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Role of Helicobacter pylori in gastric cancer: Updates.

Helicobacter pylori (H. pylori) infection is highly prevalent in human, affecting nearly half of the world's population; however, infection remains asymptomatic in majority of population. During its co-existence with humans, H. pylori has evolved various strategies to maintain a mild gastritis and limit the immune response of host. On the other side, presence of H. pylori is also associated with increased risk for the development of various gastric pathologies including gastric cancer (GC). A complex combination of host genetics, environmental agents, and bacterial virulence factors are considered to determine the susceptibility as well as the severity of outcome in a subset of individuals. GC is one of the most common cancers and considered as the third most common cause of cancer related death worldwide. Many studies had proved H. pylori as an important risk factor in the development of non-cardia GC. Although both H. pylori infection and GC are showing decreasing trends in the developed world, they still remain a major threat to human population in the developing countries. The current review attempts to highlight recent progress in the field of research on H. pylori induced GC and aims to provide brief insight into H. pylori pathogenesis, the role of major virulence factors of H. pylori that modulates the host environment and transform the normal gastric epithelium to neoplastic one. This review also emphasizes on the mechanistic understanding of how colonization and various virulence attributes of H. pylori as well as the host innate and adaptive immune responses modulate the diverse signaling pathways that leads to different disease outcomes including GC.


INTRODUCTION
In 1984 Marshall and Warren [1] identified Helicobacter pylori (H. pylori) from gastric biopsy culture. In 1994, H. pylori was recognized as definite carcinogen by Inter national agency for research on cancer. H. pylori induced gastric cancer (GC) is accountable for 5.5% of global cancer burden [2] . H. pylori is spiral shaped, gramnegative, microae rophilic, flagellated human pathogen that successfully colonizes gastric mucosa of majority of individuals [3] . Epidemiologically, the H. pylori infection is exists all over the world, but colonization rates vary considerably; high in developing compared to the developed world [4] . H. pylori acquisition thought to occurs in early childhood. Fecaloral or oraloral were considered as possible route of H. pylori transmission [4,5] . H. pylori urease is among the various virulence factors that aids in colonizing the highly acidic environment of stomach via breakdown of urea into ammonia, generating hospitable locale for its colonization [6] (Figure 1). Among the majority of H. pylori infected individuals only a small percentage of colonized individuals develop severe clinical disease such as GC.
Determing factors responsible for variation in clinical outcomes of H. pylori infection are still not well studied. For a longer period of time association between H. pylori and GC was debatable. A study from Japan on 1526 patients gives a clear evidence that H. pylori infection is significantly associated with risk of developing GC [6] . Proof that H. pylori has an influence on early stages of gastric carcinogenesis is demonstrated by randomized prospective studies which shows association between H. pylori eradication and reduction of premalignant tumors [7,8] .
Research on experimentally challenged Mongolian gerbils, provide evidence concerning H. pylori eradication with attenuation of developmental process related to GC progression [9,10] . Together these studies authenticate that H. pylori plays a key role in development of GC and indicate that H. pylori eradication provide protection against H. pyloriinduced GC. Interaction among environmental factors, host genetic polymorphism and bacterial virulence attributes collectively influence the clinical outcome of H. pylori infections [11] . This review aims to highlight recent progress in H. pylori pathogenesis, especially the bacterial and host factors that are involved in the hostpathogen interaction during persistent colonization. It also highlights the host immune response towards H. pylori colonization and its effect on diverse clinical outcomes, especially on advancement leading to GC.

