Gastric cancer: genome damaged by bugs

ABSTRACT Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide. The role of the microorganisms in gastric tumorigenesis attracts much attention in recent years.

These microorganisms include bacteria, virus, and fungi. Among them, _Helicobacter pylori_ (_H. pylori_) infection is by far the most important risk factor for GC development, with special

reference to the early-onset cases. _H. pylori_ targets multiple cellular components by utilizing various virulence factors to modulate the host proliferation, apoptosis, migration, and

inflammatory response. Epstein–Barr virus (EBV) serves as another major risk factor in gastric carcinogenesis. The virus protein, EBER noncoding RNA, and EBV miRNAs contribute to the

tumorigenesis by modulating host genome methylation and gene expression. In this review, we summarized the related reports about the colonized microorganism in the stomach and discussed

their specific roles in gastric tumorigenesis. Meanwhile, we highlighted the therapeutic significance of eradicating the microorganisms in GC treatment. SIMILAR CONTENT BEING VIEWED BY

OTHERS _HELICOBACTER PYLORI_, MICROBIOTA AND GASTRIC CANCER — PRINCIPLES OF MICROORGANISM-DRIVEN CARCINOGENESIS Article 26 February 2025 COMPARABLE GENETIC ALTERATION PROFILES BETWEEN

GASTRIC CANCERS WITH CURRENT AND PAST _HELICOBACTER PYLORI_ INFECTION Article Open access 06 December 2021 THE NOVEL ZEB1-UPREGULATED PROTEIN PRTG INDUCED BY _HELICOBACTER PYLORI_ INFECTION

PROMOTES GASTRIC CARCINOGENESIS THROUGH THE CGMP/PKG SIGNALING PATHWAY Article Open access 04 February 2021 INTRODUCTION Gastric cancer (GC) is the second leading cause of cancer-related

death in the world [1]. GC mainly occurs in Asia, Latin America, and Central and Eastern Europe, however, it is no longer a common disease in North America and part of Western Europe [2]. GC

can be separated into two types according to the locus, gastric adenocarcinomas and gastro-esophageal-junction adenocarcinomas [3]. Gastric adenocarcinoma can also be subdivided

histologically into intestinal and diffuse types by Lauren’s classification. In 2014, The Cancer Genome Atlas (TCGA) research network has described a comprehensive molecular evaluation on

295 primary gastric adenocarcinomas. They proposed a molecular classification dividing GC into four subtypes: positive for Epstein–Barr virus (EBV) (9%), microsatellite unstable tumors

(22%), genomically stable tumors (20%), and chromosomally unstable tumors (50%) [4]. In 2015, the Asian Cancer Research Group (ACRG) proposed another molecular classification associated with

clinical outcome and defined GC as four distinct molecular subtypes: microsatellite instability (MSI), microsatellite stable with epithelial-to-mesenchymal transition features (MSS/EMT),

MSS/TP53 mutant (MSS/TP53+), and MSS/TP53 wild type (MSS/TP53–) [5]. Identification of these subtypes sheds new lights on the prognosis and clinical treatment [6]. More than 15% of the tumor

cases were attributed to infectious pathogens. The proportion was even higher in less developed countries or regions (22.9%) [7]. The infectious pathogens include viruses, bacteria, and

parasites. Among the pathogens, _Helicobacter pylori_ (_H. pylori_), human papillomavirus, hepatitis B virus (HBV), and hepatitis C virus together attributed to 2 million new cancer cases

worldwide in 2012. They induced the tumorigenicity of the stomach, liver, and cervix. Of note, HBV and _H. pylori_ have most vicious contributions to the tumor burden in China [8]. _H.

pylori_ and EBV are the most well-known pathogens in gastric carcinogenesis. _H. pylori_ is an important risk factor found in 65–80% of primary GCs, while EBV leads to 10% of the GC cases.

Besides, it has been reported other microorganisms are also associated with gastric malignancies. Accompanied with the development of strategies for manipulating infectious agents,

opportunities are emerging to prevent and cure the infection-related cancers. Here, we comprehensively reviewed the role of microbiome in promoting gastric carcinogenesis. BACTERIOME IN

GASTRIC CARCINOGENESIS Because of the acid production, stomach was thought as a sterile organ previously. However, in recent years, culture independent methods have been developed to

facilitate the identification of various bacteria species in human stomach. It is believed that apart from the predominant bacteria _H. pylori_, multiple kinds of bacteria were coexisting in

human stomach, although little is known about their associations with GC progression. INFECTION OF _H. PYLORI_ _H. pylori_ infection is the most popular chronical bacterial infection

worldwide. More than 50% of the world population are infected with _H. pylori_, however, over 80% of infections are asymptomatic [9]. The transmission of _H. pylori_ is implicated with

fecal/oral, oral/oral, or gastric/oral pathways [10]. Part of the infections develop coexisting gastritis for several years, and the persistent infection might develop into gastric atrophy

followed by intestinal metaplasia, dysplasia, and eventually adenocarcinoma [6]. World Health Organization designates _H. pylori_ as a class I carcinogen because of its chronic infection as

the strongest risk factor for gastric adenocarcinoma. It was estimated that 90% of all noncardia GCs are associated with _H. pylori_ [11]. A study with 1526 Japanese population found the

