CagA Expression Research Articles

AI-2 represses CagA expression and bacterial adhesion, attenuating the Helicobacter pylori-induced inflammatory response of gastric epithelial cells.

Helicobacter pylori (H. pylori) infection of gastric epithelial cells induces inflammatory response. Outer membrane proteins (OMPs), Type 4 secretion system (T4SS) encoded by cagPAI, and the effector protein CagA are involved in the pathogenesis of H. pylori. H. pylori possesses a gene encoding LuxS which synthesizes AI-2, a quorum sensing signal molecule. The aim of this study was to investigate the role of AI-2 in the expression of virulence factors and the inflammatory response of gastric epithelial (AGS) cells induced by H. pylori. H. pylori ΔluxS mutant was constructed, and AI-2 activity was measured with Vibrio harveyi BB170. NF-κB activation, IL-8 production, expression of OMPs (outer membrane proteins), CagA, and T4SS encoded by cagPAI were investigated in H. pylori wild type, and ΔluxS with or without supplementation of AI-2. H. pylori produced approximately 7μM of AI-2 in the medium. AI-2 inhibited expression and translocation of CagA after infection of AGS cells. AI-2 upregulated the expression of CagM, CagE, and CagX, while had no effect to the interaction between T4SS and α5β1 integrin. AI-2 also reduced expression of adhesins and bacterial adhesion to AGS cells. Finally, AI-2 reduced the activation of NF-κB and expression of IL-8 in H. pylori-infected AGS. AI-2 plays an important role in the pathogenesis of H. pylori. AI-2 inhibits the bacterial adhesion, expression, and translocation of CagA, and attenuates the inflammatory response of AGS cells induced by H. pylori.

Pathways of Gastric Carcinogenesis, Helicobacter pylori Virulence and Interactions with Antioxidant Systems, Vitamin C and Phytochemicals.

Helicobacter pylori is a class one carcinogen which causes chronic atrophic gastritis, gastric intestinal metaplasia, dysplasia and adenocarcinoma. The mechanisms by which H. pylori interacts with other risk and protective factors, particularly vitamin C in gastric carcinogenesis are complex. Gastric carcinogenesis includes metabolic, environmental, epigenetic, genomic, infective, inflammatory and oncogenic pathways. The molecular classification of gastric cancer subtypes has revolutionized the understanding of gastric carcinogenesis. This includes the tumour microenvironment, germline mutations, and the role of Helicobacter pylori bacteria, Epstein Barr virus and epigenetics in somatic mutations. There is evidence that ascorbic acid, phytochemicals and endogenous antioxidant systems can modify the risk of gastric cancer. Gastric juice ascorbate levels depend on dietary intake of ascorbic acid but can also be decreased by H. pylori infection, H. pylori CagA secretion, tobacco smoking, achlorhydria and chronic atrophic gastritis. Ascorbic acid may be protective against gastric cancer by its antioxidant effect in gastric cytoprotection, regenerating active vitamin E and glutathione, inhibiting endogenous N-nitrosation, reducing toxic effects of ingested nitrosodimethylamines and heterocyclic amines, and preventing H. pylori infection. The effectiveness of such cytoprotection is related to H. pylori strain virulence, particularly CagA expression. The role of vitamin C in epigenetic reprogramming in gastric cancer is still evolving. Other factors in conjunction with vitamin C also play a role in gastric carcinogenesis. Eradication of H. pylori may lead to recovery of vitamin C secretion by gastric epithelium and enable regression of premalignant gastric lesions, thereby interrupting the Correa cascade of gastric carcinogenesis.

Evaluation of cell-penetrating peptide-peptide nucleic acid effect in the inhibition of cagA in Helicobacter pylori.

Helicobacter pylori is the most common cause of chronic infection in human and is associated with gastritis, peptic ulcer disease, and adenocarcinoma of mucosa-associated lymphoid tissue cells. Peptide nucleic acid (PNA) is a synthetic compound, which can inhibit the production of a particular gene. This study aimed to investigate the effect of PNA on inhibiting the expression of cagA. After confirmation of the desired gene by polymerase chain reaction (PCR), the antisense sequence was designed against cagA gene. The minimum inhibitory concentrations of conjugated PNA against H. pylori was determined. The effect of the compound on the expression level of the cagA was investigated in HT29 cell culture using real-time PCR. The results showed 2 and 3 log reduction in bacterial count after 8- and 24-h treatment with 4 and 8 μM of the compound, respectively. The lowest expression level of the cagA gene was observed at a concentration of 8 μM after 6 h. The results of this study showed that cell-penetrating peptide antisense can be employed as effective tools for inhibiting the target gene mRNA for various purposes. Moreover, further research is necessary to assess the potency, safety, and pharmacokinetics of CPP-PNAs for clinical prevention and treatment of infections due to H. pylori.

