Mechanisms of Disease: Helicobacter pylori- related gastric ... - Nature

REVIEW www.nature.com/clinicalpractice/gasthep

Mechanisms of Disease: Helicobacter pylorirelated gastric carcinogenesis—implications for chemoprevention Marco Romano*, Vittorio Ricci and Raffaele Zarrilli INTRODUCTION

S U M M A RY Gastric adenocarcinoma is the second most common cause of cancerrelated mortality worldwide. Infection with Helicobacter pylori is the single most common cause of adenocarcinoma of the distal stomach. Cancer risk is believed to be related to differences among H. pylori strains and inflammatory responses governed by host genetics. In particular, specific interactions between host factors that modulate the response to the infection, and bacterial virulence factors that can directly cause tissue damage seem to have a major pathogenic role in the development of gastric cancer. In addition, environmental factors can modify key growth signaling pathways within the gastric mucosa, which leads to the alteration of epithelial cell growth. Preventive strategies represent the most promising means of decreasing cancer risk, and must be aimed at the control of H. pylori infection, improvement of environmental conditions, and the identification of subjects who are genetically predisposed to the development of cancer in response to H. pylori infection. Understanding the intracellular signaling pathways that are specifically affected by H. pylori and that promote phenotypic and genotypic changes that might ultimately progress to malignant transformation could enable physicians to focus eradication therapy appropriately and design interventions targeted at the molecular level to prevent the development of gastric cancer. KEYWORDS chemoprevention, gastric cancer, Helicobacter pylori

REVIEW CRITERIA The articles cited in this review were identified in December 2005, and the reference list updated to June 2006 during revision, by searching PubMed for English-language publications. The search terms used alone and in combination were “Helicobacter pylori”, “gastric cancer”, and “chemoprevention”. No time limits were placed on the search. The records identified were scrutinized and those thought to be relevant were obtained and reviewed.

M Romano is Associate Professor of Gastroenterology at the Department of Internal Medicine–Gastroenterology Unit, Second University of Naples, V Ricci is Associate Professor of Physiology at the Department of Experimental Medicine, University of Pavia Medical School, Pavia, and R Zarrilli is Assistant Professor of Hygiene and Public Health at the Department of Preventive Medical Sciences, Medical School of the University of Naples “Federico II”, Naples, Italy. Correspondence *Dipartimento di Internistica Clinica e Sperimentale “A Lanzara e F Magrassi” – Gastroenterologia e CIRANAD, Seconda Università di Napoli, II Policlinico, Ed 3, Secondo piano, Via Pansini 5, 80131 Napoli, Italy [email protected] Received 28 January 2006 Accepted 4 August 2006 www.nature.com/clinicalpractice doi:10.1038/ncpgasthep0634

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Gastric cancer, despite its declining incidence, remains the fourth most common cancer, the second leading cause of cancer-related death, and the fourteenth most common cause of death overall worldwide, killing more than 700,000 people each year.1 Early stages of the disease are often clinically silent, so patients commonly have advanced stage disease at the time of diagnosis, and reported 5-year survival rates are approximately 20%.2 Two histologically distinct variants of gastric adenocarcinoma have been described, each with different epidemiological and pathophysiological features. Intestinaltype gastric adenocarcinoma progresses through a relatively well-defined series of histological steps, including atrophic gastritis, intestinal metaplasia, and dysplasia, whereas diffuse-type gastric adenocarcinoma consists of individually infiltrating neoplastic cells with no glandular structure, and is not associated with definite precancerous lesions, such as intestinal metaplasia.3 Helicobacter pylori is a gram-negative microaerophilic spiral bacterium that colonizes the stomachs of approximately half the world’s population. Typically acquired during childhood, the infection can persist in the gastric ecosystem throughout the life span of the host if untreated.4 Colonization of the stomach by H. pylori causes chronic gastritis, which, during the decades that follow initial infection, can remain silent or evolve into more-severe diseases, such as atrophic gastritis, peptic ulcer, or lymphoma of the mucosa-associated lymphoid tissue.5 Epidemiological studies first indicated that colonization by H. pylori increased the risk of developing distal (noncardia) gastric cancer; the earlier the age of acquisition of H. pylori infection, the higher the risk of developing cancer.6,7 H. pylori-infected subjects have at least a twofold increase in the risk of gastric cancer when compared with uninfected subjects.8 In particular, those with severe atrophy and intestinal metaplasia have a relative risk of developing cancer of 4.9 and 6.4, respectively.9 The strong association between H. pylori infection and gastric cancer led the WHO

