Maria B. Piazuelo

Arginase I Production in the Tumor Microenvironment by Mature Myeloid Cells Inhibits T-Cell Receptor Expression and Antigen-Specific T-Cell Responses

T cells infiltrating tumors have a decreased expression of signal transduction proteins, a diminished ability to proliferate, and a decreased production of cytokines. The mechanisms causing these changes have remained unclear. We demonstrated recently that peritoneal macrophages stimulated with interleukin 4 + interleukin 13 produce arginase I, which decreases the expression of the T-cell receptor CD3ζ chain and impairs T-cell responses. Using a 3LL murine lung carcinoma model we tested whether arginase I was produced in the tumor microenvironment and could decrease CD3ζ expression and impair T-cell function. The results show that a subpopulation of mature tumor-associated myeloid cells express high levels of arginase I, whereas tumor cells and infiltrating lymphocytes do not. Arginase I expression in the tumor was seen on day 7 after tumor injection. Tumor-associated myeloid cells also expressed high levels of cationic amino acid transporter 2B, which allowed them to rapidly incorporate l-Arginine (l-Arg) and deplete extracellular l-Arg in vitro. l-Arg depletion by tumor-associated myeloid cells blocked the re-expression of CD3ζ in stimulated T cells and inhibited antigen-specific proliferation of OT-1 and OT-2 cells. The injection of the arginase inhibitor N-hydroxy-nor-l-Arg blocked growth of s.c. 3LL lung carcinoma in mice. High levels of arginase I were also found in tumor samples of patients with non-small cell carcinoma. Therefore, arginase I production by mature myeloid cells in the tumor microenvironment may be a central mechanism for tumor evasion and may represent a target for new therapies.

Long term follow up of patients treated for Helicobacter pylori infection

Background:Helicobacter pylori infection induces progressive inflammatory changes in the gastric mucosa that may lead to gastric cancer. Understanding long term effects resulting from the cure of this infection is needed to design cancer prevention strategies. Methods: A cohort of 795 adults with preneoplastic gastric lesions was randomised to receive anti-H pylori treatment and/or antioxidants. At the end of six years of intervention, those who did not receive anti-H pylori treatment were offered it. Gastric biopsies were obtained at baseline, and at 3, 6, and 12 years. A histopathology score was utilised to document changes in gastric lesions. Non-linear mixed models were used to estimate the cumulative effect of H pylori clearance on histopathology scores adjusted for follow up time, interventions, and confounders. Results: Ninety seven per cent of subjects were H pylori positive at baseline, and 53% were positive at 12 years. Subjects accumulated 1703 person years free of infection. A multivariate model showed a significant regression in histopathology score as a function of the square of H pylori negative time. Subjects who were H pylori negative had 14.8% more regression and 13.7% less progression than patients who were positive at 12 years (p = 0.001). The rate of healing of gastric lesions occurred more rapidly as years free of infection accumulated, and was more pronounced in less advanced lesions. Conclusions: Preneoplastic gastric lesions regress at a rate equal to the square of time in patients rendered free of H pylori infection. Our findings suggest that patients with preneoplastic gastric lesions should be treated and cured of their H pylori infection.

Regulation of Gastric Carcinogenesis by Helicobacter pylori Virulence Factors

Helicobacter pylori is the strongest known risk factor for gastric adenocarcinoma, and strains that possess the cag secretion system, which translocates the bacterial effector CagA into host cells, augment cancer risk. H. pylori strains that express the vacuolating cytotoxin or the outer membrane protein OipA are similarly associated with severe pathologic outcomes. We previously reported that an in vivo adapted H. pylori strain, 7.13, induces gastric adenocarcinoma in rodent models of gastritis. In the current study, we used carcinogenic strain 7.13 as a prototype to define the role of virulence constituents in H. pylori-mediated carcinogenesis. Mongolian gerbils were infected with wild-type strain 7.13 or cagA(-), vacA(-), or oipA(-) mutants for 12 to 52 weeks. All infected gerbils developed gastritis; however, inflammation was significantly attenuated in animals infected with the cagA(-) but not the vacA(-) or oipA(-) strains. Gastric dysplasia and cancer developed in >50% of gerbils infected with either the wild-type or vacA(-) strain but in none of the animals infected with the cagA(-) strain. Inactivation of oipA decreased beta-catenin nuclear localization in vitro and reduced the incidence of cancer in gerbils. OipA expression was detected significantly more frequently among H. pylori strains isolated from human subjects with gastric cancer precursor lesions versus persons with gastritis alone. These results indicate that loss of CagA prevents the development of cancer in this model. Inactivation of oipA attenuates beta-catenin nuclear translocation and also decreases the incidence of carcinoma. In addition to defining factors that mediate H. pylori-induced cancer, these results provide insight into mechanisms that may regulate the development of other malignancies arising within the context of inflammatory states.

