Abstract 146: Zinc deficiency and G0s2 DNA methylation in rat esophageal carcinogenesis

Abstract

Abstract Objectives: Epigenetic processes, including DNA methylation and covalent modification of histone proteins, are key events in carcinogenesis. Several nutrients, including methionine and folate, that influence cancer susceptibility also affect epigenetic processes. Zinc deficiency (ZD) is implicated in the pathogenesis of human esophageal cancer. In the rat, a ZD diet induces esophageal cell proliferation, modulates genetic expression, and enhances carcinogenesis. Zinc-replenishment (ZR) reverses these events and inhibits tumorigenesis. Here we examined the impact of zinc on epigenetic processes during N-nitrosomethylbenzylamine (NMBA)-induced esophageal carcinogenesis and its prevention. Methods: We evaluated DNA-methyltransferase activity by S-adenosyl-L(methyl-3H) methionine incorporation in esophageal mucosa from zinc-modulated rats without NMBA treatment; at 5 weeks after the first NMBA dose; and at 15 weeks (endpoint) after 3 NMBA doses. The ZD rats had significantly higher tumor incidence (100% vs 16.6%, P<0.001) and multiplicity (11 ± 3.8 vs 0.5 ± 0.3, P<0.001) than control zinc-sufficient (ZS) rats. Replenishing zinc after the first NMBA dose led to a lower tumor incidence (28.9% vs 100%) and multiplicity (0.6 ± 0.4 vs 11 ± 3.8) in ZR than ZD rats (P<0.001). In parallel, we used Sequenom MassARRAY methylation and pyrosequencing technologies to determine if promoter region DNA CpG hypermethylation contributes to the down-regulation of 6 selected genes G0s2, P2rx2, Scl15a1, Upk1b, Tpm1, and Plag1 in hyperplastic ZD vs ZS esophagi, as identified by expression profiling (Gastroenterology, 136: 953-966, 2009). Results: DNA-methyltransferase activity was markedly higher in hyperplastic (NMBA-untreated), dypsplastic (5 weeks post NMBA dose), and neoplastic (endpoint) ZD esophagi, as compared with respective ZS controls. Among the 6 genes tested, G0s2, the G0/G1 switch gene, was the only gene with DNA CpG hypermethylation in hyperplastic, dysplastic, and neoplastic ZD esophagi, vs non-neoplastic ZS controls. ZR restored G0s2 hypermethylation to ZS levels. qRT-PCR showed that G0s2 mRNA expression was lower in hyperplastic ZD than ZS esophagi. Surprisingly, G0s2 mRNA expression was upregulated by more than 5-fold in neoplastic ZD esophagi vs non-neoplastic ZS controls, a result consistent with expression profiling data (unpublished). Conclusions: These data suggest that methylation of G0s2 promoter is not a crucial mechanism for regulation of G0s2 expression in ZD-driven esophageal neoplasia. Given that G0s2 hypermethylation is associated with head and neck cancer and overexpression occurs in disease syndromes associated with impaired immune function and ZD such as systemic lupus erythematodes, the mechanism by which G0s2 expression is regulated deserves further investigation. Supported by NIH grant R21 CA127085. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 146.