Targeted mobilization of Lrig1+ gastric epithelial stem cell populations by a carcinogenic Helicobacter pylori type IV secretion system.

Helicobacter pylori-induced gastritis is the strongest risk factor for gastric adenocarcinoma, a malignancy preceded by a series of well-defined histological stages, including metaplasia. One microbial constituent that augments cancer risk is the cag type 4 secretion system (T4SS), which translocates the oncoprotein CagA into host cells. Aberrant stem cell activation is linked to carcinogenesis, and Lrig1 (leucine-rich repeats and Ig-like domains 1) marks a distinct population of progenitor cells. We investigated whether microbial effectors with carcinogenic potential influence Lrig1 progenitor cells ex vivo and via lineage expansion within H. pylori-infected gastric mucosa. Lineage tracing was induced in Lrig1-CreERT2/+;R26R-YFP/+ (Lrig1/YFP) mice that were uninfected or subsequently infected with cag+H. pylori or an isogenic cagE- mutant (nonfunctional T4SS). In contrast to infection with wild-type (WT) H. pylori for 2 wk, infection for 8 wk resulted in significantly increased inflammation and proliferation in the corpus and antrum compared with uninfected or mice infected with the cagE- mutant. WT H. pylori-infected mice harbored significantly higher numbers of Lrig1/YFP epithelial cells that coexpressed UEA1 (surface cell marker). The number of cells coexpressing intrinsic factor (chief cell marker), YFP (lineage marker), and GSII lectin (spasmolytic polypeptide-expressing metaplasia marker) were increased only by WT H. pylori In human samples, Lrig1 expression was significantly increased in lesions with premalignant potential compared with normal mucosa or nonatrophic gastritis. In conclusion, chronic H. pylori infection stimulates Lrig1-expressing progenitor cells in a cag-dependent manner, and these reprogrammed cells give rise to a full spectrum of differentiated cells.

α-Difluoromethylornithine reduces gastric carcinogenesis by causing mutations in Helicobacter pylori cagY.

Infection by Helicobacter pylori is the primary cause of gastric adenocarcinoma. The most potent H. pylori virulence factor is cytotoxin-associated gene A (CagA), which is translocated by a type 4 secretion system (T4SS) into gastric epithelial cells and activates oncogenic signaling pathways. The gene cagY encodes for a key component of the T4SS and can undergo gene rearrangements. We have shown that the cancer chemopreventive agent α-difluoromethylornithine (DFMO), known to inhibit the enzyme ornithine decarboxylase, reduces H. pylori-mediated gastric cancer incidence in Mongolian gerbils. In the present study, we questioned whether DFMO might directly affect H. pylori pathogenicity. We show that H. pylori output strains isolated from gerbils treated with DFMO exhibit reduced ability to translocate CagA in gastric epithelial cells. Further, we frequently detected genomic modifications in the middle repeat region of the cagY gene of output strains from DFMO-treated animals, which were associated with alterations in the CagY protein. Gerbils did not develop carcinoma when infected with a DFMO output strain containing rearranged cagY or the parental strain in which the wild-type cagY was replaced by cagY with DFMO-induced rearrangements. Lastly, we demonstrate that in vitro treatment of H. pylori by DFMO induces oxidative DNA damage, expression of the DNA repair enzyme MutS2, and mutations in cagY, demonstrating that DFMO directly affects genomic stability. Deletion of mutS2 abrogated the ability of DFMO to induce cagY rearrangements directly. In conclusion, DFMO-induced oxidative stress in H. pylori leads to genomic alterations and attenuates virulence.

Carcinogenic Helicobacter pylori Strains Selectively Dysregulate the In Vivo Gastric Proteome, Which May Be Associated with Stomach Cancer Progression.

