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The Role of Desmosomal Cadherins in Colorectal Tumourigenesis

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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of Cancer Sciences University of Birmingham May 2012 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties.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 ......................
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  • 1714 Gene Comprehensive Cancer Panel Enriched for Clinically Actionable Genes with Additional Biologically Relevant Genes 400-500X Average Coverage on Tumor

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    xO GENE PANEL 1714 gene comprehensive cancer panel enriched for clinically actionable genes with additional biologically relevant genes 400-500x average coverage on tumor Genes A-C Genes D-F Genes G-I Genes J-L AATK ATAD2B BTG1 CDH7 CREM DACH1 EPHA1 FES G6PC3 HGF IL18RAP JADE1 LMO1 ABCA1 ATF1 BTG2 CDK1 CRHR1 DACH2 EPHA2 FEV G6PD HIF1A IL1R1 JAK1 LMO2 ABCB1 ATM BTG3 CDK10 CRK DAXX EPHA3 FGF1 GAB1 HIF1AN IL1R2 JAK2 LMO7 ABCB11 ATR BTK CDK11A CRKL DBH EPHA4 FGF10 GAB2 HIST1H1E IL1RAP JAK3 LMTK2 ABCB4 ATRX BTRC CDK11B CRLF2 DCC EPHA5 FGF11 GABPA HIST1H3B IL20RA JARID2 LMTK3 ABCC1 AURKA BUB1 CDK12 CRTC1 DCUN1D1 EPHA6 FGF12 GALNT12 HIST1H4E IL20RB JAZF1 LPHN2 ABCC2 AURKB BUB1B CDK13 CRTC2 DCUN1D2 EPHA7 FGF13 GATA1 HLA-A IL21R JMJD1C LPHN3 ABCG1 AURKC BUB3 CDK14 CRTC3 DDB2 EPHA8 FGF14 GATA2 HLA-B IL22RA1 JMJD4 LPP ABCG2 AXIN1 C11orf30 CDK15 CSF1 DDIT3 EPHB1 FGF16 GATA3 HLF IL22RA2 JMJD6 LRP1B ABI1 AXIN2 CACNA1C CDK16 CSF1R DDR1 EPHB2 FGF17 GATA5 HLTF IL23R JMJD7 LRP5 ABL1 AXL CACNA1S CDK17 CSF2RA DDR2 EPHB3 FGF18 GATA6 HMGA1 IL2RA JMJD8 LRP6 ABL2 B2M CACNB2 CDK18 CSF2RB DDX3X EPHB4 FGF19 GDNF HMGA2 IL2RB JUN LRRK2 ACE BABAM1 CADM2 CDK19 CSF3R DDX5 EPHB6 FGF2 GFI1 HMGCR IL2RG JUNB LSM1 ACSL6 BACH1 CALR CDK2 CSK DDX6 EPOR FGF20 GFI1B HNF1A IL3 JUND LTK ACTA2 BACH2 CAMTA1 CDK20 CSNK1D DEK ERBB2 FGF21 GFRA4 HNF1B IL3RA JUP LYL1 ACTC1 BAG4 CAPRIN2 CDK3 CSNK1E DHFR ERBB3 FGF22 GGCX HNRNPA3 IL4R KAT2A LYN ACVR1 BAI3 CARD10 CDK4 CTCF DHH ERBB4 FGF23 GHR HOXA10 IL5RA KAT2B LZTR1 ACVR1B BAP1 CARD11 CDK5 CTCFL DIAPH1 ERCC1 FGF3 GID4 HOXA11 IL6R KAT5 ACVR2A