increasing risk of GC development in patients infected with _H. pylori_ compared with the uninfected ones [12]. The eradication of _H. pylori_ significantly decreased the occurrence of GC,

suggesting that _H. pylori_ might influence early stages in gastric carcinogenesis [13]. MOLECULAR PATHOGENESIS OF _H. PYLORI_-RELATED GC Environmental factors have long been considered to

play dispensable roles in GC. High salt intake was found significantly associated with GC especially in the context of _H. pylori_ infection and atrophic gastritis [14]. It was also believed

that the risk of GC increased in the subjects with both smoking habit and _H. pylori_ infection [15]. It has been puzzling about _H. pylori_ infection, although half of the population

infected with _H. pylori_ worldwide, only a minority of colonized individuals (1–2%) develops tumors. The low morbidity indicates the impact of different strains in tumor initiation and

development. Different strains of _H. pylori_ play diverse roles in driving GC. _H. pylori_ can be subdivided into bacterial oncoprotein cytotoxin-associated gene A (CagA) positive and CagA

negative strains. In a meta-analysis, patients infected with CagA positive strains demonstrate a higher risk of GC [16], which was consistent with previous reports that individuals with CagA

antibodies have a higher risk of tumor [17,18,19,20]. Transgenic mice bearing CagA appears gastric neoplasms development, confirming that CagA is a bacteric oncoprotein [21]. However, the

mechanism seems particularly complex. _H. pylori_ injects CagA into the host gastric epithelial cells with the activation of integrin [22]. Moreover, CagA undergoes tyrosine phosphorylation

by Src family kinases or Abl kinase and subsequently activates multiple signaling pathways. For instance, phosphorylated CagA interacts with activated SHP2. CagA–SHP2 potentiates the

magnitude of Erk-MAP kinase signaling in both Ras-dependent and Ras-independent manners [23]. CagA–SHP2 also dephosphorylates focal adhesion kinase (FAK) and mediates cell–extracellular

matrix interaction. Both signaling lead to a cellular morphological change, which is called hummingbird phenotype, thus to increase the cell migration abilities [24]. In addition,

nonphosphorylated CagA impairs intracellular signaling networks. The nonphosphorylated intracellular CagA interacts with E-cadherin to disrupt the E-cadherin–β-catenin complex. It thus

induces nuclear β-catenin accumulation, allowing transcription of the target genes associated with carcinogenesis. Meanwhile, CagA was reported to directly activate β-catenin by interacting

with MET and activating PI3K–AKT signaling [25, 26]. CagA activates the signal transducer and activator of transcription 3 (STAT3) pathway. The activated STAT3 pathway is driven by the host

immune response and is associated with _H. pylori_-induced gastritis and cancer progression, independent of CagA phosphorylation [27,28,29]. In a recent study, CagA also binds to 25 known

factors in the host cells to hijack various signaling pathways related to inflammation, proliferation, genetic instability, cell polarity, and apoptosis [30]. Apart from CagA, the Cag

secretion system also delivers _H. pylori_ peptidoglycan into the host cells through outer membrane vesicles. The peptidoglycan subsequently activates PI3K–AKT and regulates cell migration,

proliferation, and apoptosis [31]. Apart from Cag, vacuolating toxin A (VacA) is another major virulence determinant of _H. pylori_. _H. pylori_ gene _vacA_ encodes the secreted protein

VacA. VacA has been reported to link to multiple cellular processes, such as vacuolation, membrane-channel formation, apoptosis, proinflammatory response, and malignancy [32]. Although all

of the _H. pylori_ strains contain _vacA_, there is variation in the _vacA_ structure. Among them, s1m1i1d1 type strains are strongly associated with gastric adenocarcinoma. Nakayama et al.

reported that VacA activates β-catenin through PI3K-dependent manner [33]. Approximately, 4% of the _H. pylori_ genome encodes integral outer membrane proteins (OMPs) [34, 35], which are

subdivided as 5 families [36]. Some of them functioned as adherence factors, such as sialic acid-binding adhesin, blood-group-antigen-binding adhesin, adherence-associated lipoprotein A and

B, outer inflammatory protein A (OipA), and _Helicobacter_ OMP Q. Most of them are linked with poor clinical outcomes. OipA was identified as a proinflammatory response inducing protein and

knockout of this gene can reduce interleukin (IL)-8 production [37]. In patient samples, it was confirmed that OipA was significantly associated with gastric inflammation and IL-8 levels

[38]. Basically, OipA is involved in the attachment of _H. pylori_ to gastric epithelial cells, which is important for the initiation and development of GC. In addition, inactivation of OipA

decreases the incidence of carcinoma by attenuating β-catenin nuclear translocation [39]. The aberrant host genetic changes are also crucial for the interaction of _H. pylori_ and gastric

epithelium cells. Polymorphisms in IL-1β and its endogenous receptor antagonist affect gastric mucosal IL-1β production in response to infection of _H. pylori_ and are associated with GC

occurrence [40,41,42]. In addition, the combination of HLA class II and IL-10–592A/C polymorphisms affect the susceptibility to GC development in _H. pylori_-infected Japanese individuals

