Society: AGA

Background and Aims: Ménétrier’s disease (MD) is a rare acquired protein-losing hypertrophic gastropathy characterized by giant gastric rugal folds, decreased acid secretion, increased gastric mucus production, and hypoalbuminemia due to protein loss in the gastric mucosa.Microscopically, it is characterized by massive foveolar hyperplasia, oxyntic gland atrophy (loss of parietal cell), and repatterning of cell specification. MD is caused by TGF-α overexpression in the stomach and it has been shown that the EGFR inhibiting antibody, cetuximab, can reverse the histopathologic phenotypes of MD, but failed to achieve long-term remission. In a proteomic analysis of gastric tissue of Ménétrier’s disease patients before and after cetuximab treatment, we observed decreased levels of the Notch ligand, Jagged1, after cetuximab treatment. We found that Notch signaling is activated in the stomach of MD exemplified by immunostaining for Hes1, a downstream target of Notch signaling. In addition, we observed that Jagged1 expression was localized in the chief cells in the MD mouse model stomach, suggesting EGFR signaling in the chief cell is important for the pathogenesis of MD. The aim of this study was to investigate the role of Egfr in the chief cell for the pathogenesis of MD and to optimize the therapeutics by combining inhibiting antibodies against Egfr, P1X/P2X, and a Notch inhibitor, DBZ. Methods: To examine the role of Egfr in the chief cell in MD, CMV-rtTA; tetO-TGFA mouse line was crossed with Mist1^CreERT2; Egfr^flox/flox mouse line. We knocked out Egfr in chief cells by tamoxifen treatment and then overexpressed TGF-α by treating mice with doxycycline. We counted the numbers of parietal, chief, neck, and foveolar cells by immunostaining them with their respective markers: HK-ATPase, GIF, GSII, and UEA1. We also tested the therapeutic effects of P1X/P2X, DBZ, and P1X/P2X in combination with DBZ in MT-TGF- α mice. Results: Egfr knock-out in chief cell rescued the phenotypes of MD. Parietal cells reappeared, and number of foveolar cells decreased. Also, combination therapy with P1X/P2X and DBZ was more effective in rescuing the phenotypes of MD compared to P1X/P2X or DBZ monotherapy. Conclusions: Our study shows that EGFR signaling in the chief cell is necessary for the pathogenesis of MD by inducing Jagged1 that activates Notch signaling. In addition, our data suggests that P1X/P2X and DBZ combination therapy is a promising treatment option for MD patients.

The transcription factor Sox2 and the adhesion molecule CD44 have been implicated in the development of inflammation-mediated gastric metaplasia and neoplasia. Lgr5 marks gastric progenitors that regulate epithelial homeostasis. We reported that inflammation and inhibition of Bone Morphogenetic Protein (BMP) signaling, achieved by expression of the BMP inhibitor noggin (Nog) in the corpus (H⁺/K⁺-Nog mice), and by crossing mice expressing Cre and GFP in Lgr5 cells to mice with floxed alleles of BMP receptor 1A (Lgr5-Cre;Bmpr1a^flox-flox mice), activate aberrant cells that give rise to SPEM, a pre-neoplastic metaplasia characterized by the expression of neck cell mucins, and CD44 at the base of glands of the corpus. We investigated the regulation, localization, and function of Sox2 in the context of inhibition of BMP signaling. Deletion of Sox2 and Cd44 in Lgr5 cells was achieved by crossing Lgr5-Cre mice to either Cd44^flox-flox mice or to Sox2^flox-flox mice to generate Lgr5-Cre;Cd44^flox-flox and Lgr5-Cre;Sox2^flox-flox mice. The role of Lgr5 cell-Cd44 and of Lgr5 cell-Sox2 in Nog-induced SPEM, was assessed by crossing Lgr5-Cre;Cd44^flox-flox and Lgr5-Cre;Sox2^flox-flox mice to H⁺/K⁺-Nog mice to generate Lgr5-Cre;Cd44^flox-flox;H⁺/K⁺-Nog and Lgr5-Cre;Sox2^flox-flox;H⁺/K⁺-Nog mice. Infection with H. felis (HF) was used to induce inflammation. Distribution of GFP-, Sox2-, and CD44-+ve cells was analyzed by immunostaining. Single cell RNA sequencing (scRNA-seq) was performed in both control and H⁺/K⁺-Nog mice. Cluster assignments relative to marker gene expression defined eighteen clusters. Transcriptomes of individual cells from these clusters were compared from these mice to identify differentially expressed genes by cluster. ChIP Enrichment Analysis (ChEA) was performed using the ChIP-X database to identify enriched transcription factors target genes. Sox2+ve staining was detected in the antrum but not in the corpus of wild type C57B/6 mice. In contrast, robust expression of Sox2 was observed in the presence of inhibition of BMP signaling in GFP+ve/Lgr5 cells located in the oxyntic mucosa of the lesser curvature of Lgr5-Cre;Bmr1a^flox-flox mice, an effect which was enhanced by HF. Similarly, Sox2 expression was seen in the corpus of H⁺/K⁺-Nog mice in CD44+ve cells. scRNA-seq analysis demonstrated an increase in the percentage of cells expressing Sox2 transcripts in the neck cell cluster of H⁺/K⁺-Nog mice. ChEA indicated over-representation of Sox2 target genes in the ChIP-X database of the mouse gastric mucosa highlighting the functional relevance of Sox2. Deletion of either Cd44 or Sox2 in Lgr5 cells attenuated the SPEM phenotype, underscoring the importance of these molecules in the development of metaplasia. Our findings demonstrate the significance of BMP signaling in the pathophysiology of metaplasia and in the regulation of Sox2 in the stomach.

