Time-extended exposure of gastric epithelial cells to secretome of Helicobacter pylori-activated fibroblasts induces reprogramming of gastric epithelium towards pre-cancerogenic and pro-invasive phenotype.

Despite of the improvement in gastric cancer (GC) therapies patients still suffer from cancer recurrence and metastasis. Recently, the high ratio of these events combined with increased chemoresistance has been related to the asymptomatic Helicobacter pylori (Hp) infections. The limited efficiency of GC treatment strategies is also increasingly attributed to the activity of tumor stroma with the key role of cancer-associated fibroblasts (CAFs). In order to investigate the influence of Hp infection within stromal gastric tissue on cancer initiation and progression, we have exposed normal gastric epithelial cells to long-term influence of Hp-activated gastric fibroblast secretome. We have referred obtained results to this secretome influence on cancer cell lines. The invasive properties of cells were checked by time-lapse video microscopy and basement membrane assays. The expression of invasion-related factors was checked by RT-PCR, Western Blot, immunofluorescence and Elisa. Hp-activated gastric fibroblast secretome induced EMT type 3-related shifts of RGM1 cell phenotype; in particular it augmented their motility, cytoskeletal plasticity and invasiveness. These effects were accompanied by Snail1/Twist activation, the up-regulation of cytokeratin19/FAP/TNC/Integrin-β1 and MMPs, and by the induction of cMethigh/pEGFRhigh phenotype. Mechanistic studies suggest that this microevolution next to TGFβ relies also on c-Met/EGFR signaling interplay and engages HGF-Integrin-Ras-dependent Twist activation leading to MMP and TNC upregulation with subsequent positive auto- and paracrine feedback loops intensifying this process. Similar shifts were detected in cancer cells exposed to this secretome. Collectively, we show that the secretome of Hp-infected fibroblasts induces reprogramming/microevolution of epithelial and cancer cells towards type 3 EMT-related invasive phenotype in a manner reciprocally reliant next to TGFβ on cMet/Integrin-β1/p-EGFR-dependent axis. Apparently, the phenotypical plasticity of Hp-activated fibroblast reprogrammed gastric epithelial cells determines their susceptibility to the pro-invasive signaling, which results in re-organization of gastric niches and provides the cues for GC promotion/progression. AJCR