Normal Ovarian Surface Epithelial Cells Research Articles

VGLL3 expression is associated with a tumor suppressor phenotype in epithelial ovarian cancer

Previous studies have implicated vestigial like 3 (VGLL3), a chromosome 3p12.3 gene that encodes a putative transcription co-factor, as a candidate tumor suppressor gene (TSG) in high-grade serous ovarian carcinomas (HGSC), the most common type of epithelial ovarian cancer. A complementation analysis based on microcell-mediated chromosome transfer (MMCT) using a centric fragment of chromosome 3 (der3p12-q12.1) into the OV-90 ovarian cancer cell line haploinsufficient for 3p and lacking VGLL3 expression was performed to assess the effect on tumorigenic potential and growth characteristics. Genetic characterization of the derived MMCT hybrids revealed that only the hybrid that contained an intact VGLL3 locus exhibited alterations of tumorigenic potential in a nude mouse xenograft model and various in vitro growth characteristics. Only stable OV-90 transfectant clones expressing low levels of VGLL3 were derived. These clones exhibited an altered cytoplasmic morphology characterized by numerous single membrane bound multivesicular-bodies (MVB) that were not attributed to autophagy. Overexpression of VGLL3 in OV-90 was achieved using a lentivirus-based tetracycline inducible gene expression system, which also resulted in MVB formation in the infected cell population. Though there was no significant differences in various in vitro and in vivo growth characteristics in a comparison of VGLL3-expressing clones with empty vector transfectant controls, loss of VGLL3 expression was observed in tumors derived from mouse xenograft models. VGLL3 gene and protein expression was significantly reduced in HGSC samples (>98%, p < 0.05) relative to either normal ovarian surface epithelial cells or epithelial cells of the fallopian tube, possible tissues of origin of HGSC. Also, there appeared to be to be more cases with higher staining levels in stromal tissue component from HGSC cases that had a prolonged disease-free survival. The results taken together suggest that VGLL3 is involved in tumor suppressor pathways, a feature that is characterized by the absence of VGLL3 expression in HGSC samples.

Elevated levels of circulating microRNA-200 family members correlate with serous epithelial ovarian cancer

BackgroundThere is a critical need for improved diagnostic markers for high grade serous epithelial ovarian cancer (SEOC). MicroRNAs are stable in the circulation and may have utility as biomarkers of malignancy. We investigated whether levels of serum microRNA could discriminate women with high-grade SEOC from age matched healthy volunteers.MethodsTo identify microRNA of interest, microRNA expression profiling was performed on 4 SEOC cell lines and normal human ovarian surface epithelial cells. Total RNA was extracted from 500 μL aliquots of serum collected from patients with SEOC (n = 28) and age-matched healthy donors (n = 28). Serum microRNA levels were assessed by quantitative RT-PCR following preamplification.ResultsmicroRNA (miR)-182, miR-200a, miR-200b and miR-200c were highly overexpressed in the SEOC cell lines relative to normal human ovarian surface epithelial cells and were assessed in RNA extracted from serum as candidate biomarkers. miR-103, miR-92a and miR -638 had relatively invariant expression across all ovarian cell lines, and with small-nucleolar C/D box 48 (RNU48) were assessed in RNA extracted from serum as candidate endogenous normalizers. No correlation between serum levels and age were observed (age range 30-79 years) for any of these microRNA or RNU48. Individually, miR-200a, miR-200b and miR-200c normalized to serum volume and miR-103 were significantly higher in serum of the SEOC cohort (P < 0.05; 0.05; 0.0005 respectively) and in combination, miR-200b + miR-200c normalized to serum volume and miR-103 was the best predictive classifier of SEOC (ROC-AUC = 0.784). This predictive model (miR-200b + miR-200c) was further confirmed by leave one out cross validation (AUC = 0.784).ConclusionsWe identified serum microRNAs able to discriminate patients with high grade SEOC from age-matched healthy controls. The addition of these microRNAs to current testing regimes may improve diagnosis for women with SEOC.

Primary human ovarian epithelial cancer cells broadly express HER2 at immunologically-detectable levels.

The breadth of HER2 expression by primary human ovarian cancers remains controversial, which questions its suitability as a universal antigen in this malignancy. To address these issues, we performed extensive HER2 expression analysis on a wide panel of primary tumors as well as established and short-term human ovarian cancer cell lines. Conventional immunohistochemical (IHC) analysis of multiple tumor sites in 50 cases of high-grade ovarian serous carcinomas revealed HER2 overexpression in 29% of evaluated sites. However, more sensitive detection methods including flow cytometry, western blot analysis and q-PCR revealed HER2 expression in all fresh tumor cells derived from primary ascites or solid tumors as well as all established and short-term cultured cancer cell lines. Cancer cells generally expressed HER2 at higher levels than that found in normal ovarian surface epithelial (OSE) cells. Accordingly, genetically-engineered human T cells expressing an HER2-specific chimeric antigen receptor (CAR) recognized and reacted against all established or primary ovarian cancer cells tested with minimal or no reactivity against normal OSE cells. In conclusion, all human ovarian cancers express immunologically-detectable levels of HER2, indicating that IHC measurement underestimates the true frequency of HER2-expressing ovarian cancers and may limit patient access to otherwise clinically meaningful HER2-targeted therapies.

