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Novel Binding Partners of PBF in Thyroid Tumourigenesis

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Novel Binding Partners of PBF in Thyroid Tumourigenesis NOVEL BINDING PARTNERS OF PBF IN THYROID TUMOURIGENESIS By Neil Sharma 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of Clinical and Experimental Medicine August 2013 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties.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 cancer is the most common endocrine cancer, with a rising incidence. The proto-oncogene PBF is over-expressed in thyroid tumours, and the degree of over-expression is directly linked to patient survival. PBF causes transformation in vitro and tumourigenesis in vivo, with PBF-transgenic mice developing large, macro-follicular goitres, effects partly mediated by the internalisation and repression of the membrane-bound transporters NIS and MCT8. NIS repression leads to a reduction in iodide uptake, which may negatively affect the efficacy of radioiodine treatment, and therefore prognosis. Work within this thesis describes the use of tandem mass spectrometry to produce a list of potential binding partners of PBF. This will aid further research into the pathophysiology of PBF, not just in relation to thyroid cancer but also other malignancies. From this list, the interaction with three proteins was further investigated and validated by GST pull-down assays and/or co-immunoprecipitation. Thyroglobulin is an essential component of thyroid hormone synthesis. Preliminary studies suggested co-localisation with PBF within intra- cellular vesicles, although further research is needed to explore this functionally. Cortactin has a role in the cellular transport of proteins, and also promotes invadopodia formation and metastases in cancer. Co-localisation was observed in vesicles and at the membrane, with PBF additionally demonstrated in cell membrane projections – indicating a potential mechanism for the shuttling of PBF to and from the cell membrane, and its secretion. SRC is a tyrosine kinase intimately linked to cancer. SRC phosphorylated PBF, an effect abrogated by treatment with specific inhibitors, including PP1. Importantly, PP1 treatment was then found to increase iodide uptake in human primary thyroid cultures over-expressing PBF. Overall, these data describe a list of possible binding partners for PBF, and specifically identify a novel potential therapeutic strategy for iodide-refractory thyroid cancer. DEDICATION For Hannah and Jake. ACKNOWLEDGEMENTS I would like to thank Professor Chris McCabe, Dr Martin Read and Professor Jayne Franklyn for their excellent supervision of this research project, and for their continued support of my academic aspirations. Thanks also to Dr Vicki Smith and Dr Kristien Boelaert for their patience, time and teaching, Dr Craig Doig for both his advice and constant questioning, and Dr Andrew Turnell and Dr Ashley Martin for their guidance and coaching through the world of proteomics. I am grateful to Rob, Greg, Craig, Gavin and Perkin for helping to keep me sane and slightly fit during my three years in the lab, as well as everyone on the 2nd floor IBR for providing a great working environment. Finally, thanks to Mr John Watkinson for his support in all aspects of my career and for giving me the opportunity to get started in research, and to Chris for helping to keep me here. This work was funded by the Get Ahead Charitable Trust, the Royal College of Surgeons of England and the Medical Research Council, to whom I am grateful for their support and confidence. Abbreviations ABBREVIATIONS °C Degrees centigrade 125I Iodine-125 ¹³¹I Iodine-131 ATA American Thyroid Association ATC Anaplastic thyroid cancer BCA Bicinchoninic acid BSA Bovine serum albumin BTA British Thyroid Association cDNA Complementary deoxyribonucleic acid CI Confidence interval cpm Counts per minute Da Dalton DIT Di-iodo-tyrosine DMEM Dulbecco Modified Eagle's Media DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DTC Differentiated thyroid cancer DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid EGFR Epidermal growth factor receptor ELISA Enzyme Linked Immunosorbent Assay ERE Oestrogen response elements ESI Electrospray ionisation FACS Fluorescence activated cell sorting FAK Focal adhesion kinase FAP Familial Adenomatous Polyposis FBS Fetal bovine serum FGF2 Fibroblast growth factor 2 FGFR Fibroblast growth factor receptor FNAC Fine needle aspiration cytology FTC Follicular thyroid cancer GST Glutathione-S-Transferase HA Haemagglutinin