[43]. The causal relationship between inflammation and cancer has been well recognized. An individual infected with _H. pylori_ has a bigger chance to develop chronic inflammation. _H.

pylori_ utilizes virulence factors CagA, VacA, and peptidoglycan to upregulate proinflammatory cytokines such as IL-1, IL-6, IL-8, TNF-α, and NF-κB, to activate NF-κB signaling cascade in

gastric epithelial cells and circulating immune cells [44]. The production of cytokine triggers activation and migration of leukocytes, and regulation cascade of cytokines, chemokine, and

adhesions. Granulocyte-macrophage colony-stimulating factor, a growth factor facilitating white cell differentiation, was found in _H. pylori_-infected antral biopsies and human gastric

epithelial cells [45]. Besides, inflammation modulators cyclooxygenase-2, which convers arachidonic acid to prostaglandins to induce inflammatory reactions, was significantly higher in _H.

pylori_-infected gastric epithelia cells [46]. Apart from the cytokine release, lipopolysaccharide (LPS), VacA, and _H. pylori_ neutrophil activating protein contribute to induce reactive

oxygen species (ROS) or reactive nitrogen species (RNS) in gastric epithelial cells and inflammatory cells. The generation of intracellular ROS and RNS are found relating to the pathogenesis

of _H. pylori_-associated GC. In addition, _H. pylori_-induced chronic inflammation leads to aberrant DNA methylation, which is the major cause of _H. pylori_-associated GC. On the other

hand, when _H. pylori_-induced inflammation was suppressed by cyclosporine A in animal model, induction of aberrant DNA methylation was also suppressed [47, 48]. Methylation on

tumor-suppressor genes can inactivate the gene expression and promotes cancer development. For example, promoter methylation in E-cadherin, an epithelial marker, has been detected in _H.

pylori_-infected stomach [49]. Regarding to these studies, _H. pylori_-induced chronic inflammation is essential for both initiation and the development of GC. The potential molecular

network of _H. pylori_ and oncogenic signaling pathways in gastric carcinogenesis are summarized in Fig. 1. HOST IMMUNITY IN _H. PYLORI_-RELATED GC The host immune system is the formidable

barrier to prevent _H. pylori_ infection. The immune system includes innate immune response and adaptive immune response. Innate immune response is the first-line defense. Epithelial cells,

dendritic cells, monocytes, macrophages, and neutrophils could play important roles in defending _H. pylori_ infection. Pathogen-associated molecular patterns of _H. pylori_, such as,

peptidoglycan, LPS, lipoproteins, and flagellins are recognized by pattern recognition receptors (PRRs). Toll-like receptors, C-type lectin receptors, NOD-like receptors, and RIG-like

receptors are members of the PRR family. The engagement of PRR then triggers the activation of multiple signaling cascades that culminate in NF-κB activation and immune effectors production.

Such an immune response could induce a chronic inflammation, which has been shown closely associated with molecular pathogenesis of _H. pylori_-related GC. However, in adaptive immune

response, _H. pylori_ can be recognized and presented by antigen-presenting cells (APCs), such as dendritic cell [50], neutrophil, macrophage, and epithelial cells [51]. The APCs produce

cytokines to stimulate naive CD4+ T cells and induce antigen-specific responses in Th1 cells [52, 53] and Th17 cells [54,55,56]. The Th1 cells and Th17 cells are critical for the control of

_H. pylori_ infection, however they are also associated with increased gastritis as well as GC [54, 57,58,59,60]. At the same times, the T regulatory (Treg) cell response is also observed,

which drives immune tolerance and suppresses Th1- and Th17-mediated immunity against _H. pylori_ infection [61, 62]. It has been reported that B cells and antibodies are not required for

clearing the _H. pylori_, rather, they might be detrimental to elimination of the bacteria [63]. DIAGNOSIS AND TREATMENT OF _H. PYLORI_ _H. pylori_ should be tested in patients with

dyspepsia if the local _H. pylori_ prevalence exceeds 10%. The testing can be performed by noninvasive and invasive methods. The noninvasive methods include the urea breath tests and fecal

antigen test. Serologic test and invasive testing strategies require upper endoscopy, biopsy urease (campylobacter-like organism) test, histologic assessment, and culture [64]. The

eradication of _H. pylori_ dramatically decreases the presence of premalignant lesions and reduce the GC risk in infected individuals. Anti-_H. pylori_ therapy is an effective means for GC

prevention and there are various proposed treatment regimens for _H. pylori_ eradication [65]. Traditional treatment regimens include standard triple therapy (PPI, amoxicillin, and

clarithromycin), bismuth quadruple PBMT therapy (PPI, bismuth, metronidazole, and tetracycline), or a treatment including PPI, clarithromycin, and metronidazole. However, with increasing

clarithromycin resistance, another regimen concomitant nonbismuth therapy PAMC (PPI, amoxicillin, metronidazole, and clarithromycin) was proposed. The first-line treatment was recommended

with a 14-day course of either concomitant PAMC therapy or bismuth quadruple PBMT therapy, according to the 2016 Toronto Consensus guidelines [66]. The 2016 Maastricht V/Florence Consensus

