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The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en Role of myosin VI in colorectal cancer By Irati Macaya Erro For the title of PhD PhD program in Biochemistry, Molecular Biology and Biomedicine Faculty of Medicine Universitat Autònoma de Barcelona The thesis was carried out at Group of Biomedical Research in Digestive Tract Tumors Center for Molecular Biology and Biochemistry Investigation in nanomedicine (CIBBIM-Nanomedicine) Vall d’Hebron Research Institute (VHIR) June 2018 DIRECTOR TUTOR Dr. Diego Arango Del Corro Dr. Anna Meseguer Navarro PhD CANDIDATE Irati Macaya Erro Lourdes eta Juanito-ri eskainia. ‘Nature was not designed to make life easy for biologists’ (Colin Tudge, 2007) INDEX INDEX OF CONTENTS INDEX OF FIGURES ........................................................................................................................ 8 INDEX OF TABLES .......................................................................................................................... 9 ABBREVIATIONS .......................................................................................................................... 10 SUMMARY................................................................................................................................... 13 Resumen .................................................................................................................................. 13 Abstract ................................................................................................................................... 15 CHAPTER 1. INTRODUCTION ...................................................................................................... 19 1. THE HUMAN GASTROINTESTINAL TRACT ....................................................................................... 19 1.1. The human intestine ................................................................................................... 19 1.1.1. Anatomy of the small intestine ........................................................................... 20 1.1.2. Anatomy of the large intestine ........................................................................... 20 1.1.3. Histology of the intestine .................................................................................... 21 1.1.4. Small intestinal epithelium .................................................................................. 23 1.1.5. Large intestinal epithelium .................................................................................. 26 2. HOMEOSTASIS OF THE HUMAN INTESTINAL EPITHELIA..................................................................... 27 2.1. Overview ..................................................................................................................... 27 2.2. The stem cell niche ...................................................................................................... 28 2.3. Morphogenic control of cell fate determination ........................................................ 29 3. HALLMARKS OF CANCER ............................................................................................................ 32 4. COLORECTAL CANCER ............................................................................................................... 34 4.1. Overview ..................................................................................................................... 34 4.2. Incidence and mortality .............................................................................................. 34 4.3. Risk factors and causes................................................................................................ 36 4.4. Histopathological classification ................................................................................... 37 4.5. Treatment .................................................................................................................... 39 4.6. The multistep nature of colorectal cancer .................................................................. 39 4.7. The origin of colorectal cancer .................................................................................... 41 4.8. Molecular pathways of colorectal cancer ................................................................... 42 4.8.1. Genomic instability .............................................................................................. 42 4.8.2. Oncogene and tumor suppressor gene mutations ............................................. 46 5. THE MYOSIN SUPERFAMILY ........................................................................................................ 51 5.1. Overview ..................................................................................................................... 51 5 INDEX 5.2. Myosin functions ......................................................................................................... 53 5.3. Myosins and cancer ..................................................................................................... 55 6. MYOSIN VI ............................................................................................................................. 57 6.1. Overview ..................................................................................................................... 57 6.2. Mechanisms of regulation ........................................................................................... 59 6.3. Functions ..................................................................................................................... 63 6.3.1. Myosin VI and endocytosis .................................................................................. 63 6.3.2. Myosin VI and secretory pathway ....................................................................... 67 6.3.3. Myosin VI and autophagy .................................................................................... 68 6.3.4. Myosin VI and cytokinesis ................................................................................... 70 6.3.5. Myosin VI and migration ..................................................................................... 71 6.3.6. Myosin VI as an anchor ....................................................................................... 75 6.3.7. Nuclear role of myosin VI .................................................................................... 76 6.4. Myosin VI and cancer .................................................................................................. 78 6.5. Myosin VI and human hereditary diseases ................................................................. 80 CHAPTER 2. AIMS OF THE STUDY ............................................................................................... 85 CHAPTER 3. MATERIALS AND METHODS ................................................................................... 89 CHAPTER 4. RESULTS ................................................................................................................ 109 1. Low MYO6 protein and mRNA expression in colorectal tumors is associated with poor patient prognosis. ................................................................................................................. 109 2. Generation of isogenic colon cancer cell models with inducible MYO6 downregulation. ....................................................................................................................................... 114 3. MYO6 does not regulate differentiation and polarization of colon cancer cells. ......... 117 4. Downregulation of MYO6 expression does not affect the growth of colon cancer cells in vitro, but it increases their growth in vivo. ........................................................................... 123 5. Downregulation of MYO6 expression does not change the metastatic potential of colon cancer cells. ........................................................................................................................... 131 6. Absence of MYO6 in deficient mice has no effects on intestinal tumorigenesis. ......... 136 7. Absence of MYO6 in mouse intestinal tumor derived organoids increases Wnt signaling. ....................................................................................................................................... 140 CHAPTER 5. DISCUSSION .......................................................................................................... 145 1. MYO6 is an independent marker of prognosis in locally advanced colorectal cancer patients. ................................................................................................................................ 145 2. Generation of isogenic MYO6 knockdown colon cancer cell models. .......................... 147 3. MYO6 does not regulate differentiation and polarization in colon cancer cells. ......... 147 4. Loss of MYO6 is important for the growth of colon cancer cells