Claudin-2 protects against colitis-associated cancer by promoting colitis-associated mucosal healing
Rizwan Ahmad, Balawant Kumar, Ishwor Thapa, Raju Lama Tamang, Santosh K. Yadav, Mary K. Washington, Geoffrey A. Talmon, Alan S. Yu, Dhundy K. Bastola, Punita Dhawan, Amar B. Singh
Rizwan Ahmad, Balawant Kumar, Ishwor Thapa, Raju Lama Tamang, Santosh K. Yadav, Mary K. Washington, Geoffrey A. Talmon, Alan S. Yu, Dhundy K. Bastola, Punita Dhawan, Amar B. Singh
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Research Article Gastroenterology

Claudin-2 protects against colitis-associated cancer by promoting colitis-associated mucosal healing

Abstract

Patients with inflammatory bowel disease (IBD) are susceptible to colitis-associated cancer (CAC). Chronic inflammation promotes the risk for CAC. In contrast, mucosal healing predicts improved prognosis in IBD and reduced risk of CAC. However, the molecular integration among colitis, mucosal healing, and CAC remains poorly understood. Claudin-2 (CLDN2) expression is upregulated in IBD; however, its role in CAC is not known. The current study was undertaken to examine the role for CLDN2 in CAC. The AOM/DSS-induced CAC model was used with WT and CLDN2-modified mice. High-throughput expression analyses, murine models of colitis/recovery, chronic colitis, ex vivo crypt culture, and pharmacological manipulations were employed in order to increase our mechanistic understanding. The Cldn2KO mice showed significant inhibition of CAC despite severe colitis compared with WT littermates. Cldn2 loss also resulted in impaired recovery from colitis and increased injury when mice were subjected to intestinal injury by other methods. Mechanistic studies demonstrated a possibly novel role of CLDN2 in promotion of mucosal healing downstream of EGFR signaling and by regulation of Survivin expression. An upregulated CLDN2 expression protected from CAC and associated positively with crypt regeneration and Survivin expression in patients with IBD. We demonstrate a potentially novel role of CLDN2 in promotion of mucosal healing in patients with IBD and thus regulation of vulnerability to colitis severity and CAC, which can be exploited for improved clinical management.

Authors

Rizwan Ahmad, Balawant Kumar, Ishwor Thapa, Raju Lama Tamang, Santosh K. Yadav, Mary K. Washington, Geoffrey A. Talmon, Alan S. Yu, Dhundy K. Bastola, Punita Dhawan, Amar B. Singh

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Figure 7

CLDN2 overexpression in the intestinal epithelium protects mice from colitis-associated cancer.

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CLDN2 overexpression in the intestinal epithelium protects mice from col...
(A) Graphical illustration of AOM/DSS tumorigenesis in Villin-Cldn2TG and WT mice. (B) Colon length (cm; WT/Villin-Cldn2TG: n = 24/31). (C) Colon edema (g/cm; WT/Villin-Cldn2TG: n = 24/31). (D and E) Tumor growth in the colon and representative images of the AOM/DSS-treated Villin-Cldn2TG and WT mouse colon (WT/Villin-Cldn2TG: n = 24/31). (F and G) H&E analysis and mucosal injury score (WT/Villin-Cldn2TG: n = 24/37). (H) Percentage of dysplasia in mice treated with AOM/DSS. (I) RNA-Seq analysis (differentially regulated genes [DEGs]) in mice subjected to AOM/DSS treatment (n = 3/group). (J) Significantly altered KEGG pathways and GO biological function in AOM/DSS-treated Villin-Cldn2TG and WT mice (n = 3/group). (K) Heatmap depicting the DEGs associated with cell cycle, apoptosis, inflammation, and immune homeostasis (n = 3/group). (L and M) Immunoblotting and densitometric analysis for Stat3 and NF-κB activation in mice subjected to AOM/DSS (n = 4/group). (N and O) High-throughput analysis for cytokine expression using total colon lysate and densitometry evaluations (n = 3/group). (P) Cartoon summarizing the role of upregulated CLDN2 in CAC. Data in B–D, G, and M are presented as the mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 by 2-tailed unpaired t test. Data in H are presented as percentage number. ****P < 0.0001 by Fisher’s exact test. Data in O are presented as the mean ± SEM. ****P < 0.0001 by 1-way ANOVA with Dunnett’s multiple comparisons. Scale bar: 200 μM.