Helicobacter pylori is the strongest risk factor for gastric cancer. Initial interactions between H. pylori and its host originate at the microbial-gastric epithelial cell interface, and contact between H. pylori and gastric epithelium activates signaling pathways that drive oncogenesis. One microbial constituent that increases gastric cancer risk is the cag pathogenicity island, which encodes a type IV secretion system that translocates the effector protein, CagA, into host cells. We previously demonstrated that infection of Mongolian gerbils with a carcinogenic cag+H. pylori strain, 7.13, recapitulates many features of H. pylori-induced gastric cancer in humans. Therefore, we sought to define gastric proteomic changes induced by H. pylori that are critical for initiation of the gastric carcinogenic cascade. Gastric cell scrapings were harvested from H. pylori-infected and uninfected gerbils for quantitative proteomic analyses using isobaric tags for relative and absolute quantitation (iTRAQ). Quantitative proteomic analysis of samples from two biological replicate experiments quantified a total of 2764 proteins, 166 of which were significantly altered in abundance by H. pylori infection. Pathway mapping identified significantly altered inflammatory and cancer-signaling pathways that included Rab/Ras signaling proteins. Consistent with the iTRAQ results, RABEP2 and G3BP2 were significantly up-regulated in vitro, ex vivo in primary human gastric monolayers, and in vivo in gerbil gastric epithelium following infection with H. pylori strain 7.13 in a cag-dependent manner. Within human stomachs, RABEP2 and G3BP2 expression in gastric epithelium increased in parallel with the severity of premalignant and malignant lesions and was significantly elevated in intestinal metaplasia and dysplasia, as well as gastric adenocarcinoma, compared with gastritis alone. These results indicate that carcinogenic strains of H. pylori induce dramatic and specific changes within the gastric proteome in vivo and that a subset of altered proteins within pathways with oncogenic potential may facilitate the progression of gastric carcinogenesis in humans.

Helicobacter pylori-induced miR-135b-5p promotes cisplatin resistance in gastric cancer.

Helicobacter pylori infection is a major risk factor for the development of gastric cancer. Aberrant expression of microRNAs is strongly implicated in gastric tumorigenesis; however, their contribution in response to H. pylori infection has not been fully elucidated. In this study, we evaluated the expression of miR-135b-5p and its role in gastric cancer. We describe the overexpression of miR-135b-5p in human gastric cancer tissue samples compared with normal tissue samples. Furthermore, we found that miR-135b-5p is also up-regulated in gastric tumors from the trefoil factor 1-knockout mouse model. Infection with H. pylori induced the expression of miR-135b-5p in the in vitro and in vivo models. miR-135b-5p induction was mediated by NF-κB. Treatment of gastric cancer cells with TNF-α induced miR-135b-5p in a NF-κB-dependent manner. Mechanistically, we found that miR-135b-5p targets Krüppel-like factor 4 (KLF4) and binds to its 3' UTR, leading to reduced KLF4 expression. Functionally, high levels of miR-135b-5p suppress apoptosis and induce cisplatin resistance. Our results uncovered a mechanistic link between H. pylori infection and miR-135b-5p-KLF4, suggesting that targeting miR-135b-5p could be a potential therapeutic approach to circumvent resistance to cisplatin.-Shao, L., Chen, Z., Soutto, M., Zhu, S., Lu, H., Romero-Gallo, J., Peek, R., Zhang, S., El-Rifai, W. Helicobacter pylori-induced miR-135b-5p promotes cisplatin resistance in gastric cancer.

Helicobacter pylori antimicrobial resistance and antibiotic consumption in the low-resource Central America setting.

BACKGROUND: Antimicrobial resistance is a global public health problem, particularly in low- and middle-income countries (LMICs), where antibiotics are often obtained without a prescription. H. pylori antimicrobial resistance patterns are informative for patient care and gastric cancer prevention programs, have been shown to correlate with general antimicrobial consumption, and may guide antimicrobial stewardship programs in LMICs. We report H. pylori resistance and antimicrobial utilization patterns for western Honduras, representative of rural Central America. METHODS: In the context of the western Honduras gastric cancer epidemiology initiative, gastric biopsies from 189 patients were studied for culture and resistance patterns. Antimicrobial utilization was investigated for common H. pylori treatment regimens from regional public (7 antimicrobials) and national private (4 antimicrobials) data, analyzed in accordance with WHO anatomical therapeutic chemical defined daily doses (DDD) method and expressed as DDD/1000 inhabitants per day (DID) and per year (DIY). RESULTS: H. pylori was successfully cultured from 116 patients (56% males, mean age: 54), and nearly all strains were cagA+ and vacAs1m1+ positive (99% and 90.4%, respectively). Unexpectedly, high resistance was noted for levofloxacin (20.9%) and amoxicillin (10.7%), while metronidazole (67.9%) and clarithromycin (11.2%) were similar to data from Latin America. Significant associations with age, gender, or histology were not noted, with the exception of levofloxacin (28%, P = 0.01) in those with histology limited to non-atrophic gastritis. Total antimicrobial usage in western Honduras of amoxicillin (17.3 DID) and the quinolones had the highest relative utilizations compared with other representative nations. CONCLUSIONS: We observed significant H. pylori resistance to amoxicillin and levofloxacin in the context of high community antimicrobial utilization. This has implications in Central America for H. pylori treatment guidelines as well as antimicrobial stewardship programs.