Cytotoxin-associated gene A increases carcinogenicity of helicobacter pylori in colorectal adenoma

This study aimed to investigate the correlation of Helicobacter pylori (Hp) infection with disease risk and severity of colorectal adenoma, also to explore the association of cytotoxin-associated gene A (CagA) positive (CagA+)-Hp infection with gastrin and ki-67 expressions in colorectal adenoma patients. There were 1000 colorectal adenoma patients and 1500 controls consecutively enrolled, then Hp infection status was determined by 14C urea breath test and rapid urease test. Also, serum CagA expression and gastrin expression of colorectal adenoma patients were determined by enzyme-linked immunosorbent assay. Ki-67 expression in adenoma tissue of colorectal adenoma patients was assessed using immunohistochemistry. Hp+ rate in colorectal adenoma patients (623 (62.3%)) was more elevated than that in controls (814 (54.3%)). Multivariate logistic regression model analysis disclosed that Hp+ was an independent risk factor for colorectal adenoma. Additionally, Hp+ was positively associated with tumor size and high-grade intraepithelial neoplasia in colorectal adenoma patients. Also, serum gastrin expression and intratumoral ki-67 expression were higher in Hp+ CagA+ patients and Hp+ CagA- patients compared to Hp- patients, and they were also higher in Hp+ CagA+ patients compared to Hp+ CagA- patients. Hp infection positively associates with higher disease risk and worse disease conditions of colorectal adenoma, and CagA enhances the carcinogenicity of Hp in colorectal adenoma.

HP-CagA+ Regulates the Expression of CDK4/CyclinD1 via reg3 to Change Cell Cycle and Promote Cell Proliferation.

Previous studies have shown that regeneration gene 3 (reg3) is significantly expressed in gastric mucosa tissues with Helicobacter pylori (HP) cytotoxin-associated gene A (CagA)-positive (HP-CagA+). CagA-positive HP increases the risk of gastric cancer. The purpose of this study was to investigate the correlation between reg3 and HP-CagA+ and explore the effects of reg3 on the proliferation of gastric cancer cells and the development of tissues and organs. We analyzed the expression of reg3 in human tissues and organs. The results showed that reg3 expression in gastric tissues was significantly higher than that in other tissues and organs. In addition, reg3 influenced the prognosis of gastric, lung, and ovarian cancers. Immunohistochemical analysis indicated that the expression of reg3 and CagA in cancerous tissues was higher than that in adjacent tissues. HP-CagA+ infection of gastric cancer cells promotes reg3 expression, suggesting that reg3 may be a target gene of CagA in gastric cancer, which together affects the formation and development of gastric cancer. reg3 and CagA promote cell proliferation, and then affect the development of mouse tissues and organs by regulating G1/S phase transition of the cell cycle via the formation of the cell cycle-dependent complex CDK4/CyclinD1. This is the first study that shows the influence of CagA on the cell cycle and induction of cell proliferation by promoting reg3 expression.

Helicobacter pylori infection, chronic atrophic gastritis and risk of stomach and esophagus cancer: Results from the prospective population-based ESTHER cohort study.