EPIDEMIOLOGY OF GC
GC is a multifactorial disease. Correa's model describes array of event beginning from chronic active gastritis, atrophic gastritis, intestinal metaplasia, dysplasia and eventually leads to GC [12] (Figure 2). Risk factors for the development of the GC include interaction among the pathogen, environmental and hostrelated factors [13] . World Health Organization recognized H. pylori as class Ⅰ carcinogen in 1994. GC is identified as the fifth most common malignancy and third leading cause of cancerrelated morbidity globally, constituting 9.7% of all cancerrelated mortality [14] . Highest agestandardized mortality rate (ASMR) is predicated for Eastern Asia (28.1 per 100000 in men, 13.0 per 100000 in women), the lowest ASMR in North America (2.8 and 1.5 per 100000, respectively) [15] . Studies reported high mortality rates are from East Asia, Central and Eastern Europe, Central and South America [15] . Developing countries have high burden of GC compared to the developed world and GC accounts for approximately 70% of both new cases and deaths [16] . Categorizing on basis of gender, 466900 cases of males were reported from developing as compared to 173700 cases from developed countries and for females the corresponding disease load was 247000 and 102000 cases, respectively. GC is associated with age incidence; commonly occurs in age group of 55 to 80 years, rare among young individual. Frequency of GC rates are two fold higher in males than females [17] . Over past decades in western nations, GC has conside rably declined. The possible reasons behind this reduction include fall in H. pylori prevalence accompanied by better hygienic practices and innovative medical diagnostic facility. Despite the decline in GC incidence in developed world, the scenario of developing world is diverse. GC incidence and mortality rate remain very high in the developing nations, particularly in regions of East Asia and South America [17] . It is expected that if appropriate measures are not implemented the number of estimated GC cases are likely to increase in future.

PATHOLOGICAL DIFFERENTIATION
Majority of gastric malignant tumors are adenocarci nomas. Histologically Lauren categorized gastric adeno carcinoma in intestinal and diffuse subtypes. Intestinal type adenocarcinoma is event dependent, start from chronic atrophic gastritis to intestinal metaplasia to dys plasia and finally carcinoma. Intestinal type adenocar cinoma is more frequent in developing world, common in male, and associated with age incidence, whereas diffuse type occurs more often in younger patients having family history of cancers, more frequent in females, background of atrophic gastritis is not prerequisite condition for its occurrence [18,19] . Anatomical site of origin is another way of differentiation of gastric adenocarcinoma. Tumors arising in the cardia region of the stomach are said to be proximal, and those from body and antrum (non cardia region) as distal. Histological subtypes represent etiological and epidemiological differences between the two tumor sub sites. Globally GC incidence is declining. However, studies show rise in incidence of cardia carcinoma which may be partly due to more accurate reporting and fall in incidence of distal cancers [20] .

H. PYLORI AS A RISK FACTOR FOR GC
Colonization of the stomach by H. pylori causes develop ment of gastritis. H. pylori is truly an "opportunistic" bacterium that uses various well defined virulence factors as tool for attachment and persistent colonization of human gastric mucosa. The possible transmission route is fecaloral, but contaminated food or water are also reported [21,22] . The most likely sources are personto person contact in families and/or exposure to a common source of infection such as contaminated water or food as supported by majority of data [23] . This notion is sup ported by studies of children in custodial care where the prevalence of infection is higher than expected and from studies of crowded families in which there is at least one infected child [24] . Before attachment of H. pylori to gastric epithelium, it has to first cross the thick mucus layer by adhering to the mucosal surface. This is aided by the presence of unipolar sheathed flagella, which allows H. pylori to quickly move from inhospitable low pH of gastric lumen to surface epithelium where pH is high and favorable for its successful colonization despites efforts made by the host to get rid of this bacterium. Nonmotile mutant H. pylori strains fail to colonize the stomach of gnotobiotic piglets [25,26] . In majority of infected individuals colonization results in development of inflammatory and immune responses against H. pylori, but in some subjects H. pylori infection becomes chronic and leads to induction of gastric inflammation which can eventually lead to destruction of normal gastric glands and their replacement by intestinaltype epithelium resulting in atrophy of gastric mucosa.
The risk for atrophic gastritis depends on pattern as well as extent of distribution of chronic active inflam mation. The individuals with lower acid output show a higher tendency towards atrophy [27] . Reduction in gland size and level of intestinal metaplasia were associated with rise in GC risk by 5 to 90folds depending on the extent and severity of atrophy [28] . Increased odds ratios were evident from case control studies that aimed to seriously study the signs of earlier H. pylori infection in GC patients and controls for development of noncardia GC in presence of H. pylori infection [29] . This fact is supported by data from animal models including Mongolian gerbil model, in which H. pylori infection induces atrophic gastritis and GC [3032] . A small number of subjects for research purposes were deliberately infected with pathogenic H. pylori strain and individuals developed acute inflammation of gastric mucosa with neutrophilic infiltration [33,34] . Volunteers after several decades when exposed repeatedly to intragastric pHelectrodes contaminated with H. pylori developed conditions called "epidemic hypochlorhydria" [34] . Such hypochlorhydric gastritis can either resolve spontaneously or change into chronic gastritis.