Report recommends first-line treatment with a 14-day course of bismuth quadruple PBMT therapy or concomitant PAMC therapy in high clarithromycin resistance areas (>15% resistance). A

standard triple therapy or bismuth quadruple PBMT therapy in low clarithromycin resistance (<15% resistance) areas is also proposed by this report [67]. OTHER BACTERIA IN GC In 2006, Bik

et al. used a small subunit 16S rDNA clone library approach identified 128 phylotypes belonging to five phyla (Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Fusobacteria) in

23 human gastric biopsies [68]. Lately, 133 phylotypes were identified by Li et al. and 59 families were detected by Delgado et al. [69, 70], which were quite similar from both phyla and

genera level. It reflects the significance of the bacterial homeostasis in stomach. Loss of bacterial homeostasis might be a reason in driving GC progression. Coker et al. reported that

microbial composition was changed, and bacterial interactions were different across stages of gastric carcinogenesis, indicating the presence of microbial dysbiosis in gastric

carcinogenesis. They also found potential roles of some microbial such as _Peptostreptococcus stomatis_, _Dialister pneumosintes_, _Slackia_ exigua, _Parvimonas micra_, and _Streptococcus

anginosus_ in GC progression [71]. It was also reported that a consistent increase of lactic acid bacteria promotes GC by a number of mechanisms such as supply of exogenous lactate,

production of ROS, and N-nitroso compounds, as well as anti-_H. pylori_ properties [72]. Notably, _H. pylori_ and other bacteria might affect each other in the stomach, but the causality has

not yet been clearly explained. As currently known, bacteria colonies in the stomach could affect the outcome of _H. pylori_ infection and the progression of GC. On the other side, _H.

pylori_ infection may influence the density of bacteria. In animal model, long-term _H. pylori_ infection affects the bacterial composition of the gastric microbiota. Maldonado-Contreras et

al. reported a higher abundance of Proteobacteria, Spirochetes, and Acidobacteria, and a decreased abundance of Actinobacteria, Bacteroidetes, and Firmicutes in _H. pylori_-positive patients

compared with _H. pylori_-negative subjects [73]. A microbial diversity analysis showed that compared with negative subjects, both of the species and Shannon index were increased in

subjects with past or current _H. pylori_-infected subjects, indicating the alterations of fecal microbiota, especially Bacteroidetes, Firmicutes, and Proteobacteria, may be involved in the

process of _H. pylori_-related gastric lesion progression [74]. However, some reports indicated that chronic _H. pylori_ infection does not alter the microbiota of stomach [68, 71, 75, 76],

suggesting the relationship between _H. pylori_ infection and the gastric microbiota dysbiosis is still controversial [77, 78]. EBV IN GASTRIC CARCINOGENESIS The mammalian virome is

constituted of viruses that infect host cells, virus-derived elements in human chromosomes, and viruses that infect the broad array of other types of organisms [79]. It was reported that

EBV, CMV, and HHV6 can be detected in gastric tumors [80]. Among them, EBV is the most prominent one. THE STRUCTURE OF EBV More than 90% of adults have been infected by EBV [81], and it is

asymptomatic in the majority of carriers. However, some of the infections can cause infectious mononucleosis. EBV is classified as a group I carcinogen by the International Agency for

Research on Cancer, since the latently infection estimated to be responsible for 200,000 cancers cases worldwide [82], such as Burkitt lymphoma, hemophagocytic lymphohistiocytosis, Hodgkin’s

lymphoma, GC, and nasopharyngeal carcinoma (NPC). Until now, approved vaccines for EBV have not been available. However, a vaccine targeting the EBV glycoprotein gp350 has been developed to

reduce the incidence of infectious mononucleosis and the efficacy has been proved [83]. EBV belongs to Herpesviridae containing an ~172 kb liner form dsDNA genome. The expression products

cover 80 proteins and 46 functional small-untranslated RNAs. EBV prefers to infect B cell and epithelial cells. After entry, like all kind of herpesviruses, EBV has two distinct life cycles:

lytic replication and latency. However, upon EBV de novo infection, it takes latency infection firstly. During latency, viral genomes exist as extrachromosomal episomes in the nucleus and

only express some latent proteins (EBV-determined nuclear antigen 1 (EBNA1), 2, 3A, 3B, 3C, and EBNA-LP; latent membrane protein 1 (LMP1) and LMP2), noncoding RNA (EBER1 and EBER2), and

viral miRNAs (BHRF1-miRNA and BART-miRNA) (Fig. 2a). EBV latency is categorized by three latency types (latency I–III), which have different latency protein expression patterns depending on

the type of cell infected. Several different kinds of latency were shown schematically (Fig. 2b). The lytic infection is triggered by several factors from the latent state. Then, nearly 80

proteins are encoded to facilitate the viral particle formation and release into the extracellular space. ESTABLISHMENT OF EBV INFECTION IN STOMACH EPITHELIAL CELLS The first puzzle about

EBV-associated gastric carcinoma (EBVaGC) is how EBV infects gastric epithelial cells, as the EBV infection often occurs in B lymphocytes and the oral epithelium. It is possible that the

EBV-contained saliva is ingested and EBV infects the epithelial cells directly. Another explanation is that EBV is reactivated somehow in B lymphocytes in stomach and released to infect

epithelial cells [84]. Ephrin receptor A2 as well as integrins and nonmuscle myosin heavy chain IIA (NMHCIIA) serve as cofactors and play an important role in EBV epithelial cell entry

