NOVEL PATHWAYS PROMOTING THYROID TUMOURIGENESIS AND GROWTH By Gregory Douglas Lewy A thesis presented to the College of Medical and Dental Sciences at the University of Birmingham for the Degree of Doctor of Philosophy Centre for Endocrinology, Diabetes and Metabolism, School of Clinical and Experimental Medicine March 2012 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. SUMMARY Thyroid cancers are the most common endocrine malignancies and their incidence continues to rise. Over-expression of the human pituitary tumor transforming gene (hPTTG) in thyroid carcinomas is a prognostic indicator of tumour recurrence. hPTTG is multifunctional with roles in mitotic control, DNA repair, genetic instability, cell transformation and apoptosis. Importantly, hPTTG transactivates expression of growth factors implicated in proliferation and angiogenesis, and represses the sodium iodide symporter (NIS), which is essential to radioiodine treatments in thyroid cancer. hPTTG interacts with a binding factor (PBF) which is an independent transforming gene and also represses iodine uptake. Work described in this thesis provides evidence for the existence of thyroidal autocrine regulatory pathways involving hPTTG and growth factors in vitro. We directly investigated the role of hPTTG in thyroid tumourigenesis through the generation and characterisation of a murine transgenic model with thyroid-targeted hPTTG over-expression (hPTTG-Tg mice). Unexpectedly, hPTTG over-expression was not sufficient for thyroid tumourigenesis. Investigations performed in hPTTG-Tg and Pttg-/- knockout mice indicated a particularly important relationship between hPTTG and the epidermal growth factor (EGF) in vivo. hPTTG and PBF were confirmed as repressors of NIS in vivo following studies in hPTTG-Tg and PBF-Tg mice respectively. The studies described in this thesis highlight the therapeutic potential of targeting hPTTG and PBF in thyroid cancer. To this effect, specific tyrosine kinase inhibitors prevented autocrine induction of hPTTG by growth factors, and siRNA depletion of PBF restored NIS function to normal levels in hPBF-Tg thyrocytes. Based on these data, hPTTG appears to play a dual role in endocrine tumourigenesis, being involved in both tumour initiation and subsequent progression towards more aggressive phenotypes. DEDICATION I would like to dedicate this Doctoral dissertation to my parents, Oliver and Sandy, and to my sister, Natalie. Thank you for your continual love and support. ACKNOWLEDGEMENTS I would like to express my sincere thanks to Dr Kristien Boelaert and Professor Chris McCabe for their inspiration, guidance, encouragement and for being all-round fantastic supervisors and also to Professor Jayne Franklyn for her support and commitment to this research. I would like to thank Dr. Martin Read for his patience and willingness to share his knowledge and expertise throughout the entire project. Further thanks to Martin and also to Gavin Ryan for their assistance during many hours of work at the Biomedical Services Unit (BMSU). Thanks to Dr. Andrea Bacon for her supporting expertise in murine transgenesis, Dr. Adrian Warfield for supporting histopathological analysis, to Mr. John Watkinson for providing thyroid specimens and to the BMSU staff for training and support. My gratitude to Dr. Margaret Eggo for her generosity in coaching me primary cell culture and associated techniques, as well as her infectious enthusiasm for this project. Thanks to all the friends I have made on IBR level 2 who have all been supportive and made my PhD an enjoyable experience. Particular thanks to the T.O.A.D Society for the banter, the practical jokes and the games, KURC FC and the exercise regimes, but most of all....the Row of Justice! I would also like to thank all of my friends outside of science who are always there, always planning something exciting and unknowingly providing much escapism and happiness. This work was funded by the Medical Research Council. ABBREVIATIONS ABT1 Activator of basal transcription 1 AFP Alpha-fetoprotein AIT Apical iodide transporter AITD Auto-immune thyroid disease AKT1 V-akt murine thymoma viral oncogene homolog 1 AP1 Activator protein 1 AP2 Activator protein 2 APC Anaphase promoting complex BRAF Serine/threonine-protein kinase CAM Chorio-allantoic membrane CAT Chloroamphenicol acetyltransferase cAMP Cyclic adenosine monophosphate CCNB1 Cyclin B CDC-2 Cyclin-dependent kinase 2 CDC-20 Cyclin-dependent kinase 20 CDE Cell cycle dependent element CDH1 