HBSS Hanks Balanced Salt Solution HPLC High performance liquid chromatography HRP Horseradish peroxidase HSP90 Heat shock protein 90 IP Immunoprecipitation kDa Kilodalton LB Lysogeny broth LC Liquid chromatography LRRK Leucine-rich repeat kinase m/z Mass to charge ratio MALDI Matrix assisted laser desorption/ionisation i Abbreviations MAPK Mitogen activated protein kinase MCT8 Monocarboxylate transporter 8 MEN Multiple endocrine neoplasia MET Hepatocyte growth factor MIFC Minimally invasive follicular carcinoma MIT Mono-iodo-tryosine MNG Multinodular goitre mRNA Messenger ribonucleic acid MS/MS Tandem mass spectrometry MTC Medullary thyroid cancer NaI Sodium Iodide NCS Newborn Calf Serum NIS Sodium-Iodide Symporter NTRK1 Neurotrophic tyrosine receptor kinase type 1 PAX8 Paired box gene 8 PBF PTTG Binding Factor (also PTTG1IP) PBF-HA Haemagglutinin-tagged PBF PBF-Tg PBF transgenic PBS Phosphate buffered saline PCR Polymerase chain reaction PDGF-R Platelet derived growth factor receptor PEI Polyethylenimine PI Protease inhibitor PP1 Src-selective kinase inhibitor PPARγ1 Peroxisome proliferator-activator receptor gamma 1 PTC Papillary thyroid cancer PTEN Phosphatase and tensin homologue PTTG Pituitary tumor transforming gene PTM Post translational modification PVDF Polyvinylidene difluoride pY174 PBF phosphorylated at Y174 RAF Serine/threonine-protein kinase RAS Rat sarcoma viral oncogene RET/PTC Rearranged during transfection/Papillary thyroid cancer RIPA Radioimmunoprecipitation assay RLN Recurrent laryngeal nerve RNA Ribonucleic acid RR Relative Risk RTK Receptor tyrosine kinase SDS Sodium dodecyl sulphate SDS-PAGE SDS-polyacrylamide gel electrophoresis SFK SRC family kinase SH2/3 Src homology domain 2/3 SILAC Stable isotope labelling by amino acid in culture SIR Standardised incidence ratio siRNA Short interfering RNA STN Solitary thyroid nodule ii Abbreviations SUMO Small ubiquitin-like modifier T/F Transfection T3 Tri-iodothyronine T4 Tetraiodothyronine TBS-T Tris buffered saline with tween TE Transfection efficiency TEMED Tetramethylethylenediamine Tg Thyroglobulin TKI Tyrosine kinase inhibitor TNM Tumour, Node, Metastases ToF Time of flight TRH Thyrotropin releasing hormone TSH Thyroid stimulating hormone Uveal autoantigen with coiled-coil domains and ankyrin UACA repeats USF1 Upstream transcription factor 1 UT Untransfected v/v Volume to volume ratio VEGFR Vascular endothelial growth factor receptor VO Vector Only w/v Weight to volume ratio WB Western blot WHO World Health Organisation WIFC Widely invasive follicular carcinoma iii Publications PUBLICATIONS RELATING TO THIS THESIS Smith VE*, Sharma N*, Watkins RJ, Read ML, Ryan GA, Kwan PP, Martin A, Watkinson JC, Boelaert K, Franklyn JA, McCabe CJ. Manipulation of PBF/PTTG1IP Phosphorylation Status; a Potential New Therapeutic Strategy for Improving Radioiodine Uptake in Thyroid and Other Tumors. J Clin Endocrinol Metab. 2013 Jul;98(7):2876-86. * = joint first author Sharma N, Martin A, McCabe CJ. Mining the proteome: the application of tandem mass spectrometry to endocrine cancer research. Endocr Relat Cancer. 2012 Jul 22;19(4):R149-61. Smith VE, Read ML, Turnell AS, Sharma N, Lewy GD, Fong JC, Seed RI, Kwan P, Ryan G, Mehanna H, Chan SY, Darras VM, Boelaert K, Franklyn JA, McCabe CJ. PTTG-binding factor (PBF) is a novel regulator of the thyroid hormone transporter MCT8. Endocrinology. 2012 Jul;153(7):3526-36. Lewy GD, Sharma N, Seed RI, Smith VE, Boelaert K, McCabe CJ. The pituitary tumor transforming gene in thyroid cancer. J Endocrinol Invest. 2012 Apr;35(4):425-33. Read ML, Lewy GD, Fong JC, Sharma N, et al. Proto-oncogene PBF/PTTG1IP regulates thyroid cell growth and represses radioiodide treatment. Cancer Res. 2011 Oct 1;71(19):6153- 64. Watkins RJ, Read ML, Smith VE, Sharma N, Reynolds GM, Buckley L, Doig C, Campbell MJ, Lewy G, Eggo MC, Loubiere LS, Franklyn JA, Boelaert K, McCabe CJ. Pituitary tumor transforming gene binding factor: a new gene in breast cancer. Cancer Res. 2010 May 1;70(9):3739-49. i Table of Contents TABLE OF CONTENTS Chapter 1 General Introduction .......................................................................................... 1 1.1 The Physiological Role of the Thyroid Gland ......................................................... 2 1.1.1 Thyroid structure and development ........................................................................ 2 1.1.2 Thyroid hormone synthesis ..................................................................................... 3 1.1.3 The role of
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