Inflammation-Induced Cell Proliferation Potentiates DNA Damage-Induced Mutations In Vivo

Mutations are a critical driver of cancer initiation. While extensive studies have focused on exposure-induced mutations, few studies have explored the importance of tissue physiology as a modulator of mutation susceptibility in vivo. Of particular interest is inflammation, a known cancer risk factor relevant to chronic inflammatory diseases and pathogen-induced inflammation. Here, we used the fluorescent yellow direct repeat (FYDR) mice that harbor a reporter to detect misalignments during homologous recombination (HR), an important class of mutations. FYDR mice were exposed to cerulein, a potent inducer of pancreatic inflammation. We show that inflammation induces DSBs (γH2AX foci) and that several days later there is an increase in cell proliferation. While isolated bouts of inflammation did not induce HR, overlap between inflammation-induced DNA damage and inflammation-induced cell proliferation induced HR significantly. To study exogenously-induced DNA damage, animals were exposed to methylnitrosourea, a model alkylating agent that creates DNA lesions relevant to both environmental exposures and cancer chemotherapy. We found that exposure to alkylation damage induces HR, and importantly, that inflammation-induced cell proliferation and alkylation induce HR in a synergistic fashion. Taken together, these results show that, during an acute bout of inflammation, there is a kinetic barrier separating DNA damage from cell proliferation that protects against mutations, and that inflammation-induced cell proliferation greatly potentiates exposure-induced mutations. These studies demonstrate a fundamental mechanism by which inflammation can act synergistically with DNA damage to induce mutations that drive cancer and cancer recurrence.Austrian Academy of Sciences (APART Fellowship)Singapore-MIT Alliance for Research and TechnologySingapore. National Research FoundationNational Institutes of Health (U.S.) (NIH R33-CA112151)National Institutes of Health (U.S.) (grant R01-CA079827

Helicobacter hepaticus cytolethal distending toxin promotes intestinal carcinogenesis in 129Rag2‐deficient mice

Multiple pathogenic Gram-negative bacteria produce the cytolethal distending toxin (CDT) with activity of DNase I; CDT can induce DNA double-strand breaks (DSBs), G2/M cell cycle arrest, and apoptosis in cultured mammalian cells. However, the link of CDT to in vivo tumorigenesis is not fully understood. In this study, 129/SvEv Rag2−/−mice were gavaged with wild-type Helicobacter hepatics 3B1(Hh) and its isogenic cdtB mutant HhcdtBm7, followed by infection for 10 and 20 weeks (WPI). HhCDT deficiency did not affect cecal colonization levels of HhcdtBm7, but attenuated severity of cecal pathology in HhcdtBm7-infected mice. Of importance, preneoplasic dysplasia was progressed to cancer from 10 to 20 WPI in the Hh-infected mice but not in the HhcdtBm7-infected mice. In addition, the loss of HhCDT significantly dampened transcriptional upregulation of cecal Tnfα and Il-6, but elevated Il-10 mRNA levels when compared to Hh at 10 WPI. Furthermore, the presence of HhCDT increased numbers of lower bowel intestinal γH2AX-positive epithelial cells (a marker of DSBs) at both 10 and 20 WPI and augmented phospho-Stat3 foci[superscript +] intestinal crypts (activation of Stat3) at 20 WPI. Our findings suggest that CDT promoted Hh carcinogenesis by enhancing DSBs and activation of the Tnfα/Il-6-Stat3 signaling pathway.National Institutes of Health (U.S.) (Grant R01-OD01141)National Institutes of Health (U.S.) (Grant T32-OD010978)National Institutes of Health (U.S.) (Grant P01 CA28842

Helicobacter hepaticus Cholesterol-α-glucosyltransferase is Essential for Establishing Colonization in Male A/JCr Mice

Background Helicobacter pylori cholesterol-α-glucosyltransferase (cgt) is essential for survival of H. pylori in mice. Enterohepatic H. hepaticus, the cause of colonic and hepatocellular carcinoma in susceptible mouse strains, contains an ortholog of the H. pylori cgt. However, the role of cgt in the pathogenesis of H. hepaticus has not been investigated. Materials and Methods Two cgt-deficient isogenic mutants of wild-type H. hepaticus (WT) 3B1 were generated and used to inoculate male A/JCr mice. Cecal and hepatic colonization levels of the mutants and WT 3B1 as well as select inflammation-associated cytokines were measured by qPCR at 4 months postinoculation. Results Both mutants were undetectable in the cecum of any inoculated mice (10 per mutant) but were detected in two livers (one for each mutant); by contrast, 9 and 7 of 10 mice inoculated with WT 3B1 were qPCR positive in the ceca and livers, respectively. The mice inoculated with the mutants developed significantly less severe hepatic inflammation (p < .05) and also produced significantly lower hepatic mRNA levels of proinflammatory cytokines Ifn-γ (p < .01) and Tnf-α (p ≤ .02) as well as anti-inflammatory factors Il10 and Foxp3 compared with the WT 3B1-inoculated mice. Additionally, the WT 3B1-inoculated mice developed significantly higher Th1-associated IgG2a (p < .0001) and Th2-associated IgG1 responses (p < .0001) to H. hepaticus infection than mice dosed with isogenic cgt mutants. Conclusion Our data indicate that the cholesterol-α-glucosyltransferase is required for establishing colonization of the intestine and liver and therefore plays a critical role in the pathogenesis of H. hepaticus.National Institutes of Health (U.S.) (Grant R010D011141)National Institutes of Health (U.S.) (Grant 01CA026731)National Institutes of Health (U.S.) (Grant R01AT004326)National Institutes of Health (U.S.) (Grant P30-ES002109