The Lymphotactin Receptor Is Expressed in Epithelial Ovarian Carcinoma and Contributes to Cell Migration and Proliferation

Chemokine receptor-ligand interactions are important to support functioning of both normal and pathologic cells. The expression and function of chemokine receptors in epithelial ovarian carcinoma (EOC) is largely unknown. Here, we report that the lymphotactin receptor (XCR1) was expressed in primary and metastatic human epithelial ovarian carcinoma (EOC) specimens and cell lines. In contrast, expression of XCR1 was not detected in the normal ovary or in human normal ovarian surface epithelial cells. Our data indicate that XCL1 and XCL2 are either present in the malignant ascites or expressed by the ovarian carcinoma cells. The addition of lymphotactin (XCL1 and XCL2) stimulated migration and proliferation of XCR1-positive cells. Reduction of XCR1 expression in ovarian carcinoma cell line SKOV-3 resulted in abrogated diaphragm and peritoneal wall tumor formation and in reduced frequency of colonic, splenetic, and liver nodules in an in vivo xenograft mouse model. Taken together, our data suggest that XCR1 is expressed early during the course of tumorigenic transformation and contributes towards increased cell migration and proliferation, which can facilitate the prometastatic behavior of EOC cells.

Overexpressing the CCL2 chemokine in an epithelial ovarian cancer cell line results in latency of in vivo tumourigenicity

The frequent loss of heterozygosity of chromosome (Chr) 17 in epithelial ovarian cancer (EOC), particularly high-grade ovarian serous carcinomas (HGOSCs), has been attributed to the disruption of known tumour suppressor genes, such as TP53 (17p13), as well as other genes on this chromosome that alone or in combination have a role in EOC. In a transcriptome analysis of Chr17 genes, we observed significant underexpression of the chemokine CCL2 (17q12) in a small set of HGOSC samples relative to normal ovarian surface epithelial cells and a significant upregulation of CCL2 in the TP53-mutated OV-90 EOC cell line rendered non-tumourigenic as a consequence of genetic manipulation. Here, we report that overexpressing CCL2 in OV-90 resulted in latency of tumour formation at intraperitoneal (i.p.) but not subcutaneous sites in a mouse xenograft model. Overexpressing CCL2 affected cell morphology and exerted modest, but not significant effects on cell viability, colony formation and cell migration. We report significant underexpression of CCL2 by transcriptome analysis (P=0.015) and by immunohistochemistry in 77% of HGOSC samples (n=65). Absent or a very low level of protein expression by immunohistochemistry was also observed in 71% of additional HGOSC samples (n=122). However, CCL2 protein expression did not significantly correlate with overall or disease-free survival. The epithelial cells of normal fallopian tubes, a purported origin of HGOSC, exhibited expression of CCL2 protein by immunohistochemistry. Our results affirm that CCL2 underexpression is a significant feature of HGOSC samples, and that CCL2 overexpression in an EOC cell line model affects tumourigenic potential in the i.p. setting.

Heterotypic Three-dimensional &lt;em&gt;In Vitro&lt;/em&gt; Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades. Whilst stage I tumors have 5-year survival rates >85%, survival rates for stage III/IV disease are <40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF) cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

MicroRNA expression in ovarian carcinoma and its correlation with clinicopathological features

