molecular pathways: involvement of helicobacter pylori...

Molecular Pathways

Molecular Pathways: Involvement of HelicobacterpyloriTriggered Inflammation in the Formation of anEpigenetic Field Defect, and Its Usefulness as CancerRisk and Exposure Markers

Toshikazu Ushijima and Naoko Hattori

AbstractInfection-associated cancers account for a large proportion of human cancers, and gastric cancer, the vast

majority of which is associated with Helicobacter pylori infection, is a typical example of such cancers.

Epigenetic alterations are known to occur frequently in gastric cancers, andH. pylori infection has now been

shown to induce aberrant DNA methylation in gastric mucosae. Accumulation of aberrant methylation in

gastric mucosae produces a field for cancerization, and methylation levels correlate with gastric cancer risk.

H. pylori infection inducesmethylation of specific genes, and such specificity is determined by the epigenetic

status in normal cells, including the presence of H3K27me3 and RNA polymerase II (active or stalled).

Specific types of inflammation, such as that induced by H. pylori infection, are important for methylation

induction, and infiltration ofmonocytes appears to be involved. The presence of an epigenetic field defect is

not limited to gastric cancers and is observed in various types of cancers. It provides translational

opportunities for cancer risk diagnosis incorporating life history, assessment of past exposure to carcino-

genic factors, and cancer prevention. Clin Cancer Res; 18(4); 9239. 2011 AACR.

Background

Infection-associated cancers account for a large propor-tion of human cancers. These include gastric cancersinduced by Helicobacter pylori (1), hepatocellular carcino-mas induced by hepatitis C virus (HCV) and hepatitis Bvirus [HBV (24)], cervical cancers induced by humanpapilloma virus [HPV (5, 6)], and lymphomas and naso-pharyngeal cancers associated with Epstein-Barr virus [EBV(7, 8)]. The carcinogenic mechanisms of these infection-associated cancers have been extensively investigated, andalthough multiple contributing mechanisms have beenclarified, they are not yet completely understood.

General mechanisms of infection-associated cancersVirus-associated cancers have complex mechanisms of car-

cinogenesis. Viral oncogenes, such as E6 and E7 of HPV and Xprotein of HBV, can be integrated into host cells and produceaberrant growth signals and inactivate tumor-suppressorgenes (6).Also, integrationof virusgenes into thehost genomecan alter the expression of nearby tumor-related genes andinduce a genomic instability that will eventually contribute to

cancer development (4). Even if the virus genes are not inte-grated, they can be persistently expressed and perturb impor-tant cellular signaling, suchas cell proliferation, apoptosis, andcytokine expression, as in the case of HCV and EBV (7).

Both bacterial and viral infections can be associated withsevere tissue damage and resultant chronic inflammation(4, 6, 7). Tissue damage itself activates cell proliferation andincreases the chance that mutations will occur. In addition,chronic inflammation is considered to be deeply involvedin cancer development and progression by multiplemechanisms, such as increased production of active oxygenspecies, induction of inflammation-mediated cell prolifer-ation, and increased cytokine production (9, 10). In addi-tion to this, induction of epigenetic alterations is nowrecognized as one of the mechanisms underlying inductionof cancer by chronic inflammation.

H. pylori infection and epigenetic alterations in gastriccancers

Gastric cancer is still the third-leading cause of death fromcancer in men and the fifth-leading cause in women world-wide, although its incidence is gradually decreasing (11).The vast majority of gastric cancers are caused by H. pyloriinfection (12), which is a Gram-negative bacterium (13). Aminor percentage (10%) of gastric cancers are associatedwith EBV infection (14). It is known that when H. pyloriinfects the human stomach, it induces severe inflammation,including ulcers, then chronic inflammation, and finallygastric cancers within tens of years. Investigators havemain-ly discussed the carcinogenic mechanisms of H. pylori from

Authors' Affiliation: Division of Epigenomics, National Cancer CenterResearch Institute, Tokyo, Japan

Corresponding Author: Toshikazu Ushijima, National Cancer CenterResearch Institute, 5-1-1-Tsukiji, Chuo-ku, Tokyo, Japan 104-0045. Phone:81-3-3542-2511; Fax: 81-3-5565-1753; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-2011

2011 American Association for Cancer Research.

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the standpoint of induction of cell proliferation,mutations,and direct activation of cellular signaling (1517).

