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Inactivation of the tumor suppressor gene Apc synergizes with H. pylori to induce DNA damage and replication stress in murine gastric stem and progenitor cells [scRNA-seq]

ABSTRACT: Helicobacter pylori infection is a major risk factor for the development of gastric cancer. The bacteria reside in close proximity to gastric surface mucous as well as stem and progenitor cells. Here, we take advantage of wild type and genetically engineered murine gastric organoids and organoid-derived monolayers to study the cellular targets of H. pylori-induced DNA damage and replication stress, and to explore possible interactions with preexisting gastric cancer driver mutations. We find using alkaline comet assay, single molecule DNA fiber assays and immunofluorescence microscopy of DNA repair factors that H. pylori induces transcription-dependent DNA damage in actively replicating, Lgr5-positive antral stem and progenitor cells and their Troy-positive corpus counterparts, but not in other gastric epithelial lineages. Infection-dependent DNA damage is aggravated by Apc inactivation, but not by Trp53 or Smad4 loss, or Erbb2 overexpression. Our data suggest that H. pylori induces DNA damage in stem and progenitor cells, especially in settings of hyperproliferation due to constitutively active Wnt signaling.

ORGANISM(S): Mus musculus

PROVIDER: GSE225379 | GEO | 2023/07/24

REPOSITORIES: GEO

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Inactivation of the tumor suppressor gene Apc synergizes with H. pylori to induce DNA damage and replication stress in murine gastric stem and progenitor cells

Project description:Helicobacter pylori infection is a major risk factor for the development of gastric cancer. The bacteria reside in close proximity to gastric surface mucous as well as stem and progenitor cells. Here, we take advantage of wild type and genetically engineered murine gastric organoids and organoid-derived monolayers to study the cellular targets of H. pylori-induced DNA damage and replication stress, and to explore possible interactions with preexisting gastric cancer driver mutations. We find using alkaline comet assay, single molecule DNA fiber assays and immunofluorescence microscopy of DNA repair factors that H. pylori induces transcription-dependent DNA damage in actively replicating, Lgr5-positive antral stem and progenitor cells and their Troy-positive corpus counterparts, but not in other gastric epithelial lineages. Infection-dependent DNA damage is aggravated by Apc inactivation, but not by Trp53 or Smad4 loss, or Erbb2 overexpression. Our data suggest that H. pylori induces DNA damage in stem and progenitor cells, especially in settings of hyperproliferation due to constitutively active Wnt signaling.

2023-07-24 | GSE225189 | GEO

Expression profiling of Troy positive gastric cells

Project description:Proliferation of the self-renewing epithelium of the gastric corpus occurs almost exclusively in the isthmus of the glands, from where cells migrate bi-directionally towards pit and base. The isthmus is therefore generally viewed as the stem cell zone. We find that the stem cell marker Troy is expressed at the gland base by a small subpopulation of chief cells. By lineage tracing using a Troy-eGFP-ires-CreERT2 allele, single marked cells are shown to generate entirely labeled gastric units over periods of months. This phenomenon accelerates upon tissue damage. Troy+ chief cells can be cultured to generate long-lived gastric organoids. Troy marks a specific, 'plastic' subset of differentiated chief cells capable of replenishing entire gastric units, essentially serving as a quiescent ‘reserve’ stem cell. An EGFP-ires-CreERT2 cassette was introduced into the transcriptional start side of Tnfrsf19 (Troy), resulting in the expression of EGFP and CreERT2 under the control of endogenous Troy-regulatory sequences. Troy is expressed in basally located chief and parietal cells. Using FACS, Troy positive chief and parietal cells can be isolated separately. Expression profiling was performed on these two cell types: three arrays of sorted chief cells, two arrays of sorted parietal cells. As a reference, two arrays of separately isolated whole gastric corpus glands were added. In addition, single sorted Troy positive chief cells can be placed in culture, forming 3D cultures called organoids. Three arrays were generated from different organoid cultures, one time in normal medium containing the factors EGF, Gastrin, FGF10, Noggin, Wnt3a and Rspondin (EGFNWR medium) and one time in medium containing only EGF and Rspondin (ER medium).

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Expression data from Helicobacter pylori-infected mouse gastric epithelial progenitor and non-progenitor cells.

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Gastric Lgr5+ cells are programmed by Rspo3 to shield the gland from infection

Project description:R-spondin (Rspo) signaling is crucial for stem cell renewal and tissue homeostasis in the gastrointestinal tract. In the stomach, Rspo is secreted from myofibroblasts and controls epithelial gland regeneration by inducing proliferation of Wnt-responsive Axin2+ cells in the isthmus of the gland. Infection with H. pylori results in increased expression of stromal Rspo, leading to an expansion of Axin2+ isthmus stem cells and gland hyperplasia. Lgr5+ cells in the gland base are exposed to Rspo3 but the effects of this are not well understood. Here we demonstrate that apart from its activity as a mitogen, endogenous Rspo3 regulates gene expression of Lgr5+ cells in the gastric gland base. Surprisingly, Rspo3 induces differentiation within the Lgr5+ compartment towards secretory deep mucous cells. Moreover, the Rspo3-Lgr5 axis turns out to be a stimulus of epithelial antimicrobial defense. Infection with H. pylori induces a strong antimicrobial response, with Lgr5+ cells expressing antimicrobial compounds that are secreted into the lumen in an Rspo3-dependent manner. Depletion of Lgr5+ cells or knockout of Rspo3 in myofibroblasts leads to hyper-colonization of gastric glands, including the stem cell compartment, whereas systemic application of recombinant Rspo clears H. pylori from the glands. We provide an intriguing, unexpected feature of the Rspo3-Lgr5 axis in the stomach, exhibiting antimicrobial self-protection of the gland to protect the stem cell compartment from invading pathogens.

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Effect of Helicobacter pylori on ATM-dependent DNA damage responses [biopsy]

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Transcription profiling of Helicobacter pylori-infected mouse gastric epithelial progenitor and non-progenitor cells.

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