BackgroundMicroRNA (miRNA) expression is known to be deregulated in ovarian carcinomas. However, limited data is available about the miRNA expression pattern for the benign or borderline ovarian tumors as well as differential miRNA expression pattern associated with histological types, grades or clinical stages in ovarian carcinomas. We defined patterns of microRNA expression in tissues from normal, benign, borderline, and malignant ovarian tumors and explored the relationship between frequently deregulated miRNAs and clinicopathologic findings, response to therapy, survival, and association with Her-2/neu status in ovarian carcinomas.MethodsWe measured the expression of nine miRNAs (miR-181d, miR-30a-3p, miR-30c, miR-30d, miR-30e-3p, miR-368, miR-370, miR-493-5p, miR-532-5p) in 171 formalin-fixed, paraffin-embedded ovarian tissue blocks as well as six normal human ovarian surface epithelial (HOSE) cell lines using Taqman-based real-time PCR assays. Her-2/neu overexpression was assessed in ovarian carcinomas (n = 109 cases) by immunohistochemistry analysis.ResultsExpression of four miRNAs (miR-30c, miR-30d, miR-30e-3p, miR-370) was significantly different between carcinomas and benign ovarian tissues as well as between carcinoma and borderline tissues. An additional three miRNAs (miR-181d, miR-30a-3p, miR-532-5p) were significantly different between borderline and carcinoma tissues. Expression of miR-532-5p was significantly lower in borderline than in benign tissues. Among ovarian carcinomas, expression of four miRNAs (miR-30a-3p, miR-30c, miR-30d, miR-30e-3p) was lowest in mucinous and highest in clear cell samples. Expression of miR-30a-3p was higher in well-differentiated compared to poorly differentiated tumors (P = 0.02), and expression of miR-370 was higher in stage I/II compared to stage III/IV samples (P = 0.03). In multivariate analyses, higher expression of miR-181d, miR-30c, miR-30d, and miR-30e-3p was associated with significantly better disease-free or overall survival. Finally, lower expression of miR-30c, miR-30d, miR-30e-3p and miR-532-5p was significantly associated with overexpression of Her-2/neu.ConclusionsAberrant expression of miRNAs is common in ovarian tumor suggesting involvement of miRNA in ovarian tumorigenesis. They are associated with histology, clinical stage, survival and oncogene expression in ovarian carcinoma.

Expression of Death Receptor 6 by Ovarian Tumors in Laying Hens, a Preclinical Model of Spontaneous Ovarian Cancer

Tumor-associated neoangiogenesis and suppression of antitumor immunity are hallmarks of tumor development and progression. Death receptor 6 (DR6) has been reported to be associated with suppression of antitumor immunity and tumor progression in several malignancies. However, expression of DR6 by malignant ovarian epithelial tumors at an early stage is unknown. The goals of this study were to determine whether DR6 is expressed by malignant ovarian epithelial tumors at an early stage and to examine whether DR6 expression is associated with ovarian cancer (OVCA) progression in a laying hen model of spontaneous OVCA. Expression of DR6 was examined in normal and malignant ovaries, normal ovarian surface epithelial (OSE) cells, or malignant epithelial cells and in serum of 3-year-old hens. The population of microvessels expressing DR6 was significantly higher in hens with early-stage OVCA than hens with normal ovaries (P < .01) and increased further in late-stage OVCA. The results of this study showed that, in addition to microvessels, tumor cells in the ovary also express DR6 with a significantly higher intensity than normal OSE cells. Similar patterns of DR6 expression were also observed by immunoblot analysis and gene expression studies. Furthermore, DR6 was also detected in the serum of hens. In conclusion, DR6 expression is associated with OVCA development and progression in laying hens. This study may be helpful to examine the feasibility of DR6 as a useful surrogate marker of OVCA, a target for antitumor immunotherapy and molecular imaging and thus provide a foundation for clinical studies.

Are CCN1 (Cysteine Rich 61-Connective Tissue Growth Factor-Nephroblastoma Overexpressed) and Integrin Receptor Subunits Expressed by KGN, an Ovarian Granulosa Tumor Cell Line?

Tumors express a variety of pro-angiogenic factors that contribute to tumor angiogenesis and cancer progression including CCN1, vascular endothelial growth factor (VEGFa), basic fibroblast growth factor (FGF2) and matrix metalloproteinases (MMPs). Interestingly, healthy tissues undergoing physiological angiogenesis, such as those comprising the female reproductive tract, also express VEGFa, FGF2 and MMPs. We have recently reported, for the first time, that the angiogenic inducer, CCN1 (CYR61), is expressed by granulosa and theca cells of normal bovine ovarian follicles. Although it has been shown that normal ovarian surface epithelial cell lines and several epithelial ovarian carcinoma cell lines express CCN1, it is not known whether or not CCN1 plays a role in the progression of human granulosa carcinomas, a malignant ovarian sex cord tumor originating from cells that surround the oocyte. These carcinomas represent between 1-5% of all ovarian cancer diagnoses. The objective of the present study was to determine the expression of CCN1, the a and β integrin subunits that form the heterodimeric receptors for CCN1 and MMP activity, in a model of human ovarian granulosa carcinoma. KGN, a steroidogenic human granulosa-like tumor cell line, was cultured to near confluence and then maintained for 24 hours with or without 10% fetal bovine serum (FBS) and in the absence or presence of testosterone (T; 10 uM). RNA and protein were extracted for analysis of CCN1 and integrin subunit expression, and conditioned medium was collected for analysis of MMPs by gelatin zymography. Our results showed baseline expression of CCN1 mRNA in KGN cells regardless of treatment while FBS increased expression of CCN1 protein. There was, however, no effect of T on either CCN1 mRNA or protein expression. A PCR array to screen for the presence of specific integrin receptor subunits was performed and revealed that KGN cells express integrin a1, a3, a5, aV, β1 and β5 mRNA. Gelatin zymography of KGN condition medium revealed an intense band (approximately 74 kD) and a weak band (approximately 92 kD), corresponding to MMP2 and MMP9 family members, respectively. A higher molecular mass doublet (approximately 128 and 123 kD), consistent with MMP9/neutrophil gelatinase-associated lipocalin (NGAL; Lipocalin 2), was also detected. In summary, we have determined that in addition to possessing aromatase activity and the ability to produce VEGFa, KGN cells also express CCN1, integrin a and β subunits and MMPs, factors that are known to contribute to tumor angiogenesis and cancer progression. Future studies are aimed at elucidating the molecular mechanisms of CCN1 action on the progression of granulosa tumor cells, including how it interacts with specific integrin receptors and other angiogenic factors such as VEGFa, to influence granulosa cell cancer progression. (Supported by NIH grant P01 CA045548 and the Stuart Weitzman Foundation to MAM, the Multistate Regional Project NE1027 to PCWT and the Department of Veterans Affairs to JSD.)

