Identification of Hypermethylated Genes Associated with Cisplatin Resistance in Human Cancers

Abstract

Cisplatin is among the most widely used cytotoxic anti-cancer agents in solid tumors, however, the development of secondary resistance remains a major obstacle to clinical efficacy. Treatment-related DNA hypermethylation may play a role in creating drug resistant phenotypes by inactivating genes that are required for cytotoxicity. We applied a pharmacologic unmasking approach to detect hypermethylated genes whose inactivation contributes to cisplatin resistance. Utilizing three pairs of isogeneic, cisplatin-sensitive and -resistant cell lines derived from two parental cell lines (KB-3-1 and SCC25), we identified several hundred genes that were down-regulated in each resistant cell line and re-activated by the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-dC). Among them, 30 genes were common to ≥ 2 cell lines, and/or reported to be down-regulated in previous studies. Bisulfite sequencing confirmed that 14 genes were hypermethylated in resistant cell lines, but not in the sensitive parental cell lines. Six of 14 genes (SAT, C8orf4, LAMB3, TUBB, G0S2, MCAM) were cisplatin-inducible in sensitive, but not in resistant cell lines. siRNA knockdown of two genes, SAT and S100P, increased cell viability with cisplatin treatment in sensitive parental cell lines. S100P knockdown significantly decreased the S-phase fraction (SPF) of parental sensitive cell lines and slowed cell proliferation, which was associated with decreased sensitivity to cisplatin. Based on these findings, we conclude that DNA methylation is a frequent event in cells that are chronically exposed to cisplatin, and that methylation-induced gene silencing may play a role in the development of resistance to cytotoxic chemotherapeutic agents.

Candidate DNA methylation drivers of acquired cisplatin resistance in ovarian cancer identified by methylome and expression profiling

“…Our results indicate that several methylation changes are directly associated with chemosensitization induced by demethylating agents combined with HDAC inhibitors. Similar observations have been reported in other tumour types (Chang et al, 2010); however, our data also reveal that epigenetic alterations associated with sensitivity to cisplatin occur at a few selected genes rather than at large numbers of loci in this cell line model (Glasspool et al, 2006). The group of epigenetically inactivated genes was upregulated by DAC and also the combined DAC and Belinostat treatment, but remained unaffected by Belinostat alone, with the exception of GLUL.…”

“…Among those changes, hypermethylation at CGI promoters and associated epigenetic silencing are prevalent in various cell line models (Dai et al, 2008;Li et al, 2009;Chang et al, 2010) and are thought to, either alone or in combination with genetic changes, account for the loss of expression of key genes in the platinum-responsive phenotype. However, our knowledge of whether these DNA methylation changes and aberrantly inactivated genes are actually driving chemoresistance is limited.…”

“…More recently, it has been proposed that, in addition to genetic changes, aberrant epigenetic marks can critically contribute to the acquisition of drug resistance (Sharma et al, 2010). In cisplatin-resistant cancer cells, in particular, multiple DNA methylation changes at promoter CpG islands and associated transcriptional gene silencing have been reported (Teodoridis et al, 2005;Dai et al, 2008;Chang et al, 2010). For instance, methylation at MLH1, a mismatch repair gene, is acquired in about 25-35% of ovarian cancer patients following platinum-based chemotherapy and has been shown to be associated with poor patient survival (Strathdee et al, 1999;Gifford et al, 2004).…”

Characterization of regulatory sequences in alternative promoters of hypermethylated genes associated with tumor resistance to cisplatin

“…However, it remains puzzling that genomic expression patterns of platinum-sensitive̸-resistant determinants in several cell lines, including squamous cell carcinoma of the head and neck, bladder and ovarian cancers, do not always correlate with the results of biochemical analyses (4)(5)(6). Additionally, epigenetic changes in the DNA methylation profiles during cancer development are associated with the acquisition of cisplatin resistance (6)(7)(8)(9). However, the problem of platinum resistance at the genomic and epigenomic levels has not been clearly defined.…”

“…The TFBS and the CGIs targeted for DNA methylation, significantly affect the functional activities of alternative promoters (6)(7)(8)(9)18,19). To date, the role and molecular characteristics of alternative promoters have not been investigated in platinum drug resistance.…”