Abstract 1245: Reactivation of growth/differentiation factor 1 contributes to the chemopreventive effect of 5-aza-2′deoxycytidine in gastric cancer

Abstract

Abstract INTRODUCTION: Helicobacter pylori (HP) is a type 1 carcinogen for gastric cancer (GC), the second most common cause of cancer-related death worldwide. The limited benefit of HP eradication or nutritional intervention in risk reduction and the dismal prognosis of GC underlie the urgent need for new preventive strategies. We have recently shown that Hp causes aberrant DNA methylation of tumor suppressor genes to promote gastric carcinogenesis (Cheng et al. 2013). However, the functional and mechanistic relationships between aberrant DNA methylation and GC remain elusive. AIMS&METHODS: We investigated the effect of 5-aza-2′-deoxycytidine (5′Aza-dC), a FDA-approved demethylating agent in a murine GC model induced by N-Nitroso-N-methylurea (MNU). Using MethylCap-microarray, quantitative RT-PCR and Western blot, we identified novel DNA methylation-controlled genes in paired GC tumors/adjacent tissues (4), 5′Aza-dC-treated (3) and -untreated (3) normal mucosa samples, followed by pyrosequencing and gene expression validation in human GC samples and cell lines. Gene functions were investigated by cell cycle and colony formation assays. RESULTS: At 52 weeks-post MNU exposure, GC was developed in 8 out of 19 mice. Administration of 5′Aza-dC for 24 weeks significantly reduced GC incidence from 42.1 to 11.1% (2/18 mice; p<0.05). Microarray analysis uncovered 12 significant and recurrent hypermethylated genes exclusive in GCs. However, only growth/differentiation factor 1 (Gdf1), a ligand for transforming growth factor-beta (TGF-β) signaling, had significantly lower mRNA expression in tumors compared to both tumor-adjacent and normal tissues (p<0.05). Notably, 5′Aza-dC treatment reactivated Gdf1 expression to the normal mucosal level. In human GCs, aberrant GDF1 methylation (>1.5-fold increase compared to tumor-adjacent tissues) was observed in 58.3% (14 out of 24) cases, which was accompanied with significant GDF1 down-regulation in both mRNA (p<0.005) and protein levels. Compared with normal mucosa, 7/8 GC cell lines exhibited GDF1 silencing, which could be reactivated by genome demethylation. Ectopic GDF1 expression increased SMAD2/3 phosphorylation and significantly suppressed GC cell proliferation at least partially through G1 phase cell cycle arrest. CONCLUSIONS: Epigenetic silencing of GDF1 may abrogate the growth-inhibitory effects of TGF-β signaling and render selective growth advantage to gastric epithelial cells during carcinogenesis. Our findings demonstrate a causal relationship between DNA methylation and GC development and lend support to demethylating drugs for GC chemoprevention trial. This study was supported by RFCID (08070172) and CUHK Direct Grant. REFERENCE: Cheng AS, et al. Helicobacter pylori causes epigenetic dysregulation of FOXD3 to promote gastric carcinogenesis. Gastroenterology 2013;144:122-33.e9. Citation Format: Wei Qin Yang, May S. M. Li, Wei Kang, Li Han Zeng, Tian Hai Wang, Anthony W. Chan, Enders K. W. Ng, Ka F. To, Francis K. L. Chan, Jun Yu, Michael W.Y. Chan, Joseph J. Y. Sung, Alfred S. L. Cheng. Reactivation of growth/differentiation factor 1 contributes to the chemopreventive effect of 5-aza-2′deoxycytidine in gastric cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1245. doi:10.1158/1538-7445.AM2014-1245