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The University of Liverpool Proteins, Which Are Upregulated at Early Time

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The University of Liverpool Proteins, Which Are Upregulated at Early Time The University of Liverpool Proteins, which are upregulated at early time points following Apc deletion, are involved in intestinal tumourigenesis and represent potential colorectal cancer biomarkers Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by Dr Shahram Ali Ibrahim November 2014 1 ACKNOWLEDGEMNTS First and foremost I offer my sincerest gratitude to my first supervisor, Dr John Jenkins, who has supported me throughout my thesis with his patience and knowledge whilst allowing me the room to work in my own way. I attribute the level of my PhD degree to his encouragement and effort and without him this thesis, too, would not have been completed or written. One simply could not wish for a better or friendlier supervisor. I would also like to show my deepest gratefulness to my second supervisor, Professor Mark Pritchard, for helping me to focus and for keeping me on track throughout the study. His support, guidance and advice throughout the research project, as well as his pain-staking effort in proof reading the drafts, are greatly appreciated. Indeed, without his guidance, it would have taken me much more time to put the topic together. Also, if I would remember someone as a role model, it would be Mark. Thanks to Dr Karen Reed from the University of Cardiff for providing me with the animal samples and thanks to Dr Paul Sutton for providing me with the clinical samples. These samples were the corner stone for this project. I am also thankful to Professor Barry Campbell who accepted to be my mentor and advisor. I benefited from his sincere advice and constructive criticism. I highly acknowledge the help that Dave Berry, Dr Abdalla Hanedi, Dr Carrie Duckworth, Sue Courtney and other staff and colleagues gave me during the course of my work in the Department of Gastroenterology. Without them things could have been much more difficult. I would also like to thank my government, our Prime Minister Mr Nicheervan Barzani and Mr Bariz Barzani for funding my studies and for supporting me and all other Kurdish students all over the world. A special thanks to my family. Words cannot express how grateful I am to my mother and my brother and sisters for all of the sacrifices that they have made on my behalf. At the end I would like to express appreciation to my beloved wife who was always my support in the moments when there was no one to answer my queries. 2 Table of contents List of abbreviations ........................................................................................ 9 Abstract .......................................................................................................... 13 1. Introduction ............................................................................................... 16 1.1 Epidemiology of colorectal cancer ..............................................................16 1.1.1 Worldwide incidence and mortality .........................................................16 1.1.2 Types of colorectal cancer .......................................................................16 1.1.3 Distribution of colorectal cancer ..............................................................17 1.1.4 Staging of colorectal cancer ....................................................................17 1.1.5 Grading of colorectal cancer ....................................................................18 1.1.6 Risk factors .............................................................................................19 1.2 Colorectal carcinogenesis ............................................................................22 1.2.1 The suppressor pathway (traditional or chromosomal instability) ............23 1.2.2 Microsatellite instability (mutator) pathway (MSI) ..................................27 1.2.3 Methylator pathway ................................................................................27 1.3 The Wnt signalling pathway .......................................................................28 1.3.1 Canonical Wnt pathway ..........................................................................29 1.3.2 Wnt activity and the cytoplasmic destruction complex ............................30 1.3.3 APC role in the Wnt pathway ..................................................................30 1.3.4 Biological role of Wnt pathway ...............................................................31 1.3.5 Wnt signalling in self-renewing tissues ...................................................31 1.3.6 APC and Wnt signalling in intestinal cancer ............................................32 1.4 Mouse models of colorectal cancer..............................................................34 1.4.1 Apc mutant mice .....................................................................................34 1.4.2 MMR (mismatch repair) and Apc mutations ............................................35 1.4.3 Cancer progression & metastasis models .................................................35 1.4.4 Chemically induced tumour formation ....................................................36 1.4.5 Mouse models of inflammatory bowel disease and colorectal cancer .......36 1.4.6 Cre and loxP mouse strains .....................................................................37 1.5. Biomarkers and CRC screening.................................................................40 1.5.1 Current CRC screening modalities ..........................................................40 1.5.2 Biomarkers of colorectal cancer ..............................................................43 3 1.5.3 Importance of molecular sub-classification of CRC .................................46 1.6 Work leading up to this project ..................................................................47 1.6.1 The AhCre+Apcfl/fl phenotype ..................................................................47 1.7 Overview on the candidate protein biomarkers .........................................48 1.7.1 Nucleosome Assembly protein1 like1 (NAP1L1) ....................................48 1.7.2 DEAD Box 5 (DDX5) or p68 ..................................................................49 1.7.3 Nucleophosmin (NPM) ...........................................................................49 1.7.4 Ribosomal protein like 6 (RPL6) .............................................................50 1.7.5 Fatty Acid Binding Protein 6 (FABP6) ....................................................50 1.7.6 Nucleolin (NCL) .....................................................................................51 1.7.7 Splicing factor, arginine/serine-rich 2 (SFRS2) or Serine/arginine rich splicing factor 2 (SRSF2) or SC35 ...................................................................51 1.7.8 High mobility group box 1 (HMGB1) .....................................................52 1.7.9 Prohibitin (PHB) .....................................................................................52 1.8 Hypothesis, aims and objectives ..................................................................54 1.8.1 Hypothesis ..............................................................................................54 1.8.2 Aims .......................................................................................................54 1.8.3 Objectives ...............................................................................................54 2. Methods ...................................................................................................... 56 2.1 Tissue samples .............................................................................................56 2.1.1 Animal models ........................................................................................56 2.1.2 Clinical samples ......................................................................................56 2.2 Immunohistochemistry ................................................................................57 2.2.1 Tissue preparation ...................................................................................57 2.2.2 Immunohistochemistry methods and materials ........................................57 2.2.3 Immunohistochemistry staining assessment .............................................59 2.3 Haematoxylin and Eosin staining methods .................................................62 2.4 Tissue culture ...............................................................................................63 2.4.1 Maintaining cell lines ..............................................................................63 2.5 Protein extraction ........................................................................................64 2.5.1 Cell lines .................................................................................................64 2.5.2 Tissues ....................................................................................................64 2.5.3 Total protein estimation...........................................................................65 4 2.6 Western Blotting (WB) ................................................................................67 2.7 siRNA transfection ......................................................................................70 2.7.1 Liposome siRNA delivery .......................................................................70 2.7.2 Mechanism of silencing...........................................................................71 2.7.3 Transfection procedure
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