ROLE OF HOST GENETICS
H. pylori infection results in three possible outcomes.  atrophic gastritis to hypochlorhydria and finally to GC. Second type results in a pangastritis having slightest impact on the host gastric acid production. Duodenal ulcer is third outcome, where an antrumpredominant gastritis leads to hyperchlorhydria. There arises con troversy that infections of H. pylori can predispose to two equally exclusive situations. The possible explanation why some people are more expected to develop GC phenotype when compared with others may be due to disparity among individual host response to H. pylori infections ( Figure 1). Initial evidence for the importance of host genetic polymorphisms was reflected in the study where a rise in incidence of atrophic gastritis and hypochlorhydria was evident from relatives of H. pylori induced GC patients than controls [35] . Proinflammatory cytokine like interleukin1beta (IL 1β) act as a powerful negative regulator of acid secretion. IL-1β gene is now considered as a potential contender for host genetic polymorphisms that may elevates GC risk. Individuals possessing IL-1β gene cluster polymorphisms have 2-3folds increased risk of noncardia cancer [36,37] ( Figure 2). Elevated levels of TNFα in gastric mucosa of H. pylori infected individuals were evident from nu merous studies. However, down regulation of anti inflammatory cytokine IL10, that suppresses the level of proinflammatory cytokines including IL1β, TNFα and interferonγ (IFNγ) is also reported [36] .

First is corpuspredominant gastritis beginning from
The risk associated with GC development in H. pylori infected individuals upsurges 27folds in individuals with three or four polymorphisms [38] . This evidently illustrates that interaction between host genetics and environment plays a key role in progression of GC, by regulating hosts adaptive immune response resulting in transformation of normal gastric mucosa to neoplastic one.

IL-8
Higher expression of chemokine IL8 and polymorphism (promoter region) has been reported in studies and linked with increased risk for GC [39] . Study on Caucasian populations proved that relationship among functional polymorphism within Toll like receptor 4, risk of GC and decrease in production of antiinflammatory cyto kine IL10 [40] . These studies reflect that host genetic polymorphisms are capable of modulating the innate immune response which results, severe inflammation and premalignant lesions in H. pylori infected individuals ( Figure 2). These studies raises a query that whether H. pylori strain characteristics are responsible for increasing cancer risk employed by host genotypes, needs to be studied further. Odds ratios for noncardia GC were highest for individuals with elevated IL1β expression, colonized by H. pylori vacAs1type strains [41] . It is evident from casecontrol studies that H. pylori successfully form a vital equations with host by its ability to send and receive signals from its hosts [42,43] .
Only certain H. pylori strains enhance the possibility of carcinogenesis because the equilibrium is likely different for each colonized individual. For example, individual infected with CagA strains leads to severe gastritis, which results in rise of proinflammatory cytokines levels that are responsible for both amplifying the mucosal inflammatory response as well as reducing the acid production. This creates a milieu encouraging growth of H. pylori that promote inflammation and continually produce oxidative stress, thus augmenting risk for trans formation of normal mucosa to neoplastic through series of events ( Figure 2).

