Oncogene (2012) 31, 4567–4576 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ONCOGENOMICS Candidate DNA methylation drivers of acquired cisplatin resistance in ovarian cancer identified by methylome and expression profiling C Zeller1, W Dai1, NL Steele2, A Siddiq3, AJ Walley3, CSM Wilhelm-Benartzi1, S Rizzo4, A van der Zee5, JA Plumb6 and R Brown1,4 1Epigenetics Unit, Department of Surgery and Cancer, Imperial College London, London, UK; 2Beatson West of Scotland Cancer Centre, Glasgow, UK; 3Department of Genomics of Common Disease, School of Public Health, Hammersmith Hospital, London, UK; 4Section of Medicine, Institute of Cancer Research, Sutton, UK; 5Department of Obstetrics and Gynecology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands and 6Cancer Research UK Beatson Laboratories, Institute of Cancer Sciences, University of Glasgow, Glasgow, UK Multiple DNA methylation changes in the cancer Keywords: drug resistance; cisplatin; DNA methylation; methylome are associated with the acquisition of drug ovarian cancer; DNMTi; HDACi resistance; however it remains uncertain how many represent critical DNA methylation drivers of chemore- sistance. Using isogenic, cisplatin-sensitive/resistant ovar- ian cancer cell lines and inducing resensitizaton with demethylating agents, we aimed to identify consistent Introduction methylation and expression changes associated with chemoresistance. Using genome-wide DNA methylation Ovarian cancer often presents at an advanced stage with profiling across 27 578 CpG sites, we identified loci at limited chances for curative treatment (Ozols, 2004). 4092 genes becoming hypermethylated in chemoresistant Common treatment strategies include debulking surgery A2780/cp70 compared with the parental-sensitive A2780 in combination with chemotherapy, which usually cell line. Hypermethylation at gene promoter regions is consists of a platinum-based compound, such as often associated with transcriptional silencing; however, cisplatin/carboplatin and a taxane, for example, pacli- expression of only 245 of these hypermethylated genes taxel. Despite responses to first-line chemotherapy in a becomes downregulated in A2780/cp70 as measured by high proportion of patients, many will relapse and microarray expression profiling. Treatment of A2780/ eventually develop resistance to currently available cp70 with the demethylating agent 2-deoxy-50-azacytidine treatment options, making the acquisition of clinical induces resensitization to cisplatin and re-expression of 41 drug resistance one of the major challenges in ovarian of the downregulated genes. A total of 13/41 genes were cancer therapy and a limiting factor in patient survival consistently hypermethylated in further independent (Cannistra, 2004). cisplatin-resistant A2780 cell derivatives. CpG sites at 9 Platinum drugs are DNA cross-linking agents exert- of the 13 genes (ARHGDIB, ARMCX2, COL1A, FLNA, ing their effect mainly via the formation of intra-strand FLNC, MEST, MLH1, NTS and PSMB9) acquired adducts between adjacent guanosines that interfere with methylation in ovarian tumours at relapse following transcription and replication, eventually leading to cell chemotherapy or chemoresistant cell lines derived at the death (Kartalou and Essigmann, 2001; Wang and time of patient relapse. Furthermore, 5/13 genes Lippard, 2005). Several mechanisms have been sug- (ARMCX2, COL1A1, MDK, MEST and MLH1) gested to participate in conferring platinum-resistant acquired methylation in drug-resistant ovarian cancer- properties to a tumour cell such as genetic alterations in sustaining (side population) cells. MLH1 has a direct role genes involved in DNA repair, drug uptake, apoptosis, cell cycle control and IGF signalling pathways (John- in conferring cisplatin sensitivity when reintroduced into cells ONCOGENOMICS in vitro. This combined genomics approach has identified stone et al., 2002; Luqmani, 2005; Edwards et al., 2008; further potential key drivers of chemoresistance whose Broxterman et al., 2009; Eckstein et al., 2009). More expression is silenced by DNA methylation that should be recently, it has been proposed that, in addition to further evaluated as clinical biomarkers of drug resistance. genetic changes, aberrant epigenetic marks can critically Oncogene (2012) 31, 4567–4576; doi:10.1038/onc.2011.611; contribute to the acquisition of drug resistance (Sharma published online 16 January 2012 et al., 2010). In cisplatin-resistant cancer cells, in particular, multiple DNA methylation changes at promoter CpG islands and associated transcriptional Correspondence: Professor R Brown, Epigenetics Unit, Department of gene silencing have been reported (Teodoridis et al., Surgery and Cancer, Imperial College