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The Role of WNT5A in the Pathogenesis of Aggressive Fibromatosis

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The Role of WNT5A in the Pathogenesis of Aggressive Fibromatosis Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2010 The role of WNT5A in the pathogenesis of aggressive fibromatosis Weisstanner, Martin Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-163911 Dissertation Published Version Originally published at: Weisstanner, Martin. The role of WNT5A in the pathogenesis of aggressive fibromatosis. 2010, University of Zurich, Faculty of Science. The Role of WNT5A in the Pathogenesis of Aggressive Fibromatosis Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Martin Weisstanner von Nufenen (GR) Promotionskomitee Prof. Dr. Josef Jiricny (Vorsitz) Prof. Dr. Bernhard Odermatt (Leitung der Dissertation) Prof. Dr. Alex Hajnal Zürich, 2010 Acknowledgments Acknowledgments I heartily thank Prof. Dr. Bernhard Odermatt and Prof. Dr. Hans Knecht for giving me the opportunity to do my PhD and for supervising and guiding my work. A special thank goes to Dr. Christa Leonard, who supervised my work in the laboratory in the first year and introduced me into different laboratory techniques. Many thanks go to Dr. Angela Broggini and Philine Zumstein for their excellent support in the lab. I am also grateful to Prof. Dr. Josef Jiricny for being my Doktorvater and Prof. Dr. Alex Hajnal for accepting to act as a co-examiner. For providing tissue samples, I like to thank Dr. Claudia Meuli, Dr. Beata Bode (who also reviewed the histological diagnosis) and Norbert Alder. My thanks also go to Prof. Dr. Gabriele Schoedon who introduced me into the Agilent microarray technology and always found time for discussions. Many thanks go to Andrea Patrignani, Dr. Hubert Rehrauer and Dr. Stefan Zoller from the FGCZ. I really appreciated their competence concerning Agilent microarray data processing, their disponibility, engagement and help whenever needed. I would like to thank Prof. Dr. Dieter Zimmermann for the sequence analyses and discussions, Dr. Laurens van der Flier for providing the TOP/FOP-luciferase constructs and Prof. Dr. Bert Vogelstein for providing the 45-β-catenin construct. I heartily thank Dr. Maria Zimmermann who supported me whenever needed. I appreciated her motivation and optimism that gave me the necessary power to afford the difficult moments. Many thanks go to all the people in the laboratory that helped me in different ways, in particular Prof. Dr. Holger Moch, Rita Moos, Silvia Behnke, André Fitsche, PD Dr. Peter Schraml, Martina Storz, Marion Bawohl, Susanne Dettwiler, Gunther Boysen, Dr. Van-Duc Luu, Dr. Manfred Beleut, Dr. Markus Rechsteiner, Cristóbal Tostado, Andreas Hollenstein, Annette Bohnert and Barbara Niederöst. I would to thank the Forschung Nottwil, the UBS acting on behalf of an anonymous client, the Krebsliga des Kantons Zürich, the Ida de Pottère-Leupold-Fonds and the Hartmann Müller-Stiftung for their financial support. A particular thank goes to my parents and the rest of my family, for their unconditional support all over the time. Contents Contents Acknowledgments Contents Summary Zusammenfassung Overview Chapter I 1. General Introduction 1 1.1. Description of the Wnt signalling pathway 1 1.1.1. Description 1 1.1.2. The role of the canonical Wnt signalling pathway 4 in embryogenesis and adult organisms 1.1.3. The role of the canonical Wnt signalling pathway 4 in tumorigenesis 1.2. Description of the TGFβββ signalling pathway 6 1.2.1. Description 6 1.2.2. The role of the TGFβ signalling pathway 7 in embryogenesis and adult organisms 1.2.3. The role of the TGFβ signalling pathway 8 in tumorigenesis 1.3. Description of the PI3K-AKT signalling pathway 10 1.3.1. Description 10 1.3.2. The role of the canonical PI3K-AKT pathway 12 in embryogenesis and adult organisms 1.3.3. The role of the PI3K-AKT signalling pathway 12 in tumorigenesis 1.4. The process of wound healing 13 2. Introduction 14 2.1. General description 14 2.2. Signalling pathways known to be involved in the 15 pathogenesis of fibromatoses 2.2.1. The canonical Wnt signalling pathway 15 2.2.2. The TGFβ signalling pathway (superficial fibromatosis) 17 2.3. Microarray-based gene expression analyses 19 2.3.1. Aggressive fibromatosis 19 2.3.2. Superficial fibromatosis 19 Contents 2.4. Aim of the study 20 3. Materials and Methods 21 3.1. Collection and characterization of fibromatoses and 21 reference fibrous tissues 3.1.1. Tissue asservation 21 3.1.2. Histology 21 3.2. Agilent 60mer-oligo microarrays 21 3.2.1. Total RNA extraction 21 3.2.2. RNA qualitiy control 21 3.2.3. Experimental setup 22 