Helicobacter pylori and gastric carcinogenesis

Gastric carcinoma is the second leading cause of cancer-related deaths in the world, accounting for more than 700,000 deaths each year. Recent studies have revealed that infection with cagA-positive Helicobacter pylori plays an essential role in the development of gastric carcinoma. The cagA-encoded CagA protein is delivered into gastric epithelial cells via the bacterial type IV secretion system, where it undergoes tyrosine phosphorylation by Src and Abl kinases. Tyrosine-phosphorylated CagA then acquires the ability to interact with and deregulate SHP-2 phosphatase, a bona-fide oncoprotein, deregulation of which is involved in a variety of human malignancies. CagA also binds to and inhibits PAR1b/MARK2 polarity-regulating kinase to disrupt tight junctions and epithelial apical-basolateral polarity. These CagA activities may collectively contribute to the transformation of gastric epithelial cells. Indeed, transgenic expression of CagA in mice results in the development of gastrointestinal and hematological malignancies, indicating that CagA is the first bacterial oncoprotein that acts in mammalian cells. The oncogenic potential of CagA may be further potentiated in the presence of chronic inflammation, which aberrantly induces activation-induced cytidine deaminase (AID), a member of the DNA/RNA-editing enzyme family. Ectopically expressed AID may contribute to H. pylori-initiated gastric carcinogenesis by increasing the risk of likelihood of epithelial cells acquiring mutations in cancer-related genes.     

The CagA protein of Helicobacter pylori is translocated into epithelial cells and binds to SHP-2 in human gastric mucosa

Recent experiments have indicated that CagA of Helicobacter pylori is injected into epithelial cells via the type IV secretion system and undergoes tyrosine phosphorylation in cells and that translocated CagA binds the SRC homology 2 domain-containing tyrosine phosphatase (SHP-2). We investigated these phenomena in in vivo human gastric mucosa. Tyrosine-phosphorylated CagA and CagA-coimmunoprecipitated SHP-2 were detected in gastric mucosa from H. pylori-positive patients with atrophic gastritis and in noncancerous tissues from H. pylori-positive patients with early gastric cancer. In contrast, CagA was not detected in gastric mucosa with either intestinal metaplasia or cancer. Our results provide the first evidence that CagA is translocated into the gastric epithelial cells, receives tyrosine phosphorylation, and binds SHP-2 in in vivo human gastric mucosa. Deregulation of SHP-2 by CagA may play a role in the acquisition of a cellular-transformed phenotype at a relatively early stage of multistep gastric carcinogenesis.     

Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation sites

Helicobacter pylori is a causative agent of gastritis and peptic ulcer. cagA(+) H. pylori strains are more virulent than cagA(-) strains and are associated with gastric carcinoma. The cagA gene product, CagA, is injected by the bacterium into gastric epithelial cells and subsequently undergoes tyrosine phosphorylation. The phosphorylated CagA specifically binds SHP-2 phosphatase, activates the phosphatase activity, and thereby induces morphological transformation of cells. CagA proteins of most Western H. pylori isolates have a 34-amino acid sequence that variably repeats among different strains. Here, we show that the repeat sequence contains a tyrosine phosphorylation site. CagA proteins having more repeats were found to undergo greater tyrosine phosphorylation, to exhibit increased SHP-2 binding, and to induce greater morphological changes. In contrast, predominant CagA proteins specified by H. pylori strains isolated in East Asia, where gastric carcinoma is prevalent, had a distinct tyrosine phosphorylation sequence at the region corresponding to the repeat sequence of Western CagA. This East Asian-specific sequence conferred stronger SHP-2 binding and morphologically transforming activities to Western CagA. Finally, a critical amino acid residue that determines SHP-2 binding activity among different CagA proteins was identified. Our results indicate that the potential of individual CagA to perturb host-cell functions is determined by the degree of SHP-2 binding activity, which depends in turn on the number and sequences of tyrosine phosphorylation sites. The presence of distinctly structured CagA proteins in Western and East Asian H. pylori isolates may underlie the strikingly different incidences of gastric carcinoma in these two geographic areas.     