Frequent overexpression of Aurora Kinase A in upper gastrointestinal adenocarcinomas correlates with potent antiapoptotic functions

Upper gastrointestinal adenocarcinomas are a common cause of cancer‐related deaths. In this study, the authors investigated the prevalence and biological significance of Aurora Kinase A (AURKA) overexpression in upper gastrointestinal adenocarcinomas.

Regulation of p53 Tumor Suppressor by Helicobacter pylori in Gastric Epithelial Cells

BACKGROUND & AIMS Infection with the gastric mucosal pathogen Helicobacter pylori is the strongest identified risk factor for distal gastric cancer. These bacteria colonize a significant part of the worlds population. We investigated the molecular mechanisms of p53 regulation in H pylori-infected cells. METHODS Mongolian gerbils were challenged with H pylori and their gastric tissues were analyzed by immunohistochemistry and immunoblotting with p53 antibodies. Gastric epithelial cells were co-cultured with H pylori and the regulation of p53 was assessed by real-time polymerase chain reaction, immunoblotting, immunofluorescence, and cell survival assays. Short hairpin RNA and dominant-negative mutants were used to inhibit activities of Human Double Minute 2 (HDM2) and AKT1 proteins. RESULTS We found that in addition to previously reported up-regulation of p53, H pylori can also negatively regulate p53 by increasing ubiquitination and proteasomal degradation via activation of the serine/threonine kinase AKT1, which phosphorylates and activates the ubiquitin ligase HDM2. These effects were mediated by the bacterial virulence factor CagA; ectopic expression of CagA in gastric epithelial cells increased phosphorylation of HDM2 along with the ubiquitination and proteasomal degradation of p53. The decrease in p53 levels increased survival of gastric epithelial cells that had sustained DNA damage. CONCLUSIONS H pylori is able to inhibit the tumor suppressor p53. H pylori activates AKT1, resulting in phosphorylation and activation of HDM2 and subsequent degradation of p53 in gastric epithelial cells. H pylori-induced dysregulation of p53 is a potential mechanism by which the microorganism increases the risk of gastric cancer in infected individuals.

Gastric cancer risk in a Mexican population: Role of Helicobacter pylori CagA positive infection and polymorphisms in interleukin‐1 and ‐10 genes

Several polymorphisms of the IL1B and IL10 gene promoters have been reported to be associated with gastric cancer risk in Caucasians. However, studies in other populations have shown differing results. We aimed to test for associations between polymorphisms in IL1B (−31 and +3954), IL10−592 and IL1RN variable number of tandem repeats (VNTR) and risk of gastric cancer in a Mexican population. DNA was extracted from sera of 183 gastric adenocarcinoma patients and 377 controls. The IL1B−31, IL1B+3954 and IL10−592 biallelic polymorphisms were discriminated using 5′ Nuclease (TaqMan) assays and Pyrosequencing. The IL1RN penta‐allelic VNTR polymorphism was genotyped using PCR followed by GeneScan analysis. A significant interaction was found between IL1B−31 and CagA status for the risk of intestinal‐type gastric cancer (p=0.023). Among CagA positive subjects, those with IL1B−31CC genotype had an increased risk of intestinal‐type gastric cancer (OR 3.19, 95%CI=1.05–9.68), compared to carriers of IL1B−31TT genotype. In contrast, among CagA negative subjects, no significant association of IL1B−31CC genotype with gastric cancer was observed. The IL10−592CC genotype was associated with more than doubling of the risk of the intestinal‐type gastric cancer (OR, 2.20, 95%CI=1.04–4.65). A nonsignificantly increased risk for intestinal‐type gastric cancer was found in IL1RN*2 carriers (OR 1.49, 95%CI=0.89–2.50). None of these polymorphisms was significantly related to the risk of diffuse‐type gastric cancer. No significant association was found between risk of gastric cancer and the IL1B+3954 polymorphism. Individuals carrying 2 or more of the risk‐associated alleles (IL1B−31C, IL1RN *2 and IL10−592C) were at increased risk for intestinal‐type gastric cancer, compared to those with 0 or 1 risk‐associated allele. The risk from multiple risk‐associated alleles was especially high in subjects infected with CagA positive H. pylori. Our results support the identification of the IL1B−31 promoter polymorphism as a useful marker for risk of intestinal type gastric cancer in persons with CagA positive H. pylori infections.

