Pathogenic Helicobacter pylori strains translocate DNA and activate TLR9 via the cancer-associated cag type IV secretion system.

Helicobacter pylori (H. pylori) is the strongest identified risk factor for gastric cancer, the third most common cause of cancer-related death worldwide. An H. pylori constituent that augments cancer risk is the strain-specific cag pathogenicity island, which encodes a type IV secretion system (T4SS) that translocates a pro-inflammatory and oncogenic protein, CagA, into epithelial cells. However, the majority of persons colonized with CagA+ H. pylori strains do not develop cancer, suggesting that other microbial effectors also have a role in carcinogenesis. Toll-like receptor 9 (TLR9) is an endosome bound, innate immune receptor that detects and responds to hypo-methylated CpG DNA motifs that are most commonly found in microbial genomes. High-expression tlr9 polymorphisms have been linked to the development of premalignant lesions in the stomach. We now demonstrate that levels of H. pylori-mediated TLR9 activation and expression are directly related to gastric cancer risk in human populations. Mechanistically, we show for the first time that the H. pylori cancer-associated cag T4SS is required for TLR9 activation and that H. pylori DNA is actively translocated by the cag T4SS to engage this host receptor. Activation of TLR9 occurs through a contact-dependent mechanism between pathogen and host, and involves transfer of microbial DNA that is both protected as well as exposed during transport. These results indicate that TLR9 activation via the cag island may modify the risk for malignancy within the context of H. pylori infection and provide an important framework for future studies investigating the microbial-epithelial interface in gastric carcinogenesis.

Matrix metalloproteinase 7 restrains Helicobacter pylori-induced gastric inflammation and premalignant lesions in the stomach by altering macrophage polarization.

Helicobacter pylori is the strongest risk factor for the development of gastric cancer. Although the specific mechanisms by which this pathogen induces carcinogenesis have not been fully elucidated, high-expression interleukin (IL)-1ß alleles are associated with increased gastric cancer risk among H. pylori-infected persons. In addition, loss of matrix metalloproteinase 7 (MMP7) increases mucosal inflammation in mouse models of epithelial injury, and we have shown that gastric inflammation is increased in H. pylori-infected MMP7(-/-) C57BL/6 mice. In this report, we define mechanisms that underpin such responses and extend these results into a genetic model of MMP7 deficiency and gastric cancer. Wild-type (WT) or MMP7(-/-) C57BL/6 mice were challenged with broth alone as an uninfected control or the H. pylori strain PMSS1. All H. pylori-challenged mice were successfully colonized. As expected, H. pylori-infected MMP7(-/-) C57BL/6 mice exhibited a significant increase in gastric inflammation compared with uninfected or infected WT C57BL/6 animals. Loss of MMP7 resulted in M1 macrophage polarization within H. pylori-infected stomachs, as assessed by Luminex technology and immunohistochemistry, and macrophages isolated from infected MMP7-deficient mice expressed significantly higher levels of the M1 macrophage marker IL-1ß compared with macrophages isolated from WT mice. To extend these findings into a model of gastric cancer, hypergastrinemic WT INS-GAS or MMP7(-/-) INS-GAS mice were challenged with H. pylori strain PMSS1. Consistent with findings in the C57BL/6 model, H. pylori-infected MMP7-deficient INS-GAS mice exhibited a significant increase in gastric inflammation compared with either uninfected or infected WT INS-GAS mice. In addition, the incidence of gastric hyperplasia and dysplasia was significantly increased in H. pylori-infected MMP7(-/-) INS-GAS mice compared with infected WT INS-GAS mice, and loss of MMP7 promoted M1 macrophage polarization. These results suggest that MMP7 exerts a restrictive role on H. pylori-induced gastric injury and the development of premalignant lesions by suppressing M1 macrophage polarization.

Increased Helicobacter pylori-associated gastric cancer risk in the Andean region of Colombia is mediated by spermine oxidase.

Helicobacter pylori infection causes gastric cancer, the third leading cause of cancer death worldwide. More than half of the world's population is infected, making universal eradication impractical. Clinical trials suggest that antibiotic treatment only reduces gastric cancer risk in patients with non-atrophic gastritis (NAG), and is ineffective once preneoplastic lesions of multifocal atrophic gastritis (MAG) and intestinal metaplasia (IM) have occurred. Therefore, additional strategies for risk stratification and chemoprevention of gastric cancer are needed. We have implicated polyamines, generated by the rate-limiting enzyme ornithine decarboxylase (ODC), in gastric carcinogenesis. During H. pylori infection, the enzyme spermine oxidase (SMOX) is induced, which generates hydrogen peroxide from the catabolism of the polyamine spermine. Herein, we assessed the role of SMOX in the increased gastric cancer risk in Colombia associated with the Andean mountain region when compared with the low-risk region on the Pacific coast. When cocultured with gastric epithelial cells, clinical strains of H. pylori from the high-risk region induced more SMOX expression and oxidative DNA damage, and less apoptosis than low-risk strains. These findings were not attributable to differences in the cytotoxin-associated gene A oncoprotein. Gastric tissues from subjects from the high-risk region exhibited greater levels of SMOX and oxidative DNA damage by immunohistochemistry and flow cytometry, and this occurred in NAG, MAG and IM. In Mongolian gerbils, a prototype colonizing strain from the high-risk region induced more SMOX, DNA damage, dysplasia and adenocarcinoma than a colonizing strain from the low-risk region. Treatment of gerbils with either α-difluoromethylornithine, an inhibitor of ODC, or MDL 72527 (N(1),N(4)-Di(buta-2,3-dien-1-yl)butane-1,4-diamine dihydrochloride), an inhibitor of SMOX, reduced gastric dysplasia and carcinoma, as well as apoptosis-resistant cells with DNA damage. These data indicate that aberrant activation of polyamine-driven oxidative stress is a marker of gastric cancer risk and a target for chemoprevention.