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to classify H. pylori as a class 1 carcinogen.10 Subsequent animal studies have shown that H. pylori fulfills Koch’s postulate: H. pylori infection in a susceptible host (in this case, Mongolian gerbils) in the absence of cocarcinogens causes a sequence of histological lesions that parallel the predominant histological pattern of intestinaltype gastric cancer found in humans, and leads to the development of gastric adenocarcinoma in up to a third of animals over the course of 1 to 2 years.11 Since then, past or present H. pylori infection has been found in up to 70% of patients with adenocarcinoma of the distal stomach,12 while it does not seem to have any role in the development of proximal gastric cancer.13 Development of gastric adenocarcinoma occurs in less than 1% of H. pylori-infected subjects.14 In addition, the incidence of gastric carcinoma in H. pylori-infected individuals can vary markedly among different geographical areas.15 This might be accounted for by H. pylori strain diversity within different geographical areas and/or within different individuals,16 and further suggests that factors other than those related to the bacterium could be involved in the carcinogenic process. Evidence increasingly indicates that H. pylorirelated gastric carcinogenesis is likely to involve three players: H. pylori (which triggers a multistep carcinogenic cascade), a genetically susceptible host, and permissive environmental factors (Table 1, Table 2 and Box 1). In this review, we focus on the bacterial virulence factors and host inflammatory responses involved in the development of gastric cancer after H. pylori infection. We also analyze the signaling pathways specifically affected by H. pylori, and try to identify possible molecular targets for intervention to prevent, and possibly treat, H. pylori-related gastric cancer. Finally, the effect of H. pylori eradication and the modification of environmental conditions on the progression of the multistep gastric carcinogenic process triggered by H. pylori are discussed. H. PYLORI VIRULENCE FACTORS

H. pylori populations are extremely diverse at the genomic level. Moreover, a single host can carry several H. pylori strains, and isolates within an individual can change over time, as endogenous mutations and/or chromosomal rearrangements or recombination between strains occur.3,17 Although this diversity has made it difficult to search for bacterial factors associated with

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Table 1 H. pylori virulence factors. Gene

Protein

Mechanism of action

cag PAI

CagA TFSS Other unidentified products

Induce cytokine production, cell proliferation, apoptosis

vacA

VacA

Induces apoptosis, immune suppression, proinflammatory effects, neoangiogenesis via VEGF

babA2

BabA

Favors colonization and inflammation

Abbreviations: PAI, pathogenicity island; TFSS, type IV secretion system; VEGF, vascular endothelial growth factor.

Table 2 Host genetic factors associated with H. pylori-related gastric carcinogenesis. Gene

Polymorphism

Mechanism of action

IL1 gene cluster IL1B IL1R

IL1B–511T IL1RN*2/*2

Hyperinflammatory response to H. pylori infection; inhibition of acid secretion

TNFA

TNFA–308A

Activation of intracellular signaling pathways related to inflammation; inhibition of acid secretion

IL8

IL8–251 A/A IL8–251 A/T

Increased IL-8 transcriptional activation in response to IL-1β and TNF

IL10

IL10 ATA/ATAa

Impaired downregulation of proinflammatory cytokines

aIL10–1082A, IL10–819T, and IL10–592A : “low IL-10 protein expression” polymorphism. Abbreviations: H. pylori, Helicobacter pylori; IL, interleukin; TNF, tumor necrosis factor.

cancer, several have been identified. Among these bacterial factors, a leading role is played by the cag pathogenicity island (see Box 2 for glossary definitions), the vacuolating cytotoxin (VacA), and the adhesin BabA (Table 1; Figure 1).14,16 The cag pathogenicity island

The most well-known H. pylori virulence determinant is the cagA gene, which encodes the 120–145 kDa immunodominant antigen cytotoxin-associated gene A product (CagA).16 H. pylori cagA+ strains are associated with higher grades of gastric inflammation and a significantly increased risk of developing gastric cancer compared with cagA– strains.16 The chromosome of cagA+ strains (i.e. about 60% of Western clinical isolates and 95–100% of East Asian strains) contains a 40 kbp insertion of foreign DNA, probably inherited by horizontal gene transfer from an unknown micro-organism.18,19 This region of foreign DNA contains 31 genes and has features that are typical of a pathogenicity island (PAI).19 In addition to encompassing the cagA gene that

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Box 1 Environmental factors associated with H. pylori-related gastric carcinogenesis. Low socioeconomic status, crowding, poor hygienic conditions ■ Favor transmission of infection at younger age High-salt diet ■ Inhibits Fas-mediated apoptosis ■ Induces gastric atrophy and hyperproliferation Few fruit and vegetables, diet high in nitroso compounds ■ Favors oxidative DNA damage ■ Induces mutagenesis and carcinogenesis

