Bacterial CagA protein compromises tumor suppressor mechanisms in gastric epithelial cells
Manikandan Palrasu, … , Richard M. Peek Jr., Alexander I. Zaika
Manikandan Palrasu, … , Richard M. Peek Jr., Alexander I. Zaika
Published April 6, 2020
Citation Information: J Clin Invest. 2020;130(5):2422-2434. https://doi.org/10.1172/JCI130015.
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Research Article Gastroenterology

Bacterial CagA protein compromises tumor suppressor mechanisms in gastric epithelial cells

Abstract

Approximately half of the world’s population is infected with the stomach pathogen Helicobacter pylori. Infection with H. pylori is the main risk factor for distal gastric cancer. Bacterial virulence factors, such as the oncoprotein CagA, augment cancer risk. Yet despite high infection rates, only a fraction of H. pylori–infected individuals develop gastric cancer. This raises the question of defining the specific host and bacterial factors responsible for gastric tumorigenesis. To investigate the tumorigenic determinants, we analyzed gastric tissues from human subjects and animals infected with H. pylori bacteria harboring different CagA status. For laboratory studies, well-defined H. pylori strain B128 and its cancerogenic derivative strain 7.13, as well as various bacterial isogenic mutants were employed. We found that H. pylori compromises key tumor suppressor mechanisms: the host stress and apoptotic responses. Our studies showed that CagA induces phosphorylation of XIAP E3 ubiquitin ligase, which enhances ubiquitination and proteasomal degradation of the host proapoptotic factor Siva1. This process is mediated by the PI3K/Akt pathway. Inhibition of Siva1 by H. pylori increases survival of human cells with damaged DNA. It occurs in a strain-specific manner and is associated with the ability to induce gastric tumor.

Authors

Manikandan Palrasu, Elena Zaika, Wael El-Rifai, Monica Garcia-Buitrago, Maria Blanca Piazuelo, Keith T. Wilson, Richard M. Peek Jr., Alexander I. Zaika

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Figure 2

Siva1 protein regulates the apoptotic response in H. pylori–infected cells.

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Siva1 protein regulates the apoptotic response in H. pylori–infected cel...
(A) Flow cytometric analysis of apoptosis using Annexin V staining in AGS cells transfected with Siva1 siRNA or scrambled siRNA and then either left uninfected or cocultured with H. pylori strains 7.13 or B128 for 18 hours. The graph panel shows the percentage of apoptotic cells (n = 3). (B) The same as A, but Western blotting was used to analyze cleaved PARP1 and caspase-3 proteins in AGS cells. Cells transfected with scrambled siRNA were used as a control. (C) Flow cytometric analysis of apoptosis using Annexin V staining in AGS cells transfected with pcDNA3-FLAG-Siva1 expression plasmid or empty pcDNA3 vector and then either left uninfected or cocultured with H. pylori strains 7.13 or B128 for 18 hours. The graph panel shows the percentage of apoptotic cells. (D) The same as C, but Western blotting was used to analyze cleaved PARP1 and caspase-3 proteins in AGS cells. Cells transfected with empty vector (pcDNA3) were used as a control. Phosphorylation of CagA was analyzed after coculture of AGS cells with the indicated H. pylori strains for 2 hours. Statistical significance was calculated using 1-way ANOVA followed by Tukey’s multiple comparison test. Data are displayed as mean ± SE and are representative of 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Representative flow cytometry scatter plots are presented in Supplemental Figure 2. Flow cytometry and Western blot analyses were performed using the same transfected cells. Apoptosis in control cells was caused by transfection. Brackets indicate low and high exposures of Western blot images.