Cyclooxygenase
H. pylori triggers numerous forms of proinflammatory cyclooxygenase (COX) enzymes. Production of endo peroxide from arachidonic acid is brought by COX en zymes. Enzymes prostaglandin synthases produces prostaglandins and various eicosanoids from endope roxide [44] . Important role is played by prostaglandins in regulating physiologic processes for instance immunity and development. Two COX isoforms (COX1 and COX2) have been categorized on the basis of variances in expression characteristics and inhibition profiles for nonsteroidal antiinflammatory drugs (NSAIDs). COX2 expression is inducible while COX1 is constitutively expressed in cells and tissues [4547] . Expression of COX2 can be stimulated by proinflammatory cytokines, growth factors such as TNFα, IFNγ and IL1. COX2 expression are raised in H. pylori infected human gastric mucosa, gastric premalignant and malignant lesions [4749] . Inhibitors of COX (aspirin and NSAIDs) are associated with reduced risk of noncardia GC [50] . Numerous studies demonstrate substantial role of COX2generated products involved in promoting neoplasia. Mechanisms like apoptosis inhibition, regulation of expression of cell surface adhesion, and production of promoting factors of neoplasia leads to malignancy [51,52] (Figure 2).  (Figure 1). Positive association of CagA was found with peptic ulcer disease [56,57] . Due to its association with several gastroduodenal pathologies, initially CagA was considered as an indicator for presence of the entire CagPAI but as research speeded up, studies demonstrated that despite its presence, CagPAI intactness and clinical outcome varied.

H. PYLORI VIRULENCE FACTORS
More or less 70% of H. pylori strains from western world and nearly 100% of East Asian strains express virulent protein CagA [54,58,59] . Majority of H. pylori strains induces superficial gastritis but the risk for chronic gas tritis, atrophic gastritis, metaplasia, and noncardia GC with intact CagPAI is much higher compared to those that lacked it [56,57,6066] . Among 32 genes of CagPAI, 18 genes are thought to code for structural parts of a T4SS, this system is responsible for exporting peptidoglycans and cagA into host gastric epithelial cells, via forming a pilus like assembly connecting bacterial and host epithelial membrane (Figure 1).

CagA
CagA is terminal gene product of the CagPAI. Classifying H. pylori strains on the basis of presence and absence of cagA into cagApositive and cagAnegative strains. After the H. pylori attachment to epithelial cell, CagA is internalized through T4SS apparatus. After translocation, CagA is tyrosine phosphorylated at glutamateproline isoleucinetyrosinealanine (EPIYA) motif, i.e., EPIYA motif which is associated with cell morphological changes known as "the hummingbird phenotype," which results in increased cellular migration [6771] . Polymorphic region of CagA, has been identified within the carboxyterminal and distinguished by different amino acid sequences. Till date, four distinct EPIYA motifs (EPIYAA, B, C and D) are known [72,73] . EPIYAA and B motifs are present in strains all over the world, whereas EPIYAC is specific to western world (Europe, North America, and Australia). Variation in number of EPIYAC sites occurs, while majority of CagA proteins contain a single EPIYAC site (ABC type). The level of phosphorylation of EPIYAC sites is greater than EPIYAA and EPIYAB sites. Risk for development of GC is found to be associated with the number of cagA EPIYAC in western strains [74] . EPIYAD motif is exclusive to East Asian strains (from Japan, South Korea, and China), and strains possessing this motif produces higher level of IL8 from gastric epithelial cells as compared to strains harboring western ABCtype CagA [72,75] .

CagA phosphorylation-dependent host cell signaling
Kinase families of Abl and Src are responsible for pho sphorylation of CagA into phosphoCagA. Interaction between phosphorylated CagA and various intracellular effectors, triggers an eukaryotic tyrosine phosphatase (SHP2), which results in continuous stimulation of extracellular signalregulated kinases 1 and 2 (ERK1/2), Crk adaptor [76] and Cterminal Src kinase in a tyrosine phosphorylationdependent manner. In East Asian ABD types, negative response is induced by interactions of phosphoCagA with Cterminal Src kinase resulting down regulation of Src signaling [77] (Figure 3).
Experimental studies on cell lines revealed that CagA internalization give rise to "hummingbird phe notype". These alterations are characterized by cell elongation and cell scattering [69,78] . Additional study also indicates that interplay among phosohorylated CagA, dephosphorylation of SHP2 and downregulation of focal adhesion kinase, causes cell elongation [69,79] . A different mechanism of cell elongation by phosphorylated CagA is by making a defect in cell retraction; yet the signaling molecules prerequisite for this phenotype remain vague [80] .

Khatoon J et al . Helicobacter pylori and gastric cancer
Phosphorylated CagA obstructs the enzymatic activity of cSrc, which leads to tyrosine dephosphorylation of actin binding proteins such as cortactin, ezrin, and vinculin, ultimately results in cell elongation [8183] (Figure 3).