[85,86,87,88]. Coculturing of epithelial cells with EBV-positive lymphocyte cells showed about 800 fold higher efficiency of infection than cell-free infection, suggesting the possibility of

direct cell-to-cell mediated virus infection [89]. It was proposed that EBV-infected lymphocytes contacts with epithelial cells via integrin β1/β2, and then promotes cell-to-cell contact by

translocating intracellular adhesion molecule-1 to the cell surface. At last, the viral particle is transmitted by clathrin-mediated endocytosis pathway [90]. After endocytosis, the EBV-DNA

is transported to nucleus, where the naked linear DNA genomes are assembled into a functional circular mini-chromosome. After circulation, viral genome chromatinization can effectively

protect it from DNA damage and offer tight regulation of gene expression [91]. The CpG motifs of viral genome are widely methylated and by this way, latent infection is successfully

established. The infection and latency processes were summarized in Fig. 3. It is well known that EBV LMP1 and nuclear antigen 2 (EBNA2) play major roles in EBV-induced oncogenesis. However

both of them were rarely detected in gastric adenocarcinoma cells [92,93,94]. Instead, EBNA1 expression was confirmed [93, 95]. It was reported that transcription of EBNA was initiated from

EBNA promoters, Qp but not Cp or Wp, which may result in the absent expression of EBNA2 [96]. In addition, BZLF1 is expressed in a proportion of the tumors, suggesting the switch from latent

to lytic infection [92]. THE HISTOPATHOLOGICAL FEATURES OF EBVAGC In 1990, Burke et al. firstly detected EBV in lymphoepithelial carcinoma of the stomach, which was similar to

undifferentiated nasopharyngeal lymphoepithelioma [97]. However, Shibata subsequently found that EBV is involved not only in the rare gastric lymphoepithelioma-like cancers, but also in

gastric adenocarcinomas. They demonstrated that the EBV genomes were specifically present within the gastric carcinoma cells and adjacent dysplastic epithelium but were absent in surrounding

normal cells [98, 99]. The result was confirmed by polymerase chain reaction (PCR) and in situ hybridization (ISH) in variety of studies [92, 94, 100,101,102,103]. Since the EBV-positive

tumor cells were from a single clonal proliferation [93, 102, 104], and EBV was not generally detected in normal stromal cells, metaplasia, gastric mucosa, and lymphocytes [93, 99, 103,

104], EBV infection was believed to occur in the dysplastic phase and related to gastric carcinogenesis. In gastric carcinoma with lymphoid stroma, all cases are EBV-positive tumors.

However, in gastric adenocarcinomas, only a small fraction of the cases shows EBV positive. It is believed that EBV plays distinct roles in etiology of these two types of GC [92, 98].

EBVaGC-associated mortality was estimated to be 70,000 worldwide each year [105]. Epidemiological studies show that male EBV-positive GC patients were twice than female [99, 106] and type 1

strain is most prevalent one in gastric carcinoma [107, 108]. EBVaGC has distinctive clinical characteristics compared with EBV-negative cases. EBVaGC often appears in the upper part of the

stomach and has a diffuse-type histology with lymphoid infiltration [109]. By analyzing individual-level data on 4599 GC patients from 13 studies, it was demonstrated that EBV positivity is

a powerful prognostic indicator of GC. In addition, the report also indicated that patients with EBV-positive GC had a better survival than EBV-negative ones [110], because of the high

degree of homogeneity in EBVaGCs compared with EBV-negative cases. Furthermore, most of the altered genes in EBVaGCs are immune response related genes leading to more immune cells to migrate

into the microenvironment, compared with EBV-negative GC. The recruitment of immune cells contributes to the better clinical outcome for EBVaGC cases [111]. Besides, CD204-positive M2-type

tumor-associated macrophages, which were associated with the aggressive behavior of tumors, exhibit low density in EBVaGCs, partly explaining the favorable outcomes [112]. Recently,

comprehensive molecular characterization of GC presents several distinct molecular features and epigenetic alterations of EBVaGC, including lack of _TP53_ mutations, frequent _PI3K_

mutations, and a high degree of CpG methylation in the tumor cell genome [113]. THE MOLECULAR PATHOGENESIS OF EBVAGC To date, the mechanism of EBVaGC has not yet been comprehensively

deciphered. In general, virologic aspects and genetic abnormalities of host cells co-potentiate the tumor development. As for virologic aspects, since EBV-positive GC is in latency type I,

only EBERs, EBNA1, and miR-BARTs are highly expressed, while LMP2A could be detected in some cases [96, 114]. Meanwhile, genetic abnormalities of host cells caused by EBV infection, such as

aberrant DNA methylation, attract more and more attention these years. The methylation of CpG DNA of the host genome is also caused by the establishment of EBV latent infection and the

expression of the EBV latent genes. PROMOTING ROLES OF VIROLOGIC GENES IN GC PATHOGENESIS EBERs are viral nonpolyadenylated RNA, which is abundantly expressed in latently EBV-infected cells.