encodes CagA, the cag PAI also contains genes that encode a so-called type IV secretion system (TFSS), through which CagA and other bacterial products (such as peptidoglycan fragments) are injected into host cells.19–21 On injection into the host cell, CagA acts as a ‘Trojan horse’ that allows H. pylori to take control of the host cell.22 Tyrosine phosphorylation of CagA at its five-amino-acid EPIYA repeat region by host Src kinase has a pivotal role in CagA action.18,20 Phosphorylated CagA binds specifically to, and activates, the tyrosine phosphatase SHP-2, which, in turn, leads to the activation of the Ras–MAPK (mitogen-activated protein kinase) kinase pathway.20 As the activation of the Ras– MAPK kinase pathway transmits a positive signal for cell growth and motility, dysregulation of SHP-2 is an important mechanism by which strains containing the cag PAI promote gastric carcinogenesis. In addition, injected CagA, independent of its phosphorylation status, interacts with components of the apical junctional complex of gastric mucosal cells, causing a disruption of the epithelial barrier function and dysplastic alterations in epithelial cell morphology.23 Moreover, CagA expression in polarized epithelial cells perturbs epithelial cell differentiation.24 In fact, CagA-expressing cells lose cell polarity and cell adhesion, and are able to invade through extracellular matrix. The above effects seem to be controlled by different domains in the CagA protein, some of which are phosphorylation dependent and involve receptor-tyrosinekinase-like signaling, whereas others, which reside in the amino terminus of the molecule, are phosphorylation independent and relate to the apical junctions.24 So, the effect of CagA on cell– cell and cell–matrix interactions might serve as an early event in H. pylori-induced carcinogenesis.


According to variation seen at its tyrosine phosphorylation–SHP-2-binding site, CagA has been subclassified into two main types—Western CagA and East Asian CagA—each of which is prevalent in well-defined geographic areas.20 East-Asian CagA binds SHP-2 more strongly than Western CagA and has greater biological activity.20 Endemic circulation of H. pylori strains carrying a more biologically active CagA might, therefore, account for the high incidence of gastric carcinoma in East Asian countries.20 The VacA toxin

VacA is a 88 kDa protein toxin that was identified by its ability to induce the formation of cytoplasmic vacuoles in cultured cells.25,26 Unlike the cag PAI, the gene encoding VacA (vacA) is present in all H. pylori strains; differences in the expression and activity of VacA between the strains are caused by sequence variation.25,26 Regions of major sequence diversity are localized to the vacA secretion signal sequence (allele types s1 or s2) and the mid-region (allele types m1 or m2).25,26 Type s1, m1 VacA strongly correlates with the presence of the cag PAI.3 H. pylori strains that contain type s1, m1 VacA are also associated with an increased risk of cancer development, compared with strains containing type s2, m2 VacA.26 In geographic areas with a high background prevalence of distal gastric cancer (such as Columbia and Japan), most H. pylori strains contain type s1 or m1 alleles, whereas bacterial strains containing type s2 or m2 VacA are more prevalent in areas with low rates of noncardia gastric adenocarcinoma.15,16 It has been suggested that VacA acts as a multifunctional toxin.26 Indeed, VacA has been reported to act both as a modulator of epithelial cell function (altering endocytosis pathways, causing mitochondrion-dependent apoptosis, triggering specific cell signal transduction pathways, and impairing the tightness of epithelial layers) and as a modulator of immune-cell function (impairing phagocytosis and antigen presentation, and exerting an immunosuppressive action through the inhibition of T-lymphocyte activation and proliferation).26 VacA has also been reported to induce the production of proinflammatory cytokines from mast cells, as well as stimulating mast-cell chemotaxis and degranulation.26-28 The BabA adhesin

BabA is a 78 kDa outer-membrane protein that is encoded by babA2, which, depending on the

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geographic region and the respective study, is present in 40–95% of H. pylori strains (a higher percentage is found in East Asian strains, compared with Western clinical isolates).16,29,30 BabA mediates bacterial binding to the fucosylated Lewis b histo-blood group antigen and related ABO antigens on gastric epithelial cells.29,30 BabA binding specificities reflect H. pylori strain adaptation to different glycosylation patterns that predominate within a particular host population, to enable maximal adherence to gastric epithelium and, therefore, promote chronic infection.17,31 BabA-mediated binding can be altered by H. pylori phase variation (i.e. “on–off ” switching of the gene) and genetic recombination.17,31 H. pylori strains that have babA2 are associated with an increased incidence of gastric adenocarcinoma.3,29 Moreover, the presence of babA2 correlates with the presence of the cag PAI and s1 allele of vacA; strains with all three carry the highest risk of gastric cancer.3,29 The gastric mucosa of patients infected with BabA+ H. pylori strains has a higher bacterial colonization density and higher levels of the proinflammatory cytokine interleukin (IL) 8.29 Transgenic Lewis b-expressing mice infected with BabA+ H. pylori are more likely to develop severe gastritis, atrophy, and antibodies to parietal cell, compared with their wild-type littermates.3 HOST FACTORS

The outcome of H. pylori infection depends on the severity and anatomical distribution of the gastritis induced by the bacterium. Individuals with corpus-predominant gastritis are more likely to develop hypochlorhydria, gastric atrophy and, eventually, gastric cancer; those with antrum-predominant gastritis have excessive acid secretion and are more likely to develop duodenal ulcer, and those with mild, mixed antrum and corpus gastritis have almost normal acid secretion and, generally, no serious disease. These clinical outcomes seem to be mutually exclusive and are largely influenced by a genetically regulated inflammatory response of the host gastric mucosa to the infection.2,14 A combination of polymorphisms in the host IL1 gene cluster (i.e. in IL1B, which encodes IL-1β—a proinflammatory cytokine and a powerful inhibitor of gastric secretion—and in IL1RN, which encodes IL-1ra, the naturally occurring IL-1 receptor antagonist) and in the genes encoding tumor necrosis factor (TNF;