Cadherin-1 CDK4 Cyclin-dependent kinase 4 CHR Cell cycle homology region CIN Chromosomal instability CRK V-crk sarcoma virus CT10 oncogene homolog CTNNB1 Catenin (cadherin-associated protein), beta 1 DB Destruction box DES Diethylstilbestrol DNA-PK DNA-dependent protein kinase ECM Extracellular matrix EGF Epidermal growth factor EGFR Epidermal growth factor receptor ERα Oestrogen receptor alpha EREs Oestrogen receptor elements ESCC Esophageal squamous cell carcinoma EST 17β-estradiol FGF-2 Fibroblast growth factor 2 FISSR-PCR Fluorescent inter-simple sequence repeat PCR FLT-1 Vascular endothelial growth factor receptor 1 FNA Fine needle aspiration FNAC Fine needle aspiration cytology FGFR1-4 Fibroblast growth factor receptor (1-4) GH Growth hormone HAT Histone acetyltransferase HCC Hepatocellular carcinoma HDAC Histone deacetylase HEK293 Human embryonic kidney 293 HGF Hepatocyte growth factor HLGAGs Heparin-like glycosaminoglycans hNUE Human upstream enhancer element hPTTG Human pituitary tumor transforming gene HRAS Harvey rat sarcoma viral oncogene HSP70 Heat shock protein 70 HUVEC Human umbilical vein endothelial cells ID3 Inhibitor of DNA-binding 3 IFNγ Interferon gamma IGF-1 Insulin-like growth factor 1 IL-1α Interleukin 1 alpha IL-1β Interleukin 1 beta IL-6 Interleukin 6 IL-8 Interleukin 8 KDR Vascular endothelial growth factor receptor 2 KRAS Kirsten rat sarcoma viral oncogene LB Lysogeny broth LH Luteneising hormone MAD2 Mitotic arrest deficient 2 MAPK Mitogen-activated protein kinase MEFs Mouse embryonic fibroblasts MEK1 Mitogen-activated protein kinase kinase 1 MEK3 Mitogen-activated protein kinase kinase 3 M-FISH Multiplex fluorescent in situ hybridisation MMP-2 Matrix metalloproteinase 2 MNG Multinodular goitre MRI Magnetic resonance imaging NF-Y Nuclear factor Y NGF Nerve growth factor NHEJ Non-homologous DNA end-joining NLS Nuclear localisation signal NIS Sodium iodide symporter NRAS Neuroblastoma rat sarcoma viral oncogene NTRK1 Neurotrophic tyrosine receptor kinase type 1 OCT-1 Octamer-binding transcription factor 1 PAX8 Paired box gene 8 PBF Pituitary tumor transforming gene binding factor PCNA Proliferating cell nuclear antigen PET Positron emission topography PI3K Phosphoinositide-3-kinase PIK3CA Phosphoinositide-3-kinase catalytic alpha polypeptide PKC Protein kinase C PKCβ1 Protein kinase C beta PLCγ Phospholipase C gamma PLK1 Polo-like kinase 1 PPARγ1 Peroxisome proliferator-activator receptor gamma 1 PRL Prolactin PTCs Papillary thyroid carcinomas PTEN Phosphatase and tensin homologue PTTG Pituitary tumor transforming gene PTTG1IP Pituitary tumor transforming gene 1 interacting protein (PBF) RAR-β Retinoic acid receptor beta RET Rearranged in transformation RET/PTC Rearranged in transformation/Papillary thyroid carcinomas shRNA Short hairpin ribonucleic acid SH-3 Src-homology-3 SP1 Specificity protein 1 T3 Triiodothyronine T4 Thyroxine TBPL1 TATA box-binding protein-like protein 1 TCF-4 Transcription factor 4 TG Thyroglobulin TGF-α Transforming growth factor alpha TGF-β Transforming growth factor beta TNF-α Tumor necrosis factor alpha TNF-β Tumor necrosis factor beta TNM Tumour size, node metastases and distant metastases TP53 Tumour protein p53 TPO Thyroid peroxidase TRα Thyroid hormone receptor alpha TRβ Thyroid hormone receptor beta TSHR Thyroid stimulating hormone receptor TSP-1 Thrombo-spondin 1 TSH Thyroid stimulating hormone TTF1 Thyroid transcription factor 1 USF1 Upstream stimulatory factor 1 VEGF Vascular endothelial growth factor WT Wild-type TABLE OF CONTENTS 1 CHAPTER ONE General Introduction ................................................................................................................... 0 1.1 Pathogenesis of thyroid cancer ............................................................................................ 1 1.1.1 Epidemiology and classification .................................................................................................1 1.1.2 Molecular genetics of thyroid cancer ..........................................................................................2 1.1.3 Oncogenes in thyroid cancer ......................................................................................................3 1.1.3.1 Tyrosine receptor kinase rearrangements................................................................................4 1.1.3.2 Nuclear receptor mutants .......................................................................................................4 1.1.3.3 RAS