3.2.4. Labeling, hybridization and data processing 22 3.3. Real-time reverse transcription PCR (RT-PCR) 24 4. Results 26 4.1. Selection of differentially expressed genes 26 on Agilent microarrays 4.2. Verification of microarray results using real-time RT-PCR 26 4.3. Hierarchical clustering of selected genes 28 4.4. Functional annotation of selected genes 29 4.4.1. Biological processes commonly differentiating the two 29 tumors from the reference fibrous tissue 4.4.2. Biological processes differentiating the two tumors 31 from each other 4.4.3. Single genes differentiating the two tumors from each other 33 4.5. Detailed analysis of signalling pathways 34 4.5.1. The canonical Wnt signalling pathway 34 4.5.2. The TGFβ signalling pathway 38 4.5.3. The PI3K-AKT signalling pathway 41 5. Discussion 44 5.1. Summary of the results 44 5.2. Comparison of own results with data published in literature 45 5.2.1. Consensus with published data 45 5.2.2. Genes belonging to nine biological processes 45 5.3. Single genes differentiating the two tumors from each other 47 5.3.1. Genes overexpressed in superficial fibromatosis 47 5.3.2. Genes upregulated in aggressive fibromatosis 48 Contents 5.4. Detailed analysis of signalling pathways and comparison 49 with published data 5.4.1. The canonical Wnt signalling pathway 49 5.4.2. The TGFβ signalling pathway 51 5.4.3. The PI3K-AKT signalling pathway 53 5.5. The process of wound healing 57 5.6. Differentially expressed genes belonging to the other 58 biological processes Chapter II 1. General Introduction 1 2. Introduction 3 2.1. Markers applicable to aggressive and superficial fibromatoses 3 2.2. Published studies using primary cultures of aggressive and 4 superficial fibromatoses 2.2.1. Aggressive fibromatosis 4 2.2.2. Superficial fibromatosis 5 2.3. Aim of the study 6 3. Materials and Methods 7 3.1. Techniques applied for the establishment of primary tumor 7 cell cultures derived from fibromatoses 3.1.1. Outgrowth of cells from small tissue pieces 7 3.1.2. Pre-treatment of small tissue pieces with collagenase 7 3.2. Mutation analysis 7 3.3. Digital pictures 8 3.4. Cell cultures of normal adult human dermal fibroblasts 8 3.5. Growth medium 8 3.6. Cytoplasmic protein extraction 8 3.7. SDS-PAGE and Western blot 8 3.8. Immunoprecipitation of WNT5A and subsequent 8 SDS-PAGE / Western blot 3.9. Agilent 60-mer-oligo microarrays 9 Contents 4. Results 10 4.1. Mutation analysis of the established primary cell cultures 10 4.2. Growth characteristics of the established primary cell cultures 11 4.3. Analysis of the endogenous WNT5A-expression 11 5. Discussion 14 5.1. Summary of the results 14 5.2. Published studies using primary cultures of aggressive and 14 superficial fibromatoses 5.3. Endogenous WNT5A-expression 15 5.4. Possible approaches to improve the rate of success for the 16 establishment of primary tumor cell cultures Chapter III 1. General Introduction 1 1.1. The non-canonical Wnt pathways 1 1.1.1. The Wnt-Ca2+ signalling pathway 1 1.1.2. The Wnt-PCP signalling pathway 3 1.1.3. The cAMP-dependent protein kinase A (PKA) 5 signalling pathway 1.1.4. The signal transducer and activator of transcription (STAT) 6 signalling pathway 1.2. The role of WNT5A in the pathogenesis of cancer 7 1.2.1. Functional studies in cell cultures and animal models 7 1.2.2. Expression studies in human tumor tissues 12 2. Introduction 1 2.1. WNT5A is highly overexpressed in aggressive fibromatosis 16 2.2. WNT5A is been implicated in the pathogenesis of many 16 types of cancer 2.3. The signalling pathways activated by WNT5A 17 2.4. Widely used methods to measure the activities of 17 WNT5A-stimulated signalling pathways 2.5. Aim of the study 18 Contents 3. Materials and methods 19 3.1. Protein extractions 19 3.1.1. Whole cell protein extracts 19 3.1.2.Subcellular phospho-protein fractions 19 3.2. SDS-PAGE and Western blot 20 3.3. TCF-reporter gene assay (Luciferase assay) 21 3.4. Detailed kinetics and treatment concentrations in 22 experiments using Western blots and Luciferase assays 3.5. Transcription factor activity assay 23 3.6. BrdU-incorporation assay 23 3.7. Collagen type I cell invasion assay 24 3.8. Agilent 60mer-oligo microarrays 24 3.9. siRNA-experiments 25 4. Results 26 4.1. Analysis of the performance of subcellular protein fractionation 26 4.2. Analysis of the impact of WNT5A on cellular signalling pathways 27 in Aggr6 and cell cultures of normal fibroblasts 4.2.1. The effect of WNT5A stimulation on the canonical 27 Wnt signalling pathway 4.2.2. The impact of recombinant WNT5A on the Wnt-Ca2+ 30 pathway in Aggr6 cells 4.2.3. The effect of WNT5A stimulation on the Wnt-PCP 30 signalling pathway 4.2.4.
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