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

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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 intracellular 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 2^nd 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

Degrees centigrade Iodine-125 Iodine-131

°C

¹²⁵I

¹³¹I

American Thyroid Association Anaplastic thyroid cancer Bicinchoninic acid Bovine serum albumin British Thyroid Association Complementary deoxyribonucleic acid Confidence interval

ATA ATC BCA BSA BTA cDNA CI

Counts per minute Dalton

cpm Da

Di-iodo-tyrosine

DIT

Dulbecco Modified Eagle's Media Dimethyl sulfoxide Deoxyribonucleic acid Differentiated thyroid cancer Dithiothreitol Ethylenediaminetetraacetic acid Epidermal growth factor receptor Enzyme Linked Immunosorbent Assay Oestrogen response elements Electrospray ionisation Fluorescence activated cell sorting Focal adhesion kinase

DMEM DMSO DNA DTC DTT EDTA EGFR ELISA ERE ESI FACS FAK FAP FBS

Familial Adenomatous Polyposis Fetal bovine serum Fibroblast growth factor 2 Fibroblast growth factor receptor Fine needle aspiration cytology Follicular thyroid cancer Glutathione-S-Transferase Haemagglutinin

FGF2 FGFR FNAC FTC GST HA

Hanks Balanced Salt Solution High performance liquid chromatography Horseradish peroxidase Heat shock protein 90

HBSS HPLC HRP HSP90 IP

Immunoprecipitation Kilodalton Lysogeny broth

kDa LB

Liquid chromatography Leucine-rich repeat kinase Mass to charge ratio

LC LRRK m/z MALDI

Matrix assisted laser desorption/ionisation i

Abbreviations

Mitogen activated protein kinase Monocarboxylate transporter 8 Multiple endocrine neoplasia Hepatocyte growth factor Minimally invasive follicular carcinoma Mono-iodo-tryosine

MAPK MCT8 MEN MET MIFC MIT

Multinodular goitre

MNG mRNA MS/MS MTC NaI

Messenger ribonucleic acid Tandem mass spectrometry Medullary thyroid cancer Sodium Iodide Newborn Calf Serum

NCS NIS NTRK1 PAX8 PBF PBF-HA PBF-Tg PBS

Sodium-Iodide Symporter Neurotrophic tyrosine receptor kinase type 1 Paired box gene 8 PTTG Binding Factor (also PTTG1IP) Haemagglutinin-tagged PBF PBF transgenic Phosphate buffered saline Polymerase chain reaction Platelet derived growth factor receptor Polyethylenimine

PCR PDGF-R PEI PI

Protease inhibitor Src-selective kinase inhibitor Peroxisome proliferator-activator receptor gamma 1 Papillary thyroid cancer

PP1

PPARγ1

PTC

Phosphatase and tensin homologue Pituitary tumor transforming gene Post translational modification Polyvinylidene difluoride PBF phosphorylated at Y174 Serine/threonine-protein kinase Rat sarcoma viral oncogene

PTEN PTTG PTM PVDF pY174 RAF RAS

Rearranged during transfection/Papillary thyroid cancer Radioimmunoprecipitation assay Recurrent laryngeal nerve

RET/PTC RIPA RLN

Ribonucleic acid Relative Risk

RNA RR

Receptor tyrosine kinase Sodium dodecyl sulphate

RTK SDS

SDS-polyacrylamide gel electrophoresis SRC family kinase

SDS-PAGE SFK

Src homology domain 2/3

SH2/3 SILAC SIR siRNA STN

Stable isotope labelling by amino acid in culture Standardised incidence ratio Short interfering RNA Solitary thyroid nodule

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Abbreviations

Small ubiquitin-like modifier Transfection Tri-iodothyronine

SUMO T/F T3

Tetraiodothyronine

T4 TBS-T TE TEMED Tg

Tris buffered saline with tween Transfection efficiency Tetramethylethylenediamine Thyroglobulin Tyrosine kinase inhibitor Tumour, Node, Metastases Time of flight

TKI TNM ToF

Thyrotropin releasing hormone Thyroid stimulating hormone

TRH TSH

Uveal autoantigen with coiled-coil domains and ankyrin repeats

UACA

Upstream transcription factor 1 Untransfected Volume to volume ratio Vascular endothelial growth factor receptor Vector Only

USF1 UT v/v VEGFR VO w/v

Weight to volume ratio Western blot World Health Organisation Widely invasive follicular carcinoma

WB WHO WIFC

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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.

