INHIBITION OF DAPK3, PINPOINTED AS A KEY FACTOR IN THE DEVELOPMENT OF COLITIS-ASSOCIATED DYSPLASIA, INCREASES SEVERITY OF DSS-INDUCED COLITIS VIA HIPPO SIGNALING

Abstract

Abstract Introduction Ulcerative colitis (UC) is a progressive disorder that elevates the risk of cancer development through a colitis-dysplasia-carcinoma sequence. Recent evidence demonstrates the necessity of Hippo-YAP/TAZ signaling, interceded by cytoskeletal remodeling, for intestinal regeneration. Death-associated protein kinase 3 (DAPK3) is a regulator of actin-cytoskeleton reorganization that controls proliferation and apoptosis. We first identified DAPK3 as a candidate gene involved in UC progression, and then examined if inhibition of DAPK3 would increase the severity of colitis. Methods Data series GSE47908 [1] comprising microarray expression profile of biopsies from left colon of healthy controls and UC patients with left-sided colitis, pancolitis, or UC-associated dysplasia was retrieved from Gene Expression Omnibus. Differentially expressed genes (DEGs) were analyzed using Ingenuity Pathway Analysis software and topGO R. C57BL/6 mice receiving 2.5% (wt./vol.) DSS or water (7 days) were treated daily s.c. with the DAPK3 pharmacological inhibitor HS38 [2] or vehicle for 12 days (pretreatment: 3 days, recovery: 2 days). Colitis was assessed by disease activity index and histopathology. Intestinal epithelial cell (IEC) proliferation was enumerated via Ki-67 immunohistochemistry (IHC). DAPK3 and YAP abundances were evaluated by immunoblot, and YAP subcellular localization was assessed by IHC. Results topGO analysis showed enrichment of inflammatory processes in pancolitis Π left-side colitis DEGs and enrichment of actin-based processes in pancolitis Π dysplasia DEGs. Pathway analysis revealed trend of Hippo signaling activation as UC progressed from left-side to pancolitis to UC-associated dysplasia. Network analysis implicated DAPK3 as a key factor in colitis-dysplasia progression. Mice treated with DSS showed decreased colonic DAPK3 abundance. Mice co-treated with DSS/HS38 showed significantly increased histological score, and decreased IEC proliferation in comparison with DSS/vehicle controls. Moreover, H2O/HS38 treated mice showed increased colonic YAP abundance versus H2O/vehicle controls while DSS/HS38 co-treatment elicited nuclear accumulation of YAP in IECs localized at the crypt apex. Conclusion DAPK3 is a key factor in intestinal regeneration and UC progression by way of Hippo-YAP signaling. This is potentially arbitrated by DAPK3-dependent reorganization of actin cytoskeleton and focal adhesion dynamics.