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Role of Helicobacter pylori infection in pathogenesis of gastric carcinoma.

Gastric cancer (GC) is one of the most common carcinoma and the second leading cause of cancer-related deaths worldwide. Helicobacter pylori (H. pylori) infection causes a series of precancerous lesions like gastritis, atrophy, intestinal metaplasia and dysplasia, and is the strongest known risk factor for GC, as supported by epidemiological, preclinical and clinical studies. However, the mechanism of H. pylori developing gastric carcinoma has not been well defined. Among infected individuals, approximately 10% develop severe gastric lesions such as peptic ulcer disease, 1%-3% progresses to GC. The outcomes of H. pylori infection are determined by bacterial virulence, genetic polymorphism of hosts as well as environmental factors. It is important to gain further understanding of the pathogenesis of H. pylori infection for developing more effective treatments for this common but deadly malignancy. The recent findings on the bacterial virulence factors, effects of H. pylori on epithelial cells, genetic polymorphism of both the bacterium and its host, and the environmental factors for GC are discussed with focus on the role of H. pylori in gastric carcinogenesis in this review.


INTRODUCTION
Gastric cancer (GC) is one of the most common mali gnancies globally [1] . The risk factors for GC consist of Helicobacter pylori (H. pylori) infection, genetic and environmental factors [1] . H. pylori mainly colonized in human stomach, has coexisted with humans for nearly sixty thousand years [2] . The outcome of infection is affected by the environmental and genetic factors, the infection in most individuals does not develop dis tinct disease or even become beneficial, leading to the hypothesis that H. pylori might be commensal [3] . However, accumulating evidences support that H. pylori infection cause a list of diseases, ranging from gastric to extragastric diseases, from chronic gastritis to gastric carcinoma, and thus this bacterium is recognized as the Class Ⅰ carcinogenic pathogen in human with less than 3% of the infected eventually suffering GC [3] .
Mechanism of H. pyloriassociated gastric carcino genesis has not been well defined. H. pylori infection commonly lasts for decades, provoking a series of histological changes including destruction of intercellular junctions, apoptosis and proliferation of epithelial cells and malignant transformation [4,5] . The genotypes of H. pylori strains, host genetic polymorphisms, envir onmental factors like high salt diet, smoking habit and certain gastric commensal organisms have been determined to be associated with occurrence of GC [6] .
H. pylori genetic polymorphisms, effects of specific H. pylori products on gastric epithelium and cellular signaling process have been intensively investigated in recent decades [6] . This review is performed to discuss the role of H. pylori in gastric carcinogenesis.

H. PYLORI VIRULENCE FACTORS
Studies on H. pylori heterogeneity have proved that the strongest virulence factors were amongst the genes within the cag pathogenicity island (PAI).

CagA
CagA, a highly immunogenic protein, is encoded at one end of the cag PAI, which encode the components to form the type Ⅳ secretion system (T4SS) [7] . As a component of T4SS, CagL protein binds to and activates the integrin α5β1 receptor on gastric epithelial cells and triggers CagA delivery into the target cells [8] , CagM, along with CagX and CagT, forms an outer membrane associated T4SS subcomplex [9] , CagX and CagT inter act directly [10] . As reported, CagA also facilitates its translocation into host epithelial cells by T4SSinduced externalization of phosphatidylserine from inner leaflet of the cellular membrane [8,11] . Recent studies demon strated that fibronectinand peptidoglycan was also transported into epithelial cells by T4SS, suggesting that T4SS might have more CagAindependent functions than its ability to inject CagA [10] . CagA and CagM are important for assessing virulence of H. pylori strains.
H. pylori strains harboring the cag PAI or producing CagA are related to enhanced inflammation and risk of ulcers and carcinoma [12] . CagA contributes to my riad signaling alterations, which profoundly affects physiology of host epithelial cells. The GluProIle TyrAla (EPIYA) motifs in CagA are phosphorylation sites and play crucial roles in pathogenesis of H. pylori infection [13,14] . Once inside host cells, CagA is tyrosine phosphorylated by Src and Abl kinases at EPIYA motifs, and binds the SH2 domain of the SHP2 phosphatase involved in transduction of signaling [15,16] . Phosphory lated CagA triggers the cellular signaling pathways leading to expression of proinflammatory cytokines and chemokines, and deregulates the signaling pathways that control host cell shape, adhesion and transfor mation [17,18] . Unphosphorylated CagA interacts with certain intracellular proteins, upregulate production of proinflammatory cytokines, provoke mitogenic responses and disrupt intercellular junctions and epithelial cell polarity [17,19] . Additionally, CagA intoxicates dendritic cells leading to impaired activation, decreased inflammatory cytokine production and Th1 immune response [20] . Re cently, it was confirmed that H. pylori infection resulted in rapid association of the virulence factor CagA with the cMet receptor, activation of signaling and epithelial proliferation [21] .

