Immortalized Ovarian Surface Epithelial Cells Research Articles

Abstract 4357: ESRP1 overexpression and its role in ovarian cancer

Abstract Epithelial splicing regulatory protein 1 (ESRP1) and 2 (ESRP2) are RNA binding proteins which are recently known to be involved in tumor progression via regulation of alternative splicing of cancer-associated genes. However, their expression and clinical significance in ovarian cancer remain unclear. Here, we found that ESRP1 and ESRP2 gene expression is upregulated in ovarian cancer cells compared with immortalized ovarian surface epithelial (IOSE) cells and their overexpression at protein level was also confirmed in ovarian cancer tissues. Consistent with TCGA data of ESRP1 gene amplification, higher ESRP1 gene copy number was observed in ovarian cancer cells compared with IOSE cells, whereas there was little correlation between gene copy number and gene expression in ovarian cancer cells. Importantly, their gene expression was inversely correlated with DNA methylation in ovarian cancer cells and ESRP2 overexpression in ovarian cancer was significantly associated with DNA hypomethylation (P= 0.036). Of note, TCGA data analysis showed that high ESRP1 gene expression is significantly associated with shorter 5-year survival of patients with ovarian cancer. Ectopic expression of ESRP1 in ESRP1 low-expressing mesenchymal type ovarian cancer cells increased cell proliferation, whereas it suppresses cell migration. Furthermore, our results revealed that the effect of ESRP1 overexpression on cell proliferation and migration is related to activation of the Akt/CyclinD pathway and increased of E-cadherin expression. In conclusion, our findings suggest that ESRP1 overexpression is associated with poor outcome of patients and ESRP1 may play a cancer-promoting role in ovarian cancer. Citation Format: Mi Jeong Kwon. ESRP1 overexpression and its role in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4357. doi:10.1158/1538-7445.AM2017-4357

Hypermethylation of the TGF-β target, ABCA1 is associated with poor prognosis in ovarian cancer patients.

BackgroundThe dysregulation of transforming growth factor-β (TGF-β) signaling plays a crucial role in ovarian carcinogenesis and in maintaining cancer stem cell properties. Classified as a member of the ATP-binding cassette (ABC) family, ABCA1 was previously identified by methylated DNA immunoprecipitation microarray (mDIP-Chip) to be methylated in ovarian cancer cell lines, A2780 and CP70. By microarray, it was also found to be upregulated in immortalized ovarian surface epithelial (IOSE) cells following TGF-β treatment. Thus, we hypothesized that ABCA1 may be involved in ovarian cancer and its initiation.ResultsWe first compared the expression level of ABCA1 in IOSE cells and a panel of ovarian cancer cell lines and found that ABCA1 was expressed in HeyC2, SKOV3, MCP3, and MCP2 ovarian cancer cell lines but downregulated in A2780 and CP70 ovarian cancer cell lines. The reduced expression of ABCA1 in A2780 and CP70 cells was associated with promoter hypermethylation, as demonstrated by bisulfite pyro-sequencing. We also found that knockdown of ABCA1 increased the cholesterol level and promoted cell growth in vitro and in vivo. Further analysis of ABCA1 methylation in 76 ovarian cancer patient samples demonstrated that patients with higher ABCA1 methylation are associated with high stage (P = 0.0131) and grade (P = 0.0137). Kaplan-Meier analysis also found that patients with higher levels of methylation of ABCA1 have shorter overall survival (P = 0.019). Furthermore, tissue microarray using 55 ovarian cancer patient samples revealed that patients with a lower level of ABCA1 expression are associated with shorter progress-free survival (P = 0.038).ConclusionsABCA1 may be a tumor suppressor and is hypermethylated in a subset of ovarian cancer patients. Hypermethylation of ABCA1 is associated with poor prognosis in these patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-014-0036-2) contains supplementary material, which is available to authorized users.

