MOLECULAR MECHANISMS OF ERBB2-DRIVEN THREE-DIMENSIONAL BREAST TUMOUR GROWTH. By Iman Aftab Khan Submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy At Dalhousie University Halifax, Nova Scotia August 2018 © Copyright by Iman Aftab Khan, 2018 Dedicated to my parents Late Dr. Aftab Ahmad Khan and Mrs. Fatima Bisquerra Khan For their unconditional love, patience and struggle to support my decisions that helped me realize my dreams. ii Table of Contents LIST OF TABLES ........................................................................................................... ix LIST OF FIGURES .......................................................................................................... x ABSTRACT .................................................................................................................... xiii LIST OF ABBREVIATIONS USED ............................................................................ xiv ACKNOWLEDGEMENTS ......................................................................................... xxv CHAPTER 1. INTRODUCTION .................................................................................... 1 Resistance to detachment-induced cell death as a critical feature of ErbB2- driven breast cancer: an overview of project goals. ...................................................... 1 1.1 The role of the cell-ECM interactions in the mammary gland development and functions................................................................................... 3 1.2 Morphology of the Human Mammary Gland. ..................................................... 4 1.3 The role of cell-ECM proteins in controlling breast epithelial cell survival. ....... 6 1.3.1 The role of laminins in breast epithelial cell survival. .................................. 6 1.3.2 The role of integrin-ECM interactions in breast epithelial cell survival. ......................................................................................................... 7 1.4 Physiological significance of ‘anoikis’ of breast epithelial cells in the mammary gland development and functions. ............................................ 9 1.4.1 The role of anoikis in lumen formation within the mammary ducts............. 9 1.4.2 The role of anoikis during involution process of the mammary gland. ...... 10 1.5 Molecular mechanisms of breast epithelial cell death after detachment from the ECM..................................................................................................... 11 1.5.1 Molecular mechanisms of anoikis. ............................................................. 11 1.5.2 The role of Bcl-2 family of proteins in regulating anoikis of breast epithelial cells. ............................................................................................ 15 a) The role of Bim in anoikis of breast epithelial cells. .............................. 15 b) The role of Bmf in anoikis of breast epithelial cells. .............................. 15 1.5.3 The mechanisms of integrin-dependent inhibition of breast epithelial cell anoikis. ................................................................................................. 16 a) The role of focal adhesion kinase in anoikis of breast epithelial cells. ... 19 b) The role of mitogen-activated protein kinase kinase in anoikis of breast epithelial cells. .............................................................................. 19 iii c) The role of Phosphatidyl inositol 3-kinase and protein kinase B in anoikis of breast epithelial cells. ......................................................... 20 d) The role of p38alpha mitogen-activated protein kinase in anoikis of breast epithelial cells. .......................................................................... 21 e) The role of Jun-N terminal kinases in anoikis of breast epithelial cells.......................................................................................................... 22 1.6 Non-apoptotic mechanisms of breast epithelial cell death after detachment from the ECM. ................................................................................ 23 1.6.1 The role of Entosis in detachment-induced death of breast epithelial cells. ............................................................................................ 23 1.6.2 The role of necrosis mechanisms in detachment-induced death of breast epithelial cells. .................................................................................. 24 1.6.3 Generation of reactive oxygen species ....................................................... 25 1.6.4 The role of lysosomal cathepsin-dependent mechanisms of death of detached breast epithelial cells. ................................................................... 29 1.7 Molecular mechanisms of anoikis resistance of breast cancer cells. ................. 31 1.7.1 The role of oncoproteins and tumour suppressor proteins in anoikis resistance of breast cancer cells. ................................................................. 31 1.7.2 The role of hypoxia in anoikis resistance of breast cancer cells. ................ 33 1.7.3 The role of cancer cell metabolism and oxidative stress in anoikis resistance of breast cancer cells. ................................................................. 34 1.7.4 The role of autophagy in anoikis resistance of breast cancer cells. ............ 37 1.7.5 The role of microRNAs in anoikis resistance of breast cancer cells. ......... 38 1.8 The role of breast tumour cell anoikis resistance in breast cancer progression. ........................................................................................................ 39 1.8.1 Breast cancer cell anoikis resistance as a hallmark of epithelial- mesenchymal transition. ............................................................................. 40 1.8.2 Breast cancer cells anoikis resistance as a critical feature of cancer stem cells. .................................................................................................... 41 1.8.3 Anoikis resistance as a critical pre-requisite for the ability of cancer cells to form tumours. ...................................................................... 42 1.8.4 Anoikis resistance as a critical pre-requisite for the ability of cancer cells to form metastases. .................................................................. 43 1.9 The role of ErbB2 receptor tyrosine kinase in development of breast cancer. ................................................................................................................ 44 1.10 ErbB2/Her2-positive breast cancer as a major breast cancer subtype. .............. 45 iv 1.11 ErbB Family of Receptor Tyrosine Kinases. ..................................................... 46 1.12 ErbB2-containing heterodimers trigger stronger survival signals. ..................... 49 1.13 Molecular Mechanisms of ErbB2 Signalling. .................................................... 51 1.14 ErbB2-mediated signaling pathways that promote breast cancer. ..................... 52 1.14.1 The Ras/MAPK/ERK Pathway ................................................................... 54 1.14.2 The Phosphatidylinositol 3-kinase/Akt/mTOR (PAM) pathway ................ 55 1.14.3 The Phospholipase C-gamma (PLC-) pathway ......................................... 56 1.14.4 The Src kinase pathway .............................................................................. 57 1.14.5 The JAK-STAT pathway ............................................................................ 58 1.14.6 The Notch-signaling pathway ..................................................................... 58 1.15 ErbB2 as an activator of the network of the anti-anoikis signals. ...................... 59 1.15.1 ErbB2-mediated upregulation of EGFR as an anti-anoikis mechanism. ................................................................................................. 59 1.15.2 ErbB2-mediated downregulation of Bim as an anti-anoikis mechanism. ................................................................................................. 60 1.15.3 ErbB2-mediated downregulation of Bmf as an anti-anoikis mechanism. ................................................................................................. 60 1.16 Concluding Remarks .......................................................................................... 61 1.17 Hypothesis and Objectives ................................................................................. 62 CHAPTER 2. MATERIALS AND METHODS ........................................................... 63 2.1 Materials ............................................................................................................. 63 2.1.1 Compounds (see Appendix B for catalogue numbers and working concentrations) ............................................................................................ 63 2.1.2 Cell culture medium and reagents (see Appendix B for catalogue numbers) ..................................................................................................... 63 2.2 Methods .............................................................................................................. 64 2.2.1 Cell culture .................................................................................................. 64 2.2.2 Western blot analysis (see Appendix B for catalogue numbers and final dilutions of antibodies). .....................................................................