Gastric carcinogenesis is correlated with the presence of metaplasia and its progression. Therefore, understanding the events that lead to metaplasia progression remains a priority. Pyloric metaplasia characterized by the presence of spasmolytic polypeptide-expressing metaplasia (SPEM) cells is the earliest type of metaplasia seen in the stomach after parietal cell loss. Using transgenic Mist1-Kras mice, we have established murine SPEM gastroid lines (Meta1) from metaplastic glands isolated at 1 month after induction of active KRas (G12D). Meta1 gastroid lines maintain their SPEM cell features across multiple passages in vitro. Moreover, single cell RNA-sequencing data revealed that Meta1 have a distinctive transcriptional profile in comparison with mouse dysplastic gastroids (Meta4), established previously. Recent investigations from our laboratory have shown an increased number of intrinsic immune cells, type 2 innate lymphoid cells (ILC2s), in pyloric metaplasia tissues of both human and mouse. ILC2s are known to release cytokines including IL-13 and we demonstrated that IL-13 could promote SPEM lineage cell development in mice. We therefore studied the effects of IL-13 in normal gastroids by treatment with either recombinant IL-13 or 0.1% BSA. IL-13-treated gastroids showed an increase in the proportion of MUC6-, CD44v9- or AQP5-positive SPEM cells (Fig 1A). Immunoblotting showed up-regulation of AQP5 and STAT6 phosphorylation (pSTAT6) in normal gastroids after the IL-13 treatment. We also observed that co-culture of Meta1 with isolated mouse ILC2s promoted gastroid growth by increasing the number of TFF2- and AQP5-co-expressing SPEM cells and proliferative Ki67-positive cells. Immunoblotting also showed upregulation of STAT6 phosphorylation in the co-culture condition. The IL-13 also increased in organoid size of Meta1 gastroids. MUC6-positive SPEM cells and proliferative Ki-67-positive cells were also increased in IL-13-treated Meta1 cells. Immunoblotting showed pSTAT6 in Meta1 gastroids treated with IL-13 in a dose-dependent manner; and RT-qPCR revealed a significant increase in the expression of SPEM-related genes. We additionally observed that IL-13 receptor a-1 (IL-13Ra-1) is expressed in the basolateral membranes of epithelial cells from the stomach corpus at 1 month after induction of active KRas (G12D) (Fig 1B). The expression is consistently observed in the stomachs with intestinal metaplasia (IM) and dysplasia at 3 and 4 months after induction, respectively. When IM and dysplastic gastroids (Meta3 and Meta4, respectively) were treated with either IL-13 or BSA, we observed that IL-13 treatment caused the phosphorylation of STAT6 (Fig 1C). These results suggest that IL-13 could play important roles in different stages of the stomach carcinogenesis.

<b>Figure 1. IL-13 effects in mouse gastroids. A)</b> Left panel, Immunofluorescence staining for SPEM markers (Muc6 and AQP5) in normal gastroids, showing an increase of SPEM cell marker in IL-13-treated gastroids. Right panel, Immunoblotting showing an up-regulation of AQP5 and phosphorylated STAT6 after treatment with IL-13. <b>B)</b> Immunofluorescence staining for IL-13Ra1 and P120 (epithelial cell marker) in Mist1-Kras mouse stomach tissues after 1, 3 or 4 months of induction. <b>C)</b> Immunoblotting showing an up-regulation of phosphorylated STAT6 after treatment with IL-13 in Meta3 (IM) and Meta4 (dysplasia) gastroids.

Figure 1. IL-13 effects in mouse gastroids. A) Left panel, Immunofluorescence staining for SPEM markers (Muc6 and AQP5) in normal gastroids, showing an increase of SPEM cell marker in IL-13-treated gastroids. Right panel, Immunoblotting showing an up-regulation of AQP5 and phosphorylated STAT6 after treatment with IL-13. B) Immunofluorescence staining for IL-13Ra1 and P120 (epithelial cell marker) in Mist1-Kras mouse stomach tissues after 1, 3 or 4 months of induction. C) Immunoblotting showing an up-regulation of phosphorylated STAT6 after treatment with IL-13 in Meta3 (IM) and Meta4 (dysplasia) gastroids.

Su-Hyung Lee

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