Influence of EPIYA-repeat polymorphism on the phosphorylation-dependent biological activity of Helicobacter pylori CagA

BACKGROUND & AIMS: Helicobacter pylori CagA-positive strain is associated with gastric adenocarcinoma. CagA is delivered into gastric epithelial cells, where it undergoes tyrosine phosphorylation at the EPIYA sites by Src family kinases (SFKs). Owing to homologous recombination within the 3'-region of the cagA gene, 4 distinct EPIYA sites, each of which is defined by surrounding sequences, are variably assembled in both number and order among CagA proteins from different clinical H pylori isolates. Tyrosine-phosphorylated CagA specifically binds and deregulates SHP-2 via the Western CagA-specific EPIYA-C or East Asian CagA-specific EPIYA-D site, and C-terminal Src kinase (Csk) via the EPIYA-A or EPIYA-B site. Here we investigated the influence of EPIYA-repeat polymorphism on the CagA activity. METHODS: A series of EPIYA-repeat variants of CagA were expressed in AGS gastric epithelial cells and the ability of individual CagA to bind SHP-2 or Csk was determined by the sequential immunoprecipitation and immunoblotting method. RESULTS: CagA proteins carrying multiple EPIYA-C or EPIYA-D sites bound and deregulated SHP-2 more strongly than those having a single EPIYA-C or EPIYA-D. Furthermore, the ability of CagA to bind Csk was correlated with the number of EPIYA-A and EPIYA-B sites. Because Csk inhibits SFK, CagA with greater Csk-binding activity more strongly inhibited Src-dependent CagA phosphorylation and more effectively attenuated induction of cell elongation caused by CagA-SHP-2 interaction. CONCLUSIONS: EPIYA-repeat polymorphism of CagA greatly influences the magnitude and duration of phosphorylation-dependent CagA activity, which may determine the potential of individual CagA as a bacterial virulence factor that directs gastric carcinogenesis.     

<Emphasis Type="Italic">Helicobacter pylori</Emphasis> CagA protein variation associated with gastric cancer in Asia

Recent molecular analysis has provided the pathological actions of CagA on gastric epithelial cells. CagA is injected into epithelial cells via the type IV secretion system and undergoes tyrosine phosphorylation in the cells. In addition, translocated CagA forms a physical complex with SHP-2. There are two major CagA subtypes; the East Asian and the Western type. The East Asian CagA protein possesses stronger SHP-2 binding activity than the Western CagA. The grades of inflammation, activity of gastritis, and atrophy are significantly higher in gastritis patients infected with the East Asian CagA-positive strain than in gastritis patients infected with the cagA-negative or Western CagA-positive strains. The prevalence of the East Asian CagA-positive strain is associated with the mortality rate of gastric cancer in Asia. Endemic circulation of H. pylori populations carrying biologically more active CagA proteins in East Asian countries, where the mortality rate of gastric cancer is among the highest in the world, may be involved in increasing the risk of gastric cancer in these populations.     

Relationship between the diversity of the cagA gene of Helicobacter pylori and gastric cancer in Okinawa, Japan

Background Helicobacter pylori CagA protein is considered to be one of the virulence factors associated with gastric cancer. CagA is injected into gastric epithelial cells, undergoes tyrosine phosphorylation, and binds to Src homology 2 domain-containing protein-tyrosine phosphatase (SHP-2). Two major subtypes of CagA have been observed in the SHP-2-binding site, the Western and East Asian types. The East Asian-type CagA binds to SHP-2 more strongly than the Western-type CagA. The diversity of CagA, which collectively determines the binding affinity of CagA to SHP-2, may be an important variable in determining the clinical outcome of infection by different H. pylori strains. Methods We investigated the relationship between the diversity of CagA and clinical outcome in Okinawa, Japan. A total 24 strains, 13 gastric cancer strains and 11 duodenal ulcer strains, were studied. We sequenced full-length cagA genes and analyzed the phylogenetic relationships between Okinawa isolates and previously characterized Western H. pylori strains. Results All isolates examined were cagA positive. The prevalence of East Asian CagA-positive strains was significantly higher in patients with gastric cancer (84.6%) than in patients with duodenal ulcer (27.3%) (χ-squared = 8.06, P = 0.011). The phylogenetic analysis showed that all gastric cancer strains with East Asian-type CagA were in the East Asian cluster, and that most duodenal ulcer strains were in the Western cluster. Conclusions The origins of H. pylori isolates are different between gastric cancer strains and duodenal ulcer strains, and East Asian CagA-positive H. pylori infection is associated with gastric cancer. The strain diversity observed in Okinawa may affect the difference in the prevalence of disease associated with H. pylori infection in Japan.     

