cancers, CTGF protein expression was frequently reduced (84 of 103 cases, 82%). Ovarian cancer tended to lack CTGF expression more frequently in the earlier stages (stages I and II) than the advanced stages (stages III and IV). CTGF protein was also differentially expressed among histologic subtypes. Exogenous restoration of CTGF expression or treatment with recombinant CTGF inhibited the growth of ovarian cancer cells lacking its expression, whereas knockdown of endogenous CTGF accelerated growth of ovarian cancer cells with expression of this gene. These results suggest that epigenetic silencing by hypermethylation of the CTGF promoter leads to a loss of CTGF function, which may be a factor in the carcinogenesis of ovarian cancer in a stage-dependent and/or histologic subtype-dependent manner.

Expression of progesterone receptor is a favorable prognostic marker in ovarian cancer

Objective Receptors for estrogen (ER), progesterone (PR), or androgen (AR) are predictive and prognostic markers of malignancy of multiple endocrine organs, including endometrial and breast cancer. However, the role of ERs, PRs, or ARs in the carcinogenesis of ovarian cancer, another sex hormone-dependent malignancy, is still controversial despite numerous studies that have attempted to determine their role. The disagreement in the findings may result from the fact that the numbers of tumor samples in studies have been small and that different immunohistochemical methods have been used that can introduce variation in the scoring of the histology. We therefore examined the pattern of expression of ERs, PRs, and ARs in a large number of samples of primary ovarian carcinoma by using a tissue microarray technique. Methods We constructed a tissue microarray with 322 samples of primary ovarian carcinoma obtained at surgery performed at The University of Texas M. D. Anderson Cancer Center between 1990 and 2000. Immunohistochemistry studies were performed by using the immunoperoxidase technique against primary antibodies (ER, PR, and AR). Results ERs, PRs, and ARs were differently expressed in different histotypes of ovarian cancer: ERs were expressed in 77.3% of all cases but more highly expressed in serous and endometrioid types; PRs were expressed in 26.2% of all cases but most highly expressed in the endometrioid type < 64.2%; and ARs were expressed in 43.7% of all cases but were most highly expressed in serous (47.5%) carcinomas. Of particular importance, the expression of PRs, but not ERs or ARs, was associated with better survival ( P < 0.0001) in univariate and multivariate analyses. Conclusions The PR is an independent marker, with its overexpression associated with a favorable prognosis in women with ovarian cancer.

A Novel Amplification at 17q21-23 in Ovarian Cancer Cell Lines Detected by Comparative Genomic Hybridization

Objective.Little is known about the molecular mechanisms involved in the pathogenesis and/or progression of ovarian cancer (OC). To investigate the genomic imbalances and identify the cancer-related genes associated with this tumor, we applied comparative genomic hybridization (CGH) in OC cell lines.Methods. Chromosomal aberrations among 17 OC cell lines were analyzed with CGH. Since novel chromosomal regions, including 17q21-23, were identified, we examined the involvement of two candidate genes, PS6K and ZNF147, mapped on this chromosomal region. We examined the status of amplification and expression by fluorescence in situ hybridization as well as by Southern blot analysis and by Northern blot analysis on two candidate genes, respectively.Results. All lines displayed numerous chromosomal imbalances; the most frequent losses were observed on 18q22-23 (29.4%), 13q22-34 (23.5%), 9p (17.6%), 4p11-14 (17.6%), and 11p14-15 (17.6%). The most common gains were noted at 20q12-13 (47.1%), 8q23-24 (35.2%), 5p15 (23.5%), 7q32-36 (23.5%), and 20p (23.5%). High-level gains (HLGs) were detected at 20q12-13 (four cell lines), 8q24 (two cell lines), 12p11-12 (two cell lines), and 17q21-23 (two cell lines). PS6K and ZNF147 genes were amplified in two cell lines exhibiting HLGs at 17q21-23, but not overexpressed.Conclusions. Our CGH data indicate that OCs have various DNA copy number changes. Among these frequent changes, 17q21-23 may harbor another tumor-associated gene(s) responsible for OC carcinogenesis.