CagA phosphorylation-independent host cell signaling
Nonphosphorylated CagA have a different way of exerting effects within the cell. CagA translocation without phosphorylation leads to aberrant catenin activation, apicaljunctional complex disruption and cellular polarity loss [8489] . Relation between nonphosphorylated CagA and epithelial tight junction scaffolding proteins, zonula occludens 1 and junctional adhesion molecule A, results in imperfect association of tight junctions at located sites of bacterial attachment. Additional molecules includes Ecadherin, hepatocyte growth factor receptor cMet, phospholipase C gamma (PL), adaptor protein Grb2, and kinase partitioning defective 1b/microtubule affinityregulating kinase 2 (PAR1b/MARK2) resulting in mitogenic responses, interruption of cellcell junctions and cell polarity destruction [84,87,88,90] (Figure 3). Recent study revealed that CagA directly binds to the cell polarity regulator such as PAR1b/MARK2. This binding prevents kinase PAR1b/MARK2 activity and deregulates the formation of mitotic spindle by cells which affects cell polarity [88,91] . Studies on transgenic mice revealed the correlation between CagA and oncogenesis by showing that CagA expression led to gastric epithelial cell proliferation and neoplastic changes. However, the following modifications were not detected in mice expressing phosphorylation resistant CagA [52] .
Presence of contradictory documentation on func tionality of CagA as a bacterial oncoprotein in mammals exists besides solid proof provided by animal models.
Pathological alterations described for transgenic CagA mice followed by absence of inflammation, which reflects disparity to what is seen in humans [52] . Although CagA act as oncoprotein, it remains to be explored why only few individuals inhabited by CagApositive H. pylori develop GC. Recent study demonstrate that H. pylori prompts the presence of a host phospholipid, phosphatidylserine where CagA can explicitly interact and gain entry into the cells [92] . Focus of future research should be to define the exact mechanism of CagA internalization in gas tric epithelial, factor responsible for regulation of this process and when during chronic infection CagA delivery in human epithelial cells.

VacA
Another important H. pylori virulence gene is vacuolating cytotoxin (VacA), which encodes a bacterial toxin (VacA) that induces series of cascades leading apoptosis of epithelial cells via induction of cytoplasmic vacuoles ( Figure 4). VacA is found throughout the H. pylori strains. The diverse polymorphic form of VacA are related with clinical outcomes [93] . Considerable genetic variations are found in: The s (signal) region with alleles s1a, slb, slc, or s2; the m (middle) region with m1 or m2 alleles; and the i (intermediate) region with type i1 or i2 alleles (Figure 4).

OTHER RISK FACTORS
Besides H. pylori, the following other environmental factors are considered to contribute in the pathogenesis of GC.

Diet
The variations in GC incidence are due to environmental inputs, particular in dietary pattern. Previous accu mulating studies have been indicated that downward trend in GC occurrence. This may be due to the advent of widespread refrigeration of foodstuff and reduction in dependency on food preservation. In addition, other studies have suggested that a preventive role of diet containing fresh vegetables and fruits. However, data from European prospective study failed to show an overall association between fresh fruits and vegetables intake and GC risk [95] . Recent studies being conducted on this field revealed that a significant association between total dietary vegetables contents (onion and garlic intake) and intestinal GC subtypes.
Additional studies are required for demonstration of positive association between H. pylori eradication and prevention of cancer. The controversy related to point of no return in case of atrophy and metaplasia is still deba table. Proposed studies on side effects and expenses of such preventive measures are required in future for proper management and treatment of GC, therefore GC prevention remains a key part of research on H. pylori.