London, Hammersmith 2005; Dai et al., 2008; Chang et al., 2010). For instance, Campus, IRDB 4th Floor, London W12 0NN, UK. E-mail: [email protected] methylation at MLH1, a mismatch repair gene, is Received 23 May 2011; revised 29 October 2011; accepted 28 November acquired in about 25–35% of ovarian cancer patients 2011; published online 16 January 2012 following platinum-based chemotherapy and has been Epigenetic drivers of resistance in ovarian cancer C Zeller et al 4568 shown to be associated with poor patient survival line model that consists of sensitive and matched (Strathdee et al., 1999; Gifford et al., 2004). Impor- isogenic platinum-resistant lines derived from A2780 tantly, reversal of MLH1 epigenetic silencing by cells by repeated exposures to increasing levels of demethylation or re-expression of the gene was demon- cisplatin (Anthoney et al., 1996). We performed strated to resensitise tumour cells to subsequent genome-wide DNA methylation profiling of resistant chemotherapeutic treatment in vitro and in vivo (Plumb A2780/cp70, A2780/MCP1 and A2780/MCP6 com- et al., 2000; Papouli et al., 2004; Steele et al., 2009). pared with non-selected A2780p5 and A2780p6 clones MLH1 might, therefore, represent one of the key genes employing Infinium HumanMethylation27 BeadArrays driving chemoresistance in ovarian cancer cell lines. that comprise 27 578 CpG sites across more than 14 000 However, although the role of methylation changes at genes. In a first step, differentially methylated genes MLH1 has been well characterized, for the majority of were extracted as showing a significantly increased or aberrant DNA methylation events it is not particularly decreased difference of beta (7Db7) corresponding to a clear whether they are associated with response to false discovery rate (FDR) o0.05 estimated from bio- chemotherapy or are just occurring by chance due to a logical replicates within the study (7Db7B0.1) between methylator phenotype or simply as random methylation A2780/cp70 versus A2780 at X1 associated CpG site events (Issa, 2004) during platinum selection or DNA (see Materials and methods). Using these criteria, we damage induction. In analogy to the concept of identified multiple methylation changes between A2780/ ‘driver and passenger’ mutations emerging during cp70 versus A2780 (Figure 1): X1 CpG sites at 4092 carcinogenesis, methylation changes could either repre- genes were hypermethylated, whereas only 1289 genes sent ‘drivers’ of chemoresistance based on their potential became hypomethylated following exposure to cisplatin, to provide the cell with a selective advantage or suggesting that hypermethylation occurs more fre- ‘passenger’ events, with no substantial impact on quently than hypomethylation during the process of chemosensitivity (Greenman et al., 2007). selection for acquired cisplatin chemoresistance. We hypothesized that, in analogy to driver mutations, there might be a subset of epigenetic changes that are causally associated with the acquisition of chemoresis- Methylation changes in cisplatin-resistant lines associate tance. In order to identify the proportion of epigeneti- with gene expression changes in only a subset of genes cally altered genes driving platinum resistance in ovarian In a second step, we examined the expression profiles of cancer, we have analysed acquired DNA methylation sensitive A2780 and resistant A2780/cp70 cell clones changes in a human ovarian cancer cell line model of using Affymetrix HG-U133 Plus 2.0 Arrays and cisplatin resistance and expression changes associated identified differentially expressed genes by applying with acquired resistance or following resensitization rank products analysis (Breitling et al., 2004). Aberrant with demethylating agents. We then examined the DNA methylation at CpG island (CGI) has been shown generality of the changes observed in ovarian tumours to critically affect gene expression and is strongly at relapse following chemotherapy. associated with transcriptional repression (Jones and Baylin, 2002; Esteller, 2008). We assumed that expres- sion changes associating with hypermethylation in chemoresistant A2780/cp70 as compared with A2780 Results would likely represent silencing events associated with the drug-resistant phenotype. We identified a total of Cisplatin selects preferentially for DNA hypermethylation 1370 genes showing significant gene expression changes, in A2780-chemoresistant cell lines with 687 genes going up and 683 genes going down in To identify DNA methylation changes associated with resistant versus sensitive cell lines. We further filtered for changes in gene expression and differential cisplatin those genes where hypermethylation associated with chemosensitivity, we used the human A2780