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Gastroduodenal
Precancerous nature of intestinal metaplasia with increased chance of conversion and accelerated DNA methylation
  1. Chihiro Takeuchi1,2,3,
  2. Satoshi Yamashita1,4,
  3. Yu-Yu Liu1,2,
  4. Hideyuki Takeshima1,2,
  5. Akiko Sasaki1,5,
  6. Masahide Fukuda1,6,
  7. Taiki Hashimoto7,
  8. Tomoaki Naka7,
  9. Kenichi Ishizu8,
  10. Shigeki Sekine7,
  11. Takaki Yoshikawa8,
  12. Akinobu Hamada9,
  13. Nobutake Yamamichi3,10,
  14. Mitsuhiro Fujishiro3,
  15. Toshikazu Ushijima1,2
  1. 1 Division of Epigenomics, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan
  2. 2 Department of Epigenomics, Institute for Advanced Life Sciences, Hoshi University, Shinagawa-ku, Tokyo, Japan
  3. 3 Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
  4. 4 Department of Biotechnology, Maebashi Institute of Technology, Maebashi, Gunma, Japan
  5. 5 Gastroenterology Medicine Center, Shonan Kamakura General Hospital, Kamakura, Kanagawa, Japan
  6. 6 Department of Gastroenterology, Faculty of Medicine, Oita University, Oita, Oita, Japan
  7. 7 Department of Diagnostic Pathology, National Cancer Center Hospital, Chuo-ku, Tokyo, Japan
  8. 8 Department of Gastric Surgery, National Cancer Center Hospital, Chuo-ku, Tokyo, Japan
  9. 9 Division of Molecular Pharmacology, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan
  10. 10 Center for Epidemiology and Preventive Medicine, The University of Tokyo Hospital, Bunkyo-ku, Tokyo, Japan
  1. Correspondence to Dr Toshikazu Ushijima, Hoshi University, Shinagawa-ku, Tokyo, Japan; tushijima142{at}hoshi.ac.jp

Abstract

Objective The presence of intestinal metaplasia (IM) is a risk factor for gastric cancer. However, it is still controversial whether IM itself is precancerous or paracancerous. Here, we aimed to explore the precancerous nature of IM by analysing epigenetic alterations.

Design Genome-wide DNA methylation analysis was conducted by EPIC BeadArray using IM crypts isolated by Alcian blue staining. Chromatin immunoprecipitation sequencing for H3K27ac and single-cell assay for transposase-accessible chromatin by sequencing were conducted using IM mucosa. NOS2 was induced using Tet-on gene expression system in normal cells.

Results IM crypts had a methylation profile unique from non-IM crypts, showing extensive DNA hypermethylation in promoter CpG islands, including those of tumour-suppressor genes. Also, the IM-specific methylation profile, namely epigenetic footprint, was present in a fraction of gastric cancers with a higher frequency than expected, and suggested to be associated with good overall survival. IM organoids had remarkably high NOS2 expression, and NOS2 induction in normal cells led to accelerated induction of aberrant DNA methylation, namely epigenetic instability, by increasing DNA methyltransferase activity. IM mucosa showed dynamic enhancer reprogramming, including the regions involved in higher NOS2 expression. NOS2 had open chromatin in IM cells but not in gastric cells, and IM cells had frequent closed chromatin of tumour-suppressor genes, indicating their methylation-silencing. NOS2 expression in IM-derived organoids was upregulated by interleukin-17A, a cytokine secreted by extracellular bacterial infection.

Conclusions IM cells were considered to have a precancerous nature potentially with an increased chance of converting into cancer cells, and an accelerated DNA methylation induction due to abnormal NOS2 expression.

  • methylation
  • gastric cancer
  • Helicobacter pylori
  • nitric oxide
  • molecular biology

Data availability statement

Data are available in a public, open access repository. The data of DNA methylation (accession #GSE220511; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE220511),29 ChIP-seq (accession #GSE220681; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE220681)39 and single-cell ATAC-seq (accession #GSE221292; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE221292)43 are deposited in the Gene Expression Omnibus database (GEO) database. Genome-wide DNA methylation profiles of small intestinal crypts (accession #GSE141254; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE141254),18 blood cells (accession #GSE35069; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE35069),19 biopsy samples (accession #GSE103186; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE103186),20 human cell lines (accession #GSE68379; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE68379),21 primary gastric cancers and the corresponding adjacent mucosa (accession #GSE127857; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE127857, and #GSE164988; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE164988)22 23 were obtained from the GEO. Single-cell RNA-seq data of IM mucosa samples (accession #GSE134520; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE134520)24 was obtained from the GEO.

https://doi.org/10.1136/gutjnl-2023-329492

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    Data availability statement

    Data are available in a public, open access repository. The data of DNA methylation (accession #GSE220511; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE220511),29 ChIP-seq (accession #GSE220681; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE220681)39 and single-cell ATAC-seq (accession #GSE221292; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE221292)43 are deposited in the Gene Expression Omnibus database (GEO) database. Genome-wide DNA methylation profiles of small intestinal crypts (accession #GSE141254; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE141254),18 blood cells (accession #GSE35069; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE35069),19 biopsy samples (accession #GSE103186; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE103186),20 human cell lines (accession #GSE68379; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE68379),21 primary gastric cancers and the corresponding adjacent mucosa (accession #GSE127857; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE127857, and #GSE164988; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE164988)22 23 were obtained from the GEO. Single-cell RNA-seq data of IM mucosa samples (accession #GSE134520; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE134520)24 was obtained from the GEO.

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    Footnotes

    • Contributors Conception and design: CT, SY and TU. Development of methodology: CT, SY, Y-YL, HT and MF. Collecting clinical samples: CT, AS, TH, TN, KI, SS, TY and AH. Acquisition of data: CT, Y-YL and HT. Analysis and interpretation of data: CT, Y-YL and TU. Writing and review of the manuscript: CT, NY, MF and TU.

    • Funding This research was supported by AMED-CREST [JP23gm1310006], AMED [JP23ck0106804], and JSPS KAKENHI [21K15941].

    • Competing interests None declared.

    • Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.