Box 2 Glossary definitions. Horizontal gene transfer The transfer of DNA sequences from one bacterium to another Pathogenicity island A contiguous stretch of chromosomal DNA that includes a cluster of genes the products of which are associated with virulence Type IV secretion system A syringe-like protein machinery at the bacterial surface through which gram-negative bacteria can deliver effector molecules into host cells ERK/MAPK Extracellular-signal-regulated kinase/mitogen-activated protein kinase; a protein kinase transmits signals from the cell surface to the nucleus CD95/FAS A cell-surface receptor that mediates apoptosis after binding to FAS ligand (FASL)

β-Catenin A protein that mediates cell adhesion, cytoskeleton formation, and the transcription of genes involved in proliferation and tumorigenesis Chemoprevention The use of drugs or natural substances to inhibit carcinogenesis Nutraceuticals A subgroup of products with biological activity that bridge the gap between food products and drugs

formerly known as TNF-α) and IL-10 result in elevated levels of IL-1β and TNF and low levels of IL-10, which, in turn, inhibit production of proinflammatory cytokines, conferring a 27-fold increased risk of developing gastric cancer (Table 2).32 In addition, it has been shown that polymorphisms in the promoter of the IL-8 gene, which enhance the transcriptional activity in response to IL-1β or TNF, are associated with increased risk of gastric cancer in patients with H. pylori infection.33,34 It is likely that the profound inhibition of acid secretion that is associated with these proinflammatory genotypes favors a shift from an antrum-predominant to a corpuspredominant gastritis with the onset of gastric atrophy. Moreover, H. pylori-induced inflammation and hypochlorhydria markedly reduce the levels of vitamin C in gastric juice, facilitating the formation of mutagenic N-nitroso compounds and reactive oxygen species.3,14,32 It is of note that several combinations of highvirulence bacterial genotypes with high-risk host genetic polymorphisms result in a marked increase in cancer risk. For example, the combination of the bacterial vacA s1 allele with the host IL1B–511*T allele confers a 87-fold increased risk of gastric cancer.35



Antioxidant agents

Herceptin Cetuximab

GGT

FASL

VacA

Gefitinib EKB569

Ca gA

EGF-related growth factors

H. pylori

ROS

EGFR

PGN

Cytokines

TFSS

CD95/FAS P13K

TNFR

RAS CagA P P

RAF

SHP-2 p38 MAPKs

VacA

AKT

CARD4 β-catenin Aspirin NSAIDs

MAPKK

MAPK

Cytochrome c release

PGN

Caspase activation

LEF–TCF

IKK

IκB and/or NFκB

Caspase activation

c-jun Apoptosis Growth Factors

c-myc Cyclin D

PGE2 VEGF

Apoptosis

COXIBs

COX2

MMP7 and/or MMP9

NFκB

Cytokines Chemokines

Proliferation, invasion, survival

Figure 1 Signal transduction pathways triggered by H. pylori virulence factors in gastric epithelial cells. H. pylori upregulates the expression of EGF-related growth factors, which activate EGFR and other tyrosine kinase receptors, and stimulate proliferation and survival through PI3K and MAPK. PI3K, MAPK, and p38 MAPKs are activated by ROS generated by bacterial secretion of GGT in the presence of glutathione and transferrin. H. pylori injects CagA through a TFSS into host cells. Following translocation, CagA undergoes tyrosine phosphorylation and subsequently binds to and activates SHP-2 phosphatase. This results in the activation of a MAPK pathway and induces a growth-factor-like response. CagA translocation induces β-catenin-dependent transcriptional activation of genes that regulate proliferation, invasion and survival of gastric epithelial cells. H. pylori PGN delivered by TFSS is recognized by CARD4 intracellular pathogen recognition molecule, which subsequently activates NFκB. Cytokines produced during H. pylori infection can also activate NFκB or caspases. NFκB activation can either induce or prevent gastric epithelial cell apoptosis. Increased cell apoptosis through direct or cytochrome-c-mediated activation of caspase is also contributed to by CD95–FAS-mediated and VacA-mediated apoptosis. Stimulatory and inhibitory effects are indicated by arrows and bars, respectively. Putative chemoprevention strategies are indicated in red. Abbreviations: CARD4, caspase recruitment domain-containing protein 4 (also known as Nod1); CD95/FAS, FAS ligand receptor; COX2, cyclo-oxygenase 2; COXIBs, cyclo-oxygenase inhibitors; EGF, epidermal growth factor; EGFR, EGF receptor; EKB569, irreversible inhibitor of EGFR tyrosine kinase; FASL, FAS ligand; GGT, γ-glutamyltranspeptidase; H. pylori, Helicobacter pylori; IκB, inhibitor of nuclear factor κB; IKK, IκB kinase; LEF–TCF, lymphocyte enhancer factor–T-cell factor; MAPK, mitogen-activated protein kinase, MAPKK, mitogen-activated protein kinase kinase; MMP, matrix metalloprotease; NFκB, nuclear factor κB; NSAIDs, nonsteroidal anti-inflammatory drugs; P, phosphorylation; PGE2, prostaglandin E2; PGN, peptidoglycan; ROS, reactive oxygen species; TFSS, type IV secretion system; TNFR, tumor necrosis factor receptor; VacA, vacuolating cytotoxin; VEGF, vascular endothelial growth factor.

