Characterization of the Regulation and Function of Epithelial Cell Adhesion Molecule EpCAM in Mouse Embryonic Stem Cells

• Applicant: Professor Dr. Olivier Gires
• Subject Area: Otolaryngology, Phoniatrics and Audiology
• Term: since 2012
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 231703341

Epithelial cell adhesion molecule EpCAM is a membrane-tethered protein with a selective and restricted expression during embryonic development and tumor progression. Cell-specific expression of EpCAM during physiological and pathological differentiation is achieved through dynamic regulation at the mRNA and protein level. We and others have investigated timing and rationale for this selective EpCAM expression in cancer and embryonic development. In cancer, EpCAM promotes proliferation and tumor growth through regulated intramembrane proteolysis (RIP) and binding of its intracellular domain EpICD to promoters of cell cycle regulators. In pluripotent embryonic stem cells (ESC) EpCAM is strongly expressed. Selective regulation of EpCAM occurs during epithelial-to-mesenchymal transition at the initiation of gastrulation, in the metastatic cascade, and in therapy-resistant tumor cells. Within the last funding period we demonstrated that early in murine embryonic development, cells of the mesodermal lineage strictly turn off EpCAM, while it is maintained in endodermal progeny. Gain- and loss-of-function experiments disrupted the tight EpCAM regulation and inhibited the formation of endodermal and mesodermal-differentiated cells, including cardiomyocytes. To generate a system with which we can study the role of EpCAM regulation during differentiation in more detail, we have generated CRISPR/Cas9-engineered EpCAM reporter cell lines that remain fully functional in terms of pluripotency marker expression and differentiation potential. Additionally, EpCAM-knockout ESC lines and RNAseq-based transcriptome comparisons of wild-type and EpCAM-knockout ESC during differentiation were generated.Up to now, insights into molecular mechanism of selective EpCAM regulation and its impact on cell differentiation remain fragmentary. Therefore, the proposed project will address the following aspects:1. Is EpCAM functional in maintaining a pluripotent ground state of ESCs?2. How does EpCAM support pluripotency exit during early differentiation of ESCs?3. What role does EpCAM play in activating/sustaining the Wnt pathway?4. What role does EpCAM play in the development of endodermal and mesodermal cell and tissue types in later stages of differentiation?To this end, we will use defined endo- and mesodermal cell populations and EpCAM reporter cell lines to address the regulation and function of EpCAM in 3D models of ESC differentiation. While RNA sequencing approaches aim to identify down-stream target genes of EpCAM, a whole genome CRISPR-knockout library screen will be applied to investigate upstream regulators, which play a role during ESC differentiation. In summary, major aims of this project are the elucidation of molecular mechanisms of selective EpCAM regulation and its function in the regulation of ESC differentiation.

• DFG Programme: Research Grants