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Background & Aims: Helicobacter pylori CagA-positive strain is associated with gastric adenocarcinoma. CagA is delivered into gastric epithelial cells, where it undergoes tyrosine phosphorylation at the EPIYA sites by Src family kinases (SFKs). Owing to homologous recombination within the 3′-region of the cagA gene, 4 distinct EPIYA sites, each of which is defined by surrounding sequences, are variably assembled in both number and order among CagA proteins from different clinical H pylori isolates. Tyrosine-phosphorylated CagA specifically binds and deregulates SHP-2 via the Western CagA-specific EPIYA-C or East Asian CagA-specific EPIYA-D site, and C-terminal Src kinase (Csk) via the EPIYA-A or EPIYA-B site. Here we investigated the influence of EPIYA-repeat polymorphism on the CagA activity. Methods: A series of EPIYA-repeat variants of CagA were expressed in AGS gastric epithelial cells and the ability of individual CagA to bind SHP-2 or Csk was determined by the sequential immunoprecipitation and immunoblotting method. Results: CagA proteins carrying multiple EPIYA-C or EPIYA-D sites bound and deregulated SHP-2 more strongly than those having a single EPIYA-C or EPIYA-D. Furthermore, the ability of CagA to bind Csk was correlated with the number of EPIYA-A and EPIYA-B sites. Because Csk inhibits SFK, CagA with greater Csk-binding activity more strongly inhibited Src-dependent CagA phosphorylation and more effectively attenuated induction of cell elongation caused by CagA–SHP-2 interaction. Conclusions: EPIYA-repeat polymorphism of CagA greatly influences the magnitude and duration of phosphorylation-dependent CagA activity, which may determine the potential of individual CagA as a bacterial virulence factor that directs gastric carcinogenesis.

G A A b st ra ct s strains were less able to activate Gli1 and Snail. Snail expression in Gli1 overexpressed gastric cell was higher than that of Gli1 unexpressed gastric epithelial cell and Gli1 overexpressed gastric cell was 8 fold increased snail promoter activity. In this study, we showed that Hh signaling was clearly upregulated in gastric cancer cell in cagA depedent manner and activated Gli1 upregulate Snail to promote epithelial-to-mesenchymal transition.

Helicobacter pylori has a profound influence upon the development of gastric adenocarcinoma, however the molecular mechanisms are not clearly understood. Much attention has been devoted to the H. pylori Cytotoxin-associated antigen A (CagA), a 120-145kDa protein that is injected into gastric epithelial cells and disrupts intracellular signaling pathways including those utilised by IL-6 family cytokines. Current evidence shows that unbalancing of the two major IL-6 cytokine signaling cascades, resulting in the constitutive activation of the signal transducer and activator of transcription 3 (STAT3) and disruption of the Src-homology tyrosine phosphatase 2 (SHP2)-Ras-ERK pathway, emanating from the common gp130 coreceptor leads to gastric tumour development and establishes co-ordinated IL-6 family cytokine signalling as a paradigm for gastric homeostasis. This is intriguing because translocated CagA binds SHP-2 in a phosphorylation dependent manner and alters SHP-2-ras-ERK signalling leading to characteristic changes in cell morphology, motility and proliferation. We hypothesized that sequestration of intracellular SHP-2 by CagA may imbalance coordinated signalling downstream of gp130 leading to the inappropriate activation of oncogenic STAT3, as implicated by our recent work showing increased STAT3 phosphorylation in CagA-positive compared with CagA-negative H. pylori infections in precancerous lesions of humans. To clarify the mechanism by which CagA interferes with STAT3 signalling we have developed transient and inducible CagA expression systems in gastric epithelial cell lines. Overexpression of CagA in AGS cells led to a ~50% reduction in available phosphorylated SHP-2 as a consequence of its interaction with CagA, yet consistent with our expectations, we also observed a concomitant two-fold increase in STAT3 phosphorylation by western blotting. Overexpression of a phosphorylation-resistant CagA mutant had no discernable effect on either SHP-2 or STAT3 phosphorylation indicating that STAT3 activation in this context is dependent upon CagA tyrosine phosphorylation. These results reveal a novel mechanism by which CagA might provoke cellular transformation by the inappropriate activation of oncogenic STAT3 and thus constitute an early event in H. pylori mediated gastric cancer progression.