Helicobacter pylori (H. pylori) infection is considered as principal cause of gastric cancer. It is further associated with a reduced risk of esophageal adenocarcinomas. In a large prospective population-based cohort study including 9,949 subjects with average observation time of 13.8 years, we assessed the risk of invasive gastric and esophageal cancer according to H. pylori infection and presence of chronic atrophic gastritis (CAG). Incidence rates and hazard ratios (HR) derived by Cox proportional hazards models and adjusted for relevant confounders were derived by seroprevalence of H. pylori and cytotoxin-associated gene A (CagA) antibodies and presence of CAG based on serological markers at baseline, respectively. During follow-up, 30 cases of noncardia gastric cancer and 33 cases of esophageal cancer were observed. Infection by H. pylori without and with expression of CagA was associated with a 5.2-fold (95% confidence interval 1.00-27.1) and an 18.2-fold (4.3-77.4) increase of noncardia gastric cancer incidence. A 0.65-fold decreased risk of esophageal adenocarcinomas (HR 0.35, 0.12-0.97) was observed among H. pylori-infected individuals. In participants infected with CagA expressed H. pylori, the presence of mild/moderate and severe CAG was associated with a 6.4-fold (1.3-31.0) and an 11.8-fold (3.1-45.4) increase of gastric cancer incidence, respectively. The results of this prospective population-based cohort study may contribute relevant evidence to the ongoing research of H. pylori-related cancers. The results may furthermore enhance the empirical basis for risk stratification among H. pylori-infected people and for recommendations regarding H. pylori screening and treatment among older adults in a Western population.

Abstract 4269: Regulation and functional roles of autophagy in Helicobacter pylori CagA-mediated gastric cancer

Abstract Autophagy is a catabolic process for the autophagosomic-lysosomal degradation of bulk cytoplasmic components and plays a crucial role in infection, inflammation and various cellular functions. Helicobacter pylori (H. pylori) was recently indicated to be able to induce autophagy. The cytotoxinA (CagA) of H.pylori was demonstrated to be degraded by autophagic pathway, but the the dynamic role of mechanism by which CagA affects autophagic signaling has not been identified. This study was determined to elucidate the functional role and regulatory mechanism of H. pylori induced autophagy in gastric cancer cells. In this study, we tested that during the H.pylori 60190(CagA+, VacA+) infection, where the nutrient starvation induced a high level of autophagy, in gastric cancer cell lines(AGS, MKN45, MKN28 and GES-1) through CagA and LC3 I/II(autophagy marker) protein expression level. The gastric cancer cells infected with H.pylori 60190, ΔCagA and ΔVacA(60190 isogenic mutant strain) were incubated with a serum free medium containing antibiotic(KM,Kanamycin) to kill extracellular H.pylori, intracellular CagA level decreased and LC3 I converted to LC3 II. The quantification of LC3 by ImageJ increased in the gastric cancer cells. Autophagy induction associated with intracellular CagA stability. CagA was essential and sufficient to induce autophagy in gastric cancer cells, with the induction of autophagy responsible for a decreased in the level of intracellular CagA. The gastric cancer cells infected with or without H.pylori were performed immunofluorescence and detected by confocal microscopy to examine the co-localization of CagA and LC3. CagA located with LC3, therefore, CagA was related to the mechanism of autophagy. The intracellular cagA decreased and LC3 I to LC3 II conversion was detected. To evaluate whether autophagy was specifically associated with H.pylori infection, we used LysoTracker® Red. H.pylori infection affected lysosomal degradation. The autophagy inhibitor (Cychloheximide, Bafilomycin A1, Chloroquine) and PI3K/AKT inhibitor (LY294002), GSK inhibitor (Lithium Chloride) in gastric cancer cells were used to examine the mechanism of CagA degradation. The cagA was affected by those inhibitors. The effects of CagA in promoting autophagy blocked autophagy. The autophagy contributed to CagA degradation in gastric cancer cells. To examine ectopic expression of CagA affects serum starvation induced autophagy, transfection of plasmid vector to AGS were carried out. Our findings showed that the autophagy play some important roles in H. pylori infected gastric cancer cells by the degradation of intracellular CagA leading to altered epithelial cellular signal transduction pathways. This indicate that studies investigating the host-pathogen autophagic interaction in H. pylori infection will provide the platform for bio-marker research for gastric cancer chemo-preventive strategy. Citation Format: BoRam Hwang, SoDam Lee, ChangYun Jung, ByeongMin Yu, YongChan Lee. Regulation and functional roles of autophagy in Helicobacter pylori CagA-mediated gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4269.

Systematic review with meta-analysis: association between Helicobacter pylori CagA seropositivity and odds of inflammatory bowel disease.

