Helicobacter pylori and gastric carcinogenesis
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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.
KeywordsHelicobacter pylori CagA Gastric carcinoma SHP-2 PAR1b
Gastric carcinoma is the fourth most common cancer worldwide, with approximately 930,000 new cases diagnosed each year, and is the second leading cause of cancer-related deaths, with approximately 700,000 deaths worldwide in 2002 . It is the most common cancer in several areas of the world, most notably Japan, Korea and China. In Japan, the incidence of gastric carcinoma is almost ten times higher than in the United States. In most areas, the incidence of gastric carcinoma in men is almost twice as high as in women. Histologically, there are two major types of gastric carcinoma, the intestinal type, which is associated more commonly with environmental perturbations, and the diffuse type, which is ascribed etiologically more often to host genetic factors. Intestinal-type carcinoma cells show irregular tubular structures, harboring pluristratification, multiple lumens and reduced stroma, often associated with intestinal metaplasia in neighboring mucosa. Diffuse-type carcinoma cells are poorly differentiated and are characterized by the production of discohesive and secrete mucus, which is delivered into the interstitium. If the mucus remains inside the tumor cell, it pushes the nucleus at the periphery (therefore called signet-ring cell carcinoma). A small portion of diffuse-type gastric carcinomas is of familial origin, caused by mutations in the E-cadherin gene . Most cases of stomach cancer are diagnosed between the ages of 50 and 70 years, but younger cases are more frequently seen in families with a hereditary risk of stomach cancer.
Recent epidemiological studies have indicated that Helicobacter pylori plays a key role in the development of both intestinal-type and diffuse-type gastric carcinomas [3, 4, 5]. H. pylori is a gram-negative, spiral-shaped bacterium that infects in the stomach of about half of the world’s population. The acidic environment in the stomach usually prevents the survival of viruses, bacteria and other microorganisms, but H. pylori has evolved to uniquely overcome this harsh environment. H. pylori secretes a special enzyme, urease, which converts urea to ammonia to neutralize the acidity of the stomach, making the stomach a more hospitable place for H. pylori. Upon acquisition of the ability to survive, the stomach provides H. pylori with a special living niche. Host inflammatory/immune cells that would normally recognize and attack invading bacteria are unable to cross from blood vessels through the stomach epithelial mucosa. Instead, the ineffective host cells continue to respond to the site of infection, where they die and release nutrients that feed the gastric pathogen. H. pylori infection is primarily acquired during childhood, and the transmission occurs through a fecal–oral or oral–oral mode primarily within families. In the majority of cases, H. pylori infection is life-long in the absence of eradication with antibiotics.
H. pylori CagA and type IV secretion system
Delivery of CagA into gastric epithelial cells
Interaction of CagA with SHP-2 phosphatase
Analysis using a series of EPIYA mutants of CagA revealed that SHP-2 specifically binds to the tyrosine-phosphorylated EPIYA-C or EPIYA-D segment (Fig. 3) . The sequence flanking the tyrosine phosphorylation site of the EPIYA-D segment perfectly matches the consensus high-affinity binding sequence for the SH2 domains of SHP-2 (Fig. 3a), whereas that flanking the tyrosine phosphorylation site of the EPIYA-C segment differs from the consensus sequence by a single amino acid at the pY+5 position. As a result, East Asian CagA, which contains the EPIYA-D segment, exhibits stronger SHP-2 binding than does Western CagA, which contains the EPIYA-C segment [24, 27]. Within Western CagA species, those having a greater number of EPIYA-C segments exhibit stronger activity to interact with SHP-2 and are more closely associated with precancerous lesions and gastric cancer [28, 29]. Furthermore, stable complex formation between tyrosine-phosphorylated CagA and SHP-2 requires CagA multimerization, most probably homodimerization . CagA multimerization is independent of CagA tyrosine phosphorylation and is mediated by a 16-amino-acid stretch termed CagA-multimerization (CM) sequence that is present in the C-terminal EPIYA-repeat region.