ENVIRONMENTAL FACTORS

The main environmental factors that influence the development of gastric cancer are indicated in Box 1. Diet, in particular, has been linked to the etiology of gastric cancer in several international studies.2 Gastric cancer risk is increased in individuals who have a high salt and nitrate intake, and in those who consume large amounts of salt-preserved food.36 Another


study37 has shown that there is no additive effect of high-salt ingestion on H. pylori-induced tumorigenesis in wild-type mice, however. Mounting evidence supports the protective effect of fresh fruits and either cooked or raw vegetables against gastric cancer; this is apparently related to the effects of antioxidants, such as ascorbic acid and β-carotene.38 Ascorbic acid inhibits H. pylori growth in both human



and animal studies, as well as prostaglandin E2 (PGE2) synthesis induced by H. pylori.39 H. PYLORI-INDUCED CELL SIGNALLING IN GASTRIC CARCINOGENESIS

The host response to H. pylori infection might contribute to gastric carcinogenesis by promoting a chronic inflammatory response that contributes to mucosal cell damage, or by interfering with the mechanisms of proliferation and/or survival that regulate epithelial cell homeostasis.3,5,14,16 Inflammatory response

The immune response associated with H. pylori infection is characterized by a proinflammatory profile of the TH1 type, with increased production of IL-1β, TNF, and IL-8, but not IL-4 and IL-10.14,16,40,41 Variation in the ability of H. pylori strains to trigger the production of chemokines from gastric epithelium depends on the presence of a functional TFSS, which is encoded by the cag PAI. Although the cag PAI facilitates the translocation of CagA, the effect of this bacterial protein on cytokine synthesis is controversial. Most reports show that CagA has no effect on cytokine synthesis, which suggests, therefore, that other effectors are involved in the epithelial cytokine and chemokine response to H. pylori infection.14,16,19,20 Indeed, it has been shown that the induction of proinflammatory responses in epithelial cells infected by H. pylori is mediated by the host protein caspase recruitment domaincontaining protein 4 (CARD4; also known as Nod1), an intracellular pathogen-recognition molecule, and that the effect is dependent on the delivery of peptidoglycan to host cells by the TFSS.21 Consistent with involvement of CARD4 in host defense, Card4-deficient mice are more susceptible to infection by H. pylori strains containing the cag PAI than wild-type mice.21 Two recent papers, however, demonstrate that CagA directly induces IL-8 release from gastric mucosal cells.42,43 Although H. pylori elicits both innate and acquired immune responses, the host is unable to eliminate the organism from the gastric mucosa, and chronic infection is the usual outcome of exposure to H. pylori.17,44 A number of factors might be crucial for the evasion of the immune response, including frequent antigenic variation of H. pylori (i.e. modification of bacterial antigenic determinants caused by mutation or intragenomic homologous recombination), and its mimicry of host antigens (i.e. bacterial


expression of antigens similar to those expressed by the host).44 In addition, the induction of apoptosis by H. pylori in dendritic-cell precursor monocytes41 and the intracellular persistence of the bacteria in gastric epithelial progenitor cells45 might be necessary for immune evasion. It has also been demonstrated that H. pylori evades the innate immune response mediated by Toll-like receptor 5.46 In fact, although it is a flagellated organism, H. pylori does not release flagellin, and recombinant H. pylori flagellin is much less active than that of Salmonella typhimurium in activating the secretion of IL-8 mediated by Toll-like receptor 5.46 Although the inflammatory response induced by H. pylori infection fails to eliminate the organism, it increases cellular damage; activation of the TNF receptor by TNF results in the induction of apoptosis and mucosal cell damage. Increased cell apoptosis through direct or cytochrome-cmediated activation of caspases is also contributed to by CD95/FAS-mediated and VacA-mediated apoptosis of gastric epithelial cells.14,16 Other ways in which proapoptotic pathways are induced during H. pylori infection include superoxide (O2–) production by infiltrating neutrophils, elevated nitric oxide production by inducible nitric oxide synthase (iNOS), which is overexpressed in infected gastric mucosa),14,16,47 and generation of reactive oxygen species by bacterial secretion of γ-glutamyltranspeptidase (GGT) in the presence of glutathione and transferrin, as a source of iron.48 In fact, it has been shown that GGT is important for H. pylorimediated apoptosis of gastric epithelial cells from the AGS cell line (derived from a human gastric adenocarcinoma).49 It is also interesting to note that inflammatory stimuli that activate cytokine receptors and p38 (a stress-activated MAPK) can induce apoptosis. Conversely, activation of cytokine receptors and p38 might also inhibit apoptosis through nuclear factor κB (NFκB) and c-Jun activation.3,14 Both the inhibition and induction of apoptosis could be relevant to H. pylori-related carcinogenesis. Induction of apoptosis might favor the development of atrophic gastritis and gastricgland recruitment of bone-marrow-derived precursor cells that might ultimately develop into intraepithelial cancer.50 Inhibition of apoptosis, on the other hand, represents the loss of a physiological safeguard against the continual acquisition of DNA damage that can lead to the malignant transformation of cells.3,14



