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 6

Inhibiting EGFR signaling in WT mice recovering from DSS-induced colitis inhibits CLDN2 upregulation and impairs mucosal healing.

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Inhibiting EGFR signaling in WT mice recovering from DSS-induced colitis...
(A) Differentially expressed genes (DEGs; RNA-Seq analysis) in mice recovering from DSS-induced colitis show upregulated Cldn2 expression, along with Egf and Ereg (EGFR ligands) (n = 3/group). (B) Immunoblot analysis using Caco-2 cells subjected to DSS-induced injury/repair with or without inhibitors of EGFR activation (n = 3 independent experiments). (C) Km analysis for survival in mice receiving EGFR inhibitor (gefitinib) during recovery from colitis (vehicle/gefitinib: n = 9/13). (D) Colon thickness (g/cm; vehicle/gefitinib: n = 8/11). (E) H&E images showing profound mucosal injury/impaired regeneration in gefitinib-treated mice and (F) mucosal injury score (vehicle/gefitinib: n = 8/9). (G and H) Immunoblot analysis using total colon lysate. pErk1/2/Erk1/2 and CLDN2 expression served as a marker of EGFR activation (water/DSS/vehicle/gefitinib: n = 2/3/3/3). (I–K) Immunoblot analysis for the markers of inflammation, cell survival, and apoptosis in gefitinib- and vehicle-treated mice during recovery from colitis (vehicle/gefitinib: n = 4/5). (L) Proliferative index (vehicle/gefitinib: n = 4/5). (M) Epithelial regenerative index in gefitinib-treated mice versus vehicle-treated WT mice (vehicle/gefitinib: n = 8/9). (N) Graphical summary depicting integration between EGFR and CLDN2 in colitis-associated epithelial restitution/healing. Survival data in C were assessed by log-rank (Mantel-Cox) test. Data in D, F, and K–M are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 2-tailed unpaired t test. Scale bar: 100 μM.