Casein kinase I epsilon interacts with mitochondrial proteins for the growth and survival of human ovarian cancer cells

Epithelial ovarian cancer is the leading cause of death among gynaecologic cancers in Western countries. Our studies have shown that casein kinase I-epsilon (CKIε), a Wnt pathway protein, is significantly overexpressed in ovarian cancer tissues and is associated with poor survival. Ectopic expression of CKIε in normal human ovarian surface epithelial cells and inhibition of CKIε in ovarian cancer cells and in xenografts demonstrated the importance of CKIε in regulating cell proliferation and migration. Interestingly, CKIε function did not seem to involve β-catenin activity. Instead, CKIε was found to interact with several mitochondrial proteins including adenine nucleotide translocase 2 (ANT2). Inhibition of CKIε in ovarian cancer cells resulted in suppression of ANT2, downregulation of cellular ATP and the resulting cancer cells were more susceptible to chemotherapy. Our studies indicate that, in the context of ovarian cancer, the interaction between CKIε and ANT2 mediates pathogenic signalling that is distinct from the canonical Wnt/β-catenin pathway and is essential for cell proliferation and is clinically associated with poor survival.

Allelic Transcripts Dosage Effect in Morphologically Normal Ovarian Cells from Heterozygous Carriers of a BRCA1/2 French Canadian Founder Mutation

We hypothesized that the transcriptome of primary cultures of morphologically normal ovarian surface epithelial cells could be altered by the presence of a heterozygous BRCA1 or BRCA2 mutation. We aimed to discover early events associated with ovarian carcinogenesis, which could represent putative targets for preventive strategies of this silent killer tumor. We identified the first molecular signature associated with French Canadian BRCA1 or BRCA2 founder mutations in morphologically normal ovarian epithelial cells. We discovered that wild-type and mutated BRCA2 allelic transcripts were expressed not only in morphologically normal but also in tumor cells from BRCA2-8765delAG carriers. Further analysis of morphologically normal ovarian and tumor cells from BRCA1-4446C>T carriers lead to the same observation. Our data support the idea that one single hit in BRCA1 or BRCA2 is sufficient to alter the transcriptome of phenotypically normal ovarian epithelial cells. The highest level of BRCA2-mutated allele transcript expression was measured in cells originating from the most aggressive ovarian tumor. The penetrance of the mutation and the aggressiveness of the related tumor could depend on a dosage effect of the mutated allele transcript.

Abstract 74: The role of epithelial splicing regulatory protein 1 in ovarian cancer

Abstract Epithelial splicing regulatory protein 1 (ESRP1) is a recently identified cell type-specific splicing factor which has been shown to control an alternative splicing network associated with the epithelial-mesenchymal transition (EMT). Whether ESRP1 play any roles in the development and progression of epithelial ovarian cancer is unknown. To answer this question, we first examined its expression by RT-PCR in a panel of cultured cells including normal ovarian surface epithelial cells (HOSE), immortalized OSE (IOSE398 and IOSE120T) as well as ovarian cancer cell lines A2780, OVCA3, OVCAR5, OVCAR8 and SKOV3. We found that ESRP1was expressed at low levels in HOSE, IOSEs and A2780 cells, at intermediate levels in OVCAR8 and SKOV3 cells and at high levels in OVCAR3 and OVCAR5 cells. Examination of ESRP1 target gene FGFR2 showed that cells with low to intermediate levels of ESRP1 exclusively expressed mesenchymal form of FGFR2 (FGFR2IIIc) while cells with high levels of ESRP1 exclusively expressed epithelial form of FGFR2 (FGFR2IIIb). These results suggest two notions: 1) the switch of FGFR2 splicing occurs in an all-or-none format dependent on the threshold of ESRP1 expression levels; 2) normal ovarian surface epithelial cells may have mesenchymal properties. We also examined the expression of ESRP1 in seven pairs of matched ovarian tumor and adjacent normal tissues and found that ovarian tumors expressed higher levels of ESRP1 than normal tissues in all pairs. In the attempt to clone the entire coding sequence of ESRP1 from SKOV3 cells, we have identified 11 ESRP1 splice variants (SVs). Ectopic expression of four ESRP1 SVs in 293T and A2780 cells, which both have very low levels of endogenous ESRP1, triggered profound apoptosis. The two SVs with exons 14 and 15 skipped appeared more cytotoxic than full-length ESRP1. We are currently investigating the effects of knockdown of ESRP1 on the transformation properties of ovarian cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 74. doi:1538-7445.AM2012-74