Salt
H. pylori is not the only the culprit for the development of GC; other influential causes include host polymorphisms and environmental elements (Figure 2). High dietary salt intake was found to be uniformly been associated with an increased risk of GC [12,96] . Two studies, one study from Japan and other a casecontrol study from South Korea stated that H. pylori-infected subjects taking high salt diet had an greater risk of GC than those with lower levels of salt [97,98] . Association between the frequency of H. pylori infection and amount of dietary salt intake is reported in another study [99] . Research on Mongolian gerbils had shown that the H. pylori presence and usage of a more salt containing diet applied concerted effects on development of precan cerous satge [100,101] . Additional study on H. pyloriinfected gerbils demonstrates that there is a positive association between level of severity of gastric inflammation and rate of proliferation of epithelial cells in gerbils consuming highsalt diet than those consuming a normal diet [100] . Similar studies on gerbils infected with H. pylori, when treated with carcinogen (NmethylNnitroso urea) shows that higher frequency of GC related with animals consuming highsalt diet as compared to animals with a normal diet [101,102] .
Mechanisms behind the highsalt diet increases the risk of development of GC in humans remains unclear. Among various explanations, one plausible hypothesis is that salt may lower the threshold for malignant transfor mation by altering the physiology of gastric epithelium thus allowing entry of carcinogens into gastric tissue and resulting in damage to gastric mucosa. Another possibility is that high salt intake might be regulating the gene expression in H. pylori. Two independent studies suggested that consumption of excess amount of salts in diet leads to higher expression of H. pylori virulence factors [103,104] .

Dietary antioxidants
Many studies had proved the antioxidants present in food in green vegetables and fruits plays a preventive role against progression of GC [105] .  recommended that consistent excessive consumption of vitamin C and carotene might be able to curtail the casual for developing GC in subjects having infection of H. pylori [106] . A randomized study on population susceptible for GC development demonstrated that combination of vitamin C and carotene dietary supplements and H. pylori eradication increases the preneoplastic lesions regression at 6 years of followup; at another 6 years followup lacking dietary supplements, the protective role of vitamin C and carotene gradually end up [7] . These results were also validated by other studies [95,106] . A similar study from Hawaii proof that consumption of fresh vegetable among H. pylori infected individuals provided a little protection against GC occurrence [107] .
On the contrary, other studies fail to provide a positive association between H. pylori infection and plasma vitamin C level, with risk of GC incidence [108] . Additional research is required to determine whether antioxidants are capable of providing protection against GC among H. pylori infected patients.

Cigarette smoking
It is evident from various studies that cigarette smoking is associated with risk of developing GC in H. pylori infected subjects. In Japan, cigarette smoking and H. pylori infection together are considered as potential threat for developing GC [109] . Swedish and German popu lationbased case control studies also demonstrated combination of cigarette smoking and infection by CagA positive H pylori strains increased the risk of developing GC (Figure 2). Los Angeles study also reported a tendency toward increased risk of GC in smokers [59,110] .
On collectively analyzing studies, it emerges that there exists relationship between H. pylori infection and smoking with increased risk of developing GC.

Helminth infection
H. pylori coinfection with helminths may have some impact in disease pathogenesis. Reduced Th1 response associated with higher levels of Th2 cytokines was reported in one study [111] . Another study on Colombian children from a coastal region having infection of both helminths and H. pylori, showed a higher Th2 associated IgG1 response [112] . Further studies are needed to assess the impact of H. pylori and helminthes coinfection in disease pathogenesis.

FOCUS OF FUTURE ENDEAVORS
Gastric cancer remains a major threat to mankind. Improvement in living standards, increase awareness in sanitation and hygiene practices, reduction in intake of salted food products and advent of refrigeration in households resulted in measurable decline both in incidence of H. pylori infection and GC. Although both H. pylori infection and GC are showing decreasing trends in the developed world, they still remain a major threat to human population in the developing countries. Therefore, there is a need for improvement in early diagnosis, iden tification of risk factors, and development of preventive strategies and initiation of timely therapeutic interven tions, especially focused for the developing countries. Further, it remains to be investigated why a small fraction of individuals colonized by H. pylori develop GC, and future research should focus on bacterial, host genetics, environmental and dietary factors.
There is need to formulate clear cut recommendation for screening and timely intervention of high risk popula tion with family history of GC. Whether all highrisk areas should undergo routine screening of H. pylori infection is still questionable. Since the patients having atrophic gastritis or dysplasia in the gastric mucosa are at increased risk of developing GC, there is a need for special recommendations including endoscopic surveil lance for such patients.