Because of their abundance, EBERs serve as the most reliable and sensitive target by ISH to detect EBV infection in tissues. It plays a role in cell proliferation, apoptosis, and antiviral

innate immunity. However, only a few studies investigated the roles of EBERs in EBV-mediated oncogenesis. EBER1 upregulates the expression of insulin growth factor 1, which promotes

proliferation of EBVaGC cells [115]. Another work showed that EBERs induce chemoresistance and enhance cellular migration in coordination with IL-6-STAT3 signaling pathway [116]. EBERs as

well as BARF0, EBNA1, and LMP2A contribute to the downregulation of miR-200 family, resulting in E-cadherin expression reduction, which is a crucial step in the carcinogenesis of EBVaGC

[117]. EBNA1 is an essential molecule for EBV latency infection. It binds to viral oriP sequence in a sequence dependent manner and tethers EBV episomes onto host cell chromosomes, which is

essential for episomal maintenance. EBNA1 also functions as a transactivator of the viral genes. In EBVaGC, EBNA1 enhances tumorigenicity in mouse model [118]. It was also reported to cause

loss of promyelocitic leukemia (PML) nuclear bodies (NBs), resulting in impaired responses to DNA damage and promotion of cell survival [119]. In addition, EBNA1 induces ROS accumulation

mediated by miR-34a and NOX2 to regulate the tumor cell viability [120]. LMP2A was detected in half of the EBVaGC cases [121]. Fukayama et al. found that LMP2A activates the NF-κB-survivin

pathway to rescue EBV-infected epithelial cells from serum deprivation, which may play a role in the progression of EBV-infected GC [122]. By using a recombinant adenoviral expression

vector, Liu et al. found that LMP2A plays an important role in pathogenesis of EBVaGC through regulating cyclin E expression and S phase cell ratio [123]. Besides, LMP2A mediates Notch

signaling to elevate mitochondrial fission and promote cellular migration [124]. In addition, LMP2A could also downregulate HLA to evade the immune response of the malignant cells [125]. It

can activate PI3K/Akt pathway to mediate the transformation process and inhibit TGFβ1-induced apoptosis, which provides a clonal selective advantage for EBV-infected cells during tumor

development [126, 127]. LMP2A upregulates miR-155–5p though NF-κB pathway and this will lead to the inhibition of Smad2 and p-Smad2 [128]. Apart from the direct modulating effects on

tumorigenesis, LMP2A also promotes malignancy by inducing epigenetic modifications of the host genome [129]. Recent studies imply that miR-BARTs contribute to EBV-associated epithelial

carcinogenesis. The miR-BARTs are abundantly expressed in EBV-infected GCs cell line, but not in EBV-transformed lymphocytes [4, 130]. By using EBV-infected AGS cell line (AGS-EBV), the

expression of miR-BARTs was quite rich but the expression of the viral protein was limited [131, 132]. EBV miRNAs contribute to the initiation and development of EBVaGC by targeting multiple

host proteins to mediate cell proliferation, transformation, senescence, apoptosis, and immune response. A comprehensive profiling of EBV miRNAs in EBVaGC was constructed by Tsai et al. and

they found the deletion of miR-BART9 could increase E-cadherin expression and decrease proliferative and invasive ability [133]. BART3–3p plays an important role in inhibiting the

senescence of GC cells by targeting _TP53_ [134]. As for apoptosis, it was reported that BART5–3p directly targets _TP53_, leading to acceleration of the cell cycle progress and inhibition

of cell apoptosis [135]. Besides, EBV encoded miR-BART5 could target p53 upregulated modulator of apoptosis (PUMA), which is a proapoptotic protein belonging to the Bcl-2 family, to

counteract apoptosis and promote cellular survival [136]. In addition to PUMA, it was reported that miR-BART9, 11, and 12 strongly downregulate Bim, which is also a member of Bcl-2 family

[137]. By comprehensively profiling the expression of EBV miRNAs in EBVaGC tissues, EBV-miR-BART4–5p was found to play a role in gastric carcinogenesis through apoptosis regulation by

suppressing the proapoptotic protein Bid (the BH3-interacting domain death agonist) [138]. MiR-BART20–5p contributes to tumorigenesis of EBVaGC by directly interacting with 3′UTR of BAD

[139]. Different from proteins, EBV-microRNAs could escape immune recognition as well as inhibit the immune response by directly suppressing the function of some antiviral host factors to

facilitate the establishment of latent EBV infection. For example, EBV miRNA BART16 have been reported to suppress type I IFN signaling [140]. The oncogenic proteins and miR-BARTs in EBVaGC

were summarized in Table 1. GENETIC AND EPIGENETIC ABNORMALITIES OF HOST CELLS IN EBVAGC Multiple abnormalities of the EBVaGC cells have been identified. Among them, high frequency and

nonrandom DNA methylation attract most attentions [141, 142]. However, the mechanisms are not fully elucidated yet. LMP2A was confirmed to mediate this process. LMP2A induces the STAT3

phosphorylation followed by DNMT1 transcriptionally activation and _PTEN_ promoter methylation, indicating LMP2A plays an essential role in the development and maintenance of EBV-associated

cancer [143]. Besides, a resistance factor against DNA methylation namely TET2 was suppressed to contribute to DNA methylation acquisition during EBV infection [144]. Variety of

tumor-suppressor genes have been identified to be methylated during EBV infection, such as _p16_, _p14_, _APC_, _SSTR1_, _FHIT_, _CRBP1_, _WWOX_, _DLC-1_, _AQP3_, _REC8_, _TP73_, _BLU_,