Abstract 1476: Hypermethylation of a TGF-β regulated micro-RNA, miR-193a, predicts prognosis in ovarian cancer

Abstract Ovarian cancer is one of the most lethal cancers in the female reproductive system. One of the hypotheses suggests that ovarian cancer arises from cancer stem/progenitor cell with surface antigen CD117 (or c-kit). In order to identify microRNAs (miRNAs) that can regulate the expression of c-kit, bioinformatic prediction was performed and identified miR-193a as one of the regulators. Expression analysis showed that miR-193a was up-regulated upon TGF-β treatment in immortalized ovarian surface epithelial cells (IOSE) cells. On the contrary, expression of miR-193a cannot be detected in a panel of ovarian cancer cell lines (SKOV3, A2780, CP70, MCP2, MCP3). Bisulphite pyrosequencing revealed that promoter CpG island of miR-193a was not methylated in IOSE cell but hypermethylated in the ovarian cancer cells showing down-regulation of miR-193a. Treatment of 5azaDC, a DNMT inhibitor, partially restored miR-193a expression in the ovarian cancer cells, thus suggesting DNA hypermethylation may be responsible for the down-regulation of miR-193a in ovarian cancer. Further clinical studies using bisulphite pyrosequencing found that promoter region of miR-193a was heavily methylated in 109 ovarian cancer patient samples but not in normal OSE samples. Kaplan-meier analysis revealed that patients with higher methylation of miR-193a was significantly associated with shorter overall survival and progression-free survival. Taken together, miR-193a may be a TGF-β regulated tumor suppressor micro-RNA and is epigenetically silenced in ovarian cancer. Methylation of miR-193a may be able to predict prognosis in ovarian cancer patients. Citation Format: Frank Hsueh-Che Cheng, Gary C.W. Chen, Jian-Liang Chou, Ya-Wen Lin, Lin-Yu Chen, Hung-Cheng Lai, Chin Li, Michael W.Y. Chan. Hypermethylation of a TGF-β regulated micro-RNA, miR-193a, predicts prognosis in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1476. doi:10.1158/1538-7445.AM2014-1476

Abstract 3285: Functional analysis of the 9p22 locus implicates the transcriptional regulation of BNC2 as a mechanism in ovarian cancer predisposition

Abstract An ovarian cancer Genome Wide Association Study (GWAS) identified 9p22.2 as a novel susceptibility locus with the most statistically significant single nucleotide polymorphisms (SNPs) located in an intergenic region near the Basonuclin 2 (BNC2) gene, which codes for a putative transcription regulator containing three pairs of zinc finger (ZF) domains. The minor alleles are protective in terms of ovarian cancer susceptibility. However, the molecular mechanisms by which these SNPs modify susceptibility remain largely unknown. The significant SNPs in the 9p22.2 locus lie in non-coding regions and therefore are hypothesized to affect the activity of regulatory elements and modify the expression of a target gene(s). In order to test this hypothesis we conducted fine mapping for the locus which delimited a ∼74kb region containing SNPs with a p-value less than 10-8. FAIRE-Seq and ChIP-Seq experiments for histone markers conducted in immortalized ovarian surface epithelial cells (IOSE) and fallopian tube epithelial cells (IFTE) were used to prioritize functional SNP candidates which overlap with regulatory elements. Luciferase assays tested the ability of these regulatory elements to activate transcription. Chromosome conformation capture (3C) experiments demonstrate a physical interaction between the BNC2 promoter and candidate regulatory elements containing risk-associated SNPs. Methylation Quantification at Trait Loci (mQTL) revealed an association between decreased methylation at the BNC2 promoter and the protective minor alleles. Expression analysis shows decreased expression of BNC2 in cancer versus normal tissue implicating tumor suppressor function of BNC2. Therefore the protective minor allele likely increases expression of BNC2 which in turn contains tumor suppressing properties that decrease ovarian cancer risk. BNC2 protein is in complex with transcriptional regulatory proteins, in particular the NURD complex components. Moreover, BNC2 acts as a transcriptional repressor in in vitro transfection assays indicating that it functions in transcriptional repression. We then used protein binding microarrays and CHIP-Seq experiments to identify its putative DNA binding motifs and its downstream target genes. Analysis of this data suggests that BNC2 functions in a regulatory transcription network that impacts on genes implicated in ovarian cancer with enrichment for genes in the TGF-beta response pathway. Citation Format: Melissa A. Buckley, Howard C. Shen, Gustavo A. Mendoza-Fandino, Nicholas T. Woods, Anxhela Gjyshi, Juliet French, Kate Lawrenson, Honglin Song, Jonathan Tyrer, Renato S. Carvalho, Alexandra Valle, Ann Chen, Sean Yoder, Gregory Bloom, Ya-Yu Tsai, Ally Yang, Timothy R. Hughes, Xiaotao Qu, Mine Cicek, Melissa Larson, Ellen Goode, Brooke Fridley, Susan Ramus, Georgia Chenevix-Trench, Paul Pharoah, Thomas A. Sellers, Simon Gayther, Alvaro N.A. Monteiro, Ovarian Cancer Association Consortium. Functional analysis of the 9p22 locus implicates the transcriptional regulation of BNC2 as a mechanism in ovarian cancer predisposition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3285. doi:10.1158/1538-7445.AM2014-3285

