Tumour-Stroma Signalling in Cancer Cell Motility and Metastasis by Valbona Luga A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy, Department of Molecular Genetics, University of Toronto © Copyright by Valbona Luga, 2013 Tumour-Stroma Signalling in Cancer Cell Motility and Metastasis Valbona Luga Doctor of Philosophy Department of Molecular Genetics University of Toronto 2013 Abstract The tumour-associated stroma, consisting of fibroblasts, inflammatory cells, vasculature and extracellular matrix proteins, plays a critical role in tumour growth, but how it regulates cancer cell migration and metastasis is poorly understood. The Wnt-planar cell polarity (PCP) pathway regulates convergent extension movements in vertebrate development. However, it is unclear whether this pathway also functions in cancer cell migration. In addition, the factors that mobilize long-range signalling of Wnt morphogens, which are tightly associated with the plasma membrane, have yet to be completely characterized. Here, I show that fibroblasts secrete membrane microvesicles of endocytic origin, termed exosomes, which promote tumour cell protrusive activity, motility and metastasis via the exosome component Cd81. In addition, I demonstrate that fibroblast exosomes activate autocrine Wnt-PCP signalling in breast cancer cells as detected by the association of Wnt with Fzd receptors and the asymmetric distribution of Fzd-Dvl and Vangl-Pk complexes in exosome-stimulated cancer cell protrusive structures. Moreover, I show that Pk expression in breast cancer cells is essential for fibroblast-stimulated cancer cell metastasis. Lastly, I reveal that trafficking in cancer cells promotes tethering of autocrine Wnt11 to fibroblast exosomes. These studies further our understanding of the role of ii the tumour-associated stroma in cancer metastasis and bring us closer to a more targeted approach for the treatment of cancer spread. iii Acknowledgements My journey as a PhD student has been an amazing learning experience. This would not have been possible without a number of people who were there for me to share both the challenges and rewards of my scientific endeavours. Jeff, it has been an honour and privilege to be your student. I am deeply grateful to you for believing in me and helping me realize that science is my call in life. Your passion, brilliance and mastery of science will always be an inspiration to me. Lil, thank you for your support and advice. You are an important role model to me and I am very grateful for your mentorship. Thanks to my committee members, Dr. Igor Jurisica, Dr. David Kaplan and Dr. Rama Khokha, for their suggestions and support. Thank you to all the past and current members of the Wrana and Attisano labs. I spent the last nine years of my life mainly in the lab and many of you became my friends and family. John, thank you for your mentorship during my first year in the lab when I barely knew about science. Christine, thank you for teaching me as a young student, aka your “little One”, how to properly design and carry out experiments. You are a remarkable scientist and I am particularly proud to be your friend. Miriam, I will always be thankful to you for your support and encouragment during my search for the “Luga factors”. Your wisdom and advice have been greatly helpful to me throughout my PhD career. I deeply cherish our friendship and will sorely miss our laughs. Abi, thanks for being a good friend and I will miss our chats by the phosphoimager. Liang, thank you for your help; it was a pleasure working with you. Alicia, thank you for your help and friendship. To everyone else, thank you for your support and friendship. I will miss all of you. To my family, thank you for your unconditional love and helping me become the person I am today. Dad, you taught me to dream big and believe in myself. Rovi, your intelligence, talent and search for perfection have been deeply inspirational to me. Koli, your bigheartedness and sense of justice are your best virtues and I look up to you for that. Calvin, Jason, Brandon, Laetitia and Delora, I thank you for bringing pure joy to my life. Mom, your love and devotion iv to your children is unparalleled. You taught me early in my life to work hard and be thorough; these lessons served me well during my PhD. I dedicate this thesis to you. Finally, Rohit, thank you for being by my side for the last eleven years. Your wisdom, patience and love have been crucial for my professional and personal growth. Words cannot express my gratitude and love for you and I look forward to our life journey together. v Table of Contents Abstract .......................................................................................................................... ii Acknowledgements ...................................................................................................... iv Table of Contents .......................................................................................................... vi List of Figures and Tables ........................................................................................... ix List of Abbreviations and Symbols .............................................................................. x Chapter 1 : General Introduction .................................................................................. 1 1.1. Cell Motility and Cancer .................................................................................................. 2 1.1.1. Overview of cancer hallmarks .................................................................................................... 2 1.1.2. Autonomous cancer hallmarks ................................................................................................... 2 1.1.3. Neovascularization and suppression of the immune system ..................................................... 5 1.1.4. Apical-basolateral cell polarity as a barrier to tumour cell invasiveness .................................... 8 1.1.5. Tumour cell migration .............................................................................................................. 12 1.1.6. Metastasis – colonization of distant organs ............................................................................. 22 1.1.7. The tumour microenvironment ................................................................................................. 27 1.1.8. Fibroblasts as components of the tumour microenvironment .................................................. 30 1.1.9. Identification and function of cancer stem-like cells ................................................................. 32 1.2. Noncanonical Wnt-Planar Cell Polarity Pathway ........................................................ 33 1.2.1. Planar cell polarity and its manifestations ................................................................................ 33 1.2.2. Molecular PCP components in the fly ...................................................................................... 36 1.2.3. Models of PCP signalling in the fly ........................................................................................... 37 1.2.4. PCP signalling in vertebrates ................................................................................................... 39 1.2.5. Wnt structure and post-translational modifications .................................................................. 44 1.2.6. Wnt signalling pathways .......................................................................................................... 46 1.2.7. Role of PCP in cell motility and cancer .................................................................................... 49 1.3. Exosomes as Potent Mediators of Intercellular Communication .............................. 50 1.3.1. Modes of intercellular communication ...................................................................................... 50 1.3.2. Introduction to exosomes ......................................................................................................... 50 1.3.3. Exosome biogenesis ................................................................................................................ 51 1.3.4. Exosome components ............................................................................................................. 54 1.3.5. Exosome functions ................................................................................................................... 55 1.3.6. Exosomes as biomarkers for disease ...................................................................................... 57 1.3.7. Exosomes as therapeutic tools ................................................................................................ 57 vi 1.3.8. Tetraspanins – masters of multiple biological functions ........................................................... 58 1.3.9. Cd81 – an exemplary tetraspanin ............................................................................................ 63 1.4. Overview of Thesis ........................................................................................................ 67 Chapter 2 : Fibroblast-secreted Exosomes Regulate Breast Cancer Cell Motility and Metastasis ............................................................................................................. 69 2.1. Introduction .................................................................................................................... 70 2.2. Materials