Epidermal growth factor receptor inhibition downregulates Helicobacter pylori-induced epithelial inflammatory responses, DNA damage and gastric carcinogenesis.

OBJECTIVE: Gastric cancer is the third leading cause of cancer death worldwide and infection by Helicobacter pylori is the strongest risk factor. We have reported increased epidermal growth factor receptor (EGFR) phosphorylation in the H. pylori-induced human carcinogenesis cascade, and association with DNA damage. Our goal was to determine the role of EGFR activation in gastric carcinogenesis. DESIGN: We evaluated gefitinib, a specific EGFR inhibitor, in chemoprevention of H. pylori-induced gastric inflammation and cancer development. Mice with genetically targeted epithelial cell-specific deletion of Egfr (EfgrΔepi mice) were also used. RESULTS: In C57BL/6 mice, gefitinib decreased Cxcl1 and Cxcl2 expression by gastric epithelial cells, myeloperoxidase-positive inflammatory cells in the mucosa and epithelial DNA damage induced by H. pylori infection. Similar reductions in chemokines, inflammatory cells and DNA damage occurred in infected EgfrΔepi versus Egfrfl/fl control mice. In H. pylori-infected transgenic insulin-gastrin (INS-GAS) mice and gerbils, gefitinib treatment markedly reduced dysplasia and carcinoma. Gefitinib blocked H. pylori-induced activation of mitogen-activated protein kinase 1/3 (MAPK1/3) and activator protein 1 in gastric epithelial cells, resulting in inhibition of chemokine synthesis. MAPK1/3 phosphorylation and JUN activation was reduced in gastric tissues from infected wild-type and INS-GAS mice treated with gefitinib and in primary epithelial cells from EfgrΔepi versus Egfrfl/fl mice. Epithelial EGFR activation persisted in humans and mice after H. pylori eradication, and gefitinib reduced gastric carcinoma in INS-GAS mice treated with antibiotics. CONCLUSIONS: These findings suggest that epithelial EGFR inhibition represents a potential strategy to prevent development of gastric carcinoma in H. pylori-infected individuals.

Dynamics of Helicobacter pylori infection as a determinant of progression of gastric precancerous lesions: 16-year follow-up of an eradication trial.

OBJECTIVE: To evaluate the long-term effect of cumulative time exposed to Helicobacter pylori infection on the progression of gastric lesions. DESIGN: 795 adults with precancerous gastric lesions were randomised to receive anti-H. pylori treatment at baseline. Gastric biopsies were obtained at baseline and at 3, 6, 12 and 16 years. A total of 456 individuals attended the 16-year visit. Cumulative time of H. pylori exposure was calculated as the number of years infected during follow-up. Multivariable logistic regression models were used to estimate the risk of progression to a more advanced diagnosis (versus no change/regression) as well as gastric cancer risk by intestinal metaplasia (IM) subtype. For a more detailed analysis of progression, we also used a histopathology score assessing both severity and extension of the gastric lesions (range 1-6). The score difference between baseline and 16 years was modelled by generalised linear models. RESULTS: Individuals who were continuously infected with H. pylori for 16 years had a higher probability of progression to a more advanced diagnosis than those who cleared the infection and remained negative after baseline (p=0.001). Incomplete-type IM was associated with higher risk of progression to cancer than complete-type (OR, 11.3; 95% CI 1.4 to 91.4). The average histopathology score increased by 0.20 units/year (95% CI 0.12 to 0.28) among individuals continuously infected with H. pylori. The effect of cumulative time of infection on progression in the histopathology score was significantly higher for individuals with atrophy (without IM) than for individuals with IM (p<0.001). CONCLUSIONS: Long-term exposure to H. pylori infection was associated with progression of precancerous lesions. Individuals infected with H. pylori with these lesions may benefit from eradication, particularly those with atrophic gastritis without IM. Incomplete-type IM may be a useful marker for the identification of individuals at higher risk for cancer.