Abstract 1828: Identification of MUC16/CA125-specific therapeutics through dual fluorescence high throughput screening

Abstract BACKGROUND: The MUC16 gene encodes the CA125 ovarian cancer antigen. This glycoprotein is expressed in the majority of Epithelial Ovarian Cancers (EOC) but has limited expression elsewhere in the adult human. Expression of the carboxy-portion of MUC16 results in increased invasion, soft agar growth and in vivo growth. OBJECTIVES:. We seek to identify MUC16 specific small molecules as chemotherapy candidate drugs for development in ovarian cancer. METHODS: Our previously reported study of dual fluorescence isogenic high content screening for MUC16/CA125 selective agents (Mol.Can.Ther, 2011) was extended to a library of 74,942 compounds from NCI, to identify small molecules which have a differential effects against MUC16 positive cells compared to isogenic MUC16 negative ovarian cancer cell lines Carboxy-terminus portions of the human MUC16 gene were introduced into A2780 EOC cell lines labeled with mCherry and compared to GFP labeled isogenic controls in a 384 well co-culture assay for cellular viability by computerized fluorescence intensities and compared for the relative toxicity against MUC16+/MUC16- isogenic cell lines. Compounds with differential activity were selected, resupplied and examined by Vi Cell counting and Alamar Blue cytotoxic assays for confirmation in SK-OV-3 cell line pairs. RESULTS: A2780 isogenic pair was initially used and thirty compounds which selectively targeted MUC16 positive cells were identified. These 30 compounds were tested for growth inhibition with a secondary SKOV3 isogenic screen for confirmation. Two compounds were among the screen identified in the secondary SKOV3 isogenic pair testing. These compounds were studied using Alamar Blue Assay with a panel of ovarian cell lines including CAOV3; OV433; OV432; SKOV8 (WT and MUC16 knockdown and an isogenic T80 MUC16+/− pair (in vitro transformed normal ovarian surface epithelial cell line) and IGROV1-R10. We are in the process of examining the Structural Activity Relationship around these 2 compounds and will present their response. CONCLUSIONS: These MUC16 +/− dual fluorescence screens can be used as an example of single gene target, high throughput screening strategies to identify potentially specific therapeutics for key oncogenic targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1828. doi:1538-7445.AM2012-1828

Abstract 5206: Systems biology identification of processes/pathways facilitating the development of high-grade serous ovarian cancer

Abstract Previously, based on well-known stem cell phenotypes (sphere formation, potent tumorigenicity, embryonic gene expression, and chemoresistance), we used patient tumors to isolate high-grade serous (HGS) ovarian cancer-initiating cells (OCICs) that express the cell surface markers CD44 (a hyaluronate receptor) and CD117 (c-kit) (Zhang et al., 2008, Cancer Res 68:4311). Here, to identify biological pathways that underlie HGS ovarian tumorigenesis, including the specific role of OCICs in that process, we performed integrated analyses of global gene expression, DNA copy number, and DNA methylation patterns. To that end, OCICs and “bulk” (i.e., disaggregated but unsorted) tumor cells were compared to primary human normal ovarian surface epithelial (nOSE) cells that, along with fallopian tube epithelial (FTE) cells, represent hypothesized cells-of-origin for HGS pelvic cancers. Principle components analysis and unsupervised hierarchical clustering revealed distinct DNA methylation and gene expression profiles for replicate (n=3) samples of each cell type (i.e., OCIC, bulk tumor, and nOSE). Gene ontology and additional unsupervised analyses of “gene expression signatures” corresponding to the epithelial-to-mesenchymal transition (82 of 159 signature genes), TGF-beta (47 of 86 genes), and Wnt (75 of 151 genes) pathways demonstrated well-defined segregation of the three cell types, with mesenchymal and Wnt signaling hierarchies being nOSE &amp;gt; OCIC &amp;gt; bulk tumor cells. To examine early events in HGS ovarian tumorigenesis, previously published (Tone et al., 2008, Clin Cancer Res 14:4067) gene expression datasets were employed to show that nOSE, OCIC, and bulk ovarian tumor cell transcriptomes distinctly segregate from normal (FTE) and malignant fallopian epithelial transcriptomes. Interestingly, the largest divergence was between the nOSE and FTE gene expression profiles. Finally, DNA methylation and gene expression patterns were integrated, implicating specific networks/circuits in ovarian carcinogenesis. Taken together, these results support a mesenchymal-to-epithelial transition during ovarian tumorigenesis, reveal legitimate therapeutic targets (at the pathway level), and raise uncertainty that surface epithelial cells of the fallopian tube and ovary are reciprocal cells-of-origin for HGS pelvic carcinomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5206. doi:1538-7445.AM2012-5206

