The epithelial cell adhesion molecule gene (EPCAM, previously known as TACSTD1 or TROP1) encodes a membrane-bound protein that is localized to the basolateral membrane of epithelial cells and is overexpressed in some tumors. Biallelic mutations in EPCAM cause congenital tufting enteropathy (CTE), which is a rare chronic diarrheal disorder presenting in infancy. Monoallelic deletions of the 3’ end of EPCAM that silence the downstream gene, MSH2, cause a form of Lynch syndrome, which is a cancer predisposition syndrome associated with loss of DNA mismatch repair. Here we report 13 novel EPCAM mutations from 17 CTE patients from two separate centers, review EPCAM mutations associated with CTE and Lynch syndrome, and structurally model pathogenic missense mutations. Statistical analyses indicate that the c.499dupC (previously reported as c.498insC) frameshift mutation was associated with more severe treatment regimens and greater mortality in CTE, whereas the c.556–14A>G and c.491+1G>A splice site mutations were not correlated with treatments or outcomes significantly different than random simulation. These findings suggest that genotype-phenotype correlations may be useful in contributing to management decisions of CTE patients. Depending on the type and nature of EPCAM mutation, one of two unrelated diseases may occur, congenital tufting enteropathy or Lynch Syndrome.

Congenital tufting enteropathy (CTE) is an autosomal recessive disease characterized by severe intestinal failure in infancy and mutations in the epithelial cell adhesion molecule ( EPCAM) gene. Previous studies of CTE in mice expressing mutant EpCAM show neonatal lethality. Hence, to study the cellular, molecular, and physiological alterations that result from EpCAM mutation, a tamoxifen-inducible mutant EpCAM enteroid model has been generated. The presence of mutant EpCAM in the model was confirmed at both mRNA and protein levels. Immunofluorescence microscopy demonstrated the reduced expression of mutant EpCAM. Mutant enteroids had reduced budding potential as well as significantly decreased mRNA expression for epithelial lineage markers ( Mucin 2, lysozyme, sucrase-isomaltase), proliferation marker Ki67, and secretory pathway transcription factors ( Atoh1, Hnf1b). Significantly decreased numbers of Paneth and goblet cells were confirmed by staining. These findings were correlated with intestinal tissue from CTE patients and the mutant mice model that had significantly fewer Paneth and goblet cells than in healthy counterparts. FITC-dextran studies demonstrated significantly impaired barrier function in monolayers derived from mutant enteroids compared with control monolayers. In conclusion, we have established an ex vivo CTE model. The role of EpCAM in the budding potential, differentiation, and barrier function of enteroids is noted. Our study establishes new facets of EpCAM biology that will aid in understanding the pathophysiology of CTE and role of EpCAM in health and disease. NEW & NOTEWORTHY Here, we develop a novel ex vivo enteroid model for congenital tufting enteropathy (CTE) based on epithelial cell adhesion molecule ( EPCAM) gene mutations found in patients. With this model we demonstrate the role of EpCAM in maintaining the functional homeostasis of the intestinal epithelium, including differentiation, proliferation, and barrier integrity. This study further establishes a new direction in EpCAM biology that will help in understanding the detailed pathophysiology of CTE and role of EpCAM.

Congenital tufting enteropathy (CTE) is a rare chronic diarrheal disease of infancy caused by mutations in epithelial cell adhesion molecule (EpCAM). Previously, a murine CTE model showed mis-localization of EpCAM away from the basolateral cell surface in the intestine. Here we demonstrate that mutant EpCAM accumulated in the endoplasmic reticulum (ER) where it co-localized with ER chaperone, GRP78/BiP, revealing potential involvement of ER stress-induced unfolded protein response (UPR) pathway in CTE. To investigate the significance of ER-localized mutant EpCAM in CTE, activation of the three UPR signaling branches initiated by the ER transmembrane protein components IRE1, PERK, and ATF6 was tested. A significant reduction in BLOS1 and SCARA3 mRNA levels in EpCAM mutant intestinal cells demonstrated that regulated IRE1-dependent decay (RIDD) was activated. However, IRE1 dependent XBP1 mRNA splicing was not induced. Furthermore, an increase in nuclear-localized ATF6 in mutant intestinal tissues revealed activation of the ATF6-signaling arm. Finally, an increase in both the phosphorylated form of the translation initiation factor, eIF2α, and ATF4 expression in the mutant intestine provided support for activation of the PERK-mediated pathway. Our results are consistent with a significant role for UPR in gastrointestinal homeostasis and provide a working model for CTE pathophysiology.

Background & Aims: Congenital Tufting Enteropathy (CTE) is an intractable diarrheal disease of infancy caused by mutation of Epithelial Cell Adhesion Molecule (EpCAM). The cellular and molecular basis of CTE pathology has been elusive. We hypothesized that the loss of EpCAM in CTE results in altered lineage differentiation and defects in absorptive enterocytes thereby contributing to CTE pathogenesis. Methods: Intestine from CTE mice was evaluated for specific markers by RT-qPCR, western blotting and immunostaining. Body weight, blood glucose and intestinal enzyme activity were also investigated. A CTE enteroid model was used to assess whether the decreased census of secretory cells could be rescued. Results: CTE mice exhibited alterations in brush-border function, disaccharidase activity and glucose absorption, potentially contributing to nutrient malabsorption and impaired weight gain. Altered cell differentiation in CTE mice led to decreased secretory cells and increased numbers of absorptive cells, though the absorptive enterocytes lacked key features, causing brush border malfunction. Further, treatment with Notch signaling inhibitor, DAPT, increased the numbers of major secretory cell types in CTE enteroids. Conclusions: Alterations in intestinal epithelial cell differentiation in CTE mice favor an increase in absorptive cells at the expense of secretory cells. Although the proportion of absorptive enterocytes is increased, they lack key functional properties. We conclude that these effects underlie pathogenic features of CTE such as malabsorption and diarrhea, and ultimately the failure to thrive seen in patients. The ability of DAPT to reverse aberrant differentiation suggests a possible therapeutic strategy.

Eosinophilic esophagitis (EoE) is a chronic type 2 allergic disease, with esophageal tissue remodeling as the mechanism behind clinical dysphagia and strictures. IL-13 is thought to be a central driver of disease, but other inflammatory factors, such as IFNs and TNF superfamily members, have been hypothesized to play a role in disease pathogenesis. We recently found that the cytokine TNFSF14/LIGHT is upregulated in the esophagus of patients with EoE and that LIGHT promotes inflammatory activity in esophageal fibroblasts. However, the global effects of LIGHT on EoE pathogenesis in vivo remain unknown. We investigated the impact of a LIGHT deficiency in a murine model of EoE driven by house dust mite allergen. Chronic intranasal challenge with house dust mite promoted esophageal eosinophilia and increased CD4+ T cell numbers and IL-13 and CCL11 production in wild-type mice. Esophageal remodeling was reflected by submucosal collagen accumulation, increased muscle density, and greater numbers of fibroblasts. LIGHT−/− mice displayed normal esophageal eosinophilia, but exhibited reduced frequencies of CD4 T cells, IL-13 expression, submucosal collagen, and muscle density and a decrease in esophageal accumulation of fibroblasts. In vitro, LIGHT increased division of human esophageal fibroblasts and selectively enhanced IL-13–mediated expression of a subset of inflammatory and fibrotic genes. These results show that LIGHT contributes to various features of murine EoE, impacting the accumulation of CD4 T cells, IL-13 production, fibroblast proliferation, and esophagus remodeling. These findings suggest that LIGHT may be, to our knowledge, a novel therapeutic target for the treatment of EoE.