Pan-genomic analyses identify key Helicobacter pylori pathogenic loci modified by carcinogenic host microenvironments.

OBJECTIVE: Helicobacter pylori is the strongest risk factor for gastric cancer; however, the majority of infected individuals do not develop disease. Pathological outcomes are mediated by complex interactions among bacterial, host and environmental constituents, and two dietary factors linked with gastric cancer risk are iron deficiency and high salt. We hypothesised that prolonged adaptation of H. pylori to in vivo carcinogenic microenvironments results in genetic modification important for disease. DESIGN: Whole genome sequencing of genetically related H. pylori strains that differ in virulence and targeted H. pylori sequencing following prolonged exposure of bacteria to in vitro carcinogenic conditions were performed. RESULTS: A total of 180 unique single nucleotide polymorphisms (SNPs) were identified among the collective genomes when compared with a reference H. pylori genome. Importantly, common SNPs were identified in isolates harvested from iron-depleted and high salt carcinogenic microenvironments, including an SNP within fur (FurR88H). To investigate the direct role of low iron and/or high salt, H. pylori was continuously cultured in vitro under low iron or high salt conditions to assess fur genetic variation. Exposure to low iron or high salt selected for the FurR88H variant after only 5 days. To extend these results, fur was sequenced in 339 clinical H. pylori strains. Among the isolates examined, 17% (40/232) of strains isolated from patients with premalignant lesions harboured the FurR88H variant, compared with only 6% (6/107) of strains from patients with non-atrophic gastritis alone (p=0.0034). CONCLUSION: These results indicate that specific genetic variation arises within H. pylori strains during in vivo adaptation to conditions conducive for gastric carcinogenesis.

BET Inhibition Attenuates Helicobacter pylori-Induced Inflammatory Response by Suppressing Inflammatory Gene Transcription and Enhancer Activation.

Helicobacter pylori infection causes chronic gastritis and peptic ulceration. H. pylori-initiated chronic gastritis is characterized by enhanced expression of many NF-κB-regulated inflammatory cytokines. Brd4 has emerged as an important NF-κB regulator and regulates the expression of many NF-κB-dependent inflammatory genes. In this study, we demonstrated that Brd4 was not only actively involved in H. pylori-induced inflammatory gene mRNA transcription but also H. pylori-induced inflammatory gene enhancer RNA (eRNA) synthesis. Suppression of H. pylori-induced eRNA synthesis impaired H. pylori-induced mRNA synthesis. Furthermore, H. pylori stimulated NF-κB-dependent recruitment of Brd4 to the promoters and enhancers of inflammatory genes to facilitate the RNA polymerase II-mediated eRNA and mRNA synthesis. Inhibition of Brd4 by JQ1 attenuated H. pylori-induced eRNA and mRNA synthesis for a subset of NF-κB-dependent inflammatory genes. JQ1 also inhibited H. pylori-induced interaction between Brd4 and RelA and the recruitment of Brd4 and RNA polymerase II to the promoters and enhancers of inflammatory genes. Finally, we demonstrated that JQ1 suppressed inflammatory gene expression, inflammation, and cell proliferation in H. pylori-infected mice. These studies highlight the importance of Brd4 in H. pylori-induced inflammatory gene expression and suggest that Brd4 could be a potential therapeutic target for the treatment of H. pylori-triggered inflammatory diseases and cancer.

Helicobacter pylori-induced cell death is counteracted by NF-κB-mediated transcription of DARPP-32.