However, tumor-suppressor genes such as CDKN2A,CDH1,MLH1, and RUNX3 are inactivated more frequentlyby aberrant DNA methylation than by mutations, indicat-ing that gastric cancer is an epigenetic disease (18). Inaddition to methylation silencing of driver tumor-suppres-sor genes, recent genome-wide analyses have revealed thathundreds of passenger genes are also methylated in gastriccancers (19). The fact that H. pylori infection induces epi-genetic alterations provides the missing link between thecausal role ofH.pylori infection ingastric carcinogenesis andthe deep involvement of epigenetic alterations in gastriccancers. Gastric cancer is a typical example of a disease inwhich infection, chronic inflammation, and epigeneticalterations are interconnected.

Induction of epigenetic alterations byH. pylori and theformation of field defects

The first hint that the presence of aberrant DNA methyl-ationmight be associated withH. pylori infection came fromthe observation that promoter methylation of CDH1 wasdetected more frequently in the gastric mucosae of indivi-duals with H. pylori infection than in those without theinfection (20). By quantifying the methylation levels of 8marker CpG islands, Maekita and colleagues (21) convinc-ingly showed that individuals with H. pylori infection havemuchhighermethylation levels in their gastricmucosae (5.4-to 303-fold) than those without (P < 0.0001). In addition tothe 8 marker CpG islands associated with protein-codinggenes,CpG islandsofmicroRNAgenes are alsomethylated inassociation with H. pylori infection (22, 23).

In one study, patients with gastric cancer who had pre-viously had an H. pylori infection but were currently notinfected had lower methylation levels of the 8 marker CpGislands in the gastric mucosae compared with patients whowere currently infectedwithH. pylori (21). This suggests thatthe methylation level is very high when active H. pyloriinfection is present in the stomach and decreases to certainlevels when the infection is discontinued. In other studies,various degrees of decrease were observed in individualswho received eradication therapy forH. pylori (24, 25), andthe methylation level after the decrease was considered torepresent the degree of epigenomic damage to the individ-ual. This decrease ofmethylation could be due to a turnoverof gastric epithelial cells withmethylation or to the removalof 5-methylcytosine, which is present in individuals withactive H. pylori infection.

Of importance, among individuals without current H.pylori infection, the methylation levels of the 8 marker CpGislands in gastric mucosae were shown to correlate withgastric cancer risk (21, 26). Patients with gastric cancers had2.2- to 32-fold higher methylation levels in gastric mucosaecompared with healthy individuals (21), and patients withmultiple gastric cancers had significantly higher methyla-tion levels than those with a single gastric cancer (26). Thiscorrelation strongly supports the notion that the accumu-lation of aberrant methylation in gastric mucosae produces

anepigenetic field for cancerization, i.e., afielddefect (Fig. 1;ref. 27).

Epigenetic field for cancerizationIn the epigenetic field for gastric cancers, tumor-suppres-

sor genes that are causally involved in gastric cancer devel-opment (i.e., driver genes), such as CDKN2A, CDH1,MLH1, and RUNX3, are methylated only at very low levels,showing that such events are present only in a very smallfraction of cells (21). In contrast, many other genes that areunlikely to be causally involved in gastric carcinogenesis(i.e., passenger genes), such as HAND1 (a transcriptionfactor involved in heart morphogenesis), are methylatedat high levels, showing that their methylation is present in alarge fraction of cells. Most of the genes that are highlymethylated in gastric cancers are either unexpressed orexpressed only at low levels in normal cells (28). Generally,genes with low expression are susceptible to methylationinduction (29), and it is considered that most of the genesthat are methylated in the epigenetic field were methylatedas a consequence of gastric carcinogenesis. In addition toaccumulation of aberrant methylation, an epigenetic fieldinvolves hypomethylation of the Alu and Sata repeatsequences (30), which potentially can be involved in geno-mic instability.

