THE ROLE OF DESMOSOMAL CADHERINS IN COLORECTAL TUMOURIGENESIS By Katherina Yasmin McEvoy A thesis submitted to the University of Birmingham For the degree of DOCTOR OF MEDICINE School of Cancer Sciences University of Birmingham May 2012 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT In cancer, loss of intercellular contact contributes to tumour progression and invasion. Desmosomal cadherins are essential constituents of desmosomes – intercellular junctions that confer significant adhesive strength to epithelial tissues and cardiac muscle. Although changes in desmosomal components have been noted in a variety of cancers previously, this investigation has shown for the first time altered desmocollin expression in colorectal cancer. Real-time PCR and western blotting were used to assess desmocollin expression in a series of colorectal cancer and matched normal tissue samples. Loss of desmocollin 2 expression was observed in the cancer samples. In addition, de novo expression of desmocollins 1 and 3, which are not normally expressed in the colon, was observed. Desmoglein gene expression was also altered in the cancer samples. Although classical cadherin switching is a hallmark of the epithelial-mesenchymal transition, desmocollin switching has not previously been reported. Further experiments, to investigate the effect of loss of desmocollin 2 and desmoglein 2 on the behaviour of cultured cells were performed. In addition, experiments were carried out to identify those transcription factors that regulate desmosomal cadherin gene expression in the colon. Transcription factors of the CCAAT/enhancer-binding proteins family act as transcriptional activators of desmosomal cadherin promoters in colonic cells. For Guy, Eve and Aiden, Despite not knowing anything about desmosomes, you knew enough to know this work matters to me. I could not have done it without you. ACKNOWLEDGEMENTS I would like to thank my supervisors, Dr Martyn Chidgey and Professor Dion Morton all the help, patience and support, throughout the duration of this work. I am also extremely grateful to my colleagues, Denise Youngs, Dr Carolyn Jones and Dr Germaine Caldwell, for all their advice and assistance, as well as for steering me in the right direction in the lab. Table of Contents INTRODUCTION ............................................................................................. 1 1.1 Colorectal Cancer ............................................................................... 1 1.1.1 Epidemiology ....................................................................................... 1 1.1.2 Aetiology.............................................................................................. 2 1.1.2.1 Acquired Risk Factors for Colorectal Cancer ................................ 2 1.1.2.2 Co-Morbid Conditions ................................................................... 6 1.1.2.3 Inherited Colorectal Cancer Syndromes ........................................... 8 1.2 Models of colorectal cancer pathogenesis ........................................... 12 1.2.1 The adenoma-carcinoma sequence ............................................. 12 1.2.2 Chromosomal instability ................................................................. 16 1.2.3 Microsatellite Instability ................................................................. 19 1.2.4 Serrated pathway of colorectal tumourigenesis ............................ 21 1.3 Role of cell adhesion in the adenoma-carcinoma sequence ................ 22 1.4 Desmosomes ....................................................................................... 23 1.4.1 Desmosome structure .................................................................... 23 1.4.2 Desmosomal cadherins ................................................................. 24 1.4.3 Armadillo proteins .......................................................................... 30 1.4.4 Desmoplakin .................................................................................. 31 1.4.5 Regulation of desmosomal gene expression ................................. 31 1.4.6 Desmosomes and disease ............................................................. 32 1.4.7 Desmosomes and cancer .............................................................. 33 1.5 Aims ..................................................................................................... 37 MATERIALS AND METHODS ....................................................................... 38 2.1 Human tissue samples ......................................................................... 38 2.3 Tissue culture ....................................................................................... 41 2.4 RNA and protein extraction .................................................................. 42 2.5 Analysis of RNA ................................................................................... 44 2.5.1 Quantitative reverse-transcriptase mediated ................................. 44 2.5.2 Semi-quantitative reverse transcriptase-mediated PCR ................ 46 2.5.3 Formaldehyde agarose gel electrophoresis ................................... 48 2.5.4 Northern blotting ............................................................................ 48 2.5.5 Northern blot hybridisation probes ................................................. 49 2.6 Western blot analysis ........................................................................... 50 2.7 Plasmids ............................................................................................... 52 2.7.1 pBATEM2 vector ............................................................................ 52 2.7.2 pGL2 and pGL3 reporter vectors ................................................... 53 2.7.3 pRL-TK .......................................................................................... 55 2.7.4 pcDNA3 based plasmids ................................................................ 56 2.8 DNA manipulation ................................................................................ 57 2.8.1 Restriction enzyme digests ............................................................ 57 2.8.2 Agarose gel electrophoresis .......................................................... 57 2.8.3 DNA isolation by electroelution ...................................................... 57 2.8.4 Ethanol precipitation of DNA .......................................................... 58 2.8.5 Ligation reactions ........................................................................... 58 2.8.6 Transformation of competent bacteria with DNA ........................... 58 2.8.7 Plasmid DNA isolation ................................................................... 59 2.8.8 DNA sequencing ............................................................................ 59 2.9 Cloning of DSC2 and DSG2 antisense DNA ........................................ 60 2.10 Cloning of the human DSC2 and DSG2 promoters ............................ 61 2.11 Transfection of cultured mammalian cells .......................................... 64 2.11.1 Stable transfection of antisense plasmids into LS174T cells ....... 64 2.11.2 Transient transfection of luciferase reporter plasmids into Caco2 cells ........................................................................................................ 64 2.12 Measurement of luciferase activity ..................................................... 65 2.13 Statistics ............................................................................................. 66 DESMOSOMAL CADHERIN EXPRESSION IN COLORECTAL CANCER ... 67 3.1 Introduction ........................................................................................... 67 3.2 DSC2 protein expression in colorectal cancer ...................................... 68 3.3 DSC1 and DSC3 protein expression in colorectal cancer .................... 72 3.5 DSC1 and DSC3 transcription in colorectal cancer .............................. 77 3.6 Desmoglein expression in colorectal cancer specimens ...................... 78 3.7 Discussion ............................................................................................ 83 INVESTIGATION OF A FUNCTIONAL ROLE FOR DESMOSOMAL CADHERINS IN COLORECTAL TUMOURIGENESIS .................................. 89 4.1 Introduction ........................................................................................... 89 4.2 Selection of cell lines ............................................................................ 91 4.3 Construction of DSC2 antisense plasmids ........................................... 95 4.4 Construction of DSG2 antisense plasmids ......................................... 102 4.5 Transfection of LS174T cells with DSG2 and DSG2 antisense plasmids ................................................................................................................. 104 4.6 DSC2 and DSG2 RNA expression in transfected cells ......................