A Combination of Sulindac and Antimicrobial Eradication of H. pylori Prevents Progression of Gastric Cancer in Hypergastrinemic INS-GAS Mice

Author Manuscript: 2010 October 15Helicobacter pylori infection causes severe dysplasia manifested as gastrointestinal intraepithelial neoplasia (GIN) after 28 weeks post–H. pylori infection (WPI) in cancer-prone, hypergastrinemic male INS-GAS mice. We examined the efficacy of the nonsteroidal anti-inflammatory drug sulindac (400 ppm in drinking water) alone, the CCK2/gastrin receptor antagonist YM022 (45 mg/kg/wk) alone, and sulindac or YM022 combined with H. pylori eradication therapy to prevent H. pylori–associated gastric cancer in male INS-GAS mice. Treatments started at 22 WPI, and mice were euthanized at 28 WPI. In uninfected mice, all treatments significantly delayed development of spontaneous GIN (P < 0.05). In H. pylori–infected mice, sulindac alone or YM022 alone had no protective effect on H. pylori–associated GIN. Importantly, sulindac exacerbated the severity of H. pylori–associated gastritis despite decreased gastric prostaglandin E2 levels. However, sulindac combined with H. pylori antimicrobial eradication reduced the incidence of GIN (P < 0.05), whereas YM022 combined with antimicrobial eradication did not reduce GIN. In infected mice, sulindac or YM022 treatment did not alter gastric expression of the proinflammatory cytokines Ifn-γ and Tnf-α and mucosal cell proliferation. Sulindac or YM022 combined with antimicrobial eradication down-regulated mRNA levels of Ifn-γ and Tnf-α and mucosal cell proliferation (P < 0.05). We conclude that sulindac enhances H. pylori gastritis and may promote inflammation-mediated gastric carcinogenesis. The combination of sulindac and antimicrobial H. pylori eradication was beneficial for reducing proinflammatory cytokine mRNA in the stomach and preventing progression from severe dysplasia to gastric cancer in H. pylori–infected INS-GAS mice. [Cancer Res 2009;69(20):8166–74]National Institutes of Health (U.S.) (Grant R01AI37750)National Institutes of Health (U.S.) (Grant P01CA26731)National Institutes of Health (U.S.) (Grant P30ES02109)National Institutes of Health (U.S.) (Grant R01CA093405-07A1

Helicobacter hepaticus Infection Promotes Hepatitis and Preneoplastic Foci in Farnesoid X Receptor (FXR) Deficient Mice

Farnesoid X receptor (FXR) is a nuclear receptor that regulates bile acid metabolism and transport. Mice lacking expression of FXR (FXR KO) have a high incidence of foci of cellular alterations (FCA) and liver tumors. Here, we report that Helicobacter hepaticus infection is necessary for the development of increased hepatitis scores and FCA in previously Helicobacter-free FXR KO mice. FXR KO and wild-type (WT) mice were sham-treated or orally inoculated with H. hepaticus. At 12 months post-infection, mice were euthanized and liver pathology, gene expression, and the cecal microbiome were analyzed. H. hepaticus induced significant increases hepatitis scores and FCA numbers in FXR KO mice (P<0.01 and P<0.05, respectively). H. hepaticus altered the beta diversity of cecal microbiome in both WT and FXR KO mice compared to uninfected mice (P<0.05). Significant upregulation of β-catenin, Rela, Slc10a1, Tlr2, Nos2, Vdr, and Cyp3a11 was observed in all FXR KO mice compared to controls (P<0.05). Importantly, H. hepaticus and FXR deficiency were necessary to significantly upregulate Cyp2b10 (P<0.01). FXR deficiency was also a potent modulator of the cecal microbiota, as observed by a strong decrease in alpha diversity. A significant decrease in Firmicutes, particularly members of the order Clostridiales, was observed in FXR KO mice (P<0.05 and FDR<5%, ANOVA). While FXR deficiency strongly affects expression of genes related to immunity and bile acid metabolism, as well as the composition of the microbiome; however, its deficiency was not able to produce significant histopathological changes in the absence of H. hepaticus infection.National Institutes of Health (U.S.) (NIH R01 OD011141)National Institutes of Health (U.S.) (NIH T32 OD010978)National Institutes of Health (U.S.) (NIH P30 ES002109)National Institutes of Health (U.S.) (P01 CA026731