Notch activation augments nitric oxide/soluble guanylyl cyclase signaling in immortalized ovarian surface epithelial cells and ovarian cancer cells

Nitric oxide (NO) is generated by tumor, stromal and endothelial cells and plays a multifaceted role in tumor biology. Many physiological functions of NO are mediated by soluble guanylyl cyclase (sGC) and NO/sGC signaling has been shown to promote proliferation and survival of ovarian cancer cells. However, how NO/sGC signaling is modulated in ovarian cancer cells has not been studied. The evolutionarily conserved Notch signaling pathway plays an oncogenic role in ovarian cancer. Here, we report that all three ovarian cancer cell lines we examined express a higher level of GUCY1B3 (the β subunit of sGC) compared to non-cancerous immortalized ovarian surface epithelial (IOSE) cell lines. Interestingly, the highest expression of GUCY1B3 in ovarian cancer OVCAR3 cells is concurrent with the expression of Notch3. In IOSE cells, forced activation of Notch3 increases the expression of GUCY1B3, NO-induced cGMP production, and the expression of cGMP-dependent protein kinase (PKG), thereby enhancing NO- and cGMP-induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP, a direct PKG substrate protein). In contrast, inhibition of Notch by DAPT reduces GUCY1B3 expression and NO-induced cGMP production and VASP phosphorylation in OVCAR3 cells. Finally, we confirmed that inhibition of sGC by ODQ decreases growth of ovarian cancer cells. Together, our work demonstrates that Notch is a positive regulator of NO/sGC signaling in IOSE and ovarian cancer cells, providing the first evidence that Notch and NO signaling pathways interact in IOSE and ovarian cancer cells.

Abstract 83: Hypomethylation of TGF-beta target gene, ABCA1 in ovarian cancer and cancer initialing cell and is associated with poor prognosis in cancer patients

Abstract Dysregulation of TGF-β signaling plays a crucial role in ovarian carcinogenesis and maintaining cancer stem cell properties. We have previously identified the TGF-β responsive targets in immortalized ovarian surface epithelial cells (IOSE) by expression microarray and found that ABCA1, a family members of ATP-binding cassette is up-regulated following TGF-β treatment (Qin et al, BMC Syst Biol 2009). Thus, we hypothesize that ABCA1 may be involved in ovarian cancer and its initiation. First, we compared the expression level of ABCA1 in IOSE and a panel of ovarian cancer cell lines and found that ABCA1 was up-regulated in HeyC2, SKOV3, MCP3, and MCP2 ovarian cancer cell lines but down-regulated in A2780 and CP70 ovarian cancer cells. The down-regulation in A2780 and CP70 cells was associated with promoter hypermethylation as demonstrated by bisulphite sequencing and demethylation treatment. To investigate if ABCA1 is involved in ovarian cancer initiating cells (OCIC), we compared the methylation level of ABCA1 in ovarian tumor samples and their corresponding spheroids which is previously found to be enriched with OCIC. Lower methylation of ABCA1 could be detected in 2/6 OCIC samples as compared to their original tumors. We further analyzed the expression and methylation level of ABCA1 in CP70 spheroid (CP70sp) derived from suspension culture of CP70 ovarian cancer cells. Comparing to CP70, up-regulation of stem cell markers Nanog (3.8 fold) and Oct4 (12 fold) was detected in CP70sp cells suggesting that CP70sp may contain OCIC. Interestingly, up-regulation of ABCA1 (5.2 fold) concomitant with promoter hypomethylation was detected in CP70sp cells. Additionally, active demethylation may be involved in the hypomethylation of ABCA1 as expression of the methylcytosine dioxygenase, TET1 was up-regulated in the CP70sp cells (5.3 fold). Furthermore, ABCA1 was involved in drug resistance of ovarian cancer which is a feature of cancer initiating cells, as lenti-viral knockdown of ABCA1 in a platinum resistant MCP2 ovarian cancer cells resensitized the cells to cisplatin (IC50: shABCA1 vs shGFP: 0.267 ug/ml vs 0.402 ug/ml). We further analyzed the methylation level of ABCA1 in 97 ovarian cancer, 46 benign, and 4 normal samples using real-time quantitative MSP assay and found that higher methylation level of ABCA1 was detected in cancer than benign (P=0.051) and normal tissues (P<0.05). Importantly, cancer patients showing lower or no methylation of ABCA1 has significantly shorter progression free survival than patients with higher methylation (log rank, P=0.0463). In conclusion, ABCA1 is hypomethylated in a sub-set of ovarian cancer and its cancer initiating cells and is associated with poor prognosis in cancer patients. The role of ABCA1 in OCIC and the involvement of 5-hydroxymethylcytosine (5hmC) in the active demethylation of ABCA1 deserves further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 83. doi:10.1158/1538-7445.AM2011-83

