Abstract 4269: Regulation and functional roles of autophagy in Helicobacter pylori CagA-mediated gastric cancer

Abstract

Abstract Autophagy is a catabolic process for the autophagosomic-lysosomal degradation of bulk cytoplasmic components and plays a crucial role in infection, inflammation and various cellular functions. Helicobacter pylori (H. pylori) was recently indicated to be able to induce autophagy. The cytotoxinA (CagA) of H.pylori was demonstrated to be degraded by autophagic pathway, but the the dynamic role of mechanism by which CagA affects autophagic signaling has not been identified. This study was determined to elucidate the functional role and regulatory mechanism of H. pylori induced autophagy in gastric cancer cells. In this study, we tested that during the H.pylori 60190(CagA+, VacA+) infection, where the nutrient starvation induced a high level of autophagy, in gastric cancer cell lines(AGS, MKN45, MKN28 and GES-1) through CagA and LC3 I/II(autophagy marker) protein expression level. The gastric cancer cells infected with H.pylori 60190, ΔCagA and ΔVacA(60190 isogenic mutant strain) were incubated with a serum free medium containing antibiotic(KM,Kanamycin) to kill extracellular H.pylori, intracellular CagA level decreased and LC3 I converted to LC3 II. The quantification of LC3 by ImageJ increased in the gastric cancer cells. Autophagy induction associated with intracellular CagA stability. CagA was essential and sufficient to induce autophagy in gastric cancer cells, with the induction of autophagy responsible for a decreased in the level of intracellular CagA. The gastric cancer cells infected with or without H.pylori were performed immunofluorescence and detected by confocal microscopy to examine the co-localization of CagA and LC3. CagA located with LC3, therefore, CagA was related to the mechanism of autophagy. The intracellular cagA decreased and LC3 I to LC3 II conversion was detected. To evaluate whether autophagy was specifically associated with H.pylori infection, we used LysoTracker® Red. H.pylori infection affected lysosomal degradation. The autophagy inhibitor (Cychloheximide, Bafilomycin A1, Chloroquine) and PI3K/AKT inhibitor (LY294002), GSK inhibitor (Lithium Chloride) in gastric cancer cells were used to examine the mechanism of CagA degradation. The cagA was affected by those inhibitors. The effects of CagA in promoting autophagy blocked autophagy. The autophagy contributed to CagA degradation in gastric cancer cells. To examine ectopic expression of CagA affects serum starvation induced autophagy, transfection of plasmid vector to AGS were carried out. Our findings showed that the autophagy play some important roles in H. pylori infected gastric cancer cells by the degradation of intracellular CagA leading to altered epithelial cellular signal transduction pathways. This indicate that studies investigating the host-pathogen autophagic interaction in H. pylori infection will provide the platform for bio-marker research for gastric cancer chemo-preventive strategy. Citation Format: BoRam Hwang, SoDam Lee, ChangYun Jung, ByeongMin Yu, YongChan Lee. Regulation and functional roles of autophagy in Helicobacter pylori CagA-mediated gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4269.