OBJECTIVE: DARPP-32 is a frequently amplified and overexpressed gene that promotes several oncogenic functions in gastric cancer. Herein, we investigated the relationship between Helicobacter pylori infection, proinflammatory NF-κB activation and regulation of DARPP-32. DESIGN: The study used in vivo and in vitro experiments. Luciferase reporter, quantitative real-time PCR, immunoblot, chromatin immunoprecipitation (ChIP), cell viability, H. pylori infection, tissue microarrays and immunohistochemical assays were used. RESULTS: Our results indicated that H. pylori infection increased the DARPP-32 mRNA and protein levels in gastric cancer cell lines and gastric mucosa of mice. H. pylori infection increased the activity of NF-κB reporter and p-NF-κB (S536) protein level in vitro and in vivo. To investigate the transcriptional regulation of DARPP-32, we cloned a 3019 bp of the DARPP-32 promoter into the luciferase reporter (pGL3-Luc). Both H. pylori infection and tumour necrosis factor-α treatment induced DARPP-32 reporter activity (p<0.01). Using deletion constructs of DARPP-32 promoter and ChIP assay, we demonstrated that the sequence -996 to -1008 bp containing putative NF-κB-binding sites is the most active region. The induction of DARPP-32 expression by H. pylori infection counteracted H. pylori-induced cell death through activation of serine/threonine-specific protein kinase (AKT), as determined by ATP-Glo and clonogenic survival assays. Immunohistochemistry analysis demonstrated a significant positive correlation between NF-κB and DARPP-32 expression levels in gastric cancer tissues (r2=0.43, p<0.01). CONCLUSIONS: Given the high frequency of DARPP-32 overexpression and its prosurvival oncogenic functions, the induction of DARPP-32 expression following H. pylori infection and activation of NF-κB provides a link between infection, inflammation and gastric tumourigenesis.

Characterization of progressive metaplasia in the gastric corpus mucosa of Mongolian gerbils infected with Helicobacter pylori.

Spasmolytic polypeptide-expressing metaplasia (SPEM) and intestinal metaplasia are considered neoplastic precursors of gastric adenocarcinoma in humans. Loss of parietal cells causes the development of SPEM in the gastric corpus and then chronic inflammation drives SPEM toward a more proliferative lineage. Mongolian gerbils infected with Helicobacter pylori develop chronic gastritis and metaplasia, mimicking aspects of human gastritis with H. pylori infection. We therefore examined metaplastic lineages in the gastric corpus mucosa of gerbils infected by H. pylori strain 7.13, which produces rapid onset of severe inflammation. Six weeks following H. pylori infection, Griffonia simplicifolia lectin II (GSII)-positive SPEM developed in the base of oxyntic glands in association with parietal cell loss and inflammation. In association with severe inflammation, SPEM glands evolved into aberrant phenotypes, including branched lesions, dilated lesions, and penetrating invasive glands. Mucin 4 (MUC4) was up-regulated in SPEM and progressive SPEM. Clusterin was expressed in the tips of branched and dilated lesions and throughout regions of invasive glands. Intriguingly, clusterin-positive regions in these lesions expressed Ki67 and matrix metalloproteinase 7 (MMP-7). These same regions were also positive for expression of phospho-IkBα, suggestive of activated NFkB signalling. These findings suggest that clusterin-positive regions in progressive phenotypes of SPEM have invasive characteristics. Thus, H. pylori infection in gerbils induces SPEM, which then can progress to further aberrant and invasive metaplastic phenotypes. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Human and Helicobacter pylori coevolution shapes the risk of gastric disease.

Helicobacter pylori is the principal cause of gastric cancer, the second leading cause of cancer mortality worldwide. However, H. pylori prevalence generally does not predict cancer incidence. To determine whether coevolution between host and pathogen influences disease risk, we examined the association between the severity of gastric lesions and patterns of genomic variation in matched human and H. pylori samples. Patients were recruited from two geographically distinct Colombian populations with significantly different incidences of gastric cancer, but virtually identical prevalence of H. pylori infection. All H. pylori isolates contained the genetic signatures of multiple ancestries, with an ancestral African cluster predominating in a low-risk, coastal population and a European cluster in a high-risk, mountain population. The human ancestry of the biopsied individuals also varied with geography, with mostly African ancestry in the coastal region (58%), and mostly Amerindian ancestry in the mountain region (67%). The interaction between the host and pathogen ancestries completely accounted for the difference in the severity of gastric lesions in the two regions of Colombia. In particular, African H. pylori ancestry was relatively benign in humans of African ancestry but was deleterious in individuals with substantial Amerindian ancestry. Thus, coevolution likely modulated disease risk, and the disruption of coevolved human and H. pylori genomes can explain the high incidence of gastric disease in the mountain population.