Proliferative response

Several signal transduction pathways are activated during the proliferative response of gastric epithelial cells to H. pylori-induced cell damage3,5,14 (Figure 1). The compensatory hyperproliferative response of gastric epithelial cells during H. pylori infection might be sustained by hypergastrinemia, increased expression of epidermal growth factor (EGF)-related peptides, and activation of the EGF receptor (EGFR) signal transduction pathway in gastric epithelial cells.3,5,14,51–53 In addition, translocation of CagA into gastric epithelial cells induces a growth-factorlike response through activation of the Ras–MAPK kinase pathway.20 Moreover, it has been shown that H. pylori upregulates the expression of cyclooxygenase2 (COX2), the inducible isoform of the enzyme responsible for prostaglandin production, in human gastric epithelial cells in vitro, and in human gastric mucosa in vivo.47,54 H. pylori-induced upregulation of COX2 and EGF-related peptide expression in human gastric epithelial cells depends on the bacterial production of GGT.48 Activation of phosphatidylinositol-3 kinase (PI3 kinase)dependent and/or p38-dependent pathways is responsible for H. pylori GGT-induced upregulation of COX2 and EGF-related peptide expression in gastric mucosal cells.48 Another study, however, demonstrates that upregulation of heparin-binding EGF-like growth factor (HB-EGF) by H. pylori in human gastric epithelial cells is dependent on MAPK kinase activation, which suggests that EGF-related peptide expression might be regulated by different signal transduction pathways.53 In keeping with this, it has been demonstrated that COX2 expression in gastric epithelial cells is also regulated by ERK/MAPK activation.54,55 The upregulation of growth factor and COX2 expression might increase the mitogenic activity of H. pylori-infected gastric mucosa and protect cells from H. pylori-induced cell damage, and, therefore, this upregulation might be regarded as an early event in the development of H. pylori-associated gastric carcinogenesis.3,5,14 Upregulation of COX2 and iNOS expression might also contribute to the high levels of oxidative damage done to DNA during H. pylori infection, which could increase the mutation rate in infected hyperproliferative gastric mucosa.14,16,47 The activation of a pathway mediated by EGFR, MAPK, and COX2 is also responsible for the induction of vascular endothelial growth factor


(VEGF) expression in H. pylori-infected gastric epithelial cells.55 This effect is specifically related to the VacA toxin of H. pylori,55 and is associated with a significant increase in blood vessel formation, which suggests that neoangiogenesis might contribute to tumor growth in H. pylori-related gastric carcinogenesis.56 H. pylori infection also promotes gastric epithelial cell invasion by inducing the production of matrix metalloproteinase (MMP) 7 through MAPK activation,57 and MMP9 and VEGF expression through a pathway mediated by NFκB and COX2.58 Another host effector that is aberrantly activated during H. pylori-induced gastric carcinogenesis is β-catenin, a ubiquitously expressed molecule that regulates the expression of several genes, including c-myc, the cyclin D genes, MMP7, and PTGS2 (prostaglandinendoperoxide synthase 2, which encodes COX2).59 In fact, an oncogenic H. pylori strain can induce nuclear translocation of β-catenin and activation of the LEF–TCF transcription factor that regulates the expression of β-cateninresponsive genes.59 Interestingly, β-catenin activation is dependent on the translocation of CagA into host epithelial cells, reinforcing the evidence that cagA+ H. pylori strains induce stronger activation of the signal transduction pathways that regulate the proliferation, invasion, and survival of gastric epithelial cells.59 CHEMOPREVENTION OF H. PYLORIRELATED GASTRIC CANCER

The development of gastric cancer usually takes decades, and its different precancerous stages, at least in the intestinal-type adenocarcinoma, have been well characterized.3,5,14 Moreover, the main etiologic factors are known, and are amenable to control measures. Preventive strategies, therefore, offer the best opportunity to reduce gastric cancer incidence and mortality (Box 3). Eradicating H. pylori infection

