Abstract 3737: Absence of p57kip2 as a new biomarker of chemoresponsiveness in epithelial ovarian cancer

Abstract

Abstract Epithelial ovarian cancer (EOC) is the most lethal gynaecological malignancy with a 5 year survival rate of around 25%. A major factor in the poor survival of patients with EOC is the development of drug resistant disease, whereby agents used first-line such as carboplatin and paclitaxel become ineffective. In an effort to understand the factors that may underlie chemoresistant disease we have developed novel models of platinum-resistant EOC. Microarray analysis of SKOV-3CR cells (with acquired carboplatin-resistance), PEO1CisR (with acquired cisplatin-resistance) and PEO1CarboR (acquired carboplatin-resistance) showed a marked decrease in a number of genes including the CDK inhibitor p57kip2. Q-PCR and western-immunoblotting verified the expression levels of p57kip2 seen in the drug resistant cells in comparison with their parental, drug sensitive counterparts. For PEO1CarboR cells for example the levels of p57kip2 were virtually undetectable. We carried out a technique of methylation reversal microarray whereby parental and resistant cells were treated with the methylation reversal agents aza-cytidine and zebularine and then subjected to DNA microarray analysis. Such a technique provided us with prima facia evidence that a number of genes may be silenced by epigenetic modification as a consequence of acquired drug resistance. We were able to identify p57kip2 amongst our candidate gene list. Methylation specific PCR (MSP) was carried out on the CpG island in the 5′ region of the p57kip2 gene and we found a clear increase in methylation in the PEO1CisR and PEO1CarboR cell lines compared with the PEO1 parental cells in which no methylated DNA was detected under the same experimental conditions. Using an SiRNA approach, we have silenced p57kip2 in both parental SKOV-3 and PEO1 cells and seen significant changes in chemoresponse to the clinically relevant agents carboplatin and cisplatin. We assessed the apoptotic response of ovarian cancer cells to the effects of both agents following a 48h treatment using annexinV and propidium iodide with flow cytometry. We saw a marked decrease in apoptotic response to platinum agents in both the SKOV-3 and PEO1 cell lines following silencing of the p57kip2 gene. Importantly, we have gone on to look at a series of primary EOC cases whereby we have extracted mRNA from surgically obtained ovarian tumours. Measurement of p57kip2 mRNA in a cohort of 20 cases of EOC showed a statistically significant reduction in cases who gave a partial or no response to platinum versus complete responders p = 0.034 (using a one way ANOVA). We are currently expanding our studies using matched serum and tumour tissue from a much larger cohort of EOC patients (>100 cases) and will be extracting DNA for the presence of methylated p57kip2. We hypothesize that the presence of methylated p57kip2 can be used as a marker of platinum response in EOC. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 3737.