Characterization of epigenetic changes during the metaplastic process by single-cell RNA sequencing

Abstract

Adenocarcinomas, the most common cancers in adults, typically arise from precursor lesions that are induced by chronic injury or inflammation. As normal cells first become metaplastic and then progress to dysplasia and neoplasia, they dramatically change phenotype. Cellular identity and lineage determination throughout development are in large part regulated by epigenomic changes. Given tumorigenesis is a type of abnormal development, it follows that similar epigenetic changes occur during metaplasia; however, research on this remains relatively scant. Here, we investigate epigenetic changes in mature, digestive-enzyme-secreting gastric chief cells as they reprogram to progenitor-like, mucinous Spasmolytic Polypeptide Expressing Metaplasia (SPEM) cells. The reprogramming process occurs via a stereotypical, evolutionarily conserved sequence of cellular and molecular events known as paligenosis. We can induce chief cell paligenosis across the whole body of the stomach by intraperitoneal injections of high-dose tamoxifen (HDT), which kills acid-pumping parietal cells and causes SPEM within 48 to 72 hous. During paligenosis, cells undergo massive ultrastructural remodeling including recycling of the vast majority of the endoplasmic reticulum (ER) that they used for secreting zymogenic enzymes, and they induce expression of a number of mucins as well as progenitor-associated genes like Sox9. Thereafter, they re-enter the cell cycle to help regenerate injured tissue. To begin to identify epigenetic factors regulating the large changes in cell structure and gene expression, we are contrasting single-cell RNA-sequencing analysis of HDT-treated mouse gastric corpus versus mice without HDT treatment that does not cause SPEM. The sc-RNA-seq confirms the loss of parietal cells in both models and SPEM predominantly in HDT. We observed the induction of multiple chromatin-modifying genes during paligenosis with few such epigenetic genes expressed in mature chief cells at homeostasis, suggesting epigenetic processes might be more inert within the fully differentiated chief cells than during the dynamic paligenosis process as cells are actively rearranging phenotype. Complementing sc-RNA-seq with proteomic analysis by mass spectrometry, we have identified the DNA methyltransferase DNMT1 as a key epigenetic factor upregulated during paligenosis. DNMT1 has been previously shown to be associated with metaplasia in the stomach of patients infected with H. pylori, where both metaplasia and infection are major risk factors for gastric cancer. We confirm with immunostaining that DNMT1 is absent in chief cells at homeostasis but undergoes dramatic increase after HDT treatment. In summary, using single-cell transcriptomic methods, we are demonstrating active epigenetic-related gene expression changes during paligenosis and are following up on one DNA-modifying gene in particular, the DNA methyltransferase, DNMT1, that emerges during this process a This work was supported by funding from the NIH through the National Institute of Diabetes and Digestive and Kidney Diseases and the National Cancer Institute. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.