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Helsinki 2018 Supervisor Academy Professor Kari Alitalo Research Program Unit Translational Cancer Biology University of Helsinki Finland Thesis committee Associate Professor Pekka Katajisto Professor Timo Otonkoski Institute of Biotechnology Molecular Neurology and Biomedicum University of Helsinki Stem Cell Center Finland University of Helsinki Finland Reviewers appointed by the Faculty Professor Cecilia Sahlgren Docent Keijo Viiri Turku Center for Biotechnology Faculty of Medicine and Life Sciences Åbo Akademi and University of Turku University of Tampere Finland Finland Opponent appointed by the Faculty Associate Professor Kim Jensen Biotech Research & Innovation Centre University of Copenhagen Denmark ISBN 978-951-51-4672-4 (paperback) ISBN 978-951-51-4673-1 (PDF) ISSN 2342-3161 (print) ISSN 2342-317X (online) Dissertationes Scholae Doctoralis Ad Sanitatem Investigandam Universitatis Helsinkiensis No. 87/2018 Cover layout by Anita Tienhaara Hansaprint Oy Helsinki 2018 2 “It’s not whether you get knocked down, it’s whether you get up” - Vince Lombardi Dedicated to my beloved family 3 TABLE OF CONTENTS TABLE OF CONTENTS ................................................................................................................... 4 LIST OF ORIGINAL PAPERS ........................................................................................................ 5 ABBREVIATIONS ............................................................................................................................ 6 ABSTRACT ........................................................................................................................................ 8 INTRODUCTION .............................................................................................................................. 9 REVIEW OF THE LITTERATURE ............................................................................................. 10 1. SIGNALLING PATHWAYS DYSREGULATED IN COLORECTAL CANCER........................................... 10 1.1 The Wnt pathway ................................................................................................................ 10 1.2 KRAS AND MAPK/ERK signalling .................................................................................... 12 1.3 p53 pathway ........................................................................................................................ 12 1.4 The Notch pathway ............................................................................................................. 13 1.5 The TGFβ pathway ............................................................................................................. 15 2. ORGANIZATION OF THE INTESTINE ............................................................................................. 16 2.2 Regulation of stemness and differentiation ........................................................................ 17 2.2.1 The stem cell niche .......................................................................................................... 17 2.2.2 Cell differentiation ........................................................................................................... 18 2.3 Plasticity of intestinal stem cells ........................................................................................ 19 2.3.1 Crypt base columnar cells ............................................................................................... 19 2.3.2 Quiescent stem cells......................................................................................................... 21 2.4 Cancer stem cells ................................................................................................................ 22 2.5 Intestinal organoid cultures as model system of the intestine ............................................ 23 3. HOMEOBOX TRANSCRIPTION FACTOR PROX1 IN CELL FATE DECISIONS AND CANCER .............. 23 3.1 Structure of PROX1 ............................................................................................................ 23 3.2 Function of PROX1 in cell fate decisions ........................................................................... 24 3.3 Oncogenic and tumor suppressor functions of PROX1 ...................................................... 24 AIM OF THE STUDY ..................................................................................................................... 27 MATERIALS AND METHODS..................................................................................................... 28 1. MATERIALS ................................................................................................................................ 28 2. METHODS ................................................................................................................................... 37 RESULTS AND DISCUSSION....................................................................................................... 45 ONCOGENIC MUTATIONS IN INTESTINAL ADENOMAS REGULATE BIM-MEDIATED APOPTOSIS INDUCED BY TGF-Β (I) ................................................................................................................... 45 PROX1 PROMOTES EXPANSION OF THE COLORECTAL CANCER STEM CELL POPULATION TO FUEL ... 48 TUMOR GROWTH AND ISCHEMIA RESISTANCE (II) .......................................................................... 48 TRANSCRIPTION FACTOR PROX1 SUPPRESSES NOTCH PATHWAY ACTIVATION VIA THE NUCLEOSOME REMODELLING AND DEACETYLASE COMPLEX IN COLORECTAL CANCER STEM-LIKE CELLS (III) ...................................................................................................................................... 52 CONCLUSIONS AND FUTURE PROSPECTS ........................................................................... 56 ACKNOWLEDGMENTS ............................................................................................................... 58 REFERENCES ................................................................................................................................