Structural and functional characterisation of EpCAM's key signaling complex

Epithelial Cell Adhesion Molecule (EpCAM) is a central molecule in proper regulation of epithelial cell-cell adhesion and signaling. Its key signaling pathway is initiated by Regulated Intramembrane Proteolysis (RIP). During the process of RIP, EpCAM's intracellular domain (EpIC) is released to form a signaling complex with key components of Wnt signaling pathway, ß-catenin and Lef1. Together with its binding partners FHL2 and ß-catenin it is translocated to the nucleus where they interact with Lef1 to form the EpIC-FHL2-ß-catenin-Lef1 signaling complex. This in turn leads to transcription of oncogenes such as cyclin A, D1, E and c-myc, that are responsible for EpCAM-mediated carcinoma cell proliferation. This is also one of the main reasons for why is EpCAM overexpression often linked to poor prognosis. What is EpIC's function in this signaling complex is still unknown. Although RIP was extensively studied in the past, researchers mainly focused on RIP initiation and its proteolytic cleavages. EpIC's role is an even greater mystery because the other three interacting partners have been reported to induce transcription of the same oncogenes without its presence. Deciphering its mechanism of action is furthermore hindered by the lack of any structural information on the EpIC-FHL2-ß-catenin-Lef1 signaling complex and sparse structural knowledge of ß-catenin/Lef1 signaling in general. In this project, we will express and purify the whole EpIC-FHL2-ß-catenin-Lef signaling complex. With integrative modeling, we will combine Cryo-EM, SAXS and XL-MS data to determine the high-resolution structure of this complex with and without EpIC and bound and not bound to its target DNA. Finally, we will validate our structures in cellular settings. This will not only enable us to describe the role of EpIC in EpCAM signaling in great detail, but will also provide the most complete high-resolution structure of any ß-catenin/Lef1 signaling complex. This knowledge will provide the basis for rational design of a new generation of carcinoma treatment strategies.