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Abstract

The canonical Wnt signalling pathway is required for stem cell maintenance and tissue homeostasis, and its misregulation can lead to cancer. Two important negative regulators of Wnt signalling in stem cells are the E3 ubiquitin-protein ligases ZNRF3 and RNF43, which target the Wnt receptors Frizzled and LRP5/6 to degradation. Since ZNRF3/RNF43 are often mutated in Wnt-associated cancers, research on how they are regulated has gained more attention in recent years. It has been shown that binding to R-spondin and LGR4/5/6 antagonises ZNRF3/RNF43, leading to their auto- ubiquitination and internalisation. However, how ZNRF3/RNF43 are stabilised at the plasma membrane remains unknown. In this project, I identified the deubiquitinase USP42 as a novel negative regulator of Wnt signaling. Using different cell culture techniques and methods in molecular biology, I showed that USP42 interacts with and deubiquitinates ZNRF3/RNF43 at the plasma membrane. I also found that USP42 prevents the internalisation of ZNRF3/RNF43 upon binding to R-spondin and LGR, thereby inducing clearance of the Wnt receptors Frizzled and LRP5/6. Furthermore, I investigated the USP42 roles in adult stem cells and cancer cells by generating Usp42 knockout mouse intestinal organoids and colorectal cancer cell lines. First, I found that loss of Usp42 promoted survival upon Wnt and R-spondin withdrawal in mouse intestinal stem cells, thereby phenocopying RNF43/ZNRF3 double knockout. Second, together with my colleague Anchel and our collaborators from the Genomics Core Facility (EMBL Heidelberg), I identified that USP42 functions as a roadblock for proliferation and epithelial-to-mesenchymal transition in colorectal cancer cells. In conclusion, my work uncovered a new role for USP42 in Wnt signalling as a regulator of ZNRF3/RNF43 with relevance in tissue homeostasis and cancer.