Genetic Evolution of a Helicobacter pylori Acid-Sensing Histidine Kinase and Gastric Disease.

Helicobacter pylori is the strongest risk factor for gastric adenocarcinoma, which develops within a hypochlorhydric environment. We sequentially isolated H. pylori (strain J99) from a patient who developed corpus-predominant gastritis and hypochlorhydia over a 6-year interval. Archival J99 survived significantly better under acidic conditions than recent J99 strains. H. pylori arsRS encodes a 2-component system critical for stress responses; recent J99 isolates harbored 2 nonsynonymous arsS mutations, and arsS inactivation abolished acid survival. In vivo, acid-resistant archival, but not recent J99, successfully colonized high-acid-secreting rodents. Thus, genetic evolution of arsS may influence progression to hypochlorhydia and gastric cancer.

TLR9 activation suppresses inflammation in response to Helicobacter pylori infection.

Helicobacter pylori (H. pylori) induces chronic gastritis in humans, and infection can persist for decades. One H. pylori strain-specific constituent that augments disease risk is the cag pathogenicity island. The cag island encodes a type IV secretion system (T4SS) that translocates DNA into host cells. Toll-like receptor 9 (TLR9) is an innate immune receptor that detects hypo-methylated CpG DNA motifs. In this study, we sought to define the role of the H. pylori cag T4SS on TLR9-mediated responses in vivo. H. pylori strain PMSS1 or its cagE- mutant, which fails to assemble a T4SS, were used to infect wild-type or Tlr9-/- C57BL/6 mice. PMSS1-infected Tlr9-/- mice developed significantly higher levels of inflammation, despite similar levels of colonization density, compared with PMSS1-infected wild-type mice. These changes were cag dependent, as both mouse genotypes infected with the cagE- mutant only developed minimal inflammation. Tlr9-/- genotypes did not alter the microbial phenotypes of in vivo-adapted H. pylori strains; therefore, we examined host immunological responses. There were no differences in levels of TH1 or TH2 cytokines in infected mice when stratified by host genotype. However, gastric mucosal levels of IL-17 were significantly increased in infected Tlr9-/- mice compared with infected wild-type mice, and H. pylori infection of IL-17A-/- mice concordantly led to significantly decreased levels of gastritis. Thus loss of Tlr9 selectively augments the intensity of IL-17-driven immune responses to H. pylori in a cag T4SS-dependent manner. These results suggest that H. pylori utilizes the cag T4SS to manipulate the intensity of the host immune response.

Bacterial CagA protein induces degradation of p53 protein in a p14ARF-dependent manner.

OBJECTIVE: Infection with Helicobacter pylori is the strongest known risk factor for adenocarcinoma of the stomach. Tumorigenic transformation of gastric epithelium induced by H. pylori is a highly complex process driven by an active interplay between bacterial virulence and host factors, many aspects of which remain obscure. In this work, we investigated the degradation of p53 tumour suppressor induced by H. pylori. DESIGN: Expression of p53 protein in gastric biopsies was assessed by immunohistochemistry. Gastric cells were co-cultured with H. pylori strains isolated from high-gastric risk and low-gastric risk areas and assessed for expression of p53, p14ARF and cytotoxin-associated gene A (CagA) by immunoblotting. siRNA was used to inhibit activities of ARF-BP1 and Human Double Minute 2 (HDM2) proteins. RESULTS: Our analysis demonstrated that H. pylori strains expressing high levels of CagA virulence factor and associated with a higher gastric cancer risk more strongly suppress p53 compared with low-risk strains in vivo and in vitro. We found that degradation of p53 induced by bacterial CagA protein is mediated by host HDM2 and ARF-BP1 E3 ubiquitin ligases, while the p14ARF protein counteracts H. pylori-induced signalling. CONCLUSIONS: Our results provide novel evidence that tumorigenicity associated with H. pylori infection is linked to inhibition of p53 protein by CagA. We propose a model in which CagA-induced degradation of p53 protein is determined by a relative level of p14ARF. In cells in which p14ARF levels were decreased due to hypermethylation or deletion of the p14ARF gene, H. pylori efficiently degraded p53, whereas p53 is protected in cells expressing high levels of p14ARF.