Abstract 2928: Functional effects of SNPs in non-coding RNAs at the 3q25 ovarian cancer susceptibility locus

Abstract Genome-wide association studies have identified six common, low penetrance susceptibility loci for epithelial ovarian cancer (EOC). The functional mechanisms underlying disease susceptibility at these loci are unknown. SNPs at the 3q25 locus confer a 1.2-fold risk of serous EOC (P=7.1x10-8). To identify causal SNP(s) we used the 1000 genomes project to identify 952 correlated SNPs within a 884kb linkage disequilibrium region. Of these, 786 tagging SNPs were genotyped in ∼20,000 invasive EOC cases and 20,000 controls. Fine mapping limited the size of the risk region to ∼130kb and 43 possible causal SNPs. This region contains four protein-coding genes; none of the 43 SNPs was predicted to have a functional effect on the translated protein. Genomic profiling data from The Cancer Genome Atlas analysis of ∼500 serous EOCs indicated one gene in the region (TIPARP) was significantly down regulated compared to normal tissues (P=0.005). Using RT-PCR expression analysis, we found significantly lower TIPARP expression in EOC cell lines (N=37) compared to normal ovarian surface epithelial (OSE) and fallopian tube epithelial (FTE) cell lines (N=39) (P=1.57x10-8). TIPARP was also significantly downregulated in an in vitro model of early stage transformation of OSE cells (P=0.0003). Collectively, these data suggest that TIPARP is a novel tumor suppressor gene (TSG) in EOC development. TIPARP is a poly(ADP-ribose) polymerase known to catalyze histone ribosylation, an epigenetic modification that plays a role in DNA repair. We evaluated its functional significance in more detail using stable lentiviral-mediated shRNA knockdown of TIPARP in normal OSE/FTE cells. TIPARP knockdown induced cellular senescence similar to the effects of many classical TSG knockdowns in normal cells. We hypothesized that TIPARP may be involved in cellular response to DNA damage; when normal OSE/FTE cells were exposed to α-irradiation TIPARP was significantly upregulated in a dose-dependent manner. We examined the functional link between susceptibility SNPs and TiPARP expression. Of 43 putative causal SNPs, four fell within two non-coding RNAs- LOC730091 and TIPARP-AS1. There is no homology between LOC730091 and genes at the 3q25 locus; but TIPARP-ASI is a non-coding RNA with antisense sequence similarity to exon 1 of TIPARP. LOC730091 and TIPARP-AS1 were variably expressed in both normal ovarian and EOC cell lines. We found no correlation between LOC730091 and TIPARP expression; but cell lines expressing TIPARP-AS1 showed significantly reduced TIPARP expression. In summary, fine mapping at the 3q25 has identified multiple possible causal SNPs. Functional studies implicate TIPARP as a plausible target TSG in this region. EOC risk associated SNPs were located in a non-coding RNA that causes reduced expression of TIPARP suggesting a mechanism by which susceptibility SNPs may confer neoplastic transformation of ovarian epithelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2928. doi:1538-7445.AM2012-2928

Abstract 1271: Aberrant activation of Spleen Tyrosine Kinase in ovarian cancer identified through a global phosphorylation profiling of protein tyrosine kinases

