Society: AGA

Introduction. Our previous study reported a significant increase in metachronous recurrence of early gastric cancer after endoscopic submucosal dissection due to CD44v9-positive cancer stem-like cells in H. pylori-infected stomach tissue (Br. J. Cancer 109:379, 2013). Additionally, we demonstrated development of CD44v9-positive cells due to overexpression of capping actin protein of muscle Z-line alpha subunit 1 (CAPZA1) by cells that accumulated H. pylori-derived CagA oncoprotein (Autophagy 15:242, 2019; Cell. Mol. Gastroenterol. Hepatol. 8:319, 2019); CAPZA1 expression is significantly increased by histone deacetylase (HDAC) inhibitors (Autophagy 15:242, 2019). Short-chain fatty acids (SCFAs), a subset of key gut-commensal metabolites, inhibit HDAC (J. Biol. Chem. 253:3364, 1978) and may induce CAPZA1 overexpression in H. pylori-infected gastric mucosa, resulting in the development of CD44v9-positive cells. To test this, we examined whether SCFAs mediate production of CD44v9-positive cells via upregulation of CAPZA1 expression in H. pylori infection, and searched for SCFA-producing bacteria in gastric cancer patients by a Taq amplified (TA) cloning and sequencing technique using a 16S rRNA library of gastric commensal bacteria.
Methods. A 16S rRNA library derived from gastric juice was constructed by TA cloning and sequenced by the Sanger method. H. pylori G27 and G27 cagPAI-deletion mutant strains were used. Gastric epithelial monolayers (mucosoids) were made from gastric organoids (Gut 68:400, 2019).
Results. Butyrate enhanced CAPZA1 expression in AGS cells and mucosoids via enhanced histone acetylation at the CAPZA1 promoter region. CD44v9 expression was induced in CAPZA1-overexpressing cells accumulating CagA secreted by H. pylori. CD44v9-positive cells expressed LGR5, KLF5, and SALL4, suggesting that butyrate imparts stem-cell properties to CD44v9-expressing cells. Specific SCFA-producing bacteria, Streptococcus spp., in stomachs of gastric cancer patients were identified as non-Helicobacter bacteria and their 16S rRNA levels were significantly higher in gastric cancer patients than in controls (AUROC = 0.83 [95% CI, 0.65–1.00]). Butyrate synthesizing gene butCoAT expression in Streptococcus spp. was significantly greater by 2-fold compared with that in E. coli, indicating that Streptococcus spp. have a superior ability to produce butyrate. Culture supernatant of Streptococcus spp. increased expression of CAPZA1 in AGS cells, suggesting that co-infection by Streptococcus spp. and H. pylori increases the risk of CD44v9-positive cancer-stem cell production.
Conclusion. Butyrate produced by Streptococcus spp. fosters development of CD44v9-positive cancer stem-like cells in H. pylori-infected stomach tissue through the enhancement of CAPZA1 expression.

Helicobacter pylori is the strongest known risk factor for gastric cancer, and pathologic outcomes are mediated via complex interactions among H. pylori virulence determinants, host immune responses, and the exposome. One virulence determinant that augments cancer risk is the cag pathogenicity island (PAI), and strains harboring this constituent induce more severe gastric injury. The cag PAI encodes a type IV secretion system (T4SS) that translocates the oncoprotein, CagA, into host cells and activates signaling cascades that mediate neoplastic transformation. One host factor that is a key transcriptional regulator of immunity and carcinogenesis is HIF-1α. To define the role of this effector in gastric carcinogenesis in humans, levels of HIF-1α were assessed in gastric biopsies from patients with non-atrophic gastritis (N=8), multifocal atrophic gastritis (N=10), intestinal metaplasia (N=14), and gastric cancer (N=10). Levels of HIF-1α significantly increased in parallel with increasing severity of gastric lesions, with highest expression levels observed in patients with gastric cancer. In interventional studies targeting HIF-1α in H. pylori-induced inflammation and injury in vivo, C57BL/6 mice were treated with or without dimethyloxalylglycine (DMOG), a cell-permeable prolyl hydroxylase (PHD) inhibitor that can decrease HIF-1α-induced inflammation, for one week prior to infection with the H. pylori cag⁺ strain PMSS1. Mice were euthanized eight weeks post-challenge and gastric tissue was harvested for quantitative culture, immunohistochemistry, histopathology, and chemokine/cytokine analyses. DMOG treatment had no effect on H. pylori colonization. As expected, H. pylori induced gastric inflammation in vehicle-treated mice; however, this was significantly attenuated (P<0.05) in H. pylori-infected mice pretreated with DMOG. Similarly, H. pylori significantly increased mucosal levels of inflammatory chemokines (KC: 1.5-fold; MIP-1β: 1.9-fold; RANTES: 1.6-fold) and cytokines (INF-γ: 1.8-fold; IL-1β: 3.8-fold; IL-6: 1.9-fold) in vehicle-treated mice relative to uninfected controls, but this response was significantly inhibited (P<0.05) in DMOG-treated mice infected with H. pylori. Further, DMOG treatment significantly attenuated H. pylori cag T4SS function in vivo, whereby 50% of DMOG-adapted isolates lost the ability to translocate CagA compared to only 20% of isolates from vehicle-treated mice. In vitro mechanistic studies demonstrated that DMOG treatment also significantly reduced (P<0.01) cag T4SS-mediated NF-κB activation and IL-8 induction. Collectively, these data indicate that PHD inhibition targeting HIF-1α is protective against the development of pathologic responses that arise within the context of H. pylori infection, and is mediated, in part, via attenuation of H. pylori virulence as well as suppression of host proinflammatory responses.

Cormac Taylor

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