Abstract 1501: Aberrant JAK/STAT signaling orchestrates global promoter methylation and promotes TGF-β mediated EMT through epigenetic silencing of miR-193a in gastric cancer
Authors: Lin Jora M.j. , Chou Jiang-Liang , Frankhouser David E. , Chuang Yu-Ming , Chang Alex Liang-Yu , Zeng Li-Han , Chen Szu-Shan , Lin Ru-Inn , Wu Cheng-Shyong , Wei Kuo-Liang , Ng Enders K.W. , Yan Pearlly S. , Cheng Alfred S.L. , Li Chin , Chan Michael W. Y. 
Published: July 2016

Abstract
Gastric cancer is the fourth leading cause of cancer-related death worldwide. Previous studies demonstrated that activation of the JAK/STAT3 signaling pathway is frequently observed in H. pylori infected gastric cancer. However, the role of aberrant JAK/STAT signaling in the global epigenetic changes remains unclear. In this regard, we compared the global methylomic changes in AGS gastric cancer cells showing constitutive activation of STA3 vs its STAT3 knock-down subclone by MBDcap-Seq followed by PrEMeR-CG analysis. Together with RNA-Seq, we identified 97 targets showing concomitant hypomethylation and over-expression while 76 targets showing concomitant hypermethylation and down-regulation after STAT3 knock down. Genes showing hypomethylaton/over-expression were subjected to transcription factor binding site analysis by MEME CentriMo. Interestingly, the transcriptional repressors binding site for ETS1 (p = 2.90E-06) and EHF (p = 3.50E-06) were overrepresented in those identifed STAT3 targets suggesting the cooperative binding with STAT3 in the epigenetic silencing of the targets. Further gene ontology analysis by DAVID showed that genes involved in cell cycle and apoptosis were significantly enriched in the hypomethylated/over-expressed targets while genes involved in protein degradation and ubiquitination were found among the hypermethylation/down-regulated targets. To experimentally confirm our result, we analyzed the functional role of one of the hypomethylated targets, miR-193a in gastric cancer. Concomitant with MBDcap-Seq, bisulphite pyrosequencing confirmed that promoter region of miR-193a was hypomethylated in AGS cells depleted with STAT3 but hypermethylated in MKN28 gastric cancer cells overexpressed with constitutive activated STAT3. Cell lines studies also found that promoter region of miR-193a was hypermethylated in gastric cancer cells which did not express miR-193a. Over-expression of miR-193a in AGS cells inhibited cell proliferation (p&lt;0.001) and migration (p &lt;0.01) by colony formation assay and wound-healing assay respectively. Clinically, significantly higher promoter methylation of miR-193a was observed in gastric cancer patient samples (Hong Kong, n = 70; Taiwan, n = 38) as compared to gastritis (n = 9, p&lt;0.05). Interestingly, gastritis with H. pylori infection (p &lt;0.05) had higher methylation of miR-193a than that without H. pylori infection. Patients with higher methylation of miR-193a tended to have shorter overall survival. Importantly, overexpression of miR-193a suppressed the expression of a predicted miR-193a target, YWHAZ (14-3-3ζ). As YWHAZ has been previously found to be a positive regulator in TGF-β-mediated EMT in human cancer, the role of JAK/STAT signaling in promoting TGF-β-mediated EMT program deserves further investigation.
Citation Format: Jora M.j. Lin, Jiang-Liang Chou, David E. Frankhouser, Yu-Ming Chuang, Alex Liang-Yu Chang, Li-Han Zeng, Szu-Shan Chen, Ru-Inn Lin, Cheng-Shyong Wu, Kuo-Liang Wei, Enders K.W. Ng, Pearlly S. Yan, Alfred S.L. Cheng, Chin Li, Michael W. Y. Chan. Aberrant JAK/STAT signaling orchestrates global promoter methylation and promotes TGF-β mediated EMT through epigenetic silencing of miR-193a in gastric cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1501.