One chemopreventive measure is to eradicate H. pylori infection. This can be achieved in approximately 70–80% of infected subjects by combining a PPI (i.e. omeprazole 20 mg twice daily) with two antibiotics (i.e. amoxicillin 1 g twice daily plus clarithromycin 500 mg or metronidazole 500 mg twice daily) for 7 days.60 The role of H. pylori eradication in the prevention of gastric cancer remains controversial, however. Intervention studies that have gastric cancer incidence as a primary outcome measure are not



easily performed because they require numerous individuals to be followed up for several decades. Most studies look at changes in preneoplastic lesions (i.e. intestinal metaplasia and gastric atrophy) as a surrogate endpoint. A large, prospective, randomized, placebocontrolled, population-based study of 1,630 carriers of H. pylori infection has shown that, during a period of 7 years, the incidence of gastric cancer development is similar between participants who receive H. pylori eradication and those who receive placebo.61 Subgroup analysis of 988 patients who did not have precancerous lesions— not including those with atrophy or intestinal metaplasia on presentation—was, however, more promising. In this group, no patients (of 483) developed gastric cancer after H. pylori eradication, compared with 6 (of 503) who received placebo.61 This suggests that H. pylori eradication is an effective way to decrease the incidence of gastric cancer in infected subjects who do not have atrophy or intestinal metaplasia. This hypothesis is partially supported by a review of indexed literature evaluating the dynamics of atrophy and intestinal metaplasia after treatment for H. pylori.62 Although most studies indicate that there is no progression of atrophy or intestinal metaplasia in subjects in whom H. pylori was eradicated, regression of atrophy after H. pylori eradication was observed in only 11 out of 25 studies; intestinal metaplasia showed an improvement in only 4 out of 28 studies.62 A report on the long-term (12-year) follow-up of patients treated for H. pylori infection indicates that subjects who were free of H. pylori after treatment had 14.8% more regression and 13.7% less progression of preneoplastic lesions than patients who were positive for H. pylori.63 The regression of atrophy or intestinal metaplasia largely depends on the underlying molecular alteration responsible for the atrophy or metaplasia. If a change in the DNA sequence (i.e. a genetic alteration) is responsible, it is likely to represent a ‘point of no return’ and is, therefore, unlikely to regress after eradication of the infection. On the other hand, should the precancerous lesion be the result of the accumulation of epigenetic alterations (i.e. without any change in the DNA sequence), elimination of the triggering agent (i.e. H. pylori) could potentially reverse the lesion,64 and interrupt the carcinogenic process. It remains to be determined whether a searchand-treat strategy can decrease the incidence of


Box 3 Preventive strategies in H. pylori-related gastric carcinogenesis. H. pylori eradication ■ Decreases cancer incidence in infected subjects with no precancerous lesions at presentation Diet supplementation with antioxidant micronutrients ■ Decreases the progression of precancerous lesions Blockade of COX2, EGFR, VEGFR2 signaling pathways ■ Might restore H. pylori-dependent alteration of cell cycle and angiogenesis homeostasis in the gastric mucosa Abbreviations: COX2, cyclo-oxygenase 2; EGFR, epidermal growth factor receptor; H. pylori, Helicobacter pylori; VEGFR2, vascular endothelial growth factor receptor 2.

gastric cancer in humans, and be cost-effective. Parsonnet et al.65 estimated the prevention of gastric cancer cases to range from 30% to 50%. Moreover, studies modeling H. pylori screening and treatment for the prevention of gastric cancer concluded that the search-and-treat strategy is cost-effective.12,65 Evidence suggests that a focused search-and-treat strategy should be followed for first-degree relatives of patients with gastric cancer, and that a more widespread search-and-treat strategy should be used for high-risk populations.66 Preventing oxidative stress

There has been some focus on the possibility that ‘oxidative stress’ might be crucial for the development of H. pylori-related carcinogenesis.14,16,67 Observational studies have shown that a diet rich in salted, pickled, or smoked food, dried fish, and cooking oil are linked to an increased gastric cancer risk, whereas diets rich in fresh fruit and vegetables are associated with a low risk of gastric cancer.68 The beneficial effect of a certain type of diet (in particular, one that contains lots of fresh fruit and vegetables) on the mucosa of the gastrointestinal tract is mainly contributed by the antioxidant potential of micronutrients, the intake of which has an inverse correlation with the risk of gastric cancer.68 In addition, a study by Correa et al. has shown that dietary supplementation with antioxidant micronutrients, such as ascorbic acid or β-carotene, in a population at a high risk of gastric cancer significantly increased the rate of regression of precancerous lesions to a similar extent to that observed after H. pylori eradication.38



