Helicobacter pylori potentiates epithelial:mesenchymal transition in gastric cancer: links to soluble HB-EGF, gastrin and matrix metalloproteinase-7

Abstract

Background and aims: Helicobacter pylori (H pylori) infection is a major risk factor in the development of distal gastric adenocarcinoma. Development of the invasive phenotype is associated with the phenomenon of epithelial:mesenchymal transition (EMT). Soluble heparin-binding epidermal growth factor (HB-EGF) has been implicated in this process. A study was undertaken to investigate the possibility that matrix metalloproteinase (MMP)-7 is upregulated in H pylori infection as a result of hypergastrinaemia, which may enhance shedding of HB-EGF and contribute towards EMT in gastric adenocarcinoma cell lines.

Methods: Three gastric epithelial cell lines (AGS, MGLVA1 and ST16) were co-cultured with the pathogenic H pylori strain 60190 and non-pathogenic strain Tx30a in an in vitro infection model. Gene expression was quantified by real-time PCR, HB-EGF shedding by ELISA and protein expression by immunofluorescence or immunohistochemistry. The INS-GAS mouse, a transgenic mouse model of gastric carcinogenesis which overexpresses amidated gastrin, was used to investigate the in vivo relationship between HB-EGF, MMP-7, gastrin and EMT.

Results: The pathogenic strain of H pylori significantly upregulated EMT-associated genes Snail, Slug and vimentin in all three gastric cell lines to a greater degree than the non-pathogenic strain. Pathogenic H pylori also upregulated HB-EGF shedding, a factor implicated in EMT, which was partially dependent on both gastrin and MMP-7 expression. Gastrin and MMP-7 siRNAs and MMP-7 neutralising antibody significantly reduced upregulation of HB-EGF shedding in H pylori infected gastric cell lines and reduced EMT gene expression. The effect of H pylori on EMT was also reversed by gastrin siRNA. Neutralisation of gastrin in the INS-GAS mouse model reduced expression of MMP-7, HB-EGF and key EMT proteins.

Conclusion: The upregulation of MMP-7 by pathogenic H pylori is partially dependent on gastrin and may have a role in the development of gastric cancer, potentially through EMT, by indirectly increasing levels of soluble HB-EGF.

Figure 1

Epithelial:mesenchymal transition gene expression following co-culture of gastric epithelial cells with Helicobacter pylori pathogenic (60190) and non-pathogenic (Tx30a) strains. (a) Snail gene expression: *significantly higher gene expression level compared with untreated control cells (strain 60190 treated: AGS p<0.001, MGLVA1 p<0.05, ST16 p<0.005; strain Tx30a treated: AGS p<0.005, MGLVA1 p<0.05, ST16 p<0.02); #significantly higher gene expression level compared with Tx30a treated cells (AGS p<0.05, ST16 p<0.05). (b) Slug gene expression: *significantly higher gene expression level compared with untreated control cells (strain 60190 treated: p<0.001 in all cell lines; strain Tx30a treated: AGS p<0.001, MGLVA1 p<0.005, ST16 p<0.005); #significantly higher gene expression level compared with Tx30a treated cells (AGS p<0.04, MGLVA1 p<0.05, ST16 p<0.002). (c) Vimentin gene expression: *significantly higher gene expression level compared with untreated control cells (strain 60190 treated: AGS p<0.05, MGLVA1 p<0.001, ST16 p<0.005; strain Tx30a treated: MGLVA1 p<0.05); #significantly higher gene expression level compared with Tx30a treated cells (MGLVA1 p<0.04). (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence).

Figure 2

Effect of co-culture of gastric epithelial cells with Helicobacter pylori strain 60190 on heparin-binding epidermal growth factor (HB-EGF) expression. (a) HB-EGF gene expression following co-culture of gastric epithelial cells with H pylori pathogenic (60190) and non-pathogenic (Tx30a) strains; *p<0.001 (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence). (b) Cell-associated HB-EGF expression following co-culture of gastric epithelial cells with H pylori pathogenic (60190) and non-pathogenic (Tx30a) strains. Cells were stained for HB-EGF (red fluorescence) and nuclei were counterstained with Hoechst (blue fluorescence); (i) AGS, (ii) AGS/H pylori 60190, (iii) AGS/H pylori Tx30a. Magnification ×20. (c) HB-EGF protein expression level quantified by image analysis following co-culture of gastric epithelial cells with H pylori pathogenic (60190) and non-pathogenic (Tx30a) strains; *significantly higher HB-EGF protein expression level compared with untreated control cells (p<0.03). (d) HB-EGF shedding level following co-culture of gastric epithelial cells with H pylori pathogenic (60190) and non-pathogenic (Tx30a) strains; *significantly higher HB-EGF shedding level compared with untreated control cells (strain 60190 treated: p<0.01 in all cell lines; strain Tx30a treated: p<0.05 in AGS and ST16); #significantly higher HB-EGF shedding level compared with Tx30a treated (p<0.05 in AGS and ST16). (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence).

