Multiple DNA methylation changes in the cancer methylome are associated with the acquisition of drug resistance; however it remains uncertain how many represent critical DNA methylation drivers of chemoresistance. Using isogenic, cisplatin-sensitive/resistant ovarian cancer cell lines and inducing resensitizaton with demethylating agents, we aimed to identify consistent methylation and expression changes associated with chemoresistance. Using genome-wide DNA methylation profiling across 27 578 CpG sites, we identified loci at 4092 genes becoming hypermethylated in chemoresistant A2780/cp70 compared with the parental-sensitive A2780 cell line. Hypermethylation at gene promoter regions is often associated with transcriptional silencing; however, expression of only 245 of these hypermethylated genes becomes downregulated in A2780/cp70 as measured by microarray expression profiling. Treatment of A2780/ cp70 with the demethylating agent 2-deoxy-5 0 -azacytidine induces resensitization to cisplatin and re-expression of 41 of the downregulated genes. A total of 13/41 genes were consistently hypermethylated in further independent cisplatin-resistant A2780 cell derivatives. CpG sites at 9 of the 13 genes (ARHGDIB, ARMCX2, COL1A, FLNA, FLNC, MEST, MLH1, NTS and PSMB9) acquired methylation in ovarian tumours at relapse following chemotherapy or chemoresistant cell lines derived at the time of patient relapse. Furthermore, 5/13 genes (ARMCX2, COL1A1, MDK, MEST and MLH1) acquired methylation in drug-resistant ovarian cancersustaining (side population) cells. MLH1 has a direct role in conferring cisplatin sensitivity when reintroduced into cells in vitro. This combined genomics approach has identified further potential key drivers of chemoresistance whose expression is silenced by DNA methylation that should be further evaluated as clinical biomarkers of drug resistance.

Platinum-based chemotherapy, with cytoreductive surgery, is the cornerstone of treatment of advanced ovarian cancer; however, acquired drug resistance is a major clinical obstacle. It has been proposed that subpopulations of tumor cells with stem cell-like properties, such as so-called side populations (SP) that overexpress ABC drug transporters, can sustain the growth of drug-resistant tumor cells, leading to tumor recurrence following chemotherapy. The histone methyltransferase EZH2 is a key component of the polycomb-repressive complex 2 required for maintenance of a stem cell state, and overexpression has been implicated in drug resistance and shorter survival of ovarian cancer patients. We observed higher percentage SP in ascites from patients that have relapsed following chemotherapy compared with chemonaive patients, consistent with selection for this subpopulation during platinum-based chemotherapy. Furthermore, ABCB1 (P-glycoprotein) and EZH2 are consistently overexpressed in SP compared with non-SP from patients' tumor cells. The siRNA knockdown of EZH2 leads to loss of SP in ovarian tumor models, reduced anchorageindependent growth, and reduced tumor growth in vivo. Together, these data support a key role for EZH2 in the maintenance of a drug-resistant, tumor-sustaining subpopulation of cells in ovarian cancers undergoing chemotherapy. As such, EZH2 is an important target for anticancer drug development.