BACKGROUND: Recent work has suggested a role for nuclear factor jB (NF-jB) in the propagation of ovarian cancer cell lines, but the significance and mechanism of NF-jB in ovarian cancer is unknown. The authors hypothesized that the NF-jB pathway is over activated in aggressive ovarian cancers. METHODS: The levels of 3 NF-jB transcription factors, the activating inhibitors of NF-jB (IjB) kinases, and the NF-jB target matrix metalloproteinase 9 (MMP9) were assessed by immunohistochemistry in specimens of ovarian cancer that were obtained at diagnosis from a cohort of 33 patients who subsequently received combined paclitaxel, cisplatin, and cyclophosphamide. Associations were made between NF-jB pathway proteins and outcome. The validation of coexpression was performed at the gene level in 2 independently collected cohorts of 185 and 153 ovarian cancers. RESULTS: The presence of NF-jB proteins in newly diagnosed advanced ovarian cancers was established, and a potential association with overall survival was identified. Transcription factors p65 and v-rel reticuloendotheliosis viral oncogene homolog B (RelB) were coexpressed with IjB kinase a, 1 component of a key trimolecular regulatory complex. Coexpression of the NF-jB machinery suggested activity of NF-jB signaling in these ovarian tumors. A significant association of p50 with poor overall survival was observed (P ¼ .02). MMP9 expression had the opposite association, in which patients who had tumors without MMP9 staining had the poorest prognosis (P ¼ .01), and this association held true at the gene expression level in an independently collected cohort of 185 ovarian cancers. CONCLUSIONS: The deregulation of NF-jB activity may influence outcome in women who receive standard therapy for advanced ovarian cancer. Modification of the NF-jB pathway may present an opportunity to improve outcome in the subset of women who have pathway activity. Cancer 2010;116:3276-84.

MicroRNAs (miRNAs) are small non-coding RNAs that exert a regulatory effect post-transcriptionally by binding target mRNAs and inhibiting gene translation. miRNA expression is deregulated in cancer. The aim of this study was to characterize the differences in miRNA expression pattern and the miRNA-regulating machinery between ovarian carcinoma (OC) cells in primary tumours versus effusions. Using miRNA array platforms, we analysed a set of 21 tumours (13 effusions, 8 primary carcinomas) and identified three sets of miRNAs, one that is highly expressed in both primary carcinomas and effusions, one overexpressed in primary carcinomas and one overexpressed in effusions. Levels of selected miRNAs were analysed using quantitative PCR in an independent set of 45 additional tumours (30 effusions, 15 primary carcinomas). Reduced miR-145 and miR-214 and elevated let-7f, miR-182, miR-210, miR-200c, miR-222 and miR-23a levels were found in effusions in both sets. In silico target prediction programs identified potential target genes for some of the differentially expressed miRNAs. Expression of zinc finger E-box binding homeobox (ZEB)1 and c-Myc, targets of miR-200c, as well as of p21 protein (Cdc42/Rac)-activated kinase (PAK)1 and phosphatase and tensin homologue deleted on chromosome 10 (PTEN), predicted targets of miR-222, were analysed. Inverse correlations between expression levels of the indicated miRNAs and of the predicted target genes were found. In addition, higher expression of the miRNA-processing molecules Ago1, Ago2 and Dicer was observed in effusions compared to primary carcinomas. In conclusion, our data are the first to document different miRNA expression and regulation profiles in primary and metastatic OC, suggesting a role for these molecules in tumour progression.

TNXB was previously identified as a gene that is more highly expressed in malignant mesothelioma compared to ovarian/peritoneal serous carcinoma based on gene expression array analysis. The objective of the present study was to validate this finding at the mRNA and protein level. Effusions (n=91; 71 ovarian carcinomas, 10 breast carcinomas, 10 malignant mesotheliomas) were assayed for TNXB mRNA expression using quantitative PCR. Tenascin-X protein expression was studied in 183 effusions (137 carcinomas of different origin, 37 mesotheliomas, 9 reactive effusions) and 178 solid lesions (122 ovarian/ peritoneal carcinomas, 56 mesotheliomas) using immunohistochemistry. Quantitative PCR analysis showed significantly higher TNXB mRNA level in mesotheliomas compared to ovarian and breast carcinomas (p<0.001). By immunohistochemistry, tenascin-X protein expression was significantly higher in malignant mesothelioma compared to metastatic carcinoma in effusions (34/37 vs. 31/137 positive cases; sensitivity = 92%, specificity = 77%; p<0.001). Reactive mesothelial cells had focal or no tenascin-X expression. Tenascin-X protein was detected 41/56 mesothelioma biopsy specimens, and was uniformly absent from all 122 ovarian carcinomas (sensitivity = 73%, specificity = 100%; p<0.001). Our data suggest that tenascin-X may be a new diagnostic marker of malignant mesothelioma in the differential diagnosis of cancers involving the serosal cavities, particularly in the differential diagnosis between this tumor and ovarian/peritoneal serous carcinoma.

The present study investigated the intracellular regulation of E-cadherin in ovarian carcinoma. E-cadherin expression and regulation by Snail and Pak1 were studied in ES-2 and OVCAR-3 ovarian cancer cells in vitro. Twist1, Zeb1 and Vimentin mRNA expression and HIF-1alpha protein expression were analyzed in 80 and 189 clinical specimens, respectively. OVCAR-3 cells incubated with an anti-E-cadherin antibody formed smaller and looser spheroids compared to controls. Snail silencing using Small Hairpin RNA in ES-2 cells reduced invasion and MMP-2 activity, with unaltered cellular morphology. Using dominant negative (DN) and constitutively active (CA) Pak1 constructs, we found that DN Pak1 ES-2 and OVCAR-3 clones had reduced attachment to matrix proteins, invasion and MMP-2 activity compared to CA and wild-type cells. DN Pak1 ES-2 cells also bound less to LP9 mesothelial cells. DN Pak1 OVCAR-3 cells had lower Vimentin levels. Snail expression was lower in cultured effusions compared to primary carcinomas, and was cytoplasmic rather than nuclear. Twist1 (P < 0.001), Zeb1 (P = 0.003) and Vimentin (P = 0.03) mRNA expression was significantly higher in solid metastases compared to primary carcinomas and effusions. HIF-1alpha protein expression was lower in effusions compared to primary carcinomas and solid metastases (P = 0.033). Our data suggest that the previously reported E-cadherin re-expression in ovarian carcinoma effusions is regulated by Pak1. The transient nature of E-cadherin expression during ovarian carcinoma progression is probably the result of partial epithelial-to-mesenchymal transition (EMT) and the reverse process of mesenchymal-to-epithelial-like transition (MET). Expression of the EMT-related molecules Twist, Zeb1, Vimentin and HIF-1alpha is anatomic site-dependent in ovarian carcinoma.

No abstract