The morphogenic function of E-cadherin-mediated adherens junctions in epithelial ovarian carcinoma formation and progression

E-cadherin expression is unusually regulated in epithelial ovarian carcinoma. It is not expressed in poorly cohesive ovarian surface epithelial (OSE) target cells, but is expressed in cohesive pre-malignant lesions and in highly cohesive, well-differentiated tumors where it is membrane associated, presumably in adherens junctions. E-cadherin expression is subsequently suppressed, or its function is disrupted, in late-stage invasive tumors. Here, we observed that increased E-cadherin expression in ovarian carcinoma cells was associated with increased E-cadherin promoter activity, increased adherens junction formation, decreased β-catenin signaling-dependent LEF-1 activity, and the generation of cohesive spheroids in basement membrane gel culture. Forced expression of wild-type E-cadherin in immortalized OSE cells initiated adherens junction formation, decreased LEF-1 activity, decreased the mesenchymal migration that is a characteristic of OSE cells that have been maintained in monolayer culture, and induced the formation of cohesive spheroids in basement membrane gels. Conversely, forced expression of a dominant-negative E-cadherin mutant in ovarian carcinoma cells disrupted adherens junctions, increased mesenchymal cell migration, and prevented spheroidal morphogenesis without altering LEF-1 signaling. Therefore, in addition to suppressing late-stage tumor progression, E-cadherin-mediated adherens junctions may also contribute to the initial emergence of a cohesive morphogenic phenotype that is a hallmark of differentiated epithelial ovarian carcinoma.

Immortalized ovarian surface epithelial cells acquire tumorigenicity by Acrogranin gene overexpression

Malignant transformation is caused by multi-step genetic mutations, and growth factors are believed to play important roles in developing and maintaining malignant phenotype. However, there is no direct evidence that a specific growth factor contributes to malignant transformation of phenotypically normal cells. In order to assess the function of Acrogranin (also known as granulin epithelial precursor; GEP) in ovarian carcinogenesis, ovarian surface epithelial (OSE) cells, which are supposed to be the origin of primary ovarian epithelial cancer, were transfected with combined genes of hTERT, SV40 LT, and Acrogranin. Introduction of hTERT and SV40 LT was sufficient for immortalizing OSE cells but not enough for tumor formation in nude mice. In contrast, transfection and overexpression of Acrogranin in immortalized OSE cells showed augmented clonogenicity in soft agar and obvious tumorigenicity in nude mice. This is the first study showing evidence that a specific growth factor plays a direct role in malignant transformation in ovarian cancer development.

4‐HPR modulates gene expression in ovarian cells

Ovarian cancer has a high rate of recurrence and subsequent mortality following chemotherapy despite intense efforts to improve treatment outcomes. Recent trials have suggested that retinoids, especially 4-(N-hydroxyphenyl) retinamide (4-HPR), play an important role as a chemopreventive agent and are currently being used in clinical trials for ovarian cancer chemoprevention as well as treatment. This study examines the mechanism of its activity in premalignant and cancer cells. We investigated the modulation of gene expression by 4-HPR in immortalized ovarian surface epithelial (IOSE) cells and ovarian cancer (OVCA433) cells with DNA microarray. Real time RT-PCR and western blotting were used to confirm the microarray results and metabolic changes were examined with optical fluorescence spectroscopy. 4-HPR resulted in an up-regulation of expression of proapoptotic genes and mitochondrial uncoupling protein in OVCA433 cells and modulation of the RXR receptors in IOSE cells, and down-regulation of mutant BRCA genes in both IOSE and OVCA433 cells. 4-HPR had a larger effect on the redox in the 433 cells compared to IOSE. These findings suggest that 4-HPR acts through different mechanisms in premalignant ovarian surface cells and cancer cells, with a preventive effect in premalignant cells and a treatment effect in cancer cells.