_FSD1_, _BCL7A_, _MARK1_, _SCRN1_, and _NKX3.1_ [129, 145,146,147,148,149,150,151]. The developed high-throughput sequencing makes it possible to reveal the EBV-induced DNA hypermethylation

comprehensively. Using methyl-DNA immunoprecipitation microarray assays, Zhao et al. found 886 genes involved in cancer-related pathways were aberrantly promoter-hypermethylated in

EBV-positive AGS cells [152]. They also employed whole-genome, transcriptome, and epigenome sequence analyses of EBV-infected or noninfected AGS cells together with primary samples to

comprehensively reveal that EBV infection alters host gene expression through methylation and affects five prominent networks [153]. Apart from the methylation of host cells, EBV could

promote vasculogenic mimicry formation, a new tumor vascular paradigm independent of endothelial cells, in NPC and GC cells through the PI3K/AKT/mTOR/HIF-1α axis [154]. EBV infects ~95% of

people, however only part of the population develops tumors, indicating that molecular abnormalities of host cells are also equally important in the EBV-associated tumorigenesis. As for

EBVaGC, high-frequency mutations of _PIK3CA_, _ARID1A_, and _BCOR_ have been identified. Interestingly, _TP53_ mutation, which counts the most frequent mutation type in cancers, is extremely

rare [113]. The amplification of _JAK2_, _PD-L1_, and _PD-L2_ were also revealed as prominent molecular features [155, 156]. HOST IMMUNITY IN EBV-POSITIVE GC By using gene expression

profile analysis, it was found that the prominent changes in EBVaGCs are immune response genes, which might allow EBVaGC to recruit reactive immune cells [111]. In fact, EBVaGC is

characterized with the high density of CD8+ T cells and low density of CD204+ macrophages [112, 157, 158]. The robust present of infiltrating immune cells and specific microenvironments

partially contribute to antitumor immunity [159]. However, the tumor cells in EBVaGC evade the immune response through multiple strategies. It was reported that indoleamine 2,3-dioxygenase

(IDO1), a potent immune-inhibitory molecule, was upregulated in EBVaGC to resistance tumor immune response [130, 160]. In addition, Tregs were recruited by CCL22 produced by EBVaGC cells to

counteract the antitumor response of CD8+ T cells [161]. EBVaGC also exhibits higher levels of programmed death ligand 1 (PD-L1) expression in carcinoma cells and the infiltrated immune

cells [162, 163]. As tumor cells employ PD-L1 to evade antitumor immunity through interaction with programmed cell death protein 1 on the surface of T cells, the high expression of PD-L1 in

EBVaGC is thought to contribute to the tumor progression [164]. THE DIAGNOSIS AND TREATMENT OF EBVAGC By measuring immune-related proteins in plasma of patients with EBV-positive tumors and

EBV-negative tumors, Camargo et al. found some chemokines and PD-L1 in plasma that could be used for the diagnosis of EBV status [165]. The plasma EBV-DNA load in EBVaGC patients decreases

when the patients show response to the treatment, while load increases when the disease progresses, suggesting that plasma EBV-DNA serves as an ideal marker in predicting recurrence and

chemotherapy response [166]. EBVaGC, MSI-high GC, intestinal type GC as a surrogate for chromosomal instability, diffuse type as a surrogate for genomically stable was classified as four

different subtypes of GC proposed by TCGA [113]. The molecular subtypes of GC are also correlated with the immune subtype [167, 168], suggesting the TCGA classification could be further

employed in future immunotherapy trials. The ACRG classification also revealed four molecular subtypes with clinical outcome. MSI subtype has the best prognosis and lowest recurrence rate

followed by MSS/TP53+ and MSS/TP53−, while the MSS/EMT subtype demonstrates the worst prognosis and highest recurrence rate among the four subtypes. In ACRG classification, EBVaGCs are more

frequently found in the MSS/TP53+ group than in the other groups, indicating a modest survival and recurrence [5]. Patients with EBV-positive tumors showed high responses to pembrolizumab

treatment in a phase II trial of metastatic GC [169]. The satisfied response might rely on that EBVaGC expresses high levels of PD-L1 [165, 170] and exhibits more tumor infiltrating

lymphocytes (TILs) [163, 167, 171, 172]. The amount of TILs has been reported to be associated with improved overall survival in GC patients [173]. In a research of advanced GC patients

treated with nivolumab, only 25% of patients (1/4) demonstrated good response, and this might be because not all EBV-positive tumors show high PD-L1 expression [174]. Evaluating both EBV

status and PD-L1 expression is necessary for predicting clinical benefit of anti-PD-L1 therapy [175]. To some extent, the result indicates that EBV is a potential biomarker for selecting

patients with better response to PD-L1 treatment [176]. In addition to PD-L1, Kim et al. combined PI3K/mTOR dual inhibitor CMG002, together with the autophagy inhibitor CQ, to provide

enhanced therapeutic efficacy against EBVaGC [177]. FUNGUS IN GASTRIC CARCINOGENESIS Fungus is a kind of eukaryotic microorganism, which is widely distributed worldwide. It was identified

that more than 400 species of fungus associated with human beings. These years, the incidence of invasive fungal infections has experienced a dramatic increase globally. Fungus is detectable

in the digestive tract of about 70% of healthy adults in an analysis by using culture dependent methods. Most of them belong to Candida genus, and the number of fungus in the human stomach

is 0–102 CFU/mL [178, 179]. Another research using PCR amplification of bacterial 16S ribosomal RNA genes and fungal internal transcribed spacers identified two fungal genera, _Candida_ and