Accumulating data support a protective role of Helicobacter pylori against inflammatory bowel diseases (IBD), which might be mediated by strain-specific constituents, specifically cagA expression. To perform a systematic review and meta-analysis to more clearly define the association between CagA seropositivity and IBD. We identified comparative studies that included sufficient detail to determine the odds or risk of IBD, Crohn's disease (CD) or ulcerative colitis (UC) amongst individuals with vs without evidence of cagA expression (eg CagA seropositivity). Estimates were pooled using a random effects model. Three clinical studies met inclusion criteria. cagA expression was represented by CagA seropositivity in all studies. Compared to CagA seronegativity overall, CagA seropositivity was associated with lower odds of IBD (OR 0.31, 95% CI 0.21-0.44) and CD (OR 0.25, 95% CI 0.17-0.38), and statistically nonsignificant lower odds for UC (OR 0.68, 95% CI 0.35-1.32). Similarly, compared to H pylori non-exposed individuals, H pylori exposed, CagA seropositive individuals had lower odds of IBD (OR 0.26, 95% CI 0.16-0.41) and CD (OR 0.23, 95% CI 0.15-0.35), but not UC (OR 0.66, 0.34-1.27). However, there was no significant difference in the odds of IBD, CD or UC between H pylori exposed, CagA seronegative and H pylori non-exposed individuals. We found evidence for a significant association between CagA seropositive H pylori exposure and reduced odds of IBD, particularly CD, but not for CagA seronegative H pylori exposure. Additional studies are needed to confirm these findings and define underlying mechanisms.

Helicobacter pylori CagA promotes the malignant transformation of gastric mucosal epithelial cells through the dysregulation of the miR-155/KLF4 signaling pathway.

The Helicobacter pylori (H. pylori) cytotoxin-associated gene A (CagA) and Krüppel-like transcription factor (KLF4) were both closely associated with the development and progression of gastric cancer (GC). However, the nature of theinteractions between CagA and KLF4 in GC development has not been elucidated. Therefore, we focused on the CagA-mediated promotion of the malignant transformation of gastric epithelial cells. Herein, we first examined the expression of KLF4 in both human cancer and paracarcinoma tissues with or without H. pylori infection and found that KLF4 expression was significantly decreased in H. pylori-positive GC cells compared with the H. pylori-negative GC cells. Further functional studies revealed that the increased expression of CagA could suppress KLF4 expression and promote the malignant transformation of normal epithelial cells. Subsequently, we found that CagA could upregulate miR-155 and further restrict the expression of downstream KLF4. More importantly, the overexpression of miR-155 in GES-1 promoted epithelial-mesenchymal transition and eventually facilitated tumor growth in vivo. Overall, the identification of the CagA/miR-155/KLF4 signaling pathway provided a new insight into the development and treatment of GC.

Role of the protein CagA from Helicobacter pylori in the modifications of the human B lymphocyte cytoskeleton

Abstract The cytotoxin-associated gen A (CagA) is an oncogenic virulence factor of Helicobacter pylori that is related with the development of gastric cancer and gastric MALToma: a B cell non-Hodgkin’s lymphoma. CagA has an important role in the development of cancer due to its promiscuous interaction with different signaling-mediators. The association of CagA with cytoskeleton regulators generates an effect that gives invasive characteristics to epithelial cells, thus explaining the evolution to late stages of the gastric carcinoma. Although the translocation of CagA has been demonstrated in B lymphocytes, its impact on MALToma development and its effects over B cell cytoskeleton have not been characterized. Human Raji B cells were transiently transfected to express an EGFP-tagged CagA fusion protein. In culture, these cells displayed altered morphological and adhesion properties when compared with control cells. Fibronectin induced-spreading and adhesion assays were performed to assess the changes observed. The transfected cells exhibited atypical size and shape descriptors, determined by analyses of confocal microscopy images. Concomitantly, the adhesion of CagA+ Raji cells was observed reduced. These phenotypes could be explained by the diminished basal levels of ezrin that we detect in the CagA+ cells, determined by Western blot, that contrast with a high expression of this actin-regulatory molecule in Raji control cells. Taken together, our results show that the expression of CagA produces important changes in B cell morphology as well as cell adhesion due to alteration in actin cytoskeleton regulation; changes that could explain an invasive and aggressive B lymphocyte profile.

