Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2018 Elucidating the roles of Wnt-secretion and ฀-catenin’s interaction partners in development and disease Zimmerli, Dario Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-165192 Dissertation Published Version Originally published at: Zimmerli, Dario. Elucidating the roles of Wnt-secretion and ฀-catenin’s interaction partners in develop- ment and disease. 2018, University of Zurich, Faculty of Science. Elucidating the Roles of Wnt-Secretion and b-Catenin’s Interaction Partners in Development and Disease Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Dario Zimmerli von Oftringen, Aargau Promotionskommission Prof. Dr. Konrad Basler (Vorsitz und Leitung der Dissertation) Prof. Dr. Maries van den BroeK Prof. Dr. Sabine Werner Zürich, 2018 Summary ........................................................................................................................... 3 Zusammenfassung ............................................................................................................ 5 Introduction ...................................................................................................................... 7 Review ............................................................................................................................ 10 Pharmacological interventions in the Wnt pathway: Inhibition of Wnt secretion versus disrupting the protein-protein interfaces of nuclear factors ..................................................... 10 Manuscripts .................................................................................................................... 22 WNT ligands control initiation and progression of human papilloma-virus-driven squamous cell carcinoma ................................................................................................................................ 22 Pax6-dependent, but b-catenin-independent, function of Bcl9 proteins in mouse lens development ............................................................................................................................ 50 Mutations in the Wnt/b-catenin cofactors Bcl9 and Pygo Cause Congenital Heart Malformations ................................................................................................................................................ 58 A regulatory receptor network directs range and output of the Wingless signal ..................... 101 Brief summaries, discussions and outlooks .................................................................... 113 WNT ligands control initiation and progression of human papilloma-virus-driven squamous cell carcinoma .............................................................................................................................. 113 Pax6-dependent, but β-catenin-independent function of Bcl9 proteins in mouse lens development .......................................................................................................................... 115 Mutations in the Wnt/b-catenin cofactors Bcl9 and Pygo Cause Congenital Heart Malformations .............................................................................................................................................. 115 A regulatory receptor network directs the range and output of the Wingless signal ............... 118 Conclusion ..................................................................................................................... 120 Appendix ....................................................................................................................... 122 1 A regulatory receptor network directs the range and output of the Wingless signal ............................................................................................................... 118 Conclusion ........................................................................................................... 120 Appendix ............................................................................................................. 122 β-catenin binding to either C- or N-terminal co-factors alone is sufficient to drive Wnt/β-catenin signaling in hair follicle regeneration ........................................ 122 β-catenin’s signaling function is necessary for HPV-induced cSCC growth ......... 129 Appendix Materials and Methods .................................................................... 133 Bibliography ........................................................................................................ 134 Acknowledgments ............................................................................................... 136 2 Summary The discovery of the connection between Wnt/β-catenin signaling, APC and colon cancer two decades ago created fertile ground for research in the field of Wnt/β-catenin signaling. The hope was that by elucidating all the elements of the cascade, one might discover inhibitors of this pathway that help treating cancer patients. This work has revealed a picture of Wnt/β-catenin signaling’s pivotal involvement in virtually every developmental process as well as in adult tissue homeostasis; moreover, Wnt-signaling is involved in many forms of cancer. Unfortunately, one aspect that also became clear over the years was that targeting the pathway is not a simple matter. Apart from the fact, that inhibition of the pathway is associated with severe side effects due to its crucial role in gut homeostasis, it is also a notoriously difficult cascade to target. This difficulty arises mainly because there are no pathway specific kinases known against which an inhibitor could be developed. Another problematic aspect is the dual role the main pathway effector b-catenin has in transducing the Wnt signal as well as maintaining adherens junctions. Nevertheless, the last years have shown several possible ways to tackle these issues. Besides the discovery of Porcupine inhibitors, which hinder palmitoylation of the Wnt ligands, thereby blocking secretion and shutting down the pathway, the idea to block b-catenin’s interaction with transcriptional co- factors has gained some merit. In this work, we set out to test the advantages and draw- backs of Porcupine inhibitors. Furthermore, we wanted to clarify the tissue specific roles of b-catenin’s transcriptional co-factors with regards to their ability to transduce the signal. This information will be crucial to judge the feasibility of using an inhibitor in a specific setting. Here we show that Porcupine is overexpressed in a murine cutaneous squamous cell carcinoma model (cSCC) model, as well as in human cSCCs. This gave us the incentive to test Porcupine inhibitors in this cSCC model, where we found that this hinders tumor development and reduces the cancer stem cell compartment of established tumors. This leads to increased differentiation and a reduction of markers for tumor invasiveness. 3 In an effort to further characterize the specific roles of b-catenin’s transcriptional co-factors, we used mouse models with specific domain deletions inhibiting the binding of b-catenin to the N-terminal co-factors (Bcl9 and Pygopus) and the co-factors to each other. We found that the integrity of these interactions is specifically needed in the development of the embryonic heart and limbs. Loss of Bcl9-Pygo-b-catenin binding in mouse embryos phenocopies human congenital heart disorders by disrupting a molecular cascade necessary for the correct specification of the heart outflow tract and valves. We thus uncovered a novel role for Wnt/b-catenin signaling in heart development and possibly human congenital heart disorders. 4 Zusammenfassung Die Erkenntnis in den Neunzigerjahren, dass zwischen der Wnt/b-catenin Signalkaskade, APC und Darmkrebs ein Zusammenhang besteht, liess die Forschung im Wnt/b-catenin Gebiet aufleben. Das Ziel dieser Forschungstätigkeit war, durch besseres Verständnis aller Elemente der Signalkaskade neue Inhibitoren zu entdecken, um Darmkrebs zu heilen. Die Studien damals haben gezeigt, dass die Wnt/b-catenin Signalkaskade in praktisch allen entwicklungsbiologischen Schritten als auch in der Gewebehomöostase eine Rolle spielt. Unglücklicherweise wurde auch immer klarer, dass es nicht einfach werden würde diese Signalkaskade zu inhibieren. Abgesehen davon, dass die Inhibition der Kaskade schwere Nebenwirkungen hervorrufen kann wegen der wichtigen Rolle, die die Wnt/b-catenin Signalkaskade in der Homöostase des Darmes spielt, ist es generell eXtrem schwierig diese Kaskade zu inhibieren. Dies kommt vor allem daher, dass keine Kaskaden-spezifischen Kinasen bekannt sind, welche als Ziel eines Inhibitors verwendet werden könnten. Ein anderer sehr problematischer Aspekt der Kaskade ist die Doppelrolle des Haupteffektors b- catenin als Signalgeber sowie als Teil der Adhärenzverbindungen zwischen zwei Zellen. Trotzdem wurden in den letzten Jahren verschiedene Möglichkeiten gezeigt, wie das Problem angegangen werden könnte. Einerseits wäre da die Entdeckung von Porcupine Inhibitoren zu erwähnen, welche die Palmytoylierung der Wnt-Liganden verhindern, was zur kompletten Blockade jeglicher Sekretion der Wnt-Moleküle führt, womit die Kaskade blockiert wird. Des Weiteren wird die Idee in Erwägung gezogen, die Bindung von b-catenin