Interaction of Helicobacter pylori with gastric epithelial cells is mediated by the p53 protein family

BACKGROUND & AIMS Although the p53 tumor suppressor has been extensively studied, many critical questions remain unanswered about the biological functions of p53 homologs, p73 and p63. Accumulating evidence suggests that both p73 and p63 play important roles in regulation of apoptosis, cell differentiation, and therapeutic drug sensitivity. METHODS Gastric epithelial cells were cocultured with Helicobacter pylori, and the roles of p63 and p73 proteins were assessed by luciferase reporter, real-time polymerase chain reaction, immunoblotting, and cell survival assays. Short hairpin RNA and dominant-negative mutants were used to inhibit activity of p73 and p63 isoforms. Human and murine gastric tissues were analyzed by immunohistochemistry with p73 and p63 antibodies and modified Steiners silver method. RESULTS Interaction of H pylori with gastric epithelial cells leads to robust up-regulation of p73 protein in vitro and in vivo in human gastritis specimens and H pylori-infected mice. The p73 increase resulted in up-regulation of pro-apoptotic genes, NOXA, PUMA, and FAS receptor in gastric epithelial cells. Down-regulation of p73 activity suppressed cell death and Fas receptor induced by H pylori. Bacterial virulence factors within the cag pathogenicity island, c-Abl tyrosine kinase, and interaction with p63 isoforms control the activity of p73. CONCLUSION Our findings implicate p73 in H pylori-induced apoptosis and more generally suggest that the p53 family may play a role in the epithelial cell response to H pylori infection.

Systems Modeling of the Role of Interleukin-21 in the Maintenance of Effector CD4+ T Cell Responses during Chronic Helicobacter pylori Infection