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Table of Contents

TABLE OF CONTENTS

Chapter 1 General Introduction .......................................................................................... 1

• 1.1
• The Physiological Role of the Thyroid Gland .........................................................2

1.1.1 1.1.2 1.1.3
Thyroid structure and development ........................................................................2 Thyroid hormone synthesis.....................................................................................3 The role of the thyroid gland in health....................................................................4

• 1.2
• Thyroid Cancer..........................................................................................................6

1.2.1 1.2.2 1.2.3 1.2.4 1.2.5 1.2.6
Incidence of Thyroid Cancer ..................................................................................6 Risk factors for developing DTC............................................................................7 Types of thyroid cancer ..........................................................................................9 Diagnosing thyroid cancer....................................................................................11 Management of differentiated thyroid cancer.......................................................13 Prognosis...............................................................................................................16

1.3 1.4
The molecular basis of thyroid cancer ...................................................................17

• 1.3.1
• Oncogenes and thyroid cancer..............................................................................17

PTTG-Binding Factor (PBF) ..................................................................................30

1.4.1 1.4.2
Physiology ............................................................................................................30 PBF in thyroid cancer ...........................................................................................35

• 1.5
• Mass spectrometry and protein-protein interactions ...........................................46

1.5.1 1.5.2 1.5.3 1.5.4 1.5.5
Role of protein-protein interactions in the cell .....................................................46 General principles.................................................................................................47 Protein complexes.................................................................................................49 Protein quantitation...............................................................................................49 Mass spectrometry and thyroid cancer .................................................................52

• 1.6
• Hypothesis and aims ................................................................................................57

Chapter 2 Materials and Methods ..................................................................................... 60

2.1 2.2
Cell lines....................................................................................................................61 Transfection..............................................................................................................62

2.2.1 2.2.2 2.2.3
Plasmid purification..............................................................................................62 Transfection of bacterial plasmids........................................................................66 siRNA transfection ...............................................................................................67

• 2.3
• Western blotting.......................................................................................................67

2.3.1 2.3.2
Protein Extraction and quantification ...................................................................67 Western blotting....................................................................................................68

2.4 2.5
Co-immunoprecipitation.........................................................................................69 GST-tagged pull down assays .................................................................................70

2.5.1 2.5.2 2.5.3
Expression of GST-tagged PBF............................................................................70 Translation of L-α-[³⁵S]-methionine labelled protein...........................................71 Binding assay........................................................................................................71

• 2.6
• Fluorescence immunocytochemistry ......................................................................72

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Table of Contents

2.7 2.8
Human primary thyroid cultures...........................................................................73 Statistical analysis....................................................................................................74

Chapter 3 Optimisation of tandem mass spectrometry ................................................... 75

3.1 3.2
Introduction..............................................................................................................76 Methods.....................................................................................................................78

3.2.1 3.2.2 3.2.3 3.2.4 3.2.5
Cell culture............................................................................................................78 Bacterial plasmids.................................................................................................78 Fluorescence-activated cell sorting (FACS) .........................................................78 Western blotting....................................................................................................79 Tandem mass spectrometry (MS/MS) ..................................................................79

3.3 3.4
Results.......................................................................................................................81

3.3.1 3.3.2 3.3.3
Optimisation of transfection efficiencies for K1 and TPC1 cells .........................81 Optimisation of MS/MS........................................................................................85 Optimum conditions for MS/MS ..........................................................................93

Discussion .................................................................................................................94

Chapter 4 Novel binding partners of PBF identified by MS/MS.................................... 98

4.1 4.2
Introduction..............................................................................................................99 Methods.....................................................................................................................99