Jennifer M. Noto

Iron deficiency accelerates Helicobacter pylori–induced carcinogenesis in rodents and humans

Gastric adenocarcinoma is strongly associated with Helicobacter pylori infection; however, most infected persons never develop this malignancy. H. pylori strains harboring the cag pathogenicity island (cag+), which encodes CagA and a type IV secretion system (T4SS), induce more severe disease outcomes. H. pylori infection is also associated with iron deficiency, which similarly augments gastric cancer risk. To define the influence of iron deficiency on microbial virulence in gastric carcinogenesis, Mongolian gerbils were maintained on iron-depleted diets and infected with an oncogenic H. pylori cag+ strain. Iron depletion accelerated the development of H. pylori-induced premalignant and malignant lesions in a cagA-dependent manner. H. pylori strains harvested from iron-depleted gerbils or grown under iron-limiting conditions exhibited enhanced virulence and induction of inflammatory factors. Further, in a human population at high risk for gastric cancer, H. pylori strains isolated from patients with the lowest ferritin levels induced more robust proinflammatory responses compared with strains isolated from patients with the highest ferritin levels, irrespective of histologic status. These data demonstrate that iron deficiency enhances H. pylori virulence and represents a measurable biomarker to identify populations of infected persons at high risk for gastric cancer.

Helicobacter pylori Perturbs Iron Trafficking in the Epithelium to Grow on the Cell Surface

Helicobacter pylori (Hp) injects the CagA effector protein into host epithelial cells and induces growth factor-like signaling, perturbs cell-cell junctions, and alters host cell polarity. This enables Hp to grow as microcolonies adhered to the host cell surface even in conditions that do not support growth of free-swimming bacteria. We hypothesized that CagA alters host cell physiology to allow Hp to obtain specific nutrients from or across the epithelial barrier. Using a polarized epithelium model system, we find that isogenic ΔcagA mutants are defective in cell surface microcolony formation, but exogenous addition of iron to the apical medium partially rescues this defect, suggesting that one of CagAs effects on host cells is to facilitate iron acquisition from the host. Hp adhered to the apical epithelial surface increase basolateral uptake of transferrin and induce its transcytosis in a CagA-dependent manner. Both CagA and VacA contribute to the perturbation of transferrin recycling, since VacA is involved in apical mislocalization of the transferrin receptor to sites of bacterial attachment. To determine if the transferrin recycling pathway is involved in Hp colonization of the cell surface, we silenced transferrin receptor expression during infection. This resulted in a reduced ability of Hp to colonize the polarized epithelium. To test whether CagA is important in promoting iron acquisition in vivo, we compared colonization of Hp in iron-replete vs. iron-deficient Mongolian gerbils. While wild type Hp and ΔcagA mutants colonized iron-replete gerbils at similar levels, ΔcagA mutants are markedly impaired in colonizing iron-deficient gerbils. Our study indicates that CagA and VacA act in concert to usurp the polarized process of host cell iron uptake, allowing Hp to use the cell surface as a replicative niche.

The Hyaluronic Acid Receptor CD44 Coordinates Normal and Metaplastic Gastric Epithelial Progenitor Cell Proliferation

Background: Gastric parietal cell atrophy causes metaplasia, reactive stem cell proliferation, and increased risk for cancer. Results: Atrophy induces proliferation of CD44-positive epithelial cells that requires ERK → CD44 → STAT3 signaling. Conclusion: CD44 is a putative gastric stem cell marker that regulates normal and metaplasia-associated proliferation. Significance: Targeted pharmacological inhibition of ERK/CD44/STAT3 signaling may help block or reverse proliferation in precancerous atrophic/metaplastic lesions. The stem cell in the isthmus of gastric units continually replenishes the epithelium. Atrophy of acid-secreting parietal cells (PCs) frequently occurs during infection with Helicobacter pylori, predisposing patients to cancer. Atrophy causes increased proliferation of stem cells, yet little is known about how this process is regulated. Here we show that CD44 labels a population of small, undifferentiated cells in the gastric unit isthmus where stem cells are known to reside. Loss of CD44 in vivo results in decreased proliferation of the gastric epithelium. When we induce PC atrophy by Helicobacter infection or tamoxifen treatment, this CD44+ population expands from the isthmus toward the base of the unit. CD44 blockade during PC atrophy abrogates the expansion. We find that CD44 binds STAT3, and inhibition of either CD44 or STAT3 signaling causes decreased proliferation. Atrophy-induced CD44 expansion depends on pERK, which labels isthmal cells in mice and humans. Our studies delineate an in vivo signaling pathway, ERK → CD44 → STAT3, that regulates normal and atrophy-induced gastric stem/progenitor-cell proliferation. We further show that we can intervene pharmacologically at each signaling step in vivo to modulate proliferation.