Vacuolating cytotoxin A
Vacuolating cytotoxin A (VacA) contributes to multiple structural and functional alterations of epithelial cells.
After secretion by the bacterium through the type Ⅴ secretion system, VacA binds to host cells interfering with endosomal maturation and leading to vacuolation, enhances leakage of nutrients by destruction of barrier function at tight junctions of epithelial cells, provokes mitochondrial damage and cell apoptosis, which improves H. pylori growth [2224] . Recent studies proved that VacA could disrupt phagocytosis, interfere with antigen presentation, restrain T cell activation in vitro and inhibit T cell proliferation independing of NFAT (nuclear factor of activated T cells) activation or IL2 expression [2527] . These effects of VacA on the immune system may explain how H. pylori evades adaptive immune responses to establish persistent infection.

BabA and SabA
Adherence to epithelial cells is important for H. pylori colonization and delivery of virulence factors to host cells [6] . BabA and SabA are two sialic acidbinding adhesins variably expressed by H. pylori. Among babA and babB genes, only the babA2 allele possesses active Zhang RG et al . H. pylori and gastric carcinoma function [6,28] . BabA can bind to sialylLewis x/a antigens and the Lewis b ABO blood group antigen (Leb), which are mainly distributed in red blood cells and certain epithelial cells [29] , and this binding activity is commonly present in CagA positive strains [30] . Adherence mediated by BabA enhances the ability of T4SS to contact host cells, thus strengthen inflammatory response [31] . BabA binding to Leb contributes to gene mutations through formation of double stranded DNA breaks in host cell lines [32] . SabA can facilitate colonization in patients lacking Leb by binding to the sialylLewis antigens [33] , and mediate binding of H. pylori to sialylated structures of neutrophils [34] . The data suggest that BabA and SabA might be involved in carcinogenesis as abundance of sialyl-Lewis antigens is commonly enhanced in inflamed or cancerous gastric tissues [35] .

OipA
OipA is an inflammationrelated outer membrane protein, and the functional oipA gene is associated with more severe clinic outcomes [36,37] . OipA is commonly expressed together with CagA in cagA positive strains, which make it rather difficult to identify the effects of oipA alone in H. pylori infected human body or animal modules [38] . OipA expression is linked to increased production of proinflammatory cytokines like IL8, IL1, IL17 and TNFα [39] as well as other host effector proteins including those associated with GC [40] . OipA can activate βcatenin, and mutant H. pylori strains lacking OipA decrease nuclear translocation of βcatenin, while tumorigenesis can be depressed by inactivating oipA of H. pylori strain in experimental animals [33,37] . These data suggest that OipA might take part in gastric carcinogenesis [33] .

