Helicobacter pylori CagA protein induces factors involved in the epithelial to mesenchymal transition (EMT) in infected gastric epithelial cells in an EPIYA‐ phosphorylation‐dependent manner

Abstract

As a result of Helicobacter pylori adhesion to gastric epithelial cells, the bacterial effector cytotoxin-associated gene A (CagA) is translocated intracellularly, and after hierarchical tyrosine phosphorylation on multiple EPIYA motifs, de-regulates cellular polarity and contributes to induction of an elongation and scattering phenotype that resembles the epithelial to mesenchymal transition (EMT). Stromelysin-1/matrix metalloproteinase-3 (MMP-3) has been reported to induce a sequence of molecular alterations leading to stable EMT transition and carcinogenesis in epithelial cells. To identify the putative role of CagA protein in MMP-3 induction, we exploited an experimental H. pylori infection system in gastric epithelial cell lines. We utilized isogenic mutants expressing CagA protein with variable numbers of EPIYA and phosphorylation-deficient EPIFA motifs, as well as cagA knockout and translocation-deficient cagE knockout strains. Increased levels of MMP-3 transcriptional activation were demonstrated by quantitative real time-PCR for strains with more than two terminal EPIYA phosphorylation motifs in CagA. MMP-3 expression in total cell lysates and the corresponding culture supernatants was associated with CagA expression and translocation and was dependent on CagA phosphorylation. A CagA EPIYA phosphorylation-dependent increase in gelatinase and caseinolytic activity was also detected in culture supernatants by zymography. A significant increase in the transcriptional activity of the mesenchymal markers Vimentin, Snail and ZEB1 and the stem cell marker CD44 was observed in the case of CagA containing phosphorylation-functional EPIYA motifs. Our data suggest that CagA protein induces EMT through EPIYA phosphorylation-dependent up-regulation of MMP-3. Moreover, no significant increase in EMT and stem cell markers was observed following infection with H. pylori strains that cannot effectively translocate CagA protein.

CDH1 (E-cadherin) expression independently affects clinical outcome in acute myeloid leukemia with normal cytogenetics

“…Accumulating evidence has indicated that EMT is a critical process not only in normal development but also in oncogenesis in recent years [25][26][27]. Cancer cells were endowed with increased motility and invasiveness during this process.…”

Proteolysis in Helicobacter pylori-Induced Gastric Cancer

“…MMP-3 was shown to promote EMT and was suggested to be a natural tumor promoting factor [80,81]. MMP-3 expression in response to H. pylori was suggested to depend on the presence of phosphorylated CagA EPIYA motifs in gastric adenocarcinoma cell lines and is regulated alike the EMT markers ZEB1 (zinc finger E-box-binding homeobox 1), vimentin, snail, and CD44 [82]; However, this dependency on CagA was not observed in mouse infection models [75]. It is conceivable, that the different regulation in vivo might be attributed to IL-1β dependent secretion of MMP-3 [79].…”

Microbial carcinogenic toxins and dietary anti-cancer protectants

“…Sougleri et al [42] observed the morphological and polarity changes characteristic of EMT in response to Caga. Using mutant gastric epithelial cells expressing variant forms of Caga with differing numbers of EPIYA (Glu-Pro-Ile-Tyr-Ala)-binding sites, they noted that phosphorylation of those sites mediates the interaction of Caga with SHP-2 noted above, leading to an elongated cell structure resembling that of the EMT and known as the ‘hummingbird’ phenotype.…”