Abstract Aberrations in protein kinase activity and the consequent deregulation of downstream signaling pathways are considered a hallmark of oncogenesis. The purpose of this study was to investigate the phosphorylation state, the ultimate readout of dysregulated activity, of protein tyrosine kinases in ovarian tumors. An innovative Luminex multiplex platform was employed to systematically determine phosphorylation status of protein tyrosine kinases in a total of 69 protein lysates derived from normal human ovarian surface epithelial (HOSE) cells, ovarian tumors, and cancer cell lines. Significance Analysis of Microarrays (SAM) was performed to identify the signature of phosphorylated kinases with statistical significance. Profiling results were confirmed by using phosphor-specific kinase antibodies in immunoblot analysis of the profiled lysates and immunohistochemistry on formalin-fixed paraffin-embedded archived ovarian and fallopian tissues, as well as immunofluorescence of ovarian cancer cells growing as monolayer or 3D Matrigel cell cultures. A signature of twenty-two phosphorylated protein tyrosine kinases was identified by SAM analysis of the profiling data. Spleen Tyrosine Kinase (SYK), a novel tyrosine kinase included in the signature, was found significantly phosphorylated in lysates from ovarian cancer tissues compared to normal tissues by both Western blot analysis and immunohistochemistry on 94 clinical ovarian tissues (P=0.024). SYK phosphorylation was also identified in tubal intraepithelial carcinomas, the hypothesized precursors of high-grade serous ovarian carcinomas. However, SYK activation was not observed in ovarian cancer cell lines growing as attached monolayer cells, but was present when they grew as mouse xenografts or in 3D Matrigel in vitro cultures. We are the first to report that SYK, an extensively investigated tyrosine kinase in hematopoietic cells, is aberrantly phosphorylated and activated in ovarian cancer. SYK phosphorylation in ovarian cancer might be induced by extracellular stimuli that are exclusively present in the tumor microenvironment. These findings provide additional options in targeted therapy for ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1271. doi:1538-7445.AM2012-1271

Abstract 3915: Insulin-like growth factor 1 promotes cancer cell growth via RAC1 activation in ovarian epithelial cancer cells

Abstract Ovarian cancer is the most lethal of all gynecological malignancies, due in part to the diagnosis at an advanced stage caused by the lack of specific signs and symptoms and the absence of reliable tests for screening and early detection. Furthermore, survival is reduced by recurrence of chemo-resistant tumors. The insulin-like growth factor 1 (IGF1)/IGF1 receptor (IGFR) signaling system exerts a broad anti-apoptotic function and plays a crucial role in human cancer progression, including ovarian cancers. The small GTP-binding protein RAC1 regulates a diverse array of cellular events, including the control of cell growth, cytoskeleton reorganization for motility, and the activation of protein kinases. Although RAC1 is expressed in most tissues, it can be elevated in tumors at various stages of neoplastic progression. This study was conducted to explore the role of RAC1 in IGF1-stimulated ovarian cancer cell growth. While IGFR was expressed in normal human ovarian surface epithelial (HOSE) cells and all OSE cancer cell lines examined, the IGF1R was over expressed in IGROV1 ovarian cancer cells. Exposure of IGROV1 cells to IGF1 rapidly (&amp;lt;0.5 min) induced IGFR phosphorylation which triggered the phosphorylation of AKT (S473) (≥0.5 min), ERK1[[Unsupported Character - &amp;#8260;]] 2 (&amp;gt;1.0 min) and p70S6K (T389) (≥1.0 min); indicating that IGF1 actives PI3K and MAPK signaling pathways that are known to be important in cell growth. We observed a maximal effect of IGF1 on IGROV1 cell growth at 50 ng/ml (2.2 fold increase, p&amp;lt;0.001) after 24 h. In contrast, exposure of IGROV1 cells to the PI3K inhibitor LY294002 or the MEK1/2 inhibitor UO126 decreased cell viability. Pull-down assays revealed that IGF1 stimulated GTP loading of RAC1 in less than 1.0 min with maximal increases observed after 5 min (2.6 fold, p&amp;lt;0.05). Treatment with the PI3K inhibitor LY294002 significantly reduced RAC1 activation in response to IGF1. Pre-treatment of IGROV1 cells with the RAC1 inhibitor (NSC23766) for 1 h inhibited IGF1-induced activation of RAC1; and decreased cell survival in a concentration-dependent manner (5- 50 µM NSC23766, p&amp;lt;0.05). The reduction in viability after 48 hr was accompanied by an increase in apoptotic cells, cleaved PARP and elevated cytosolic cytochrome C. In contrast, treatment of HOSE cells with NSC23766 did not alter cell viability. Of interest, following 6 h treatment of IGROV1 cells with 20-50 µM NSC23766 we observed increases in the phosphorylation of Akt (S473), GSK3β (S9), and p70S6K (T389), suggesting the activation of PI3K/AKT pathway was not sufficient to rescue cells from death. Furthermore, treatment with NSC23766 (50 µM) and cisplatin (20 µM) reduced cell viability to an extent greater than achieved by either agent alone (p&amp;lt;0.05). Thus, disruption of RAC1 activation reduces IGF1-stimulated cell growth, induces apoptosis, and enhances cell death by anti-cancer agents in ovarian cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3915. doi:1538-7445.AM2012-3915