Gamma-glutamyl transpeptidase
Gammaglutamyl transpeptidase (GGT) is mainly found in outer membrane vesicles of H. pylori, and has been proved to be related to enhanced levels of hydrogen peroxide and IL8 production in epithelial cells and H. pyloriassociated diseases [4143] . GGT accelerates glutathione degradation, prooxidant compounds and reactive oxygen production [41] . GGT adjusts IL8 exp ression by depletion of glutamine [41] . These findings indicate that GGT plays a significant role in H. pylori related chronic inflammation and tissue damage.
It should be noted that interaction of the factors commonly exists in vivo, actions that a virulence factor take under the conditions with presence of other viru lence factors might be different from those observed in vitro [44] . The influence of interactions between virulence factors in multifactorial pathogenesis is still uncertain. The main virulence factors in pathogenesis of H. pylori infection are shown in Figure 1 [6] .

EPITHELIAL CELLS
Beside responsibility for digestive processes, the gastric epithelium has the function to protect underlying tissues from infection by pathogens [45] . H. pylori take specialized mechanisms to avoid host defense and adaptive imm une for persistent colonization in human body, such as disruption of epithelial junctions, stimulation of cytokine production, overproliferation, DNA damage, apoptosis and cell transformation.

H. pylori disrupts junctions and polarity of epithelial cells
Intercellular apical junctions of epithelial cells are critical in keeping integrity of gastric epithelial barrier and essential cellular functions [25] . H. pylori disrupts epithelial tight junctions through binding to specific cellular receptors and stimulating the signaling pathways. As transported into epithelial cells through T4SS, CagA interacts with junction proteins like Ecadherin and ZO1, and alters the tight or adherence junctions [25,46] . It has been confirmed that E-cadherin, a transmembrane protein, localizes at celltocell junctions and interacts with βcatenin to form the Ecadherin/βcatenin comp lex, which play a key role in interaction of epithelial cells and stabilization of cellular architecture [46] . However, the complex is destabilized by translocated CagA in a phosphorylation independent manner during H. pylori infection [46] . As reported, CagA translocation is rele vant to mislocalization of ZO1 in epithelial cells [47,48] . Studies revealed that H. pylori altered expression and localisation of claudin7, a cancerassociated tight junction protein, in gastroids and human epithelial cells, which was mediated by βcatenin and snail activation [49] .
A recent study demonstrated that H. pylori diminished acidinduced tightening of cell junctions, affected the response of epithelial cells to acid, which took effects in inflammatory response and alteration of the barrier function [50] .
H. pylori cause defect of epithelial cell polarity by targeting the epithelial adhesion receptors like Ecadherin and β1integrin to modulate formation of cytoskeleton [51] . CagA disrupts polarity of epithelial cells through interaction with PAR1/MARK kinase [52] . As proved, an atypical protein kinase C (aPKC) contributes to disaggregation of PAR1 from tight junctions by phosphorylation of PAR1 at the junctions [52] , and PAR1b binding to CagA restrains PAR1b activity and phosp horylation by aPKC to promote disruption of cellular polarity [48,52] .

Induction of gastric epithelial cell autophagy or apoptosis
H. pylori not only colonize the mucus layer covering gastric mucosa, but also invade gastric epithelial cells, and even immunocytes [53] . Recent studies demonstrated that H. pylori induced autophagy of epithelial cells and phagocytes [53] . The autophagy of epithelial cells is modulated by H. pylori, and can be inducted by acute VacA exposure, and prolonged exposure to the toxin disrupts autophagy by preventing maturation of the autolysosome. The evidences support that H. pylori to TRAILmediated apoptosis through regulation of cellular FLICEinhibitory protein activity and assembly of deathinducing signaling complex [61] .