Helicobacter pylori targets cancer-associated apical-junctional constituents in gastroids and gastric epithelial cells

Objective Helicobacter pylori strains that express the oncoprotein CagA augment risk for gastric cancer. However, the precise mechanisms through which cag+ strains heighten cancer risk have not been fully delineated and model systems that recapitulate the gastric niche are critical for understanding pathogenesis. Gastroids are three-dimensional organ-like structures that provide unique opportunities to study host-H. pylori interactions in a preclinical model. We used gastroids to inform and direct in vitro studies to define mechanisms through which H. pylori modulates expression of the cancer-associated tight junction protein claudin-7. Design Gastroids were infected by luminal microinjection, and MKN28 gastric epithelial cells were cocultured with H. pylori wild-type cag+ strains or isogenic mutants. β-catenin, claudin-7 and snail localisation was determined by immunocytochemistry. Proliferation was assessed using 5-ethynyl-2′-deoxyuridine, and levels of claudin-7 and snail were determined by western blot and flow cytometry. Results Gastroids developed into a self-organising differentiation axis and H. pylori induced mislocalisation of claudin-7 and increased proliferation in a CagA- and β-catenin-dependent manner. In MKN28 cells, H pylori-induced suppression of claudin-7 was regulated by β-catenin and snail. Similarly, snail expression was increased and claudin-7 levels were decreased among H. pylori-infected individuals. Conclusions H. pylori increase proliferation in a strain-specific manner in a novel gastroid system. H. pylori also alter expression and localisation of claudin-7 in gastroids and human epithelial cells, which is mediated by β-catenin and snail activation. These data provide new insights into molecular interactions with carcinogenic potential that occur between H. pylori and epithelial cells within the gastric niche.

β-Catenin and p120 Mediate PPARδ-Dependent Proliferation Induced by Helicobacter pylori in Human and Rodent Epithelia

BACKGROUND & AIMS Colonization of gastric mucosa by Helicobacter pylori leads to epithelial hyperproliferation, which increases the risk for gastric adenocarcinoma. One H pylori virulence locus associated with cancer risk, cag, encodes a secretion system that transports effectors into host cells and leads to aberrant activation of β-catenin and p120-catenin (p120). Peroxisome proliferator-activated receptor (PPAR)δ is a ligand-activated transcription factor that affects oncogenesis in conjunction with β-catenin. We used a carcinogenic H pylori strain to define the role of microbial virulence constituents and PPARδ in regulating epithelial responses that mediate development of adenocarcinoma. METHODS Gastric epithelial cells or colonies were co-cultured with the H pylori cag(+) strain 7.13 or cagE(-), cagA(-), soluble lytic transglycosylase(-), or cagA(-)/soluble lytic transglycosylase(-) mutants. Levels of PPARδ and cyclin E1 were determined by real-time, reverse-transcription polymerase chain reaction, immunoblot analysis, or immunofluorescence microscopy; proliferation was measured in 3-dimensional culture. PPARδ and Ki67 expression were determined by immunohistochemical analysis of human biopsies and rodent gastric mucosa. RESULTS H pylori induced β-catenin- and p120-dependent expression and activation of PPARδ in gastric epithelial cells, which were mediated by the cag secretion system substrates CagA and peptidoglycan. H pylori stimulated proliferation in vitro, which required PPARδ-mediated activation of cyclin E1; H pylori did not induce expression of cyclin E1 in a genetic model of PPARδ deficiency. PPARδ expression and proliferation in rodent and human gastric tissue was selectively induced by cag(+) strains and PPARδ levels normalized after eradication of H pylori. CONCLUSIONS The H pylori cag secretion system activates β-catenin, p120, and PPARδ, which promote gastric epithelial cell proliferation via activation of cyclin E1. PPARδ might contribute to gastric adenocarcinoma development in humans.

The Helicobacter pylori cag Pathogenicity Island

The cag pathogenicity island is a well-characterized virulence determinant. It is composed of 32 genes that encode a type IV bacterial secretion system and is linked with a more severe clinical outcome. The following chapters will explore the manipulation of bacterial factors in order to understand their role in gastric mucosal disease.