Trefoil factor 1 expression suppresses Helicobacter pylori-induced inflammation in gastric carcinogenesis.

BACKGROUND: Infection with Helicobacter pylori, a high-risk factor for gastric cancer, is frequently associated with chronic inflammation through activation of nuclear factor κB (NF-κB). Trefoil factor 1 (TFF1) is a constitutively expressed protein in the stomach that has tumor-suppressor functions and plays a critical role in maintaining mucosal integrity. This study investigated the role of TFF1 in regulating the proinflammatory response to H. pylori infections. METHODS: For in vitro studies, immunofluorescence, luciferase reporter assays, Western blots, and quantitative real-time polymerase chain reaction were performed to investigate the activation of NF-κB and its target genes in response to infections with H. pylori strains J166 and 7.13. In addition, Tff1-knockout (KO) and Tff1-wild-type mice were used for infections with the H. pylori strain called premouse Sydney strain 1. RESULTS: The reconstitution of TFF1 expression in gastric cancer cells significantly suppressed H. pylori-mediated increases in NF-κB-p65 nuclear staining, transcriptional activity, and expression of proinflammatory cytokine genes (tumor necrosis factor α, interleukin 1ß, chemokine [C-X-C motif] ligand 5, and interleukin 4 receptor) that were associated with reductions in the expression and phosphorylation of NF-κB-p65 and IκB kinase α/ß proteins. The in vivo studies using the Tff1-KO mouse model of gastric neoplasia confirmed the in vitro findings. Furthermore, they demonstrated increases in chronic inflammation scores and in the frequency of invasive gastric adenocarcinoma in the Tff1-KO mice infected with H. pylori versus the uninfected Tff1-KO mice. CONCLUSIONS: These findings underscore an important protective role of TFF1 in abrogating H. pylori-mediated inflammation, a crucial hallmark of gastric tumorigenesis. Therefore, loss of TFF1 expression could be an important step in H. pylori-mediated gastric carcinogenesis.

Identification of alanyl aminopeptidase (CD13) as a surface marker for isolation of mature gastric zymogenic chief cells.

Injury and inflammation in the gastric epithelium can cause disruption of the pathways that guide the differentiation of cell lineages, which in turn can cause persistent alterations in differentiation patterns, known as metaplasia. Metaplasia that occurs in the stomach is associated with increased risk for cancer. Methods for isolating distinct gastric epithelial cell populations would facilitate dissection of the molecular and cellular pathways that guide normal and metaplastic differentiation. Here, we identify alanyl aminopeptidase (CD13) as a specific surface marker of zymogenic chief cells (ZCs) in the gastric epithelium. We show that 1) among gastric epithelial cells alanyl aminopeptidase expression is confined to mature ZCs, and 2) its expression is lost en route to metaplasia in both mouse and human stomachs. With this new marker coupled with new techniques that we introduce for dissociating gastric epithelial cells and overcoming their constitutive autofluorescence, we are able to reliably isolate enriched populations of ZCs for both molecular analysis and for the establishment of ZC-derived ex vivo gastroid cultures.

Pathogenic bacterium Helicobacter pylori alters the expression profile of p53 protein isoforms and p53 response to cellular stresses.