Cytokines secretion
The secretion of proinflammatory cytokines by gastric epithelial cells plays significant roles in pathogenesis of H. pylorirelated gastric diseases. The cytokines involved in H. pylori infection include IL8, IL6, MCP1, TNFα, MIF, IL1α, TGFβ, IL1β and GMCSF [17] . The pro duction of IL8, a chemokine mediating accumulation of neutrophils, is related to the expression of CagA [17] .
Further study confirmed that IL-8 and NF-κB expression was activated by urease B subunit [62] , and the urease stimulates gastric epithelial cells to produce TNFα and IL6 [63] . As reported, both cag PAI and OipA upregulate IL6 production in gastric epithelial cells [64] . Additionally, Th17 subsets are enriched in H. pylori infected muc osa [65] . The expression level of interleukin17 (IL17) has been observed to be upregulated in gastric tissues of both human and animal during H. pylori infection, while suppressed autophagy facilitates intracellular survival of this bacterium and generates an environment favoring carcinogenesis [54] . Rapid turnover of epithelial cells contributes to protect the epithelium from infection. H. pylori disrupts the balance of the proliferation and turnover of gastric epithelium to facilitate its survival [55] . Apoptosis is a regulated and conserved process in tissue, and takes the key role in tissue homeostasis [56] . H. pylori regulates the balance of epithelial cell apoptosis and proliferation for its reproduction [57,58] . The mechanism for this phenomenon remains to be well defined. The damage of gastric mucosa, stimulation of inflammatory immune responses by the enzymes like urease and VacA contribute to cellular apoptosis. The elevated level of free radicals produced by neutrophils and TH1 cytokines like IFNγ in inflammatory response can damage DNA and induce apoptosis of epithelial cells [5,59] . H. pylori adhering to the epithelial surface also stimulate cellular apoptosis [60] . Studies demonstrated that human gastric epithelial cells sensitized to H. pylori confer susceptibility 100 February 15, 2016|Volume 7|Issue 1| WJGP|www.wjgnet.com  Figure 1 The roles of the main virulence factors in pathogenesis of Helicobacter pylori infection [6] . Adherence of Helicobacter pylori to gastric epithelial cells is mediated by BabA and SabA binding Leb and Lewis x/a respectively. CagA is translocated into epithelial cells through T4SS, and then tyrosine phosphorylated at EPIYA sites by Src and Abl kinases. CagA contributes to alteration of myriad signaling transduction, which affects host cell physiology with disruption of intercellular junctions, loss of cell polarity, promotion of inflammation, dysregulation of cellular apoptosis and proliferation. VacA inducts cytoplasmic vacuolation, apoptosis and immune suppression [6] .
On the other hand, elevated levels of IL21 and IL23 expression in gastric mucosa induce and sustain IL17 production [66] . Recently, a new clue for the pathogenesis of H. pylorirelated gastric inflammation and GC is impairment of ghrelin synthesis in H. pyloricolonized stomach. Ghrelin, the ligand of growth hormone secre tagogue receptor 1a, has immunoregulatory properties and function of certain inflammatory pathways inhi bitor [67] . The defective ghrelin synthesis may contribute to sustain the ongoing inflammatory response in the gastric diseases [67] .

Pro-carcinogenic responses
The mechanism underlying H. pylor-related gastric carcinogenesis remains unclear. CagA interacts with Ecadherin, deregulates βcatenin signal transduction and promotes gastrictointestinal transdifferentiation [47] . As observed, CagA translocated intracellularly binds to PAR1, destroys cellular junctions and polarity, and fosters carcinogenesis [48] . Development of gastric and hematological carcinoma has been observed in the mice that were genetically modified to express CagA [68] . Studies revealed that cagA+/vacAs1+/vacAm1+H. pylori strains promoted pathogenesis of intestinal metaplasia and gastric carcinoma [69] . Aditionally, H. pylori regulates expression of such toll like receptors as tolllike receptor (TLR) 4 and TLR9 in epithelial cells during gastric carcinogenesis [70] . Caveolin1 plays a protective role in immunologic injury caused by H. pylori [71] . Rapid association of the virulence factor CagA with the cMet receptor, activation of signaling and induction of epithelial proliferation have been observed by using pluripotent stemcellderived gastric organoids [21] .