Aag-initiated base excision repair promotes ischemia reperfusion injury in liver, brain, and kidney

Inflammation is accompanied by the release of highly reactive oxygen and nitrogen species (RONS) that damage DNA, among other cellular molecules. Base excision repair (BER) is initiated by DNA glycosylases and is crucial in repairing RONS-induced DNA damage; the alkyladenine DNA glycosylase (Aag/Mpg) excises several DNA base lesions induced by the inflammation-associated RONS release that accompanies ischemia reperfusion (I/R). Using mouse I/R models we demonstrate that Aag[superscript −/−] mice are significantly protected against, rather than sensitized to, I/R injury, and that such protection is observed across three different organs. Following I/R in liver, kidney, and brain, Aag[superscript −/−] mice display decreased hepatocyte death, cerebral infarction, and renal injury relative to wild-type. We infer that in wild-type mice, Aag excises damaged DNA bases to generate potentially toxic abasic sites that in turn generate highly toxic DNA strand breaks that trigger poly(ADP-ribose) polymerase (Parp) hyperactivation, cellular bioenergetics failure, and necrosis; indeed, steady-state levels of abasic sites and nuclear PAR polymers were significantly more elevated in wild-type vs. Aag[superscript −/−] liver after I/R. This increase in PAR polymers was accompanied by depletion of intracellular NAD and ATP levels plus the translocation and extracellular release of the high-mobility group box 1 (Hmgb1) nuclear protein, activating the sterile inflammatory response. We thus demonstrate the detrimental effects of Aag-initiated BER during I/R and sterile inflammation, and present a novel target for controlling I/R-induced injury.National Institutes of Health (U.S.) (Grant R01-CA055042)National Institutes of Health (U.S.) (Grant R01-CA149261)National Institutes of Health (U.S.) (Grant P30-ES02109)Ellison Medical Foundatio

Interferon-γ inhibits gastric carcinogenesis by inducing epithelial cell autophagy and T cell apoptosis

Author Manuscript 2012 June 15.IFN-γ mediates responses to bacterial infection and autoimmune disease, but it is also an important tumor suppressor. It is upregulated in the gastric mucosa by chronic Helicobacter infection; however, whether it plays a positive or negative role in inflammation-associated gastric carcinogenesis is unexplored. To study this question, we generated an H[superscript +]/K[superscript +]-ATPase-IFN-γ transgenic mouse that overexpresses murine IFN-γ in the stomach mucosa. In contrast to the expected proinflammatory role during infection, we found that IFN-γ overexpression failed to induce gastritis and instead inhibited gastric carcinogenesis induced by interleukin-1beta (IL-1β) and/or Helicobacter infection. Helper T cell (Th) 1 and Th17 immune responses were inhibited by IFN-γ through Fas induction and apoptosis in CD4 T cells. IFN-γ also induced autophagy in gastric epithelial cells through increased expression of Beclin-1. Finally, in the gastric epithelium, IFN-γ also inhibited IL-1β- and Helicobacter-induced epithelial apoptosis, proliferation, and Dckl1[superscript +] cell expansion. Taken together, our results suggest that IFN-γ coordinately inhibits bacterial infection and carcinogenesis in the gastric mucosa by suppressing putative gastric progenitor cell expansion and reducing epithelial cell apoptosis via induction of an autophagic program. Cancer Res; 71(12); 4247–59

Gastric colonisation with a restricted commensal microbiota replicates the promotion of neoplastic lesions by diverse intestinal microbiota in the Helicobacter pylori INS-GAS mouse model of gastric carcinogenesis