Helicobacter pylori CagA targets gastric tumor suppressor RUNX3 for proteasome-mediated degradation.

Chronic infection with cagA-positive Helicobacter pylori is the strongest risk factor for the development of gastric adenocarcinoma. The cagA gene product CagA is injected into gastric epithelial cells and disturbs cellular functions by physically interacting with and deregulating a variety of cellular signaling molecules. RUNX3 is a tumor suppressor in many tissues, and it is frequently inactivated in gastric cancer. In this study, we show that H. pylori infection inactivates the gastric tumor suppressor RUNX3 in a CagA-dependent manner. CagA directly associates with RUNX3 through a specific recognition of the PY motif of RUNX3 by a WW domain of CagA. Deletion of the WW domains of CagA or mutation of the PY motif in RUNX3 abolishes the ability of CagA to induce the ubiquitination and degradation of RUNX3, thereby extinguishing its ability to inhibit the transcriptional activation of RUNX3. Our studies identify RUNX3 as a novel cellular target of H. pylori CagA and also reveal a mechanism by which CagA functions as an oncoprotein by blocking the activity of gastric tumor suppressor RUNX3.

CagA C-terminal variations in Helicobacter pylori strains from Colombian patients with gastric precancerous lesions.

The C-terminus of the Helicobacter pylori CagA protein is polymorphic, bearing different EPIYA sequences (EPIYA-A, B, C or D), and one or more CagA multimerization (CM) motifs. The number of EPIYA-C motifs is associated with precancerous lesions and gastric cancer (GC). The relationship between EPIYA, CM motifs and gastric lesions was examined in H. pylori-infected Colombian patients from areas of high and low risk for GC. Genomic DNA was extracted from H. pylori strains cultured from gastric biopsies from 80 adults with dyspeptic symptoms. Sixty-seven (83.8%) of 80 strains were cagA positive. The 3' region of cagA was sequenced, and EPIYA and CM motifs were identified. CagA proteins contained one (64.2%), two (34.3%) or three EPIYA-C motifs (1.5%), all with Western type CagA-specific sequences. Strains with one EPIYA-C motif were associated with less severe gastric lesions (non-atrophic and multifocal atrophic gastritis), whereas strains with multiple EPIYA-C motifs were associated with more severe lesions (intestinal metaplasia and dysplasia) (p <0.001). In 54 strains, the CM motifs were identical to those common in Western strains. Thirteen strains from the low-risk area contained two different CM motifs: one of Western type located within the EPIYA-C segment and another following the EPIYA-C segment and resembling the CM motif found in East Asian strains. These strains induced significantly shorter projections in AGS cells and an attenuated reduction in levels of CagA upon immunodepletion of SHP-2 than strains possessing Western/Western motifs. This novel finding may partially explain the difference in GC incidence in these populations.

Helicobacter pylori infection stimulates plasminogen activator inhibitor 1 production by gastric epithelial cells.

Chronic infection with the gastric pathogen Helicobacter pylori significantly increases the risk of developing atrophic gastritis, peptic ulcer disease, and gastric adenocarcinoma. H. pylori strains that possess the cag pathogenicity island, which translocates CagA into the host cells, augment these risks. The aim of this study was to determine the molecular mechanisms through which H. pylori upregulates the expression of plasminogen activator inhibitor 1 (PAI-1), a member of the urokinase activator system that is involved in tumor metastasis and angiogenesis. Levels of PAI-1 mRNA and protein were examined in tissues from H. pylori-infected patients and in vitro using AGS gastric epithelial cells. In vitro, cells were infected with toxigenic cag-positive or nontoxigenic cag-negative strains of H. pylori or isogenic mutants. The amount of PAI-1 secretion was measured by enzyme-linked immunosorbent assay, and mRNA levels were determined using real-time PCR. The regulation of PAI-1 was examined using the extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor and small interfering RNA. Analysis of human biopsy samples revealed an increase in both PAI-1 mRNA and protein levels in patients with H. pylori gastritis compared to those of uninfected controls. Infection of AGS cells with H. pylori significantly increased PAI-1 mRNA expression and the secretion of PAI-1 protein. Moreover, PAI-1 mRNA and protein production was more pronounced when AGS cells were infected by H. pylori strains carrying a functional cag secretion system than when cells were infected by strains lacking this system. PAI-1 secretion was also reduced when cells were infected with either cagE-negative or cagA-negative mutants. The ectopic overexpression of CagA significantly increased the levels of PAI-1 mRNA and protein, whereas blockade of the ERK1/2 pathway inhibited H. pylori-mediated PAI-1 upregulation. These findings suggest that the upregulation of PAI-1 in H. pylori-infected gastric epithelial cells may contribute to the carcinogenic process.