Abstract 2310: Epigenetic silencing of a potential tumor suppressor NR4A3 by aberrant JAK/STAT signaling predicts prognosis in gastric cancer

Abstract

Abstract Gastric cancer is the second leading cause of cancer worldwide. Epigenetic silencing of tumor-suppressors has emerged as an important underlying mechanism in the gastric carcinogenesis. Previous studies showed that infection of H. pylori activates JAK/STAT3 signaling pathway in gastric cancer. However, the role of this aberrant signaling remains unclear. We hypothesized that activation of JAK/STAT signaling leads to epigenetic silencing of STAT3 target genes in gastric cancer. To test this hypothesis, a constitutively activated mouse STAT3 mutant (STAT3c) was transfected into MKN28 gastric cancer cells in which the JAK/STAT signaling pathway is inactive. STAT3c stable transfectant (S16) showing hyperphosphorylation of STAT3 demonstrated increased cell proliferation as compared to vector control (C9). Integrative expression microarray coupled with bioinformatic analysis identified putative STAT3 targets, NR4A3 that are down-regulated in S16 cells. In association with up-regulation of DNMT1, NR4A3 exhibited increased promoter methylation in S16 but not C9 cells as demonstrated by bisulphite sequencing and demethylation treatment. Interestingly, NR4A3 was also found to be epigenetically silenced in AGS cells where JAK/STAT signaling is constitutively activated. ChIP-PCR experiment revealed that STAT3 bound to the putative STAT3 binding site in NR4A3 promoter of AGS cells. Depletion of STAT3 by lenti-viral knockdown restored NR4A3 expression in this cell. Interestingly, luciferase reporter assay using the NR4A3 promoter containing putative STAT3 binding site exhibited a further 1.6 fold increment after deleting the STAT3 binding region (P < 0.005). Ectopic expression of NR4A3 in AGS cells reduced cancer cell growth in colony formation assay (P < 0.001). In clinical specimens, quantitative MSP demonstrated a significant correlation between the degree of NR4A3 methylation and STAT3 nuclear translocation in 72 gastric tumor samples (P < 0.05). Importantly, methylation of NR4A3 was significantly associated with patients with shorter survival (P < 0.05). In conclusion, our result demonstrated that aberrant JAK/STAT3 signaling confers epigenetic silencing of a potential tumor suppressor, NR4A3 in gastric cancer. Methylation of NR4A3 may be able to serve as a prognostic indicator in gastric cancer patients. Citation Format: Michael W. Y. Chan, Li-Han Zeng, Liang-Yu Chang, Claudia Dittner, Jian-Liang Chou, Yao-Ting Huang, Alfred S.L. Cheng, Jiayuh Lin, Kun-Tu Yeh. Epigenetic silencing of a potential tumor suppressor NR4A3 by aberrant JAK/STAT signaling predicts prognosis 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 2310. doi:10.1158/1538-7445.AM2014-2310