Afadin Downregulation by Helicobacter pylori Induces Epithelial to Mesenchymal Transition in Gastric Cells

Abstract

Afadin is a cytoplasmic protein of the adherens junctions, which regulates the formation and stabilization of both the adherens and the tight junctions. Aberrant expression of Afadin has been shown in cancer and its loss has been associated with epithelial-to-mesenchymal transition (EMT). EMT is characterized by the change from an epithelial to a mesenchymal phenotype, with modifications on the expression of adhesion molecules and acquisition of a migratory and invasive cell behavior. While it is known that Helicobacter pylori disrupts the tight and the adherens junctions and induces EMT, the effect of the bacteria on Afadin is still unknown. The aim of this study was to disclose the effect of H. pylori on Afadin and its impact in the induction of an EMT phenotype in gastric cells. Using two different cell lines, we observed that H. pylori infection decreased Afadin protein levels, independently of CagA, T4SS, and VacA virulence factors. H. pylori infection of cell lines recapitulated several EMT features, displacing and downregulating multiple proteins from cell–cell junctions, and increasing the expression of ZEB1, Vimentin, Slug, N-cadherin, and Snail. Silencing of Afadin by RNAi promoted delocalization of junctional proteins from the cell–cell contacts, increased paracellular permeability, and decreased transepithelial electrical resistance, all compatible with impaired junctional integrity. Afadin silencing also led to increased expression of the EMT marker Snail, and to the formation of actin stress fibers, together with increased cell motility and invasion. Finally, and in line with our in vitro data, the gastric mucosa of individuals infected with H. pylori showed decrease/loss of Afadin membrane staining at cell–cell contacts significantly more frequently than uninfected individuals. In conclusion, Afadin is downregulated by H. pylori infection in vitro and in vivo, and its downregulation leads to the emergence of EMT and to the acquisition of an aggressive phenotype in gastric cells, which can contribute to gastric carcinogenesis.

Mechanisms of the Epithelial–Mesenchymal Transition and Tumor Microenvironment in Helicobacter pylori-Induced Gastric Cancer

“…Afadin is a cytoplasmic actin-filament-binding protein responsible for the formation and stabilization of tight and adherens junctions [192][193][194][195]. Marques et al (2018) observed that infection by H. pylori lowered the expression of Afadin protein independently of CagA and VacA in MKN74 and NCI-N87 cell lines [196]. Downregulation of Afadin resulted in dysregulation of tight junctions and adherens junctions, which caused inappropriate cell permeability, stiffness, and impaired transepithelial electrical resistance.…”

TAZ Controls Helicobacter pylori-Induced Epithelial–Mesenchymal Transition and Cancer Stem Cell-Like Invasive and Tumorigenic Properties

“…EMT is a very well-known pathophysiological trans-differentiation process that confers mesenchymal phenotype and properties to epithelial cells. In the gastric context, this EMT is characterized by the loss of epithelial polarity and cellular junctions and the acquisition of a mesenchymal, motile phenotype called the “hummingbird” phenotype [ 7 , 8 , 9 , 10 ]. The overexpression of zinc finger E-box-binding homeobox 1 (ZEB1) and Snail transcription factors and of structural components such as Vimentin, as well as migration and invasion capacities are reminiscent events of the EMT process.…”

Review: Gastric malignancies: Basic aspects

“…Deletion of Claudin‐18 in these animals led to the activation of multiple signaling pathways involved in cell proliferation and cancer stem cell development, to increased inflammation, and to the appearance of gastric intraepithelial neoplasia and tumorigenesis. In line with these observations, Marques et al showed that H pylori downregulates multiple epithelial cell junction components, including the adherens junction protein Afadin. H pylori downregulates Afadin in vitro and in vivo , and Afadin loss led to the emergence of epithelial‐mesenchymal transition (EMT) and acquisition of an aggressive phenotype in gastric cells .…”

“…In line with these observations, Marques et al showed that H pylori downregulates multiple epithelial cell junction components, including the adherens junction protein Afadin. H pylori downregulates Afadin in vitro and in vivo , and Afadin loss led to the emergence of epithelial‐mesenchymal transition (EMT) and acquisition of an aggressive phenotype in gastric cells . Accordingly, H pylori promotion of EMT in gastric carcinogenesis by activation of the YAP signaling pathway was also reported …”