Epigenetic field defects are present not only in gastriccancers but in other cancers as well (27). In the case ofhepatocellular carcinoma, aberrant DNA methylation wasfrequently observed in noncancerous tissues of cancerpatients compared with normal livers of patients withmetastatic liver tumors (31). A quantitative analysisrevealed increased methylation of multiple tumor-suppres-sor genes, such as SOCS1, RASSF1A, and CDH1, in HCV-infected, noncancerous liver tissues (32), suggesting theimportance of an epigenetic field for HCV-associated hepa-tocarcinogenesis. In the case of esophageal adenocarcino-ma, the presence of APC and CDKN2A methylation inBarretts metaplasia has been reported (33), and suchmeth-ylation was shown to be associated with progression ofBarretts metaplasia (34). Also in the case of esophagealsquamous cell carcinoma, methylation of specific genes,such as UCHL1 and HOXA9, in esophageal mucosae wasassociatedwith the riskof developing esophageal squamouscell carcinoma (35, 36). In ulcerative colitis, the driver geneCDKN2A and passenger genes such as MYOD and ESRwere shown to be methylated in colonic mucosae, whicharepredisposed to colon cancers (37, 38). In addition, in thecase of sporadic colorectal cancers, MGMT methylation incancer tissues was associated with high levels of MGMTmethylation in the background colonic mucosae (39). Thepresence of epigenetic field defects has also been indicatedfor breast (40), renal (41), and bladder cancers (42, 43).

Critical roles of specific types of inflammation inmethylation induction

The association between H. pylori infection and highlevels of DNA methylation in gastric mucosae in humansstrongly indicates that H. pylori infection induces aberrant

Ushijima and Hattori

Clin Cancer Res; 18(4) February 15, 2012 Clinical Cancer Research924




DNA methylation. This cause-consequence relationshipwas shown with the use of Mongolian gerbils, in whichH. pylori infection-induced gastritis and gastric cancers canbe recapitulated (44). Gerbils infected with H. pylori devel-oped severe gastritis and had markedly increased methyl-ation levels, showing the causal role ofH. pylori infection inmethylation induction (45). The methylation levels wereclearly decreased after eradication of the H. pylori, in agree-

ment with the decreased methylation levels observed inpatients who received eradication therapy.

In the attempt to determine how H. pylori induces meth-ylation, investigators have considered both direct and indi-rect actions of H. pylori. First, because H. pylori possessesmultiple DNA methyltransferases (46) and a type IV secre-tion system [a syringe-like structure capable of deliveringbacterial materials into a host cell (47)],H. pylori itself may

2011 American Association for Cancer Research

Normalepithelium

Acuteinflammation

Chronicinflammation

Epigeneticfield for

cancerization

Clinical tumorformation

Suppression ofmethylation induction

Removal of aberrantmethylation

UnmethylatedMethylated

Gene1234

H. pylori infection

Figure 1. Formation of epigenetic field for cancerization by chronic inflammation triggered byH. pylori infection.H. pylori infection induces acute inflammation,followed by chronic inflammation, formation of an epigenetic field for cancerization, and development of gastric cancers. Aberrant methylation is induced indriver genes (schematically represented by gene 1) and passenger genes (genes 3 and 4). Specific genes are methylated in gastric mucosae withH.pylori infection, anddriver genesusually have very lowmethylation levels. On theother hand, passenger genes that have lowor noexpression in normal cellsusually have high methylation levels. The methylation level of some passenger genes reflects the degree of accumulation of epigenomic damage, andcorrelates with gastric cancer risk. Chronic inflammation triggered by H. pylori infection is critical for methylation induction, and if data from a mousecolitismodel are combined, the importanceofmonocytes canbespeculated. As translational targets,methylation levels of specificgenes innormal-appearingtissues can be used as a cancer risk marker that reflects a person's life. The methylation signature has potential as a marker for past exposure tospecific environmental factors. Suppression of induction of aberrant DNA methylation, and possibly removal of accumulated aberrant methylation canbe used for cancer prevention (shown in red).

