Abstract
Epithelial cell-adhesion molecule (EpCAM) is a transmembrane protein that regulates cell cycle progression and differentiation and is overexpressed in many carcinomas. The EpCAM-induced mitogenic cascade is activated via regulated intramembrane proteolysis (RIP) of EpCAM by ADAM and γ-secretases, generating the signaling-active intracellular domain EpICD. Because of its expression pattern and molecular function, EpCAM is a valuable target in prognostic and therapeutic approaches for various carcinomas. So far, several immunotherapeutic strategies have targeted the extracellular domain of EpCAM. However, targeting the intracellular signaling cascade of EpCAM holds promise for specifically interfering with EpCAM's proliferation-stimulating signaling cascade. Here, using a yellow fluorescence protein-tagged version of the C-terminal fragment of EpCAM, we established a high-content screening (HCS) of a small-molecule compound library (n = 27,280) and characterized validated hits that target EpCAM signaling. In total, 128 potential inhibitors were initially identified, of which one compound with robust inhibitory effects on RIP of EpCAM was analyzed in greater detail. In summary, our study demonstrates that the development of an HCS for small-molecule inhibitors of the EpCAM signaling pathway is feasible. We propose that this approach may also be useful for identifying chemical compounds targeting other disorders involving membrane cleavage-dependent signaling pathways.
Highlights
Epithelial cell-adhesion molecule (EpCAM) is a transmembrane protein that regulates cell cycle progression and differentiation and is overexpressed in many carcinomas
The EpCAMinduced mitogenic cascade is activated via regulated intramembrane proteolysis (RIP) of EpCAM by ADAM and ␥-secretases, generating the signaling-active intracellular domain EpICD
This subsequent step leads to the release of small, soluble extracellular fragments (EpCAM-A-like) and the cytoplasmatic EpICD, which can vary in length because of differential cleavage [50]. hEpICD is part of a large protein complex together with FHL2, -catenin, and the transcription factor Lef-1 [10]
Summary
A high-content screen for small-molecule regulators of epithelial cell-adhesion molecule (EpCAM) cleavage yields a robust inhibitor. Our study demonstrates that the development of an HCS for small-molecule inhibitors of the EpCAM signaling pathway is feasible We propose that this approach may be useful for identifying chemical compounds targeting other disorders involving membrane cleavagedependent signaling pathways. Cleavage by membrane-associated ␣-secretases of the ADAM family is the prerequisite for the second step of RIP, which is conducted by ␥-secretase [10] This subsequent step leads to the release of small, soluble extracellular fragments (EpCAM-A-like) and the cytoplasmatic EpICD, which can vary in length because of differential cleavage [50]. We have addressed the development of inhibitors of EpCAM signaling through a high-content screening (HCS) approach. Identification of small molecule inhibitors of EpCAM in the newly established HCS bears potential for the future development of inhibitory substances
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