Natural variation in populations of persistently colonizing bacteria affect human host cell phenotype

The highly diverse bacterium Helicobacter pylori, which persistently colonizes the human stomach, provides models to study the role of genome plasticity in host adaptation. Within H. pylori populations from 2 colonized individuals, intragenomic recombination between cagA DNA repeat sequences leads to deletion or duplication of tyrosine phosphorylation sites in the CagA protein, which is injected by a type IV secretion system into host cells. Experimental coculture of gastric epithelial cells with the strains containing these naturally occurring CagA phosphorylation site variants induced markedly divergent host cell morphologic responses. Mutants were constructed in which a phosphorylation site was either added or deleted in the expressed CagA protein; coculture studies confirmed that the naturally occurring differences in CagA phosphorylation are responsible for the observed phenotypic variation. These findings indicate that within an individual host, intragenomic recombination between H. pylori repetitive DNA produces strain variants differing in their signals to host cells.     

Determinants and consequences of different levels of CagA phosphorylation for clinical isolates of Helicobacter pylori

BACKGROUND & AIMS: The Helicobacter pylori cag pathogenicity island encodes a secretory system that translocates CagA into epithelial cells, where it becomes tyrosine phosphorylated and induces cytoskeletal rearrangements. Strains with more CagA tyrosine phosphorylation motifs are most closely associated with gastric cancer. Here we assess whether clinical strains can deliver CagA, whether strains with different numbers of CagA phosphorylation motifs have CagA phosphorylated to different degrees, and whether this induces different amounts of epithelial cytoskeletal change. METHODS: Forty-four H. pylori strains from South African patients, all cagA gene positive, were cocultured with the gastric adenocarcinoma cell line AGS. CagA expression and phosphorylation were determined by Western blot and interleukin-8 secretion by enzyme-linked immunosorbent assay. The cagA 3' variable regions of 22 strains were sequenced and shown to possess 3-6 phosphorylation motifs. These strains were used to quantify CagA phosphorylation and cytoskeletal rearrangements. RESULTS: cagA genotype and typing of cag pathogenicity island genes were poorly predictive of phenotype. Thirty-four of 44 strains expressed CagA protein that could be delivered to and phosphorylated within AGS cells. Only these 34 strains induced interleukin-8 secretion from AGS cells. Among those strains, the number of CagA tyrosine phosphorylation motifs determined the degree of CagA phosphorylation and the level of biologic activity in terms of degree and extent of AGS cell elongation. CONCLUSIONS: H. pylori strains that deliver CagA with more phosphorylation motifs induce higher levels of CagA phosphorylation in epithelial cells, induce more cytoskeletal changes, and are more likely to be associated with gastric cancer.     

Cdx1 induced intestinal metaplasia in the transgenic mouse stomach: comparative study with Cdx2 transgenic mice

BACKGROUND AND AIMS: Gastric intestinal metaplasia, which is mainly induced by Helicobacter pylori infection, is thought to be a precancerous lesion of gastric adenocarcinoma. Intestinal metaplastic mucosa expresses intestine specific homeobox genes, Cdx1 and Cdx2, in the human gastric mucosa. We and others have reported that ectopic expression of Cdx2 in the gastric epithelium generates intestinal metaplasia in the transgenic mouse model. METHODS: To clarify the differences in the roles of Cdx1 and Cdx2 in intestinal metaplasia, we generated transgenic mice expressing Cdx1 in the gastric mucosa and compared Cdx1 induced gastric mucosal morphological changes with Cdx2 induced intestinal metaplasia. RESULTS: The gastric mucosa in Cdx1 transgenic mice was completely replaced by intestinal metaplastic mucosa, consisting of all four intestinal epithelial cell types: absorptive enterocytes, goblet, enteroendocrine, and Paneth cells. Paneth cells, which were not recognised in Cdx2 transgenic mice, were in the upper portion of the intestinal metaplastic mucosa. Pseudopyloric gland metaplasia, which was induced in Cdx2 transgenic mice, was not recognised in Cdx1 transgenic mice. Proliferating cell nuclear antigen (PCNA) positive cells were diffusely scattered in Cdx1 induced intestinal metaplastic mucosa while PCNA positive cells in Cdx2 induced intestinal metaplastic mucosa were in the base of the metaplastic mucosa. Intestinal metaplastic mucosa of Cdx1 transgenic mouse stomach was significantly thicker than that of wild-type or Cdx2 transgenic mouse stomach. CONCLUSIONS: We have confirmed that Cdx1 induced gastric intestinal metaplasia but that it differed from Cdx2 induced intestinal metaplasia in differentiation, structure, and proliferation.     