Epigenetic Field by H. pylori Infection

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induce methylation in epithelial cells by injecting its ownDNA methyltransferases. Alternatively, studies in patientswith ulcerative colitis showed that chronic inflammationplayed a role inmethylation induction (37, 38), and chron-ic inflammation triggered by H. pylori infection may havebeen responsible for the methylation induction. Niwa andcolleagues (45) addressed this issue by suppressing inflam-mation in gerbils with H. pylori infection using cyclosporinA, an immunosuppressant. Although colonization of H.pylori was not affected at all, methylation induction wasmarkedly suppressed. This clearly shows that it was theinflammation triggered by theH. pylori infection, not theH.pylori itself, that was involved in methylation induction. Atemporal analysis of the expression of inflammation-relatedgenes in gastric mucosae of infected gerbils showed that theexpression levels of Cxcl2, Il1b,Nos2, and Tnf paralleled themethylation levels.

Inflammation in the stomach can be induced not only byH. pylori infection but also by high concentrations of eth-anol (EtOH) or saturated sodium chloride (NaCl) solution.A methylation analysis of gastric mucosae exposed to thesekinds of inflammation showed that only inflammationtriggered by H. pylori infection was capable of inducingaberrant DNA methylation (48). Histologically, H. pyloriinfection induced chronic inflammation with prominentlymphocyte and macrophage infiltration, whereas EtOHand NaCl treatment induced persistent neutrophil infiltra-tion. Cell proliferation, which is known to be important formethylation induction (38), was most strongly induced inthe NaCl group and was shown to be insufficient formethylation induction. Among inflammation-relatedgenes, expression of Il1b, Nos2, and Tnf was increasedspecifically in gastric mucosae of gerbils with H. pyloriinfection. Therefore, it is considered that specific types ofinflammation are necessary for methylation induction.

Chronic inflammation is characterized by infiltration ofmononuclear cells, i.e., lymphocytes and monocytes. Toclarifywhich cell type(s) plays themajor role inmethylationinduction, Katsurano and colleagues (49) examined SCIDmice, which lack both B and T lymphocytes. Because H.pylori cannot infectmice efficiently, theyused a colitismodelinduced by dextran sulfate sodium (DSS). Even in SCIDmice,DNAmethylation and colon tumors couldbe inducedat the same levels as in wild-type mice. This shows thatlymphocytes are dispensable for methylation induction,and strongly suggests thatmonocytes are important. Expres-sion of Ifng, Il1b, and Nos2 was induced in both wild-typeand SCID mice by DSS treatment.

If we hypothesize that the same effectors are working ingerbil stomachs infected by H. pylori and mouse colonstreated with DSS, we can conclude that expression of Il1band Nos2 may be involved in methylation induction. Pro-moter polymorphisms of IL1B are reported to be associatedwith human gastric cancer susceptibility by increasing ordecreasing IL1b production in response to H. pylori infec-tion and thus the progression of gastric atrophy (50, 51).Increased production of NO in vitro is reported to increasethe enzyme activity of DNA methyltransferases without

changing their expression, and to induce DNAmethylationof specific genes (52). In the human and gerbil stomachsinfected byH. pylori andmouse colons treated with DSS, nochanges in the expression of DNAmethyltransferases 1, 3a,and 3bwere observed (28, 45, 49). It is possible that a signalfrom chronic inflammation, possibly IL1b, and elevation ofNO in epithelial cells lead to inappropriate localization ofderegulated DNA methyltransferase(s) to methylation-sus-ceptible CpG islands (see below) and induce aberrant DNAmethylation as an infrequent event.