The Role of microRNAs in Helicobacter pylori Pathogenesis and Gastric Carcinogenesis

Gastric carcinogenesis is a multistep process orchestrated by aberrancies in the genetic and epigenetic regulation of oncogenes and tumor suppressor genes. Chronic infection with Helicobacter pylori is the strongest known risk factor for the development of gastric cancer. H. pylori expresses a spectrum of virulence factors that dysregulate host intracellular signaling pathways that lower the threshold for neoplastic transformation. In addition to bacterial determinants, numerous host and environmental factors increase the risk of gastric carcinogenesis. Recent discoveries have shed new light on the involvement of microRNAs (miRNAs) in gastric carcinogenesis. miRNAs represent an abundant class of small, non-coding RNAs involved in global post-transcriptional regulation and, consequently, play an integral role at multiple steps in carcinogenesis, including cell cycle progression, proliferation, apoptosis, invasion, and metastasis. Expression levels of miRNAs are frequently altered in malignancies, where they function as either oncogenic miRNAs or tumor suppressor miRNAs. This review focuses on miRNAs dysregulated by H. pylori and potential etiologic roles they play in H. pylori-mediated gastric carcinogenesis.

Helicobacter Pylori Promotes the Expression of Krüppel-Like Factor 5, a Mediator of Carcinogenesis, In Vitro and In Vivo

Helicobacter pylori is the strongest known risk factor for the development of gastric adenocarcinoma. H. pylori expresses a repertoire of virulence factors that increase gastric cancer risk, including the cag pathogenicity island and the vacuolating cytotoxin (VacA). One host element that promotes carcinogenesis within the gastrointestinal tract is Krüppel-like factor 5 (KLF5), a transcription factor that mediates key cellular functions. To define the role of KLF5 within the context of H. pylori-induced inflammation and injury, human gastric epithelial cells were co-cultured with the wild-type cag+ H. pylori strain 60190. KLF5 expression was significantly upregulated following co-culture with H. pylori, but increased expression was independent of the cag island or VacA. To translate these findings into an in vivo model, C57BL/6 mice were challenged with the wild-type rodent-adapted cag+ H. pylori strain PMSS1 or a PMSS1 cagE− isogenic mutant. Similar to findings in vitro, KLF5 staining was significantly enhanced in gastric epithelium of H. pylori-infected compared to uninfected mice and this was independent of the cag island. Flow cytometry revealed that the majority of KLF5+ cells also stained positively for the stem cell marker, Lrig1, and KLF5+/Lrig1+ cells were significantly increased in H. pylori-infected versus uninfected tissue. To extend these results into the natural niche of this pathogen, levels of KLF5 expression were assessed in human gastric biopsies isolated from patients with or without premalignant lesions. Levels of KLF5 expression increased in parallel with advancing stages of neoplastic progression, being significantly elevated in gastritis, intestinal metaplasia, and dysplasia compared to normal gastric tissue. These results indicate that H. pylori induces expression of KLF5 in gastric epithelial cells in vitro and in vivo, and that the degree of KLF5 expression parallels the severity of premalignant lesions in human gastric carcinogenesis.

Helicobacter pylori : An Overview

Infection with Helicobacter pylori is directly responsible for substantial morbidity and mortality worldwide. This ubiquitous organism causes disease through the interaction of multiple factors including bacterial factors, host immune responses, and environmental factors. The following chapters address the bacterial specific contributions to disease.

Bacterial CagA protein induces degradation of p53 protein in a p14ARF-dependent manner

Objective Infection with Helicobacter pylori is the strongest known risk factor for adenocarcinoma of the stomach. Tumorigenic transformation of gastric epithelium induced by H. pylori is a highly complex process driven by an active interplay between bacterial virulence and host factors, many aspects of which remain obscure. In this work, we investigated the degradation of p53 tumour suppressor induced by H. pylori. Design Expression of p53 protein in gastric biopsies was assessed by immunohistochemistry. Gastric cells were co-cultured with H. pylori strains isolated from high-gastric risk and low-gastric risk areas and assessed for expression of p53, p14ARF and cytotoxin-associated gene A (CagA) by immunoblotting. siRNA was used to inhibit activities of ARF-BP1 and Human Double Minute 2 (HDM2) proteins. Results Our analysis demonstrated that H. pylori strains expressing high levels of CagA virulence factor and associated with a higher gastric cancer risk more strongly suppress p53 compared with low-risk strains in vivo and in vitro. We found that degradation of p53 induced by bacterial CagA protein is mediated by host HDM2 and ARF-BP1 E3 ubiquitin ligases, while the p14ARF protein counteracts H. pylori-induced signalling. Conclusions Our results provide novel evidence that tumorigenicity associated with H. pylori infection is linked to inhibition of p53 protein by CagA. We propose a model in which CagA-induced degradation of p53 protein is determined by a relative level of p14ARF. In cells in which p14ARF levels were decreased due to hypermethylation or deletion of the p14ARF gene, H. pylori efficiently degraded p53, whereas p53 is protected in cells expressing high levels of p14ARF.