Abstract 1620: Effects of γ-glutamyl hydrolase and folylpolyglutamyl synthase modulation on gene-specific promoter CpG island methylation

Abstract

Abstract Background: Folate mediates the transfer of one-carbon units for most biological methylation reactions including DNA methylation, which is catalyzed by DNA methyltransferase (DNMT). Genomic DNA hypomethylation and gene-specific promoter CpG island hypermethylation are important epigenetic mechanisms of carcinogenesis. DNA methylation and DNMT also are potential therapeutic targets and may modify the effect of specific chemotherapeutic agents. Intracellular folate homeostasis is maintained by folylpolyglutamate synthase (FPGS) and γ-glutamyl hydrolase (GGH) for folate-dependent one-carbon transfer reactions and antifolate-induced cytotoxic effects. We have previously shown that GGH/FPGS modulation significantly affects global DNA methylation and DNMT activity in human HCT116 colon and MDA-MB-435 breast cancer cells in a predictably manner. We investigated whether GGH/FPGS modulation would also affect gene-specific promoter CpG island methylation. Methods: We generated an in vitro model of GGH overexpression/inhibition in HCT116 and MDA-MB-435 cells by transfecting the cells with the sense GGH cDNA or GGH-targeted siRNA, respectively. An in vitro model of FPGS overexpression/inhibition in HCT116 was generated by transfecting the cells with the sense or antisense FPGS cDNA, respectively. An in vitro model of FPGS overexpression/inhibition in MDA-MB-435 cells was generated by transfecting cells with the sense FPGS cDNA or FPGS-targeted siRNA, respectively. Illumina Infinium DNA methylation assay was used to interrogate 27,578 individual CpG loci in 14,495 different gene promoters. Results: MDA-MB-435 cells showed more CpG methylation alterations in response to GGH and FPGS modulation than HCT116 cells. Sixty-one hyper- and 54 hypomethylated genes were common between HCT116 and MDA-MB-435 cell lines in GGH overexpression while 117 hyper- and 129 hypomethylated genes were common between these cell lines in GGH inhibition. In both cell lines, 64 hyper- and 52 hypomethylated genes were common in FPGS overexpression whereas 31 hyper- and 38 hypomethylated genes were common in FPGS inhibition. Preliminary analysis demonstrated that genes associated with efflux and influx of (anti)folate (ABCC1, ABCC2, ABCC3, ABCG2, SLC19A1), de novo purine synthesis, glutathione transferase (GSTM1, GSTP1, GSTT1), growth regulation (IGF2, IGFBP3), mismatch repair (MLH1), tumor suppressor (PTEN) and transcription (RUNX3) were hyper- or hypomethylated in both cell lines. Conclusions: Our data indicate that GGH/FPGS modulation can affect gene-specific promoter CpG island methylation as well as genomic DNA methylation, which might influence chemosensitivity of colon and breast cancer cells to 5FU and antifolates. Studies are underway to investigate the functional ramifications of the altered CpG methylation in response to GGH/FPGS modulation in these cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1620. doi:10.1158/1538-7445.AM2011-1620