DNA methylation changes associated with acquired platinum resistance in ovarian cancer

Abstract

Despite high responses to initial chemotherapy most patients with ovarian cancer (OC) relapse and inevitably die from their disease. Aberrant DNA methylation is frequently seen in ovarian tumours and may provide biomarkers of clinical outcome or insight into mechanisms of chemoresistance. We firstly performed Differential Methylation Hybridisation (DMH) to identify loci that gained methylation between 34 matched cisplatin sensitive and resistant OC tumour cell lines. Differentially methylated loci identified were further validated by Methylation Specific PCR (MSP) and bisulphite pyrosequencing. Selected loci were further investigated for association with clinical outcome in primary OC tumour samples and matched tumour samples from patients' pre- and post-chemotherapy. Frequent increased methylation of a CpG island at the NR2E1 gene was identified in this experiment. Increased methylation correlated with decreased gene expression and could be reversed following treatment with a demethylating agent. Increased methylation at NR2E1 was observed between matched pre- and post-treatment tumour pairs. A novel biostatistical method, methylation linear discrimination analysis (MLDA), was next used to identify differentially methylated loci in sensitive and resistant A2780 human ovarian cell lines. Eight of nine loci identified were validated by MSP. A locus at the SP5 gene was further investigated by pyrosequencing and found to show a very high level methylation in most cell lines and ovarian tumours. Increased methylation correlated with decreased gene expression and this could be reversed using decitabine treatment. Knockdown of SP5 expression caused increased apoptosis. DMH was next used to identify loci that gained methylation between 3 in vivo derived matched sensitive and resistant cell lines. KIAA1383, a gene of unknown function, was identified and methylation shown to correlate with response to chemotherapy and progression-free survival (PFS) in patients with OC. Over-expression was found to attenuate the response to cisplatin, in the PEA2 cell line, as measured by cell cycle analysis