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.

Keywords: Afadin; Helicobacter pylori; cell–cell junction disruption; epithelial to mesenchymal transition; gastric cancer.

FIGURE 1

Impact of H. pylori infection on Afadin expression. MKN74 and NCI-N87 gastric cell lines were left untreated or infected with H. pylori 26695 (A,B) or infected with H. pylori 60190, or with the respective CagA^-, CagE^- and VacA^- mutants (C), for 24 h at a MOI of 100. Afadin detection by immunofluorescence (red) and quantification of the fluorescence intensity at the membrane and the nucleus (A), and by Western blot and its quantification, using GAPDH or tubulin as loading controls (B,C). Scale bar, 10 μm. Data correspond to the mean value ± SEM and are representative of at least three independent experiments. Statistical significance was evaluated with the Student’s t-test or with one-way ANOVA.

FIGURE 2

H. pylori induces an EMT phenotype in epithelial gastric cells. MKN74 and NCI-N87 gastric cell lines were left untreated or infected with H. pylori 26695 for a period of 24 h at a MOI of 100. (A) Immunofluorescence of E-cadherin, occludin, and ZO-1. Scale bar, 10 μm. (B) Western blot and respective quantifications of the levels of apical junctional complex proteins E-cadherin, β-catenin, ZO-1, and occludin. Tubulin was used as loading control. (C) Western blot and respective quantifications of the levels of mesenchymal markers Snail, N-cadherin, ZEB1, Slug, and Vimentin. GAPDH was used as loading control. Data correspond to the mean value ± SEM and are representative of three independent experiments. Statistical significance was evaluated with the Student’s t-test.

FIGURE 3

Afadin downregulation displaces apical junctional complex proteins and promotes the formation of actin stress fibers. Double immunofluorescence of Afadin (red) with AJs proteins E-cadherin and β-catenin (green), and with TJs proteins ZO-1 and occludin (green) in MKN74 cells transfected with a non-silencing siRNA (siNS) or with a siRNA to Afadin (siAFDN). Immunofluorescence of actin (green) and of Snail (red) are also shown. Nuclei were counterstained with DAPI. Scale bar, 10 μm. White arrows represent cells with Afadin not efficiently silenced by the siRNA and that retain the epithelial morphology; Yellow arrows, lamellipodia; cyan arrows, filopodia (A). Quantification of Afadin fluorescence intensity in MKN74 cells in both membrane and the nucleus upon treatment with non-silencing siRNA or with a siRNA to Afadin (B).

FIGURE 4

Afadin downregulation does not alter the expression of apical junctional complex proteins, but upregulates the EMT marker Snail. Western blot analyses of MKN74 and NCI-N87 cells, cells transfected with a non-silencing siRNA (siNS), or with a siRNA to Afadin (siAFDN), evaluating (A) apical junctional complex proteins E-cadherin, β-catenin, ZO-1, and occludin, and (B) mesenchymal markers Snail, N-cadherin, ZEB1, Slug, and Vimentin. GAPDH was used as loading control. Numbers below the bands correspond to quantifications. Data are representative of three independent experiments. Statistical significance was evaluated with the Student’s t-test.

FIGURE 5

Afadin downregulation alters the TJ barrier function and increases cell motility and invasion. (A) Transepithelial electrical resistance (TER) measurements of MKN74 gastric cells during a period of 6 days post Afadin silencing (siAFDN) in comparison with non-silenced (siNS) and with untreated cells, and during 2 days after H. pylori infection in comparison with uninfected cells. (B) Cell monolayer permeability to 4 kDa FITC-Dextran on day 6 post Afadin silencing and on day 2 after H. pylori infection. (C–F) Single cell motility analysis: (C) Time-lapse microscopy photos of cells during the 24 h period. (D) Representative graphs of the X and Y coordinates (in pixels, px) of each cell path, on a fibronectin coated surface. (E) Cell velocity in μm/minute and (F) total distance covered by and individual cell in μm. (G,H) Cell invasion capacity on Matrigel-coated transwells for MKN74 cells transfected with siNS control or transfected with siAFDN (G), and for MKN74 cells infected or not with H. pylori 26695 (H). Data are presented as mean ± SEM. Statistical significance was evaluated with the one- and two-way ANOVA and with Student’s t-test.

FIGURE 6

H. pylori infection affects the expression and localization of the Afadin protein in the human gastric mucosa. (A) AFDN gene expression in the stomach of 181 (114 uninfected and 67 H. pylori-infected) individuals without disease, retrieved from the GTEx database. RPKM, reads per kilobase million. Significance was evaluated with Kruskal–Wallis statistics. (B–F) Immunohistochemical detection of Afadin in paraffin-embedded sections of the gastric mucosa of individuals uninfected (n = 20) or infected with H. pylori (n = 22). (B) Graphical representation of Afadin alterations in the two biological groups. Statistical significance was determined with the Fisher’s exact test. Representative micrographs of Afadin staining in an uninfected individual, showing a strong intensity at the apicolateral epithelial cell–cell contacts (black arrowheads) (C), and a belt-like pattern (red arrowheads) (D) in longitudinal and cross sections, respectively, and in H. pylori-infected individuals, presenting decreased intensity/loss of membrane staining at the cell–cell contacts (blue arrowheads) (E) or showing cytoplasmic staining in areas of epithelial tufting (F).