Novel Insights of Lymphomagenesis of Helicobacter pylori-Dependent Gastric Mucosa-Associated Lymphoid Tissue Lymphoma

Gastric mucosa-associated lymphoid tissue (MALT) lymphoma is the most common subtype of gastric lymphoma. Most gastric MALT lymphomas are characterized by their association with the Helicobacter pylori (HP) infection and are cured by first-line HP eradication therapy (HPE). Several studies have been conducted to investigate why most gastric MALT lymphomas remain localized, are dependent on HP infection, and show HP-specific intratumoral T-cells (e.g., CD40-mediated signaling, T-helper-2 (Th2)-type cytokines, chemokines, costimulatory molecules, and FOXP3+ regulatory T-cells) and their communication with B-cells. Furthermore, the reason why the antigen stimuli of these intratumoral T-cells with tonic B-cell receptor signaling promote lymphomagenesis of gastric MALT lymphoma has also been investigated. In addition to the aforementioned mechanisms, it has been demonstrated that the translocated HP cytotoxin-associated gene A (CagA) can promote B-cell proliferation through the activation of Src homology-2 domain-containing phosphatase (SHP-2) phosphorylation-dependent signaling, extracellular-signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), B-cell lymphoma (Bcl)-2, and Bcl-xL. Furthermore, the expression of CagA and these CagA-signaling molecules is closely associated with the HP-dependence of gastric MALT lymphomas (completely respond to first-line HPE). In this article, we summarize evidence of the classical theory of HP-reactive T-cells and the new paradigm of direct interaction between HP and B-cells that contributes to the HP-dependent lymphomagenesis of gastric MALT lymphomas. Although the role of first-line HPE in the treatment of HP-negative gastric MALT lymphoma remains uncertain, several case series suggest that a proportion of HP-negative gastric MALT lymphomas remains antibiotic-responsive and is cured by HPE. Considering the complicated interaction between microbiomes and the genome/epigenome, further studies on the precise mechanisms of HP- and other bacteria-directed lymphomagenesis in antibiotic-responsive gastric MALT lymphomas are warranted.

Decreased IL-17RB expression impairs CD11b+CD11c\u2212 myeloid cell accumulation in gastric mucosa and host defense during the early-phase of Helicobacter pylori infection

Interleukin-17 receptor B (IL-17RB), a member of the IL-17 receptor family activated by IL-17B/IL-17E, has been shown to be involved in inflammatory diseases. However, the regulation and function of IL-17RB in Helicobacter pylori (H. pylori) infection, especially in the early-phase is still unknown. Here, we found that gastric IL-17RB mRNA and protein were decreased in gastric mucosa of both patients and mice infected with H. pylori. In vitro experiments show that IL-17RB expression was down regulated via PI3K/AKT pathway on gastric epithelial cells (GECs) stimulated with H. pylori in a cagA-involved manner, while in vivo studies showed that the effect was partially dependent on cagA expression. IL-17E was also decreased during the early-phase of H. pylori infection, and provision of exogenous IL-17E resulted in increased CD11b+CD11c− myeloid cells accumulation and decreased bacteria colonization within the gastric mucosa. In the early-phase of H. pylori infection, IL-17E-IL-17RB promoted gastric epithelial cell-derived CXCL1/2/5/6 to attract CD11b+CD11c− myeloid cells, and also contributed to host defense by promoting the production of antibacterial protein Reg3a. This study defines a negative regulatory network involving IL-17E, GECs, IL-17RB, CD11b+CD11c− myeloid cells, and Reg3a in the early-phase of H. pylori infection, which results in an impaired host defense within the gastric microenvironment, suggesting IL-17RB as a potential early intervening target in H. pylori infection.

A Multicenter Prospective Study of First-Line Antibiotic Therapy for Early-Stage Gastric Mucosa-Associated Lymphoid Tissue (MALT) Lymphoma and Diffuse Large B-Cell Lymphoma with Histological Evidence of MALT

