Abstract 795: Epigenetics, cell cycle and drug-resistant ovarian cancer

Abstract

Abstract Epithelial ovarian cancer (EOC) is the most lethal of the gynaecological malignancies and currently stage III/IV disease is associated with a 25% 5 year survival rate. A major factor associated with advanced EOC is the development of drug resistant disease, characterised by a lack of response to platinum and taxane based agents. In an effort to clarify mechanisms underlying chemorefractory EOC we have developed a number of drug resistant cancer cell lines. DNA microarray screening of drug resistant versus drug sensitive cell lines (combining a methylation reversal step using demethylating agents) indicated down-regulation of a number of genes with evidence of epigenetic silencing by promoter methylation in many cases. We have identified P57kip2 a cyclin dependent kinase inhibitor (CDKi) as a gene that is subjected to methylation in carboplatin-resistance, as shown in the PEO1CarbR line. Using a siRNA approach, silencing of the p57kip2 gene in drug sensitive parental PEO1 or SKOV-3 cells gave rise to a reduced sensitivity to platinum agents i.e. cisplatin or carboplatin as assessed using apoptosis with the annexinV assay and flow cytometry. Using chemosensitivity testing (MTT) of PEO1 parental and PEO1CarbR lines versus seliciclib (a CDK2 inhibitory drug) we saw an association with sensitivity to this agent with down-regulation of p57kip2. The IC50 values obtained for the human ovarian cancer cell line PEO1 and PEO1CarboR were 27.4 and 19.2μM, respectively (p = <0.05). Cell cycle analysis of seliciclib treated cells indicated a prolonged S-phase as a marker of increased sensitivity to the drug and an increased apoptotic response in p57kip2 silenced cells. Our data suggest that CDK inhibitors such as seliciclib may have utility in tumours that show downregulation of the endogenous CDKi p57kip2 and identification of such patients may indicate a sub-group that respond preferentially to seliciclib. The same screening platform also enabled us to identify wee1 and stratifin (14-3-3α) as genes significantly downregulated in taxane-resistant cell lines. The 2 gene products are important in maintaining genomic stability alongside other proteins such as Chk1 and ATR. Wee1 controls G2M by keeping CDK2/Cyclin B inactive whilst binding of 14-3-3 isoforms enhances its activity. Demethylation treatment using 5′aza-cytidine and Trichostatin A unmasked gene expression, indicating that these genes are hypermethylated in taxane-resistant cell line. Current work aims to confirm further the role of these 2 genes in drug resistance by a series of knock-in and knock-out experiments. 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 795. doi:1538-7445.AM2012-795