Consistent with the role of SHP-2 in the Erk MAP kinase , expression of CagA in gastric epithelial cells evokes sustained Erk MAP kinase activation . Since prolonged Erk activation promotes G1-to-S phase progression , CagA aberrantly stimulates cell proliferation at least partly through sustained Erk activation. Deregulation of SHP-2 by CagA is of potential importance in the context of cell transformation because mutations in PTPN11, the gene encoding SHP-2, have been identified in various human malignancies, such as juvenile myelomonocytic leukemia (JMML), childhood myelodysplastic syndrome, B cell acute lymphoblastic leukemia and acute myelocytic leukemia, as well as some solid tumors [38, 39]. Most of the reported cases carry missense mutations in exons 3 and 8, which encode the N-SH2 domain and the PTPase domain, respectively. Molecular modeling of SHP-2 indicates that such mutations weaken the autoinhibitory interaction that occurs between the N-SH2 domain and the C-terminal PTPase domain and thereby constitutively activate SHP-2 phosphatase. Deregulation of SHP-2 by CagA may therefore functionally mimic the activating SHP-2 mutant that is associated with human malignancies .
Interestingly, a polymorphism in the PTPN11 gene has been shown to be associated with the risk of gastric atrophy and gastric cancer among H. pylori-infected Japanese patients . The G allele of the JST057927 SNP increases the risk of atrophy, while the A/A genotype is protective against it. These findings indicate the possibility that the PTPN11 polymorphism quantitatively or qualitatively influences the strength of signal transduction through the CagA–SHP-2 complex and thereby contributes to the development of pre-malignant mucosal lesions in the stomach.
Effect of CagA on transcription
CagA indirectly affects the activities of transcription factors through multiple distinct mechanisms. CagA activates serum responsive element (SRE)-dependent transcription in a phosphorylation-independent manner . CagA also activates NF-κB, which induces pro-inflammatory cytokines, such as interleukin (IL)-8 . CagA has recently been shown to activate the nuclear factor of activated T cells (NFAT) by eliciting nuclear translocation of the cytoplasmic NFAT in gastric epithelial cells . Again, the CagA activity toward NFAT is independent of CagA phosphorylation. Intriguingly, another H. pylori virulence factor, vacuolating toxin VacA, has been found to counteract the activity of CagA to activate NFAT . This finding indicates that VacA also plays a role in modifying the fate of a CagA-injected gastric epithelial cell (Fig. 4).
Polarity and junctional defects caused by CagA–PAR1b interaction
CagA and E-cadherin/β-catenin system
Recent studies have also provided evidence that CagA perturbs the Wnt/β-catenin signaling pathway, which is critically involved in colorectal carcinogenesis . A carcinogenic H. pylori strain in Mongolian gerbils acquired the ability to selectively activate β-catenin signaling in gastric epithelial cells . This β-catenin activation is dependent on CagA, but is independent of CagA tyrosine phosphorylation. Although the precise mechanism remains to be elucidated, it is known that activation of the β-catenin signal is induced by translocation of β-catenin from the membrane to the cytoplasm/nucleus [52, 53]. Given the recent finding that the effect of CagA on β-catenin requires the CM sequence of CagA, it is possible that the CagA–PAR1b complex directly or indirectly interacts with E-cadherin and thereby destabilizes the E-cadherin–β-catenin complex at the membrane. CagA-deregulated β-catenin transactivates several genes including cdx1, which encodes an intestine-specific transcription factor Cdx1 . Since Cdx1 governs genes involved in intestinal differentiation, this observation raises the possibility that CagA-deregulated β-catenin is involved in the development of intestinal metaplasia, a precancerous transdifferentiation of gastric epithelial cells to an intestinal phenotype.
