Targeting Hedgehog Signalling As a Drug Therapy in Aggressive Fibromatosis
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Targeting Hedgehog Signalling as a Drug Therapy in Aggressive Fibromatosis by Ronak Ghanbari Azarnier A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Laboratory Medicine and Pathobiology University of Toronto © Copyright by Ronak Ghanbari Azarnier 2012 Targeting Hedgehog Signalling as a Drug Therapy in Aggressive Fibromatosis Ronak Ghanbari Azarnier Master of Science Department of Laboratory Medicine and Pathobiology University of Toronto 2012 Abstract Aggressive fibromatosis is a benign fibroproliferative tumour that can occur as a sporadic lesion or a manifestation in patients with familial syndromes, such as familial adenomatous polyposis. Tumours are characterized by the stabilization of β-catenin and the activation of β-catenin- mediated transcription. Current treatment results are far from ideal, and recurrence rates are high. As a result, there remains a need for more effective therapeutic strategies. In this work, we demonstrate the effect of hedgehog signalling inhibition on aggressive fibromatosis tumour development and β-catenin modulation. We found that hedgehog inhibition decreased cell viability and proliferation as well as total β-catenin levels in human aggressive fibromatosis tumour cells in vitro. Furthermore, following hedgehog inhibition in Apc+/Apc1638N aggressive fibromatosis mouse model, the number and volume of the tumours formed was reduced. Together, this work suggests that hedgehog signalling inhibitor agents are potential candidates to effectively manage aggressive fibromatosis. ii Table of Contents List of Abbreviations ...................................................................................................................... v List of Figures ............................................................................................................................... vii Chapter 1 Introduction .................................................................................................................... 1 1.1 Abstract ................................................................................................................................. 1 1.2 Clinical Perspectives of Aggressive Fibromatosis ................................................................ 2 1.2.1 Overview ........................................................................................................................ 2 1.2.2 Morbidity and Mortality ................................................................................................. 3 1.2.3 Therapeutic Management of Aggressive Fibromatosis .................................................. 3 1.3 Molecular Etiology of Aggressive Fibromatosis .................................................................. 6 1.4 Overview of WNT/β-catenin Pathway .................................................................................. 7 1.4.1 Non-canonical and Canonical Wnt Pathways ................................................................ 7 1.4.2 APC ................................................................................................................................ 9 1.4.3 β-catenin ....................................................................................................................... 11 1.4.4 β-catenin Dysregulation and Tumourigenesis .............................................................. 13 1.5 Mouse Models of Aggressive Fibromatosis ........................................................................ 17 1.5.1 Beta-catenin Stabilized Mouse Model .......................................................................... 17 1.5.2 Apc1638N Mouse Model .................................................................................................. 17 1.6 Cell of Origin of AF ............................................................................................................ 18 1.7 Mesenchymal Stem/Progenitor Cells .................................................................................. 19 1.8 Hedgehog Signalling Pathway ............................................................................................ 20 1.8.1 Hh Gene Discovery ...................................................................................................... 20 1.8.2 Production, Secretion, and Reception of Hh Ligand .................................................... 21 1.8.3 Localization of Hh Pathway Components in Cilia ....................................................... 23 1.8.4 Hh Signal Transduction Downstream of Smo .............................................................. 23 1.8.5 Hh Signalling in Tumourigenesis ................................................................................. 26 1.9 Thesis Summary and Rationale ........................................................................................... 27 1.9.1 Hypothesis .................................................................................................................... 28 1.9.2 Research Aims .............................................................................................................. 28 1.10 Figures ............................................................................................................................... 29 1.11 References ......................................................................................................................... 39 Chapter 2 Targeting Hedgehog Signalling as a Drug Therapy in Aggressive Fibromatosis ........ 69 iii 2.1 Abstract ............................................................................................................................... 69 2.2 Introduction ......................................................................................................................... 70 2.3 Materials and Methods ........................................................................................................ 74 2.3.1 Human Tumour Samples .............................................................................................. 74 2.3.2 Mouse Models .............................................................................................................. 74 2.3.3 Inhibition of Hedgehog Signalling In Vivo................................................................... 75 2.3.4 Cell Culture Studies and Treatments ............................................................................ 75 2.3.5 Gene Expression Studies .............................................................................................. 76 2.3.6 Western Blot Analysis .................................................................................................. 77 2.3.7 Fibroblastic Colony-Forming Unit (CFU-F) Assay ..................................................... 77 2.3.8 Statistical Analysis ....................................................................................................... 78 2.4 Results ................................................................................................................................. 78 2.4.1 Hedgehog signalling pathway is activated in human and murine aggressive fibromatosis ........................................................................................................................... 78 2.4.2 Hedgehog pathway inhibition results in a reduction in the number and volume of aggressive fibromatosis in vivo.............................................................................................. 78 2.4.3 Hedgehog signalling regulates the number of mesenchymal progenitors .................... 80 2.4.4 Hedgehog signalling alters cell behaviours of aggressive fibromatosis in vitro .......... 80 2.4.5 Hedgehog regulates β-catenin and β-catenin regulates hedgehog activity in aggressive fibromatosis and fibroblasts................................................................................................... 81 2.5 Discussion ........................................................................................................................... 82 2.6 Figures ................................................................................................................................. 86 2.7 References ........................................................................................................................... 95 Chapter 3 Summary and Future Directions ................................................................................ 103 3.1 Summary ........................................................................................................................... 103 3.2 Future Directions ............................................................................................................... 104 3.2.1 Effects of Hedgehog Inhibition on Aggressive Fibromatosis .................................... 104 3.2.2 Mechanism of β-catenin Regulation ........................................................................... 105 3.2.3 Regulation and Expression of Wnt Target Genes ...................................................... 106 3.3 References ......................................................................................................................... 107 iv List of Abbreviations FAP familial adenomatous polyposis AF aggressive fibromatosis NSAID non-steroidal anti-inflammatory drugs IFNs interferons APC adenomatous polyposis coli PCP planar cell polarity FZ frizzled Dsh disheveled GSK-3β glycogen synthase kinase 3-beta CK1γ casein kinase 1-gamma β-TrCP beta-transducin