Background: To evaluate the efficacy of first-line Helicobacter pylori (HP) eradication (HPE) therapy against HP-positive gastric mucosa-associated lymphoid tissue (MALT) lymphoma and diffuse large B-cell lymphomas (DLBCL) with histological evidence of MALT (DLBCL[MALT]). Methods: Patients with gastric DLBCL(MALT) (n=10) and gastric MALT lymphoma (n=36) who had participated in a multicenter prospective study on first-line HPE therapy, conducted by the Taiwan Cooperative Oncology Group between December-2006 and September-2014, were enrolled. The HPE regimen consisted of omeprazole, amoxicillin, and clarithromycin for 14 days. The histological responses of tumors were re-assessed using the Groupe d'Etude des Lymphomes de l'Adult (GELA) scoring system. The primary end point was the therapeutic efficacy (complete remission, CR) of first-line HPE. The levels of expression of cytotoxin-associated gene A (CagA), BCL10, and NF-κB and the presence of t(11;18)(q21;q21) associated with HP-independence (lack of lymphoma regression after HPE) were assessed. Findings: Of the total 46 patients, 34 (73.9 %) achieved CR, including eight with DLBCL(MALT) and 26 with MALT lymphoma. The CR rates for patients with stages IE (n=40) and IIE1 (n=6) tumors were 75.0 % and 66.7 %, respectively. The frequency of CagA expression was significantly higher (85.3 % [29/34] vs 33.3 % [4/12]; p=0.001), whereas the frequency of nuclear BCL10 expression was significantly lower (8.8 % [3/34] vs 83.3 % [10/12]; p<0.0001), in HP-dependent tumors than in HP-independent tumors. The expression of nuclear NF-kB was significantly associated with HP-independence (p=0.0001). The API2-MALT1 fusion transcript was detected in 4 (33.0 %) of the 12 HP-independent MALT lymphomas, but in none of the 10 DLBCL(MALT) tumors. Interpretation: HPE is considered a first-line treatment for early-stage HP-positive DLBCL(MALT) as well as MALT lymphoma of the stomach. BCL10, and/or NF-kB are potential therapeutic targets in these lymphoma subtypes. Trial Registration Number: This trial is registered with ClinicalTrials.gov, number NCT00327132. Funding Statement: This study was sponsored, conducted and analyzed by the TCOG and following research grants: MOST 107-2314-B-002 -217-MY3 from Ministry of Science and Technology, Taiwan, and MOHW107-TDU-B-211-123002 from Ministry of Health and Welfare, Taiwan. Declaration of Interests: All authors declare they have no competing interests. Ethics Approval Statement: All experimental protocols were approved by the Institutional Review Board (IRB) of the Research Ethical Committee of National Taiwan University Hospital (NTUH) (NTUH IRB numbers: 950606).

Helicobacter pylori CagA promotes epithelial mesenchymal transition in gastric carcinogenesis via triggering oncogenic YAP pathway

BackgroundHelicobacter pylori (H. pylori) delivers oncoprotein CagA into gastric epithelial cells via the T4SS and drives activation of multiple oncogenic signalling pathways. YAP, a core effector of the Hippo tumour suppressor pathway, is frequently overexpressed in human cancers, suggesting its potential tumor-promoting role. Although CagA is a casual factor in H. pylori induced gastric carcinogenesis, the link between CagA and YAP pathway has not been identified. In this work, we investigated the regulation of oncogenic YAP pathway by H. pylori CagA.MethodsExpression of YAP and E-cadherin protein in human gastric biopsies were assessed by immunohistochemistry. H. pylori PMSS1 cagA− isogenic mutant strains were generated. Gastric epithelial cells were co-cultured with H. pylori wild-type cagA+ strains or isogenic mutants and were also treated by recombinant CagA expression. Immunofluorescence was performed for YAP localization. Immunoblot and quantitative PCR were performed for examining levels of YAP, downstream effectors and markers of epithelial-mesenchymal transition. Verteporfin and siRNA silencing were used to inhibit YAP activity.ResultsYAP is significantly upregulated in human gastric carcinogenesis. We generated PMSS1 CagA isogenic mutant strains with chloramphenicol resistance successfully. Our analysis indicated that H. pylori infection induced YAP and downstream effectors in gastric epithelial cells. Importantly, knockout of CagA in 7.13 and PMSS1 strains reduced the expression of YAP by H. pylori infection. Moreover, Inhibition of YAP suppressed H. pylori infection-induced Epithelial-mesenchymal transition (EMT).ConclusionOur results indicated that H. pylori CagA as a pathogenic protein promotes oncogenic YAP pathway, which contributes to EMT and gastric tumorigenesis. This study provided a novel mechanistic insight into why cagA+H. pylori infection is associated with a higher risk for the development of gastric cancer.