Objectives: Gastric colonisation with intestinal flora (IF) has been shown to promote Helicobacter pylori (Hp)-associated gastric cancer. However, it is unknown if the mechanism involves colonisation with specific or diverse microbiota secondary to gastric atrophy. Design: Gastric colonisation with Altered Schaedler's flora (ASF) and Hp were correlated with pathology, immune responses and mRNA expression for proinflammatory and cancer-related genes in germ-free (GF), Hp monoassociated (mHp), restricted ASF (rASF; 3 species), and specific pathogen-free (complex IF), hypergastrinemic INS-GAS mice 7 months postinfection. Results: Male mice cocolonised with rASFHp or IFHp developed the most severe pathology. IFHp males had the highest inflammatory responses, and 40% developed invasive gastrointestinal intraepithelial neoplasia (GIN). Notably, rASFHp colonisation was highest in males and 23% developed invasive GIN with elevated expression of inflammatory biomarkers. Lesions were less severe in females and none developed GIN. Gastritis in male rASFHp mice was accompanied by decreased Clostridum species ASF356 and Bacteroides species ASF519 colonisation and an overgrowth of Lactobacillus murinus ASF361, supporting that inflammation-driven atrophy alters the gastric niche for GI commensals. Hp colonisation also elevated expression of IL-11 and cancer-related genes, Ptger4 and Tgf-β, further supporting that Hp infection accelerates gastric cancer development in INS-GAS mice. Conclusions: rASFHp colonisation was sufficient for GIN development in males, and lower GIN incidence in females was associated with lower inflammatory responses and gastric commensal and Hp colonisation. Colonisation efficiency of commensals appears more important than microbial diversity and lessens the probability that specific gastrointestinal pathogens are contributing to cancer risk.National Institutes of Health (U.S.) (grant R01 AI37750)National Institutes of Health (U.S.) (grant R01 CA093405)National Institutes of Health (U.S.) (grant P30-ES02109)National Institutes of Health (U.S.) (grant P01 CA028842)National Institutes of Health (U.S.) (grant T32 RR07036

Megaesophagus in a Line of Transgenic Rats: A Model of Achalasia

Megaesophagus is defined as the abnormal enlargement or dilatation of the esophagus, characterized by a lack of normal contraction of the esophageal walls. This is called achalasia when associated with reduced or no relaxation of the lower esophageal sphincter (LES). To date, there are few naturally occurring models for this disease. A colony of transgenic (Pvrl3-Cre) rats presented with megaesophagus at 3 to 4 months of age; further breeding studies revealed a prevalence of 90% of transgene-positive animals having megaesophagus. Affected rats could be maintained on a total liquid diet long term and were shown to display the classic features of dilated esophagus, closed lower esophageal sphincter, and abnormal contractions on contrast radiography and fluoroscopy. Histologically, the findings of muscle degeneration, inflammation, and a reduced number of myenteric ganglia in the esophagus combined with ultrastructural lesions of muscle fiber disarray and mitochondrial changes in the striated muscle of these animals closely mimic that seen in the human condition. Muscle contractile studies looking at the response of the lower esophageal sphincter and fundus to electrical field stimulation, sodium nitroprusside, and L-nitro-L-arginine methyl ester also demonstrate the similarity between megaesophagus in the transgenic rats and patients with achalasia. No primary cause for megaesophagus was found, but the close parallel to the human form of the disease, as well as ease of care and manipulation of these rats, makes this a suitable model to better understand the etiology of achalasia as well as study new management and treatment options for this incurable condition.National Institutes of Health (U.S.) (Grant T32OD011141)National Institutes of Health (U.S.) (Grant P30ES002109

Lamellipodin-Deficient Mice: A Model of Rectal Carcinoma

During a survey of clinical rectal prolapse (RP) cases in the mouse population at MIT animal research facilities, a high incidence of RP in the lamellipodin knock-out strain, C57BL/6-Raph1[superscript tm1Fbg] (Lpd[superscript -/-]) was documented. Upon further investigation, the Lpd[superscript -/-] colony was found to be infected with multiple endemic enterohepatic Helicobacter species (EHS). Lpd[superscript -/-] mice, a transgenic mouse strain produced at MIT, have not previously shown a distinct immune phenotype and are not highly susceptible to other opportunistic infections. Predominantly male Lpd[superscript -/-] mice with RP exhibited lesions consistent with invasive rectal carcinoma concomitant to clinically evident RP. Multiple inflammatory cytokines, CD11b+Gr1+ myeloid-derived suppressor cell (MDSC) populations, and epithelial cells positive for a DNA damage biomarker, H2AX, were elevated in affected tissue, supporting their role in the neoplastic process. An evaluation of Lpd[superscript -/-] mice with RP compared to EHS-infected, but clinically normal (CN) Lpd[superscript -/-] animals indicated that all of these mice exhibit some degree of lower bowel inflammation; however, mice with prolapses had significantly higher degree of focal lesions at the colo-rectal junction. When Helicobacter spp. infections were eliminated in Lpd[superscript -/-] mice by embryo transfer rederivation, the disease phenotype was abrogated, implicating EHS as a contributing factor in the development of rectal carcinoma. Here we describe lesions in Lpd[superscript -/-] male mice consistent with a focal inflammation-induced neoplastic transformation and propose this strain as a mouse model of rectal carcinoma.United States. National Institutes of Health (T32-OD010978)United States. National Institutes of Health (R01-OD011141)United States. National Institutes of Health (P30-ES002109)Massachusetts Institute of Technology. Ludwig Center for Molecular Oncology (U54- CA114462)National Cancer Institute (U.S.) (P30-CA14051