Association between diversity in the Src homology 2 domain--containing tyrosine phosphatase binding site of Helicobacter pylori CagA protein and gastric atrophy and cancer

We investigated the relationship between the diversity of Helicobacter pylori CagA protein and clinical outcome. The cagA gene was sequenced in 115 clinical isolates. The binding affinity of CagA to Src homology 2 domain-containing tyrosine phosphatase (SHP-2) was examined by in vitro infection. Two major CagA subtypes were observed--the East Asian and the Western type. The grades of inflammation, activity of gastritis, and atrophy were significantly higher in patients with gastritis infected with the East Asian CagA-positive strain than in patients with gastritis infected with cagA-negative or Western CagA-positive strains. All strains isolated from patients with gastric cancer were East Asian CagA positive. East Asian CagA exhibited stronger SHP-2-binding activity than did Western CagA. These findings suggest that infection with East Asian CagA-positive H. pylori is associated with atrophic gastritis and gastric cancer and that persistent active inflammation induced by the East Asian CagA-positive strain may play a role in the pathogenesis of disease.     

Activation of Abl by Helicobacter pylori: a novel kinase for CagA and crucial mediator of host cell scattering

BACKGROUND & AIMS: The pathogenesis of Helicobacter pylori (Hp)-associated diseases depends on a specialized type IV secretion system. This type IV secretion system injects the cytotoxin-associated gene A (CagA) effector protein into target cells where CagA becomes phosphorylated on tyrosine residues by Src. Src then is inactivated rapidly, suggesting the presence of another host tyrosine kinase to ensure constant CagA phosphorylation in sustained Hp infections. We aimed to identify this kinase. METHODS: By using the AGS gastric epithelial cell model, we performed a detailed functional characterization of Abl tyrosine kinase in signaling during Hp infections. RESULTS: We showed that Abl kinase is activated and a novel crucial mediator of Hp infections. First, Abl-specific inhibitors SKI-DV2-43 or STI571 (Gleevec, Novartis) and knockdown of c-Abl/Abl-related gene Arg by small hairpin and interfering RNAs efficiently inhibit CagA phosphorylation and cell scattering. Second, during infection, Abl is activated rapidly by autophosphorylation at Y-412. Third, both Abl and Src phosphorylated Y-899, Y-918, and Y-972 of CagA. Fourth, we found that the Abl substrate CrkII is phosphorylated at Y-221 in vivo. Fifth, overexpression of kinase-dead Abl (K290M) blocked Hp-induced actin cytoskeletal rearrangements. We further showed that sustained activity of Abl is required to maintain CagA in a phosphorylated state. Moreover, phosphorylated CagA forms a physical complex with Abl and activated CrkII in vivo. CONCLUSIONS: We propose a model in which Hp has evolved a mechanism to use at least 2 tyrosine kinases, Abl and Src, for CagA phosphorylation and subsequent actin-cytoskeletal rearrangements leading to cell scattering and elongation.     