Preliminary study and bioinformatics analysis on the potential role of CagQ in type IV secretion system of H.pylori

Helicobacter pylori (H.pylori), is a major causative agent of chronic gastritis, gastric carcinoma and duodenal ulcer. Remarkably, H.pylori carries cytotoxin-associated gene pathogenicity island (CagPAI) which encodes a type IV secretion system (T4SS). T4SS is capable of forming a syringe-like structure to deliver oncoprotein cytotoxin-associated Antigen (CagA) into gastric epithelial cells and resulting in a cascade of events in host cells, such as induction of pro-inflammatory cytokines, alteration of cellular gene expression and cytoskeletal rearrangements. Among of those proteins in T4SS, CagQ still remains unknown functions. In this study, we performed analysis of protein-protein interaction and revealed that CagQ correlated with the most virulence factor CagA in T4SS. Interestingly, our data demonstrated that CagQ-deficient mutant strain had significantly lower expression in both mRNA and protein levels of CagA compared with H.pylori wild-type strain 26695. Moreover, we demonstrated that CagQ deletion also played a vital role in suppressing CagA-induced apoptosis of host gastric epithelial cells. To further investigate the role of CagQ in T4SS, we used bioinformatics analysis to provide a preliminary insight into CagQ. These results showed that CagQ possessed a transmembrane region from amino acid 50–68 which is also consistent with the prediction of hydrophobic scale and structure modeling. Thus, we conclude that CagQ is a membrane protein in T4SS and is crucial for maintaining CagA expression and CagA-induced apoptotic effects. This provides a novel specific therapeutic target for H.pylori CagA-induced gastroduodenal diseases.

Helicobacter pylori CagA protein activates Akt and attenuates chemotherapeutics-induced apoptosis in gastric cancer cells.

Infection with cagA-positive Helicobacter pylori is associated with a higher risk of gastric cancer. The cagA gene product, CagA, is translocated into gastric epithelial cells and perturbs host cellular biological functions. Etoposide, a topoisomerase II inhibitor widely used to couple DNA damage to apoptosis, is a common cytotoxic agent used for advanced gastric cancer. We investigate the effect of CagA on etoposide-induced apoptosis in gastric cancer cells to elucidate whether CagA play a role in gastric carcinogenesis via impairing DNA damage-dependent apoptosis. AGS cell lines stably expressing CagA isolated from H. pylori 26695 strain were established. In the presence of etoposide, viability of parental AGS cells was decreased in a time-and dose-dependent manner, whereas CagA-expressing AGS cells were less susceptible to etoposide induced cell-killing effect. Suppression of etoposide-induced apoptosis was shown in CagA-expressing but not in parental AGS cells by DNA fragmentation, cell cycle, and annexin-V assays. This inhibitory effect of etoposide-induced apoptosis conferred by CagA was also demonstrated in SCM1 and MKN45 gastric cancer cell lines, with two additional chemotherapeutics, 5-FU and cisplatin. The effect of Akt activation on inhibition of etoposide-induced cytotoxicity by CagA was also evaluated. CagA expression and etoposide administration activate Akt in a dose-dependent manner. Enhancement of etoposide cytotoxicity by a PI-3-kinase inhibitor, LY294002, was evident in parental but was attenuated in CagA-expressing AGS cells. CagA may activate Akt, either in the absence or presence of etoposide, potentially contributing to gastric carcinogenesis associated with H. pylori infection and therapeutic resistance by impairing DNA damage-dependent apoptosis.

32P - Helicobacter pylori CagA expression is closely associated with tumor PD-L1 expression and the better prognosis of gastric cancer patients

32P - Helicobacter pylori CagA expression is closely associated with tumor PD-L1 expression and the better prognosis of gastric cancer patients

Gastric lymphoma: association with Helicobacter pylori outer membrane protein Q (HopQ) and cytotoxic-pathogenicity activity island (CPAI) genes.