Methylation fingerprints produced by H. pyloriinfection

Target genes for methylation induction byH. pylori infec-tion are present in gastric mucosae (28). Among 48 pro-moter CpG islands whose methylation was analyzed ingastric mucosae of individuals with and without H. pyloriinfection, somewere consistentlymethylated in individualswith current or past infection and others were not methyl-ated at all. Analysis of polyclonal tissues, unlike that ofcancers, canprovide information aboutmultiple events thathave taken place independently, and the presence of targetgenes was convincingly shown in gastric mucosae (29).Similarly, in the esophagus, specific genes were methylatedin association with smoking history (35), and again thepresence of target gene specificity formethylation inductionwas shown.

The target gene specificity is defined by epigenetic factorsin the cells wheremethylation is induced (29, 53, 54) and inthe genome architecture (55, 56). Epigenetic factors thatpromote methylation induction include low transcriptionand the presence of an H3K27me3 modification. In con-trast, the presence of histone acetylation and RNA polymer-ase II (active or stalled)protectsCpG islands frombecomingmethylated. A multivariate analysis revealed that the mostinfluential factors are the promoting effect of H3K27me3and the protective effect of RNA polymerase II (54). Agenomic factor that promotes methylation induction is adistant location from repetitive elements (55, 56). It iscurrently speculated that infection by H. pylori inducesH3K27me3 and removes RNA polymerase II at its targetgenes, and that these genes then become methylated.

Clinical-Translational Advances

Cancer risk marker that reflects life historyThe importance of predicting cancer risks has been

repeatedly emphasized because the ability to select high-risk individuals enables efficient cancer screening andreduces social costs (5759). To this end, a massive efforthas been made in association studies, and many cancer riskalleles for common cancers have been identified. Most ofthese risk alleles give odds ratios between 1.5 and 2.0(51, 58, 59), and can be used to estimate cancer risk whena person is born.

At the same time, a person is exposed to various envi-ronmental carcinogenic factors, and the cancer risk of anadult will differ depending on what sort of life he or she has






spent. Therefore, a cancer risk marker that incorporatesinformation about life-to-date is important. At least someof a persons life history, such as smoking and infection byH. pylori, is imprinted on the epigenome and produces anepigenetic field for cancerization. The severity of the fieldcan be measured as methylation levels of specific markergenes, and correlates with cancer risk. The odds ratiosobtained by DNA methylation markers of gastric cancers,such as THBD, FLNc, andmiR-124a, range from 2.4 to 22.1[calculated based on our previous reports (21, 22)]. Pas-senger genes can be useful as marker genes because they areconsistentlymethylated andhavehighmethylation levels innoncancerous tissues (21, 27), whereas driver genes areonly stochastically methylated and have low methylationlevels (Fig. 1). Therefore, for evaluating the degree of epi-genomic damage that has been done in the past, methyl-ation of passenger genes is often superior to that of drivergenes.The presence of an epigenetic field defect is also known

for other types of cancers, as mentioned above (27). There-fore, investigators are now developing methods to estimateepigenetic cancer risk, taking life history into account, invarious types of cancer. For example, a multicenter studywas conducted to evaluate the validity of methylationmarkers to predict progression of Barretts esophagus, andmethylation ofHPP1,CDKN2A, andRUNX3were shown tobe informative (34).

Marker for past exposure to specific environmentalfactorsH. pylori infection is associated with methylation of a

specific set of genes, most of which are considered aspassengers, in gastric mucosae (28). A history of smokingis associated with methylation of UCHL1 and HOXA9,which are also considered to be passengers, in esophagealmucosae (35, 36). Once the specificity of methylationsignatures to various carcinogenic agents is clarified, pastexposure to such carcinogenic factors can be estimated bythe methylation signature. The methylation signature hasadvantages over other exposure markers because it persistsfor a long time and does not require any record by humans.For example, past exposure to H. pylori infection can beestimated by serum antibody, but it persists only up toseveral years afterH. pylori infection discontinues (60). Theability to estimate past exposure using a methylation sig-nature would be very helpful from an epidemiologicalviewpoint.