Helicobacter pylori and CagA under conditions of iron deficiency

Iron deficiency is the most common nutritional deficiency worldwide and compelling evidence has demonstrated that this condition heightens the risk of gastric cancer. Infection with Helicobacter pylori is the strongest known risk factor for the development of gastric adenocarcinoma. Recent work has demonstrated that, under conditions of iron deficiency, H. pylori-induced gastric carcinogenesis is augmented through increased formation of the strain-specific cag type IV secretion system and enhanced delivery of the bacterial oncoprotein CagA into host cells. Although CagA is a potent virulence factor that promotes oncogenic responses, additional studies have now demonstrated that CagA modulates host cell iron homeostasis in vitro and fundamental metabolic functions of the bacterial cell in vivo. Here we discuss these findings and describe working models by which CagA exerts its effects on gastric epithelial cells, with particular emphasis on its potential role in modulation of host iron homeostasis.     

Helicobacter pylori and CagA under conditions of iron deficiency

Iron deficiency is the most common nutritional deficiency worldwide and compelling evidence has demonstrated that this condition heightens the risk of gastric cancer. Infection with Helicobacter pylori is the strongest known risk factor for the development of gastric adenocarcinoma. Recent work has demonstrated that, under conditions of iron deficiency, H. pylori-induced gastric carcinogenesis is augmented through increased formation of the strain-specific cag type IV secretion system and enhanced delivery of the bacterial oncoprotein CagA into host cells. Although CagA is a potent virulence factor that promotes oncogenic responses, additional studies have now demonstrated that CagA modulates host cell iron homeostasis in vitro and fundamental metabolic functions of the bacterial cell in vivo. Here we discuss these findings and describe working models by which CagA exerts its effects on gastric epithelial cells, with particular emphasis on its potential role in modulation of host iron homeostasis.     

The inhibitory potential of Fc receptor homolog 4 on memory B cells

Fc receptor homolog 4 (FcRH4) is a B cell-specific member of the recently identified family of FcRHs whose intracellular domain contains three potential immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The signaling potential of this receptor, shown here to be preferentially expressed by memory B cells, was compared with the inhibitory receptor FcgammaRIIb in B cells expressing either WT FcgammaRIIb or chimeric proteins in which the intracellular domain of FcRH4 was fused to the transmembrane and extracellular domains of FcgammaRIIb. Coligation of the FcgammaRIIb/FcRH4 chimeric protein with the B cell receptor (BCR) led to tyrosine phosphorylation of the two membrane-distal tyrosines and profound inhibition of BCR-mediated calcium mobilization, whole cell tyrosine phosphorylation, and mitogen-activated protein (MAP)-kinase activation. Mutational analysis of the FcRH4 cytoplasmic region indicated that the two membrane-distal ITIMs are essential for this inhibitory potential. Phosphopeptides corresponding to these ITIMs could bind the Src homology 2 (SH2) domain-containing tyrosine phosphatases SHP-1 and SHP-2, which associated with the WT FcRH4 and with mutants having inhibitory capability. These findings indicate the potential for FcRH4 to abort B cell receptor signaling by recruiting SHP-1 and SHP-2 to its two membrane distal ITIMs.     

Carcinogenesis in mouse stomach by simultaneous activation of the Wnt signaling and prostaglandin E2 pathway

BACKGROUND & AIMS: Accumulating evidence indicates that prostaglandin E(2) (PGE(2)), a downstream product of cyclooxygenase 2 (COX-2), plays a key role in gastric tumorigenesis. The Wnt pathway is also suggested to play a causal role in gastric carcinogenesis. However, the molecular mechanism remains poorly understood of how the Wnt and PGE(2) pathways contribute to gastric tumorigenesis. To investigate the role of Wnt and PGE(2) in gastric cancer, we have generated transgenic mice that activate both pathways and examined their phenotypes. METHODS: We constructed K19-Wnt1 transgenic mice expressing Wnt1 in the gastric mucosa using the keratin 19 promoter. We then crossed K19-Wnt1 mice with another transgenic line, K19-C2mE, to obtain K19-Wnt1/C2mE compound transgenic mice. The K19-C2mE mice express COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1) in the stomach, showing an increased gastric PGE(2) level. We examined the gastric phenotypes of both K19-Wnt1 and K19-Wnt1/C2mE mice. RESULTS: K19-Wnt1 mice had a significant suppression of epithelial differentiation and developed small preneoplastic lesions consisting of undifferentiated epithelial cells with macrophage accumulation. Importantly, additional expression of COX-2 and mPGES-1 converted the preneoplastic lesions in the K19-Wnt1 mice into dysplastic gastric tumors by 20 weeks of age. Notably, we found mucous cell metaplasia in the glandular stomach of the K19-Wnt1/C2mE mice as early as 5 weeks of age, before the dysplastic tumor development. CONCLUSIONS: Wnt signaling keeps the gastric progenitor cells undifferentiated. Simultaneous activation of both Wnt and PGE(2) pathways causes dysplastic gastric tumors through the metaplasia-carcinoma sequence.     