B-cell non-Hodgkin lymphoma (B-cell NHL) is the second commonest malignancy in the stomach. We determined the distribution of Helicobacter pylori outer membrane protein Q (HopQ) allelic type, cytotoxin-associated gene (cag)-pathogenicity activity island (cag-PAI) and vacuolation activating cytotoxin A (vacA) genes, respectively, in patients with B-cell NHL. We also compared them with their distribution in non-ulcer dyspepsia (NUD). H. pylori was cultured from gastric biopsy tissue obtained at endoscopy. Polymerase chain reaction was performed. Of 170 patients enrolled, 114 (63%) had NUD and 56 (37%) had B-cell NHL. HopQ type 1 was positive in 66 (58%) in NUD compared with 46 (82%) (P = 0·002) in B-cell NHL; HopQ type 2 was positive in 93 (82%) with NUD compared with 56 (100%) (P < 0·001) in B-cell NHL. Multiple HopQ types were present in 46 (40%) in NUD compared with 46 (82%) (P < 0·001) in B-cell NHL. CagA was positive in 48 (42%) in NUD vs. 50 (89%) (P < 0·001) in B-cell NHL; cagT was positive in 35 (31%) in NUD vs. 45 (80%) (P < 0·001) in B-cell NHL; left end of the cagA gene (LEC)1 was positive in 23 (20%) in NUD vs. 43 (77%) (P < 0·001) in B-cell NHL. VacAs1am1 positive in B-cell NHL in 48 (86%) (P < 0·001) vs. 50 (44%) in NUD, while s1am2 was positive in 20 (17%) in NUD vs. 46 (82%) (P < 0·001) in B-cell NHL. H. pylori strains with multiple HopQ allelic types, truncated cag-PAI evidenced by expression of cagA, cagT and cag LEC with virulent vacAs1 alleles are associated with B-cell NHL development.

Clinical value of CagA, c-Met, PI3K and Beclin-1 expressed in gastric cancer and their association with prognosis.

Gastric cancer (GC) is the fourth most common type of malignant tumor worldwide, and causes the second highest number of cancer-associated mortalities in 2012. Gastric tumorigenesis is a multistep and multifactorial process. In the present study, tissue microarray and immunohistochemistry analysis were used to detect cytotoxin-associated gene A (CagA), c-Met, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and Beclin-1 expression in 121 GC tumors and 120 normal gastric tissues. The clinical relevance and prognostic implications of CagA, c-Met, PI3K and Beclin-1 expression in GC patients were analyzed. Furthermore, the Cox proportional hazards model was performed to indicate the independent prognostic factors for GC patients, including various clinicopathological parameters and CagA, c-Met, PI3K and Beclin-1 expression. The results indicated that CagA-positive H. pylori infection, c-Met, PI3K and Beclin-1 may have major roles in the oncogenesis, invasion and lymph node metastasis of GC. The disease-free survival rate was negatively associated with the expression of c-Met and CagA in tissues, and was positively associated with Beclin-1 expression. Overall survival was also negatively associated with the expression of c-Met and PI3K, and was positively associated with Beclin-1 expression. This indicated that c-Met and Beclin-1 may be independent and efficient biomarkers for predicting the DFS of patients with GC. Furthermore, in CagA-positive H. pylori infection-associated GC, c-Met expression was significantly upregulated and Beclin-1 expression was significantly downregulated. CagA-positive H. pylori infection therefore associated with the c-Met signaling pathway and the suppression of autophagy in the neoplasia, invasion and metastasis of GC.

The bacterial virulence factor CagA induces microbial dysbiosis that contributes to excessive epithelial cell proliferation in the Drosophila gut.

Gut microbiota facilitate many aspects of human health and development, but dysbiotic microbiota can promote hyperplasia and inflammation and contribute to human diseases such as cancer. Human patients infected with the gastric cancer-causing bacterium Helicobacter pylori have altered microbiota; however, whether dysbiosis contributes to disease in this case is unknown. Many H. pylori human disease phenotypes are associated with a potent virulence protein, CagA, which is translocated into host epithelial cells where it alters cell polarity and manipulates host-signaling pathways to promote disease. We hypothesized that CagA alone could contribute to H. pylori pathogenesis by inducing microbial dysbiosis that promotes disease. Here we use a transgenic Drosophila model of CagA expression to genetically disentangle the effects of the virulence protein CagA from that of H. pylori infection. We found that expression of CagA within Drosophila intestinal stem cells promotes excess cell proliferation and is sufficient to alter host microbiota. Rearing CagA transgenic flies germ-free revealed that the dysbiotic microbiota contributes to cell proliferation phenotypes and also elicits expression of innate immune components, Diptericin and Duox. Further investigations revealed interspecies interactions are required for this dysbiotic CagA-dependent microbiota to promote proliferation in CagA transgenic and healthy control Drosophila. Our model establishes that CagA can alter gut microbiota and exacerbate cell proliferation and immune phenotypes previously attributed to H. pylori infection. This work provides valuable new insights into the mechanisms by which interactions between a specific virulence factor and the resident microbiota can contribute to the development and progression of disease.