Targeting signaling pathways activated by H. pylori infection

Another potential preventive strategy is to target the intracellular signaling pathways that are activated by H. pylori infection (Figure 1). In particular, COX2 and EGFR could be attractive pharmacologic targets of for chemoprevention. Targeting COX2 COX2 expression is upregulated by H. pylori infection47,54 and overexpression of COX2 has been demonstrated in gastric cancer.69 There is a strong epidemiological link between chronic use of NSAIDs and a reduction in risk of gastric cancer (summary odds ratio of 0.78).70 In addition, experimental studies in rodents have shown that selective COX2 blockade significantly reduces tumor formation.71 Whether or not COX2 inhibition can prevent or delay the onset of gastric cancer in humans remains to be established. The reported increased incidence of myocardial infarction in patients taking the selective COX2 inhibitor rofecoxib on a long-term basis also has implications, and additional safety data regarding COX2 inhibitors are awaited. Targeting EGFR EGFR activation stimulates several intracellular signaling pathways that regulate gene transcription and, therefore, modulate cell proliferation, apoptosis, angiogenesis, and malignant transformation.72 H. pylori infection activates the EGFR and upregulates the expression of amphiregulin and HB-EGF, which are EGFR ligands.51 H. pylori-related activation of the EGFR is mediated by HB-EGF and seems to be independent of the cag PAI,53 even though cag PAI+ strains seem to induce greater activation of EGFR than cag PAI– strains.52 Targeting EGFR, therefore, might represent a rational chemopreventative approach to H. pylorirelated gastric tumorigenesis. Two strategies for blocking the action of EGFR include antibodies directed against the ectodomain of EGFR, and drugs that inhibit protein tyrosine kinase activity. An EGFR ectodomain-directed antibody (i.e. cetuximab or herceptin) and a small-molecule EGFR tyrosine kinase inhibitor (i.e. gefitinib or EKB569) are commercially available and have been successfully evaluated for the treatment of colorectal cancer.73 EGFR blockade is associated with a skin rash and gastrointestinal toxicity, however,72 so the success or failure of targeting EGFR as an approach to gastric cancer chemoprevention depends on the balance between efficacy and tolerability.


Targeting VEGFR2 VEGF has a pivotal role in tumor-associated neoangiogenesis and is overexpressed in human gastric carcinomas.55 H. pylori upregulates the expression of VEGF in human gastric epithelial cells.55,56 As such, blockade of VEGF receptor 2 (VEGFR2), which mediates the angiogenic effect of VEGF, might be a potential chemopreventive target. ZD6474 is an orally bioavailable, smallmolecule VEGFR2 tyrosine kinase inhibitor with additional anti-EGFR tyrosine kinase activity,74 which is currently in phase II development in patients with cancer. Nutraceuticals from red grape or green tea, such as resveratrol or epigallocatechin, have also been shown to inhibit angiogenesis, both by acting on inflammatory angiogenesis and by interfering with VEGF signaling.75,76 Targeting COX2, EGFR, and VEGFR2 There is crosstalk between EGFR, COX2, and VEGF. Activation of EGFR leads to increased MAPK activity, which results, in turn, in COX2 transcription, which is mediated by transcription factor AP-1.77 Increased COX2 transcription results in enhanced production of prostaglandins, including PGE2, and initiates a positive feedback loop by activating EGFR72 and stimulating VEGF production.55 Combination therapy, a common strategy in cancer treatment, might also be applicable to chemoprevention; it is possible to envision the combined use of COX2, EGFR, and VEGFR2 inhibitors in the chemoprevention of gastric cancer.72,78 Combined blockade of different, relevant cancer signaling pathways should be carefully evaluated, however, because this approach could increase toxicity. CONCLUSIONS

Gastric cancer is still a major health problem worldwide and its relationship with H. pylori infection is strongly supported by epidemiological, experimental, and interventional studies. Preventive strategies to decrease gastric cancer risk include eradication of the infection, amelioration of environmental factors (in particular, diet), and blockade of the intracellular signaling pathways affected by the bacterium. These factors are also of potential importance in the progression of the multistep process leading to the development of gastric cancer (Figure 1, Box 3). The main challenges in this field are to define better the genetic or functional markers of the bacterium that might predict the development of gastric cancer, as



well as the host genetic factors and environmental factors that influence the outcome of the infection. Finally, increasing knowledge of the molecular mechanisms underlying H. pylori-related gastric carcinogenesis might allow the development of safe and effective drugs that, alone or in combination, prevent the progression of H. pylori-triggered gastric carcinogenesis, by controlling inflammation and altering cell growth or differentiation.

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KEY POINTS ■

Gastric cancer is still a major health problem worldwide

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Mounting evidence indicates that H. pylori infection has a major role in the development of adenocarcinoma of the distal stomach

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H. pylori-related gastric carcinogenesis involves interactions between specific bacterial virulence factors, host genetic susceptibility factors, and environmental factors

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Preventive strategies include eradication of H. pylori infection, modification of diet to increase the intake of antioxidant micronutrients, and the identification of signaling pathways specifically affected by the bacterium, which might represent molecular targets for intervention

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Acknowledgments The work performed in authors’ laboratories is supported in part by grants from CIRANAD, and Ministero dell’Istruzione, dell’ Università e della Ricerca Scientifica e Tecnologica (PRIN 2002 and 2004 to VR and RZ), Italy. The excellent artwork of Mrs Maria Grazia Catenacci is also acknowledged. Restrictions placed on the number of references that could be cited in this review mean that, in many cases, either a single paper or a review is cited. We apologize to those authors whose work has not been cited.

Competing interests The authors declared they have no competing interests.

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