O-038 Loss of Intestinal Mucin-type O-glycans Promotes Inflammation-Associated Colon Cancer in an Inflammasome-Dependent Manner

Abstract

BACKGROUND:: Mucin-type O-linked oligosaccharides (O-glycans) are major components of mucins, which form a protective barrier against microbiota-induced inflammation in the gut. How, and to what extent, mucin-type O-glycans contribute to intestinal homeostasis is still unclear. The two major forms of mucin-type O-glycans are core 1 and 3-derived O-glycans. We previously demonstrated core 3 O-glycan-deficient mice are susceptible to chemically-induced colitis and cancer. More recently we showed mice lacking core 1 O-glycans specifically in gut epithelial cells develop spontaneous colitis resembling human ulcerative colitis (UC) due to impaired mucus barrier function. Importantly, longstanding UC is an important risk factor for colorectal cancer and is associated with O-glycosylation defects, although a causal relationship has yet to be demonstrated. We therefore investigated whether loss of intestinal mucin-type O-glycans would increase risk for spontaneous inflammation-driven colon cancer in mice.METHODS:: We utilized mice conditionally lacking core 1 O-glycans in intestinal epithelium, and generated mice deficient in both intestinal core 1 and 3-derived O-glycans (called "DKO" mice), thus missing all major gut epithelial O-glycans. For mechanistic studies, we developed DKO mice lacking caspase 1 and 11 and therefore deficient in inflammasome signaling (DKO/Casp1/11 or DKO mice). Wild-type (WT), DKO, and DKO mice were sacrificed at 3, 5 and 9 months of age (n = 3-5 per time point). Colon tissues were analyzed by gross examination of tumor burden, as well as histology, immunostaining, western blotting, and mRNA expression and ELISA for cellular and molecular markers of colitis, inflammasome activation, and cancer.RESULTS:: We found loss of intestinal epithelial core 1-derived O-glycans led to severe unremitting colitis that progressed to spontaneous colorectal cancer between 12 and 18 months of age. Colitis severity increased and cancer progression was accelerated upon additional deletion of core 3-derived O-glycans (i.e., DKO mice), likely due to complete ablation of colonic mucus, indicating a moderate protective role for core 3 O-glycans in the absence of core 1. The colitis in intestinal core 1 O-glycan-deficient and DKO mice was highly associated with extensive epithelial proliferation, neutrophil infiltration, and DNA damage. These lesions were dependent upon microbiota-driven inflammation, as antibiotic-mediated microbial depletion reduced each parameter, including adenocarcinoma. Efforts to elucidate a mechanism revealed colitis and cancer in DKO mice was associated with microbiota-dependent constitutive inflammasome activation and IL1β and IL18 production in inflamed and neoplastic tissues versus WT mice. We confirmed a role for inflammasomes using DKO mice, which showed significantly reduced colitis, DNA damage and tumor incidence relative to DKO mice. Caspase 1 and 11 expression was high in colonic epithelial cells compared to immune cells, and BM-chimera studies suggested epithelial inflammasomes were critical for colitis and cancer progression in the absence of mucin-type O-glycans.CONCLUSIONS:: Impairment of mucus barrier function caused by loss of mucin-type O-glycans leads to microbiota-dependent colitis and inflammation-associated cancer in a manner mediated in part by epithelial inflammasomes. These findings provide novel insight into the importance of mucin-type O-glycans for colonic homeostasis and consequences of defects in fundamental O-glycosylation pathways reported in UC patients.