Alterations in Keratin Levels and Modifications in Colorectal Adenomagenesis Ria Rosser January 2016 Supervisors: BM Corfe and KS Chapple Department of Oncology Thesis submitted to the University of Sheffield for the degree of Doctor of Medicine 2 Alterations in Keratin Levels and Modifications in Colorectal Adenomagenesis Table of Contents Abstract ................................................................................................................................. 7 Acknowledgments ............................................................................................................. 9 List of Figures ................................................................................................................... 11 List of Tables .................................................................................................................... 13 Abbreviations .................................................................................................................. 15 Chapter 1 Literature review ....................................................................................... 19 1.1 The History of Adenomatous polyps ................................................................. 19 1.1.1 Origins of the Adenoma ............................................................................................... 20 1.1.2 Adenoma-Carcinogenesis model ......................................................................................... 24 1.2 Field effects – Theory and Definitions .............................................................. 27 1.2.1 Difficulties in Investigating Field Effect changes in Colorectal Cancer ....... 28 1.2.2 Colorectal Field Effects and Clinical Applications .............................................. 29 1.3 Evidence for field cancerization ......................................................................... 33 1.3.1 Epigenetic evidence ...................................................................................................... 33 1.3.1.1 DNA methylation studies .................................................................................................... 33 1.3.1.2 Epigenetic inactivation of secreted frizzled-related proteins (SFRP) ............. 34 1.3.2 Protein expression and early tissue events .......................................................... 35 1.3.2.1 Expression of carcinoembryonic antigen (CEA) ....................................................... 35 1.3.2.2 Bcl-XL anti-apoptotic protein expression ..................................................................... 35 1.3.2.3 Altered mucin ........................................................................................................................... 36 1.3.2.4 Protein kinase C ...................................................................................................................... 36 1.3.2.5 Neuropilin-1 expression ...................................................................................................... 37 1.3.2.6 Proteomic analysis ................................................................................................................. 37 1.3.3 Clonality studies ............................................................................................................ 39 1.3.4 Metachronous adenoma .............................................................................................. 41 1.3.5 Summary –potential causes of field cancerization ............................................. 42 1.4 Dietary modification of colorectal cancer risk .............................................. 43 1.5 Keratins and Butyrate in Colonic Tissue ......................................................... 44 1.5.1 Keratins ............................................................................................................................ 44 1.5.2 Butyrate ............................................................................................................................ 47 1.5.3 Post-translational modifications of keratins ....................................................... 49 1.5.3.1 Phosphorylation ...................................................................................................................... 49 1.5.3.2 Glycosylation ............................................................................................................................ 49 1.5.3.3 Acetylation ................................................................................................................................ 50 1.5.3.4 Sumoylation .............................................................................................................................. 50 1.6 Summary .................................................................................................................... 51 Chapter 2 General Hypothesis and Aims ................................................................ 55 2 Hypotheses ............................................................................................................................. 55 2.1 Aims and Objectives ......................................................................................................... 55 3 Chapter 3 Metachronous Adenomas Occur Primarily at the same site with proximal drift .................................................................................................................. 59 3.1 Introduction ........................................................................................................................ 59 3.2 Hypothesis and Aims ....................................................................................................... 60 3.2.1 Hypothesis .................................................................................................................................... 60 3.2.2 Rationale ........................................................................................................................................ 61 3.2.3 Aims ................................................................................................................................................. 66 3.3 Method for Data Collection ............................................................................................ 66 3.3.1 Problems and solutions for statistical analysis ............................................................. 69 3.3.2 Whole dataset analyses ........................................................................................................... 70 3.3.3 Single adenoma analyses ........................................................................................................ 71 3.4 Results .................................................................................................................................. 73 3.4.1 Whole dataset - analysis 1 ...................................................................................................... 73 3.4.2 Whole dataset - analysis 2 ..................................................................................................... 74 3.4.3 Whole dataset - analysis 3 ...................................................................................................... 75 3.4.4 Single adenoma – analysis 1 .................................................................................................. 78 3.4.5 Single adenoma – analysis 2 .................................................................................................. 79 3.4.6 Single adenoma – analysis 3 .................................................................................................. 79 3.4 Results ................................................................................................................................................ 81 3.4.7 Single adenoma – analysis 4 .................................................................................................. 81 3.5 Discussion ............................................................................................................................ 82 3.5.1 Whole data set analysis ........................................................................................................... 82 3.5.2 Single adenoma analysis ......................................................................................................... 82 3.6 Summary .............................................................................................................................. 87 3.6.1 Conclusion ..................................................................................................................................... 87 3.6.2 Limitations .................................................................................................................................... 87 Chapter 4 Development of an integrated workflow for extraction and solubilization of intermediate filaments from colorectal biopsies for proteomic analysis ......................................................................................................... 91 4.1 Introduction ........................................................................................................................ 91 4.2 Aims and Objectives ......................................................................................................... 92 4.3 Materials and Methods .................................................................................................... 93 4.3.1 Materials .......................................................................................................................................