Platinum compounds have served as a standard therapy for post-surgical ovarian cancer patients for over two decades, as well as for other malignancies, including testicular, bladder, lung, endometrial, and head and neck cancers . Acquired or de novo resistance to platinum-based chemotherapy is commonly observed in ovarian cancer, with numerous underlying mechanisms now proposed to explain this phenomenon, including drug inactivation, elevated resistance to apoptosis, decreased recognition of DNA damage, and increased DNA repair [8, 9, 53, 54]. Accumulating evidence now shows that aberrant epigenetic alterations contribute to these chemoresistance-associated phenomena, perhaps even more so than genetic aberrations [10, 16]. By comparing and combining genome-wide gene expression and methylation changes observed in cisplatin-sensitive and -resistant ovarian cancer sublines, we discovered both novel and reported pathways and gene ontology (GO) groups likely to mediate acquired cisplatin resistance. These included cell cycle progression (G2/M checkpoint), response to DNA damage, nucleotide binding, and various cellular metabolic processes, in agreement with previous reports  and our previous study , further supporting a role for promoter CpG island (CGI) methylation in disrupting gene expression during tumor progression.
One possible explanation for our observed chemoresistance-associated changes in DNA methylation patterns is aberrant activity or substrate specificity of DNA methyltransferase (DNMT) enzymes [56, 57]. In the current study, we observed modest but highly significant upregulation of both DNMT1 and DNMT3B in the cisplatin-resistant Round5 (i.e., treated for five drug cycles) A2780 subline (Table 1), suggesting that the altered methylation profile in these cells may be associated with increased or altered DNMT activity. In support of this possibility, a number of other studies have demonstrated pharmacologic or genetic downregulation of DNMT1 and DNMT3B enhanced chemosensitivity to various platinum drugs, including cisplatin [31, 58–61]. Our current results demonstrate that 5-aza-dC and another methylation inhibitor, zebularine, dose-dependently restored chemosensitivity to Round5 cells (Figure 2), and previously we reported upregulation of DNMT1 and DNMT3B in ovarian cancer cell lines, along with a potential positive correlation between DNMT1 overexpression and tumor aggressiveness . Alterations in DNMT isoforms have also been reported in ovarian cancer, with a complex relationship between global DNA hypomethylation and regional hypermethylation [63, 64], while one study of serous endometrioid cancer actually demonstrated DNMT downregulation .
In addition to aberrant DNMT enzyme activity, drug-induced de novo promoter methylation has been hypothesized . This phenomenon is believed to be due to DNA structural distortions resulting from the formation of cisplatin-DNA adducts, allowing subsequent access of DNMTs and/or other auxiliary methylation machinery components to target DNA regions [67, 68]. It has also been demonstrated that endogenous DNA damage can alter the site selectivity of DNMT1 or recruit DNMTs to sites of repair [69, 70]. The fact that chemoresistance is reproducibly reversible by inhibition of DNA methyltransferases further suggests that patients whose cancers have methylation of multiple relevant genes might be selected as candidates for demethylating therapy, in addition to platinum-based ovarian cancer chemotherapy, an approach that our group (National Institutes of Health, NIH, Study NCT00477386) and others (NIH studies NCT00748527 and NCT00529022) are taking to the clinic .
Our promoter CGI methylation profiles from A2780 sublines representing early, intermediate and late-stage cisplatin resistance demonstrated that the total number of hypermethylated genes linearly increased (Spearman correlation 0.99) with increasing cisplatin resistance (Figure 1B). This positive correlation suggests that in concert with altered promoter DNA methylation, distinct methylation profiles progressively may emerge during the development of drug resistance. Our previous studies of methylation profiling of late stage ovarian cancer patient tumors correlated CGI methylation and disease recurrence [22, 23], further supporting an association between progressive methylation patterns and advanced disease [24, 72]. Such methylation patterns may disrupt specific intracellular signaling pathways, and while using genome-wide approaches to demonstrate direct regulation of gene expression by aberrant promoter CGI methylation has been a challenge in the epigenomics field, direct or indirect biological outcomes of epigenetic modifications commonly associate with specific cellular behavior changes.
To assess the role of promoter CGI methylation in cisplatin resistance, we examined biological pathways potentially dysregulated by hypermethylation (Table 2), showing likely DNA hypermethylation-downregulated pathways such as CAMs, tight junction formation, PPAR signaling, and leukocyte transendothelial migration pathways. CAMs and tight junctions, by affecting signal transduction pathways, are both directly involved in the regulation of cell proliferation, differentiation, and apoptosis [73, 74], and loss of functional tight junctions has been associated with tumorigenesis . Specifically, hypermethylation-associated downregulation of numerous claudins, integral membrane protein constituents of tight junctions, has been demonstrated to associate with tumorigenesis and tumor invasion in ovarian and other cancers, including those of the breast, bladder, and colon [76–80]. In addition to alterations in claudin and CAM functions, we observed hypermethylation and downregulation of gene products previously hypothesized as suppressors of ovarian tumor progression, including alpha-integrins (possible regulators of cell proliferation and adhesion), carnitine palmitoyltransferase I (CPT1A, a protein believed to play a role in histone deacetylase inhibition), and N-cadherin (CDH2), a member of the cell adhesion proteins often lost during tumor progression [81–84]. Consequently, using global approaches, our analyses identified several potential links between cell adhesion and the acquisition of chemoresistance, in the context of regulation by epigenetic modification.
By using a similar approach for hypermethylated and downregulated genes in the cisplatin-resistant cells, we identified several pathways likely regulated by promoter CGI hypomethylation, including those characteristic of other cancers, such has pancreatic, prostate, colorectal, non-small cell lung cancers, chronic myeloid leukemia, glioma, and melanoma (Table 3). Interestingly, all significantly enriched pathways were found to be cancer-related, and those broad-based cancer categories commonly included signal transduction pathways such as phosphatidylinositol kinase/Akt, transforming growth factor-beta, the E2F transcription factor family, and platelet-derived growth factor signaling (PDGFR). Specifically, the PI3K/Akt pathway has been shown to contribute to cisplatin resistance by promoting cell proliferation and increasing drug metabolism and resistance to apoptosis [85, 86], while E2F transcription factors have been previously implicated in platinum-resistant ovarian cancer [87–89]. Moreover, while regulation of TGFBR2 by promoter CGI methylation has previously been reported in lymphoma , a role for promoter hypomethylation in dysregulation of that pathway was previously unknown.
Taken together, our pathway analyses suggest significant upregulation of tumor-promoting cascades by hypomethylation and disruption of tumor-suppressive functions by hypermethylation. While many of these genes/pathways have been previously implicated in cisplatin drug response/resistance , we also identified various cellular mediators (CIPT1A, alpha-integrins) previously unreported in the action of that widely used chemotherapeutic agent. Finally, although others have reported proteomic [27, 28], chromosomal [29, 30], gene expression [13, 25, 54], or histone/DNA modification  profiles of cancer cisplatin resistance, we believe this is the first report integrating aberrations in DNA methylation with changes in gene expression to identify likely drug sensitivity-associated biological pathways.