426 Epithelial Cell Adhesion Molecule (EpCAM) regulates epithelial homeostasis via Rho-associated protein kinase (ROCK)

Abstract

EpCAM (Epithelial Cell Adhesion Molecule) is a cell surface protein that is expressed by some normal epithelial cells and many carcinomas. EpCAM is not a classical adhesion molecule and it has been reported to modulate epithelial cell physiology via several mechanisms. Some patients with a congenital diarrheal syndrome termed congenital tufting enteropathy (CTE) have EpCAM mutations, and EpCAM knockout mice have a CTE-like phenotype, documenting the importance of EpCAM in intestinal epithelial cell (IEC) homeostasis. For this reason, we are studying primary IEC to understand EpCAM function in greater detail. IEC organoids can be readily propagated from mouse intestine and they recapitulate many facets of normal IEC development and physiology. Treatment of crypts from ROSA CreER EpCAMfl/fl mice with tamoxifen for the first 3 days of culture leads to loss of EpCAM expression and organoids that exhibit smaller sizes, lower forming efficiencies, and markedly reduced passaging efficiencies as compared with control organoids. Although cellular polarity is maintained and intercellular junctions are not strikingly abnormal in EpCAM-deficient organoids, organoid architecture is disorganized and epithelial integrity is lost. Effects of EpCAM loss are selectively attenuated in EpCAM-deficient organoids treated with Rho-associated protein kinase (ROCK) inhibitors and a myosin II inhibitor. Consistent with this, phosphorylation of targets that are downstream of ROCK, including myosin light chain II (Ser19 and Thr18/Ser19), is enhanced in EpCAM-deficient organoids. Fluorescence in situ hybridization experiments suggest that stem cell numbers are decreased in EpCAM-deficient organoids but Paneth cells, an important source of Wnt in vitro, are also abnormal. Similar results have been obtained after acute EpCAM deletion in intestines of ROSA CreER EpCAMfl/fl mice treated with tamoxifen for 3 days. We are in the process of determining if our results reflect regulation of ROCK by EpCAM in stem cells, Paneth cells or perhaps both cells, and elucidating the mechanism by which this occurs.