Ginkgo May Sensitize Ovarian Cancer Cells to Cisplatin

Ginkgolide B (GB), the primary active component ofGinkgo bilobaextracts, may have antitumor properties. The objective of this study was to determine the effects and possible mechanisms of GB in ovarian cancer cells. In this study, human ovarian cancer cell lines (SKOV3 and CAOV3) were treated with different concentrations of GB alone or in combination with Cis-diaminodichloroplatinum (CDDP). An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to determine cell viability. The apoptosis rates of cells were measured by flow cytometric analysis. The expression of apoptosis-associated and proliferation-associated proteins was detected by Western blot. The cytotoxicity of GB was analyzed using a lactate dehydrogenase assay. Treatment with 100 µM GB for 3 days significantly inhibited SKOV3 and CAOV3 cell proliferation by 57.3% and 63.1% compared with control cells, respectively, as determined by MTT assay. Similarly, the apoptotic cell population was increased when treated with GB in a dose-dependent manner both in SKOV3 and CAOV3 cells. These effects were characterized by the upregulation of p21, p27, cleaved capase-3, and cleaved caspase-8 and downregulation of cyclin D1. In addition, a combined treatment of low concentrations of GB and CDDP showed an additive effect on the inhibition of SKOV3 cell proliferation. Furthermore, GB had significantly less cytotoxicity than CDDP in normal human ovarian surface epithelial cells. This study suggests that GB can be proposed as an effective antiproliferative and apoptosis-inducing agent with interesting translational application in ovarian cancers, used in addition to conventional chemotherapy.

Wild-Type Tumor Repressor Protein 53 (TRP53) Promotes Ovarian Cancer Cell Survival

Loss of Pten in the Kras(G12D);Amhr2-Cre mutant mice leads to the transformation of ovarian surface epithelial (OSE) cells and rapid development of low-grade, invasive serous adenocarcinomas. Tumors occur with 100% penetrance and express elevated levels of wild-type tumor repressor protein 53 (TRP53). To test the functions of TRP53 in the Pten;Kras (Trp53+) mice, we disrupted the Trp53 gene yielding Pten;Kras(Trp53-) mice. By comparing morphology and gene expression profiles in the Trp53+ and Trp53- OSE cells from these mice, we document that wild-type TRP53 acts as a major promoter of OSE cell survival and differentiation: cells lacking Trp53 are transformed yet are less adherent, migratory, and invasive and exhibit a gene expression profile more like normal OSE cells. These results provide a new paradigm: wild-type TRP53 does not preferentially induce apoptotic or senescent related genes in the Pten;Kras(Trp53+) cancer cells but rather increases genes regulating DNA repair, cell cycle progression, and proliferation and decreases putative tumor suppressor genes. However, if TRP53 activity is forced higher by exposure to nutlin-3a (a mouse double minute-2 antagonist), TRP53 suppresses DNA repair genes and induces the expression of genes that control cell cycle arrest and apoptosis. Thus, in the Pten;Kras(Trp53+) mutant mouse OSE cells and likely in human TP53+ low-grade ovarian cancer cells, wild-type TRP53 controls global molecular changes that are dependent on its activation status. These results suggest that activation of TP53 may provide a promising new therapy for managing low-grade ovarian cancer and other cancers in humans in which wild-type TP53 is expressed.

Chromosome 17q25 genes, RHBDF2 and CYGB, in ovarian cancer

It has been proposed that the frequent loss of heterozygosity (LOH) of an entire chromosome 17 contig in epithelial ovarian cancers (EOC) is the consequence of the inactivation of multiple tumour suppressor genes on this chromosome. We report the characterization of a 453 Kb 17q25 locus shown previously to exhibit a high frequency of LOH in EOC samples. LOH analysis further defined the minimal region of deletion to a 65 Kb interval flanked by D17S2239 and D17S2244, which contains RHBDF2, CYGB and PRCD as tumour suppressor gene candidates. Tissue specific expression excluded PRCD as a candidate. RHBDF2 was expressed at low levels in the majority of benign and low malignant potential (LMP) tumours, and in a subset of malignant ovarian tumour samples, as compared with primary cultures of normal ovarian surface epithelial cell (NOSE) samples. CYGB was expressed at low levels in the majority of LMP and malignant samples compared with benign and NOSE samples. In contrast to CYGB expression, RHBDF2 was expressed at low or undetectable levels in EOC cell lines exhibiting tumourigenic characteristics and up-regulated in a genetically modified EOC cell line rendered non-tumourigenic. DNA sequence analysis identified variants but no apparent deleterious mutations in either gene. Methylation-specific PCR analysis suggested that promoter methylation of CYGB but not RHBDF2 occurred in 6 of 31 malignant samples. The results combined suggest that RHBDF2 and CYGB may play distinctive roles in ovarian cancer and could be added to the growing roster of chromosome 17 genes implicated in this disease.