In vivo oncogenic potential of CagA
Despite accumulating in vitro evidence for the transforming potential of CagA, the exact role of CagA in in vivo tumorigenesis had remained obscure. Infection of wild-type mice with H. pylori does not result in the development of gastric carcinoma, probably due to poor host adaptation. Whereas long-term infection with H. pylori can induce gastric carcinoma in Mongolian gerbils, it remains uncertain whether CagA plays an active role in carcinogenesis in gerbils [54, 55]. Accordingly, rodent models have so far failed to demonstrate a causal link between CagA and the development of neoplasms in vivo. To challenge this important question, transgenic mice systemically expressing wild-type or phosphorylation-resistant CagA were recently generated . Mice expressing wild-type CagA showed gastric epithelial hyperplasia, and some of them developed gastric polyps as well as adenocarcinomas of the stomach and small intestine. Systemic expression of wild-type CagA also induced leukocytosis that was associated with hypersensitivity to hematopoietic cytokines, such as interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in bone-marrow cells. Some of the transgenic mice also developed myeloid leukemia’s and B cell lymphomas, hematological malignancies that are also caused by gain-of-function SHP-2 mutations. In contrast, no pathological abnormalities were observed in transgenic mice expressing phosphorylation-resistant CagA. These results provide the first direct evidence for the role of CagA as a bacterium-derived oncoprotein (bacterial oncoprotein) that acts in mammals. They further indicate the importance of CagA tyrosine phosphorylation, which enables CagA to bind and deregulate SHP-2, in the development of H. pylori-associated neoplasms.
CagA and multistep gastric carcinogenesis
Development of gastric adenocarcinoma is a multistep process that requires qualitative as well as quantitative alterations in the expression of host oncogenes and tumor suppressor genes, lasting for several decades. During infection with cagA-positive H. pylori, gastric epithelial cells are continuously exposed to the injection of CagA from the bacteria. The injected CagA binds and deregulates SHP-2 and other intracellular signaling molecules in both tyrosine phosphorylation-dependent and -independent manners, generating abnormal signals for cell growth and cell movement. CagA also disrupts apical junctions and thereby destroys normal epithelial architecture.
It is well documented that chronic infection with cagA-positive H. pylori induces progressive histopathological changes in gastric mucosa that lead to intestinal-type gastric adenocarcinoma: superficial gastritis, atrophic gastritis, intestinal metaplasia, dysplasia and adenocarcinoma . Since the CagA-SHP-2 complex is detected mainly in atrophic gastric mucosa, the complex may play a critical role in the development of atrophic gastritis and/or the transition from atrophy to intestinal metaplasia . CagA-mediated abnormal signals that cause deregulated cell growth with impaired cell-cell contact as well as elevated cell motility may enhance epithelial cell turnover as a result of increased cell proliferation and subsequent apoptosis. Such an elevated cell turnover will obviously increase the risk of gastric stem cells acquiring genetic changes that favor cell transformation . In this regard, the results of a recent study using a Helicobacter-infected mouse model have led to the surprising conclusion that gastric adenocarcinoma originates from circulating bone marrow-derived cells (BMDC), not from resident gastric epithelial cells . If this is also true in humans, then chronic mucosal inflammation caused by cagA-positive H. pylori infection may exhaust gastric stem cells and thereby lead to depletion of the resident stem cell pool, resulting in recruitment and settlement of BMCD, from which gastric carcinoma arises. This caveat obviously warrants further investigation.
It has recently been reported that activation-induced cytidine deaminase (AID) is aberrantly induced in response to infection with H. pylori via the NF-κB signaling pathway . AID is a B cell-specific DNA-editing enzyme that generates immune diversity by inducing somatic hypermutations and class-switch recombination’s in human immunoglobulin genes. Aberrant activation of AID, which could act as a genome mutator, is capable of contributing to the generation of somatic mutations in tumor-related genes, such as p53 in gastric epithelial cells. Thus, inflammation-mediated AID expression may significantly promote multistep gastric carcinogenesis triggered by cagA-positive H. pylori.
In Japan, 60 million people have already been infected with cagA-positive H. pylori. Interaction of CagA with SHP-2, the first phosphatase shown to act as a bona fide oncoprotein, is one of the key determinants for the development of H. pylori-associated gastric carcinoma. Given the results of recent studies demonstrating that eradication of H. pylori in humans lowers the risk of developing gastric carcinoma [61, 62], systemic eradication of H. pylori from a high-risk population is expected to dramatically reduce the incidence of gastric carcinoma. Furthermore, elucidation of molecular mechanisms underlying H. pylori-induced gastric carcinoma is not only important for developing revolutionary therapies against gastric carcinoma, but also for understanding general mechanisms that underlie infection/inflammation-associated cancers.
The author thanks members of Hatakeyama Laboratory for valuable discussions and comments. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.