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Characterising the spatial organisation and regulation of the Wnt receptor complex in stem cells and cancer

Periodic Reporting for period 1 - WntTraffic (Characterising the spatial organisation and regulation of the Wnt receptor complex in stem cells and cancer)

Reporting period: 2016-04-01 to 2018-03-31

The small intestine epithelium is one of the most proliferative tissues in the human body, renewing every 3-5 days. The rapid turnover is sustained by stem cells under the control of Wnt signalling, dictating critical cell behaviours during both embryonic development and adult tissue homeostasis. Deregulation of this pathway is linked to a plethora of diseases including tumorigenesis.

Leucine rich repeat G–protein coupled receptor 5 (Lgr5) is a Wnt pathway receptor that serves as a molecular determinant of stem cells with its ligand R-spondin (RSPO). Despite its importance as a marker for this critical niche, little is known about Lgr5 trafficking and it’s signalling which may be intimately linked. This information is crucial to understand how stem cells perceive and respond to their environment and ultimately for developing specific and successful anticancer strategies.

The key objective of this project was to resolve early critical molecular details that drive Wnt signalling in stem and cancer cells. Utilizing a novel tool, SNAP-tagged Lgr5, we were able to exclusively track the cell membrane subpopulation of the Lgr5 from early molecular events that occur immediately after receptor activation such as endocytosis through to late events such as lysosomal degradation. These experiments led to formulation of a trafficking model of Lgr5 in an unbound and a ligand-bound state. Moreover, insights into interactions with Wnt signalling players, such as RNF43 and Wnt receptor complex components, have been gained. Future studies based on our findings will help detailed characterization of the stem cell marker Lgr5 and may provide further understanding of the molecular mechanism of Lgr5-RSPO-induced Wnt pathway activation in stem cells and cancer.
The Fellow employed an integrated approach combining biochemistry and genome editing in intestinal organoid cultures with advanced imaging including immune-electron microscopy to develop a unique and novel set of tools to study Lgr5 receptor trafficking and turnover in complex tissues. Using SNAP-tagged Lgr5, the cell surface pool of Lgr5 was comprehensively tracked throughout the endosomal system. These experiments performed in both HEK 293T cells and small intestinal mouse organoids via fluorescence microscopy and at the ultra-structural level using electron microscopy revealed specific trafficking routes of Lgr5 in both an unbound and RSPO bound state. New post-translational modifications and binding partners of Lgr5 were identified including RNF43 and Wnt receptor components through biochemical assays and immunofluorescence experiments. The specific details are currently confidential due to manuscript preparation; however, this new information deepens our understanding on how receptor trafficking and turnover controls receptor mediated signal relay in stem cells and cancer.
The link between Wnt signalling and cancer is paramount. Currently there is a large world-wide effort to develop anti-cancer strategies and battle cancer which the European Commission is at the forefront of. The principal aim of this project was to acquire key mechanistic information on how stem cells receive and perceive signals (Wnt and RSPO) in their environment to open new avenues to interfere with inappropriate Wnt signalling in cancer. Moreover, the novel model system developed during this project to study upstream Wnt activities is of immediate use to the Wnt and wider scientific communities.
Studying the regulation of Lgr5 receptor trafficking and turnover in polarised cells of organoids has helped solve the specific endocytic pathways that are taken by Lgr5 in steady and activated states in primary stem cells of complex tissues. These results are vital to our knowledge of how stem cell receptor activity is regulated in vivo and for further understanding how stem and differentiated cells organise, process and tune signalling events. This crucial information gives clues to the therapeutic potential of stem cells in the repair of diseased tissue and facilitate improvements in cancer treatment, including new targets and systems for drug screening purposes.