_Phialemonium_, in gastric fluid from 25 clinically patients [180]. Generally, host immune system could tolerate fungus colonization and defend its invasion. However, the infection will

occur when the balance is disturbed by systemic immunosuppressive such as the acquired immune deficiency syndrome, leukemia and HSCT, solid organ transplantation and immunosuppressant

therapy, anti-microbial and steroid treatments, total parenteral nutrition, iatrogenic catheters and mechanical ventilation, malignant tumors, chemoradiotherapy, and diabetes mellitus [181,

182]. Besides, GI mucosal lesions and surgical procedures can also lead to GI fungal infection [181]. In a gastro-esophageal candidiasis detection by histological examination of biopsies

from 465 patients, it was thought that the candidiasis is usually secondary to mucosal damage [183]. Candidiasis was detected in 54.2% of the gastric ulcer cases and 10.3% of the chronic

gastritis cases. As for GC, the candidiasis was present in 20% of patients [179, 183]. Although the infection of fungal microorganisms in GC is only in rare cases, it is necessary to

eliminate opportunistic infection of Candida to reduce the significant morbidity and mortality. FUTURE DIRECTIONS Although EBV-related and _H. polyri_-related GCs are classified into

different categories, it should be reminded that the stomach is an organ with multiple microorganism coexistence, which means that disease is promoted by multiple microorganisms. In fact,

apart from direct promoting gastric carcinogenesis, _H. pylori_ potentiates the transformation of the gastric mucosa into a hypochlorhidric environment, which further allow other microbes to

colonize. In addition, coinfection with EBV and _H. pylori_ in pediatric patients are associated with more severe inflammation than those with _H. pylori_ infection alone [184]. Although

the underlying mechanism has been partially suggested, such as host SHP1 phosphatase, antagonist of CagA, is downregulated by EBV-induced promoter hypermethylation [185], the synergistic

oncogenic effects of two or more infectious agents remain to be further explored in the future studies. In recent years, the researches about the microbiota in gastrointestinal attract more

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downregulated by Epstein-Barr virus. Nat Microbiol. 2016;1:16026. CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We acknowledge the technical support from Core Utilities

of Cancer Genomics and Pathobiology of Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong. FUNDING The current manuscript is supported by Research Grants

Council of the Hong Kong Special Administrative Region, China [Project nos.: CUHK 14100019, 14118518 (for GRF projects)], and CUHK Direct Grant for Research (2018.002) from The Chinese

University of Hong Kong. AUTHOR INFORMATION Author notes * These authors contributed equally: Yanan Zhao, Jinglin Zhang AUTHORS AND AFFILIATIONS * Department of Anatomical and Cellular

Pathology, State Key Laboratory of Translational Oncology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong SAR, PR China Yanan Zhao, Jinglin Zhang, Ka Fai To & 

Wei Kang * Institute of Digestive Disease, State Key Laboratory of Digestive Disease, The Chinese University of Hong Kong, Hong Kong SAR, PR China Yanan Zhao, Jinglin Zhang, Jun Yu, Ka Fai

To & Wei Kang * Li Ka Shing Institute of Health Science, Sir Y.K. Pao Cancer Center, The Chinese University of Hong Kong, Hong Kong SAR, PR China Yanan Zhao, Jinglin Zhang, Ka Fai To 

& Wei Kang * School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong SAR, PR China Alfred S. L. Cheng * Department of Medicine and Therapeutics, The Chinese

University of Hong Kong, Hong Kong SAR, PR China Jun Yu Authors * Yanan Zhao View author publications You can also search for this author inPubMed Google Scholar * Jinglin Zhang View author

publications You can also search for this author inPubMed Google Scholar * Alfred S. L. Cheng View author publications You can also search for this author inPubMed Google Scholar * Jun Yu

View author publications You can also search for this author inPubMed Google Scholar * Ka Fai To View author publications You can also search for this author inPubMed Google Scholar * Wei

Kang View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS WK and KFT provided direction and instruction in preparing this manuscript. YZ and JZ

reviewed the literatures and drafted this manuscript. ASLC and JY reviewed the manuscript and made significant revisions on the drafts. CORRESPONDING AUTHORS Correspondence to Ka Fai To or

Wei Kang. ETHICS DECLARATIONS CONFLICT OF INTEREST The authors declare that they have no conflict of interest. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with

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http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Zhao, Y., Zhang, J., Cheng, A.S.L. _et al._ Gastric cancer: genome damaged by bugs.

_Oncogene_ 39, 3427–3442 (2020). https://doi.org/10.1038/s41388-020-1241-4 Download citation * Received: 23 December 2019 * Revised: 18 February 2020 * Accepted: 20 February 2020 *

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