H. pylori genetic variability
Genetic diversity of H. pylori has major contribution in the pathogenesis [72] . Studies have been conducted focusing on polymorphism of the main virulence factors, such as cagA, vacA, oipA, iceA and hopQ.
The highly polymorphic EPIYA motifs at the Cterminal of CagA are involved in pathogenesis of H. pylorirelated gastroduodenal diseases [73] . CagA containing EPIYA motifs can activate the STAT3 pathway and promote cell migration [74] . The EPIYA motifs are distinguished by different amino acid sequences surrounding the EPIYA motif, and an increased number of CagA EPIYAC sites confers a heightened risk for GC developing [75,76] . The sequences from Western and East Asian strains contain EPIYAC and D, respectively, and the strains with two segment C have more chances to develop GC than those with one [73] . The significantly higher prevalence of East Asian CagA in patients from Japan with H. pylori infection may be involved in the pathogenesis of GC [77] . Polymorphisms among the vacA alleles of H. pylori strains contribute to various levels of cytotoxicity, while variations in various regions can influence activity of VacA, including vacuolating activity [78,79] . It has been confirmed that vacuolating activity is highest in s1/m1 strains, vacA s1/m1 strains are closely relevant with GC in western countries [80,81] . Nevertheless, this situation is not universal worldwide, for example, s1/m1 strains in districts of Asia is irrelevant to clinical outcome [82,83] . Additionally, investigation of the prevalence of oipA and iceA1/iceA2 positive strains among patients suffering from GC or gastritis results in that the frequency of iceA1 allele in patients with GC is significantly higher than those with gastritis [72] . However, there is no significant difference in prevalence of oipA and iceA2 genes among the two groups of patients (P > 0.05), suggesting the iceA1 gene might take a role in pathogenesis of H. pyloriinduced GC [72] . Studies also indicated that certain genetic types of H. pylori hopQ were closely related to GC [84] .

Genetic polymorphism of H. pylori hosts
Polymorphisms in the genes encoding innate immune factors are involved in pathogenesis of H. pylorirelated diseases, and the polymorphisms of cytokine genes cause interindividual variation in cytokine responses which contributes to diversity of clinical outcome [85] . As reported, the risk of GC in many populations was affected by the polymorphism of the genes encoding IL1β, TNFα, IL8, IL17 and IL10 or their receptor antagonist [25] . An elevated risk of GC was observed in IL8251 AA or IL101082 G genotype carriers with H. pylori infection [86] . IL17 A/F plays critical function in inflammation and probably in cancer. Studies concluded that polymorphism of IL17F was involved in suscepti bility to GC [87] . Current evidences support that TLRs are play roles in both recognition of H. pylori and gastric carcinogenesis, and polymorphisms in genes involved in the TLR signaling pathways modulate the risk of GC [88] . Additionally, peroxisome proliferatoractivated receptors may play roles in H. pylori-related gastric carcinogenesis [89] . The G/G variant rs2076167 is relevant to increased risk of GC in an animal model. The association between G/G variants of rs2016167 and GC is close among those consuming higher salt diet [89] . The insertion/deletion polymorphism of the angiotensin Iconverting enzyme gene was recently proved to be linked to the pathogenesis and progression of human cancers [90] . As demonstrated, both bacterial and host gene polymorphisms affect oxidative stress and DNA damage, as thought to be a key mechanism in gastric carcinogenesis. The interaction of bacterial and host gene polymorphisms may become an explanation for why GC only occurs in a small proportion of H. pylori infected individuals [91] .

PYLORI-RELATED GC
There are multiple ways by which H. pylori manipulates the host to lower the threshold for carcinogenesis, gastric microbiota, highsalt diet, smoking habit, low iron levels and use of proton pump inhibitors (PPIs) may enhance risk of H. pyloriassociated carcinogenesis [92] .

