Genetic and Chemical Modulators of JAK/STAT Signalling in Cancer Sally Jane Thomas Submitted to the University of Sheffield for the Degree of Doctor of Philosophy September 2015 University of Sheffield, Department of Biomedical Science Abstract ................................................................................................................................. 6 Declaration ............................................................................................................................ 7 Publications ........................................................................................................................... 8 Acknowledgements ............................................................................................................... 9 Abbreviations ..................................................................................................................... 11 List of figures ...................................................................................................................... 13 List of tables ........................................................................................................................ 16 CHAPTER 1: INTRODUCTION ........................................................................... 17 1.1 Overview ....................................................................................................................... 17 1.2 JAK/STAT signalling ................................................................................................... 18 1.2.1 Structure of pathway and pathway components ....................................... 18 1.2.2 JAK/STAT signalling in cancer ................................................................ 27 1.2.3 JAK/STAT signalling in immunity and inflammation ............................. 39 1.2.4 The JAK/STAT pathway as a therapeutic target....................................... 42 1.3 ANKHD1 ....................................................................................................................... 45 1.3.1 ANKHD1 protein ...................................................................................... 45 1.3.2 ANKHD1 gene ........................................................................................... 47 1.3.3 ANKHD1 transcripts ................................................................................. 48 1.3.4 ANKHD1 functions .................................................................................. 49 1.3.5 ANKHD1 in cancer ................................................................................... 54 1.3.6 Rationale behind the investigation of ANKHD1 as a modulator of JAK/STAT signalling. ....................................................................................... 54 1.4 Methotrexate ................................................................................................................. 55 1.4.1 Structure .................................................................................................... 55 1.4.2 Pharmacokinetics ...................................................................................... 55 1.4.3 Methotrexate as a chemotherapy drug ...................................................... 56 1.4.4 Methotrexate in the treatment of inflammatory conditions....................... 57 1.4.5 Rationale behind the investigation of methotrexate as a suppressor of JAK/STAT signalling ........................................................................................ 58 1.5 Synopsis ......................................................................................................................... 59 CHAPTER 2: MATERIALS AND METHODS ................................................... 60 2.1 Cell culture .................................................................................................................... 60 2.1.1 Cell lines and culture conditions ............................................................... 60 2.1.2 Drug and ligand treatments ....................................................................... 63 2.1.3 Transfection of HeLa cells ........................................................................ 64 2.2 Protein techniques ........................................................................................................ 65 2.2.1 Protein extraction ...................................................................................... 65 1 2.2.2 Western Blotting ....................................................................................... 66 2.3 DNA techniques ............................................................................................................ 68 2.3.1 Bacterial culture and starting plasmids ..................................................... 68 2.3.2 Generation of plasmid encoding fragment of ANKHD1 containing first and second ankyrin repeats and the intervening region ..................................... 69 2.4 RNA techniques ............................................................................................................ 73 2.4.1 RNA extraction ......................................................................................... 73 2.4.2 cDNA synthesis ......................................................................................... 73 2.4.3 Quantitative real-time PCR ....................................................................... 73 2.5 Drosophila techniques .................................................................................................. 74 2.5.1 Luciferase assays ....................................................................................... 74 2.5.3 Preparation of fly media containing drugs ................................................ 74 2.5.4 Crosses ...................................................................................................... 74 2.5.5 Tumour assay ............................................................................................ 75 2.6 Kinase profiling ............................................................................................................ 75 2.7 Immunofluorescence .................................................................................................... 75 2.7.1 Preparation of slides .................................................................................. 75 2.7.2 Fluorescence Microscopy.......................................................................... 76 2.7.3 Image analysis ........................................................................................... 76 2.8 Source and initial processing of patient samples ....................................................... 76 2.8.1 Ethical approval ........................................................................................ 76 2.8.2 Whole blood and bone marrow aspirate.................................................... 77 2.8.3 Apheresis material ..................................................................................... 78 2.8.4 Formalin fixed paraffin embedded melanoma samples ............................ 79 2.9 Flow cytometry ............................................................................................................. 79 2.9.1 Development of flow cytometry protocol ................................................. 79 2.9.2 Fixation and permeabilisation ................................................................... 81 2.9.3 Immunostaining ........................................................................................ 82 2.9.4 Assessment of cell viability ...................................................................... 83 2.9.5 Analysis on flow cytometer ...................................................................... 83 2.9.6 Gating strategies ........................................................................................ 84 2.9.7 Flow cytometry calculations ..................................................................... 84 2.10 Immunohistochemistry .............................................................................................. 89 2.10.1 Preparation of slides ................................................................................ 89 2.10.2 Microscopy .............................................................................................. 90 2.11 Antibody validation .................................................................................................... 90 2.12 Statistical analysis .................................................................................................... 925 2 CHAPTER 3: ANKHD1 IN NORMAL BLOOD AND HAEMATOLOGICAL MALIGNANCIES .................................................................................................... 96 3.1 Introduction .................................................................................................................. 96 3.2 Results ........................................................................................................................... 97 3.2.1 Patient characteristics ................................................................................ 97 3.2.2 Quantitative real time PCR ....................................................................... 99 3.2.3 Western Blotting ....................................................................................... 99 3.2.4 ANKHD1 expression in normal blood cells ........................................... 101 3.2.5 ANKHD1 in haematopoietic stem cells .................................................