Figure 3

Effect of co-culture of gastric epithelial cells with Helicobacter pylori strain 60190 on matrix metalloproteinase 7 (MMP-7) expression. (a) MMP-7 gene expression following co-culture of gastric epithelial cells with H pylori pathogenic (60190) and non-pathogenic (Tx30a) strains. *p<0.001 (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence). (b) MMP-7 protein expression following co-culture of gastric epithelial cells with H pylori pathogenic (60190) and non-pathogenic (Tx30a) strains. Cells were stained for MMP-7 (green fluorescence) and nuclei were counterstained with Hoechst (blue fluorescence). (i) AGS, (ii) AGS/H pylori 60190, (iii) AGS H pylori Tx30a. Magnification ×20. (c) MMP-7 protein expression level quantified by image analysis following co-culture of gastric epithelial cells with H pylori pathogenic (60190) and non-pathogenic (Tx30a) strains; *significantly higher MMP-7 protein expression level compared with untreated control cells (p<0.001). (d) MMP-7 and heparin-binding epidermal growth factor (HB-EGF) co-localisation: MMP-7 expression was stained by green fluorescence (i), HB-EGF by red fluorescence (ii), nuclei were stained with Hoechst blue fluorescence (iii). Merged image shows co-localisation of MMP-7 and HB-EGF (iv). Magnification ×20. (e) MMP-7 gene expression in MMP-7 siRNA treated gastric cell lines. MMP-7 siRNA significantly reduced MMP-7 mRNA expression in all three gastric cell lines tested. *p<0.001 (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence). (f) HB-EGF gene expression in MMP-7 siRNA treated gastric cell lines exposed to H pylori 60190. MMP-7 siRNA significantly reduced HB-EGF shedding in all three gastric cell lines tested. *p<0.02, **p<0.03 (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence). (g) HB-EGF gene expression in MMP-7 neutralising antibody treated gastric cell lines exposed to H pylori 60190. *p<0.03 (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence). (h) HB-EGF shedding levels in gastric cell lines exposed to H pylori 60190 treated with an MMP-7 neutralising antibody. *p<0.04, **p<0.03 (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence). (i) EMT gene expression in MMP-7 siRNA treated gastric cell lines exposed to H pylori strain 60190: Snail, p<0.01; Slug, p<0.005; vimentin, p<0.001 (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence).

Figure 4

Effect of co-culture of gastric epithelial cells with Helicobacter pylori strain 60190 on gastrin expression and the effect of gastrin siRNA on matrix metalloproteinase 7 (MMP-7) and epithelial:mesenchymal transition (EMT). (a) Gastrin gene expression following co-culture of gastric epithelial cells with H pylori pathogenic (60190) and non-pathogenic (Tx30a) strains. *p<0.001 (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence). (b) Gastrin gene expression in gastrin siRNA treated gastric cell lines. Gastrin siRNA significantly reduced gastrin mRNA expression in all three gastric cell lines tested. *p<0.001 (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence). (c) MMP-7 expression in gastric cells exposed to H pylori and gastrin siRNA.*p<0.02 and **p<0.05 (n=3 replicates per condition and a representative graph is shown, error bars indicate 95% confidence). (d) MMP-7 protein expression in gastrin siRNA transfected gastric cell lines exposed to H pylori. Cells were stained for MMP-7 (green fluorescence) and nuclei were counterstained with Hoescht (blue fluorescence). Magnification ×20. (e) MMP-7 protein expression level quantified by image analysis in gastrin siRNA transfected gastric cell lines exposed to H pylori; *significantly higher MMP-7 protein expression level compared with untreated control cells (p<0.001). (f) Gastrin siRNA reverses H pylori induced EMT in gastric cell lines.*p<0.01 (n=3 replicates per condition and a representative graph is shown. Error bars indicate 95% confidence).

Figure 5

Effect of gastrin neutralisation on matrix metalloproteinase 7 (MMP-7), heparin-binding epidermal growth factor (HB-EGF) and epithelial:mesenchymal transition (EMT) expression in the INS-GAS transgenic mouse model of gastric carcinogenesis. (a) Haematoxylin and eosin stained INS-GAS samples treated with gastrin immunogen (i) or control immunogen (ii). Magnification ×20. (b) Quantification of levels of proliferation in INS-GAS samples treated with gastrin immunogen or control immunogen. *p<0.05, **p<0.02 (5 animals per group, error bars indicate SD). (c) MMP-7, HB-EGF and Slug expression in malignant lesions in the gastric mucosa of gastrin immunogen (i) and control immunogen (ii) treated mice. Samples were stained by immunohistochemistry for HB-EGF and MMP-7 (brown staining indicates positive expression) or by immunofluorescence for Slug (green fluorescence indicates positive expression). An area representative of staining in nine stomach sections is shown. The appearance of the other sections was similar. Magnification ×20. (d) Protein expression levels of MMP-7, HB-EGF and Slug in the INS-GAS transgenic mouse model of gastric carcinogenesis. Staining was quantified by image analysis in both gastrin immunogen and control immunogen treated mice. MMP-7, p<0.01; HB-EGF, p<0.01; Slug, p<0.02 (n=9, error bars indicate SD).