Gastric microbiota
Alterations of microbiota inhabiting human digestive tract can favor carcinogenesis [93] . Conventional wisdom espoused the dogma that pH values < 4 were able to sterilize the stomach, but since the discovery of H. pylori [94] , a complex community of noncultivatable inhabitants have been uncovered in the stomach [95] . The interaction of gastric microbiota with H. pylori likely affects gastric immunobiology and the outcome of infection [95] . Data indicate that the microbial density in the normal stomach is low (10 1 10 3 CFU/g) [94] , and the low bacterial densities within this portion of gastrointestinal tract is attributed to rapid peristalsis, low pH and/or high bile concentration [96] . The parietal cell loss caused by H. pylori infection leads to hypoch lorhydria or even achlorhydria, thereby increase the risk of bacterial overgrowth and detrimental infection [97] . Alteration of gastric microbiota may promote the deve lopment of GC by upregulating production of Nnitroso  Figure 2 The pathogenesis of Helicobacter pylori-associated gastric cancer. The pathogenesis of H. pylori-associated GC is a multi-factorial process, its development depends on a combination of host, bacterial and environmental factors, and the pathological changes might progress in steps. H. pylori: Helicobacter pylori; GC: Gastric cancer. compounds [93] .

Smoking
Studies demonstrated that virulence factors like cagA and smoking might have synergistic effect in carcino genesis of GC, and cagA genotype of H. pylori strains was closely related to activesmoking in population with H. pylori infection as shown in Table 1 [98] .

High salt diet
As evidenced, CagA expression is significantly upre gulated when certain H. pylori strains are cultured in a medium of high salt concentrations. Through seq uence analysis and sitedirected mutagenesis, it was determined that saltresponsive H. pylori strains were more likely to contain two copies of TAATGA motif within the cagA gene promoter, while the strains containing only a single copy of this motif were less likely to possess properties of saltresponsive CagA expression [92,99] . However, another study showed that the severity of gastritis in H. pylori infected population might be unassociated with highsalt diet [100] .

Iron levels
The iron level in the host has also been proved to mani pulate the virulence potential of H. pylori. The bacteria harvested from gerbils with low iron levels were found to assemble more T4SS pili per bacterium, translocate increased amounts of CagA, and augment more IL8 secretion compared to those isolated from gerbils with normal iron levels [101,102] . Furthermore, the H. pylori strains isolated from patients with low ferritin levels induce significantly higher levels of IL-8 compared to the strains from patients with the highest ferritin levels, sug gesting that iron deficiency in the host might enhance the bacterial virulence and the risk for carcinogenesis of gastric tissues [101,102] .

PPIs
It has been evidenced that longterm use of proton PPIs might aggravate corpus atrophic gastritis in H. pylori infected patients [97] . The worsening atrophic gastritis contributes to development of gastric carcinoma, particularly owing increasing production of potentially carcinogenic Nnitroso compounds by the bacteria overgrowing under conditions of hypochlorhydria [97] . Hypergastrinemia induced by PPI administration might also promote the development of GC [97] .

Di (2-ethylhexyl) phthalate
Di (2ethylhexyl) phthalate (DEHP), as an essential additive in plastic manufacturing, has been used as plasticizer for many products including plastic food packaging [103] . Recent studies confirmed that DEHP was a teratogenic compound closely related to carcino genesis [103] . DEHP may enhance H. pylori cytotoxicity, induce gastric epithelial cell apoptosis, disrupt the gastric mucosa integrity and promote pathogenesis of gastric carcinogenesis [103] .

CONCLUSION
The genomes of H. pylori are highly diverse, multiple virulence factors take effects on host epithelium in various manners, including direct action and indirect action like eliciting immune response. Genetic poly morphism of host, dietary factors, smoking, gastric microbiota and long-term consuming of PPIs influence the progression of H. pylorirelate gastric lesion. The pathogenesis of H. pyloriassociated GC is a multi factorial and multistep process, and its development depends on a combination of host, bacterial and environ mental factors as shown in Figure 2. It is important to further reveal the carcinogenesis of H. pylorirelated GC in order to develop more effective treatments for this common but deadly malignancy.