Inhibition of antigen-receptor signaling by Platelet Endothelial Cell Adhesion Molecule-1 (CD31) requires functional ITIMs, SHP-2, and p56(lck)

Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1, CD31) is a 130-kd member of the immunoglobulin gene superfamily that is expressed on the surface of platelets, endothelial cells, myeloid cells, and certain lymphocyte subsets. PECAM-1 has recently been shown to contain functional immunoreceptor tyrosine-based inhibitory motifs (ITIMs) within its cytoplasmic domain, and co-ligation of PECAM-1 with the T-cell antigen receptor (TCR) results in tyrosine phosphorylation of PECAM-1, recruitment of Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2), and attenuation of TCR-mediated cellular signaling. To determine the molecular basis of PECAM-1 inhibitory signaling in lymphocytes, the study sought to (1) establish the importance of the PECAM-1 ITIMs for its inhibitory activity, (2) determine the relative importance of SHP-2 versus SHP-1 in mediating the inhibitory effect of PECAM-1, and (3) identify the protein tyrosine kinases required for PECAM-1 tyrosine phosphorylation in T cells. Co-ligation of wild-type PECAM-1 with the B-cell antigen receptor expressed on chicken DT40 B cells resulted in a marked reduction of calcium mobilization-similar to previous observations in T cells. In contrast, co-ligation of an ITIM-less form of PECAM-1 had no inhibitory effect. Furthermore, wild-type PECAM-1 was unable to attenuate calcium mobilization in SHP-2-deficient DT40 variants despite abundant levels of SHP-1 in these cells. Finally, PECAM-1 failed to become tyrosine phosphorylated in p56(lck)-deficient Jurkat T cells. Together, these data provide important insights into the molecular requirements for PECAM-1 regulation of antigen receptor signaling.     

East Asian-type Helicobacter pylori cytotoxin-associated gene A protein has a more significant effect on growth of rat gastric mucosal cells than the Western type

BACKGROUND AND AIM: Cytotoxin-associated gene A (CagA) protein from H. pylori was reported to be injected into host gastric epithelial cells via a bacterial type IV secretion system, thereby modifying signal transduction. It is classified into two major subtypes, Western and East Asian. The present study aimed to compare the effects of East Asian-type and Western-type CagA on host cell growth. METHODS: A tetracycline (tet)-off system and cagA genes from Western and East Asian-type H. pylori (NCTC 11637 and F32) were transfected into untransformed rat gastric mucosal (RGM1) cells to establish RGM1-CagA cell lines in which CagA expression could be controlled by tetracycline. These cell lines were used to investigate the effect of CagA protein expression on cell growth with BrdU and water-soluble tetrazolium reagent (WST-8) assays. CagA expression, phosphorylation and extracellular signal-regulated kinase (ERK) activation were examined with immunoprecipitation and Western blotting analysis. RESULTS: 5-Bromo-2'deoxyuridine (BrdU) and WST-8 assays demonstrated significant increases in DNA replication and RGM1 cell growth after CagA protein expression. ERK phosphorylation was enhanced when CagA protein was expressed in RGM1-CagA cells. Moreover, the East Asian-type CagA showed a significantly greater effect on ERK activation and host cell growth than the Western type. PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor, suppressed ERK phosphorylation and the CagA protein-induced increase in RGM1-CagA cell growth. CONCLUSIONS: CagA protein expression induces an increase in RGM1-CagA cell proliferation via the mitogen-activated protein kinase (MAPK) signal pathway. The East Asian-type CagA showed a significantly greater effect on cell growth than the Western type, suggesting that the East Asian CagA-positive strain may have an important role in pathogenesis.