ABSTRACT The development of gastritis during Helicobacter pylori infection is dependent on an activated adaptive immune response orchestrated by T helper (Th) cells. However, the relative contributions of the Th1 and Th17 subsets to gastritis and control of infection are still under investigation. To investigate the role of interleukin-21 (IL-21) in the gastric mucosa during H. pylori infection, we combined mathematical modeling of CD4+ T cell differentiation with in vivo mechanistic studies. We infected IL-21-deficient and wild-type mice with H. pylori strain SS1 and assessed colonization, gastric inflammation, cellular infiltration, and cytokine profiles. Chronically H. pylori-infected IL-21-deficient mice had higher H. pylori colonization, significantly less gastritis, and reduced expression of proinflammatory cytokines and chemokines compared to these parameters in infected wild-type littermates. These in vivo data were used to calibrate an H. pylori infection-dependent, CD4+ T cell-specific computational model, which then described the mechanism by which IL-21 activates the production of interferon gamma (IFN-γ) and IL-17 during chronic H. pylori infection. The model predicted activated expression of T-bet and RORγt and the phosphorylation of STAT3 and STAT1 and suggested a potential role of IL-21 in the modulation of IL-10. Driven by our modeling-derived predictions, we found reduced levels of CD4+ splenocyte-specific tbx21 and rorc expression, reduced phosphorylation of STAT1 and STAT3, and an increase in CD4+ T cell-specific IL-10 expression in H. pylori-infected IL-21-deficient mice. Our results indicate that IL-21 regulates Th1 and Th17 effector responses during chronic H. pylori infection in a STAT1- and STAT3-dependent manner, therefore playing a major role controlling H. pylori infection and gastritis. IMPORTANCE Helicobacter pylori is the dominant member of the gastric microbiota in more than 50% of the world’s population. H. pylori colonization has been implicated in gastritis and gastric cancer, as infection with H. pylori is the single most common risk factor for gastric cancer. Current data suggest that, in addition to bacterial virulence factors, the magnitude and types of immune responses influence the outcome of colonization and chronic infection. This study uses a combined computational and experimental approach to investigate how IL-21, a proinflammatory T cell-derived cytokine, maintains the chronic proinflammatory T cell immune response driving chronic gastritis during H. pylori infection. This research will also provide insight into a myriad of other infectious and immune disorders in which IL-21 is increasingly recognized to play a central role. The use of IL-21-related therapies may provide treatment options for individuals chronically colonized with H. pylori as an alternative to aggressive antibiotics. Helicobacter pylori is the dominant member of the gastric microbiota in more than 50% of the world’s population. H. pylori colonization has been implicated in gastritis and gastric cancer, as infection with H. pylori is the single most common risk factor for gastric cancer. Current data suggest that, in addition to bacterial virulence factors, the magnitude and types of immune responses influence the outcome of colonization and chronic infection. This study uses a combined computational and experimental approach to investigate how IL-21, a proinflammatory T cell-derived cytokine, maintains the chronic proinflammatory T cell immune response driving chronic gastritis during H. pylori infection. This research will also provide insight into a myriad of other infectious and immune disorders in which IL-21 is increasingly recognized to play a central role. The use of IL-21-related therapies may provide treatment options for individuals chronically colonized with H. pylori as an alternative to aggressive antibiotics.

Analysis of cagA in Helicobacter pylori strains from Colombian populations with contrasting gastric cancer risk reveals a biomarker for disease severity

Background: Helicobacter pylori infection is a risk factor for the development of gastric cancer, and the bacterial oncoprotein CagA contributes to gastric carcinogenesis. Methods: We analyzed H. pylori isolates from persons in Colombia and observed that there was marked variation among strains in levels of CagA expression. To elucidate the basis for this variation, we analyzed sequences upstream from the CagA translational initiation site in each strain. Results: A DNA motif (AATAAGATA) upstream of the translational initiation site of CagA was associated with high levels of CagA expression. Experimental studies showed that this motif was necessary but not sufficient for high-level CagA expression. H. pylori strains from a region of Colombia with high gastric cancer rates expressed higher levels of CagA than did strains from a region with lower gastric cancer rates, and Colombian strains of European phylogeographic origin expressed higher levels of CagA than did strains of African origin. Histopathologic analysis of gastric biopsy specimens revealed that strains expressing high levels of CagA or containing the AATAAGATA motif were associated with more advanced precancerous lesions than those found in persons infected with strains expressing low levels of CagA or lacking the AATAAGATA motif. Conclusions: CagA expression varies greatly among H. pylori strains. The DNA motif identified in this study is associated with high levels of CagA expression, and may be a useful biomarker to predict gastric cancer risk. Impact: These findings help to explain why some persons infected with cagA-positive H. pylori develop gastric cancer and others do not. Cancer Epidemiol Biomarkers Prev; 20(10); 2237–49. ©2011 AACR.

ΔNp73α regulates MDR1 expression by inhibiting p53 function

The p73 protein is a transcription factor and member of the p53 protein family that expresses as a complex variety of isoforms. ΔNp73α is an N-terminally truncated isoform of p73. We found that ΔNp73 protein is upregulated in human gastric carcinoma suggesting that ΔNp73 may play an oncogenic role in these tumors. Although it has been shown that ΔNp73α inhibits apoptosis and counteracts the effect of chemotherapeutic drugs, the underlying mechanism by which this p73 isoform contributes to chemotherapeutic drug response remains to be explored. We found that ΔNp73α upregulates MDR1 mRNA and p-glycoprotein (p-gp), which is involved in chemotherapeutic drug transport. This p-gp upregulation was accompanied by increased p-gp functional activity in gastric cancer cells. Our data suggest that upregulation of MDR1 by ΔNp73α is mediated by interaction with p53 at the MDR1 promoter.