The p53 protein plays a central role in the prevention of tumorigenesis. Cellular stresses, such as DNA damage and aberrant oncogene activation, trigger induction of p53 that halts cellular proliferation and allows cells to be repaired. If cellular damage is beyond the capability of the repair mechanisms, p53 induces apoptosis or cell cycle arrest, preventing damaged cells from becoming cancerous. However, emerging evidence suggests that the function of p53 needs to be considered as isoform-specific. Here, we report that the expression profile of p53 can be shifted toward inhibitory p53 isoforms by the pathogenic bacterium Helicobacter pylori, which is known for its strong association with gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma. We found that interaction of H. pylori with gastric epithelial cells, mediated via the cag pathogenicity island, induces N-terminally truncated Δ133p53 and Δ160p53 isoforms in human cells. Induction of an orthologous p53 isoform, Δ153p53, was also found in H. pylori-infected Mongolian gerbils. The p53 isoforms inhibit p53 and p73 activities, induce NF-κB, and increase survival of infected cells. Expression of Δ133p53, in response to H. pylori infection, is regulated by phosphorylation of c-Jun and activation of activator protein-1-dependent transcription. Together, these results provide unique insights into the regulation of p53 protein and may contribute to the understanding of tumorigenesis associated with H. pylori.

The hyaluronic acid receptor CD44 coordinates normal and metaplastic gastric epithelial progenitor cell proliferation.

The stem cell in the isthmus of gastric units continually replenishes the epithelium. Atrophy of acid-secreting parietal cells (PCs) frequently occurs during infection with Helicobacter pylori, predisposing patients to cancer. Atrophy causes increased proliferation of stem cells, yet little is known about how this process is regulated. Here we show that CD44 labels a population of small, undifferentiated cells in the gastric unit isthmus where stem cells are known to reside. Loss of CD44 in vivo results in decreased proliferation of the gastric epithelium. When we induce PC atrophy by Helicobacter infection or tamoxifen treatment, this CD44(+) population expands from the isthmus toward the base of the unit. CD44 blockade during PC atrophy abrogates the expansion. We find that CD44 binds STAT3, and inhibition of either CD44 or STAT3 signaling causes decreased proliferation. Atrophy-induced CD44 expansion depends on pERK, which labels isthmal cells in mice and humans. Our studies delineate an in vivo signaling pathway, ERK → CD44 → STAT3, that regulates normal and atrophy-induced gastric stem/progenitor-cell proliferation. We further show that we can intervene pharmacologically at each signaling step in vivo to modulate proliferation.

Strain-specific suppression of microRNA-320 by carcinogenic Helicobacter pylori promotes expression of the antiapoptotic protein Mcl-1.

Helicobacter pylori is the strongest risk factor for gastric cancer, and strains harboring the cag pathogenicity island, which translocates the oncoprotein CagA into host cells, further augment cancer risk. We previously reported that in vivo adaptation of a noncarcinogenic H. pylori strain (B128) generated a derivative strain (7.13) with the ability to induce adenocarcinoma, providing a unique opportunity to define mechanisms that mediate gastric carcinogenesis. MicroRNAs (miRNAs) are small noncoding RNAs that regulate expression of oncogenes or tumor suppressors and are frequently dysregulated in carcinogenesis. To identify miRNAs and their targets involved in H. pylori-mediated carcinogenesis, miRNA microarrays were performed on RNA isolated from gastric epithelial cells cocultured with H. pylori strains B128, 7.13, or a 7.13 cagA(-) isogenic mutant. Among 61 miRNAs differentially expressed in a cagA-dependent manner, the tumor suppressor miR-320 was significantly downregulated by strain 7.13. Since miR-320 negatively regulates the antiapoptotic protein Mcl-1, we demonstrated that H. pylori significantly induced Mcl-1 expression in a cagA-dependent manner and that suppression of Mcl-1 results in increased apoptosis. To extend these results, mice were challenged with H. pylori strain 7.13 or its cagA(-) mutant; consistent with cell culture data, H. pylori induced Mcl-1 expression in a cagA-dependent manner. In human subjects, cag(+) strains induced significantly higher levels of Mcl-1 than cag(-) strains, and Mcl-1 expression levels paralleled the severity of neoplastic lesions. Collectively, these results indicate that H. pylori suppresses miR-320, upregulates Mcl-1, and decreases apoptosis in a cagA-dependent manner, which likely confers an increased risk for gastric carcinogenesis.