Epigenetic cancer preventionThe presence of an epigenetic field for cancerization

and the deep involvement of chronic inflammation inits formation provide targets for cancer prevention. Sup-pression of induction of aberrant DNA methylation isexpected to lead to a decreased incidence of cancers.This concept is supported by animal models for macro-scopic colon tumors (61, 62), lung tumors (63), andprostate cancers (64, 65). It was shown that in gerbilstomachs, administration of a demethylating agent,

5-aza-20-deoxycytidine (5-aza-dC), decreased the inci-dence of gastric cancers induced by H. pylori infectionand a mutagen, N-methyl-N-nitrosourea (unpublished).In addition to suppression of DNA methylation induc-tion, suppression of H3K27me3, a premarker for DNAmethylation induction, is also an attractive target. Thehistone methyltransferase that is responsible for thismodification, EZH2, is known to be overexpressed inaggressive tumors (66) and precancerous lesions (67),and therefore inhibitors of EZH2, such as 3-deazanepla-nocin A (66), may have preventive applications.

Anti-inflammatory drugs, especially nonsteroidal anti-inflammatory drugs (NSAIDs), are effective for preventionof at least some cancers, but their use is still limited toindividuals with high risk (68). The use of NSAIDS islimited in part because of possible side-effects, such aspeptic ulcer. To avoid such side-effects and suppress thepathways that are responsible for cancer development,researchers are actively investigating the mechanisms ofcancer induction by chronic inflammation (69). Becauseinduction of epigenetic alterations is one of these importantmechanisms, suppressionof specific components of inflam-mation that are responsible for induction of epigeneticalterations is expected to provide a good target for cancerprevention.

Lastly, DNA methylation is reversible by DNA demethy-lating agents, such as 5-aza-dC and 5-azacytidine (70).Currently available demethylating agents do not have ahigh specificity for aberrantly methylated genes, and candemethylate normally methylated sequences. Suchsequences include normally methylated CpG islands andrepetitive sequences originating from retrotransposons, andit is feared that DNA demethylating agents might inducedemethylation of these retrotransposons. Therefore, forcancer prevention using current demethylating agents, wemust carefully balance risk and benefit, and probably suchagents are not widely indicated. However, many epigeneticdrugs are beingdeveloped, and it is possible that someof thenew demethylating agents will have a specificity or prefer-ence for aberrantly methylated promoter CpG islands, andcan be used in a wider range of individuals in the future.

Conclusions

The fact that H. pylori infection induces aberrant DNAmethylation in gastric mucosae provides the missing linkbetween the major role of H. pylori infection in gastriccancers and the deep involvement of epigenetic alterationsin gastric cancers. The severity of infection correlates withgastric cancer risk and can provide a unique cancer riskmarker that reflects a persons life. H. pylori infection hasbeen shown to induce methylation of specific genes, andthere are underlying mechanisms. The methylation signa-ture has potential as a marker for past exposure to H. pyloriinfection. Specific types of inflammation, such as thatinduced by H. pylori infection, are capable of inducingaberrantmethylation, andmonocytes appear to be involvedin the induction. Suppression of methylation induction,






specific inflammatory processes, and reversal of epigeneticalterations are targets for cancer prevention.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

Third-Term Comprehensive Cancer Control Strategy, Ministry of Health,Labor and Welfare, Japan.

ReceivedAugust 16, 2011; revisedDecember 11, 2011; acceptedDecember13, 2011; published OnlineFirst December 28, 2011.

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2012;18:923-929. Published OnlineFirst December 28, 2011.Clin Cancer Res Toshikazu Ushijima and Naoko Hattori Usefulness as Cancer Risk and Exposure Markers

ItsInflammation in the Formation of an Epigenetic Field Defect, and TriggeredHelicobacter pyloriMolecular Pathways: Involvement of

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