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Functional Analysis of ANKRD11 and FBXO31

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Functional Analysis of ANKRD11 and FBXO31 Functional Analysis of ANKRD11 and FBXO31: Two Candidate Tumour Suppressor Genes from the 16q24.3 Breast Cancer Loss of Heterozygosity Region Paul Matthew Neilsen B.Sc. B.Biomed.Sc. (Hon) Thesis submitted for the Degree of Doctor of Philosophy Breast Cancer Genetics Group Faculty of Health Sciences, School of Medicine, Discipline of Medicine, University of Adelaide, South Australia. Hanson Institute, Adelaide, South Australia. January 2008 This thesis is dedicated to my brother; Although you are no longer with us, your undying determination and passion for life remains with us all. You have always be my guiding light in times of dark, my guardian angel in the presence of evil. I know you will always be with me, and together we will walk, hand in hand, along the road towards a cure. II Table of Contents ABSTRACT VII DECLARATION IX LIST OF PUBLICATIONS X ABBREVIATIONS XI ACKNOWLEDGEMENTS XV CHAPTER 1: INTRODUCTION 1 1.1 – Cancer: a genetic disease 1 1.1.1 – Oncogenes and proto-oncogenes 1 1.1.2 – Tumour suppressor genes 2 1.1.2.1 – Inactivation of tumour suppressor genes 2 1.1.2.2 – Retinoblastoma as a model for Knudson’s “two-hit” hypothesis 7 1.1.2.3 – Epigenetic silencing of tumour suppressor genes 8 1.1.2.4 – Haploinsufficient tumour suppressor genes 9 1.2 – Breast cancer 11 1.2.1 – Estrogens and breast cancer 12 1.2.2 – Molecular basis of breast tumorigenesis 12 1.2.2.1 – Breast cancer oncogenes: ERBB2 / HER2 13 1.2.2.2 – Breast cancer oncogenes: CCND1 14 1.2.2.3 – Breast cancer oncogenes: MYC 15 1.2.2.4 – Breast Cancer Tumour Suppressor Genes: TP53 15 1.2.2.5 – Breast Cancer Tumour Suppressor Genes: BRCA1, BRCA2 and BARD1 17 1.3 – Identification of breast cancer tumour suppressor genes 19 1.3.1 – Identification of the smallest region of overlap for 16q LOH in breast cancer 19 1.3.1.1 – CDH1: the 16q22.1 breast cancer tumour suppressor gene 20 1.3.1.2 - Identification of the 16q24.3 breast cancer tumour suppressor gene(s) 21 1.3.1.2.1 - FBXO31 (F-box protein 31) 22 1.3.1.2.2 - CBFA2T3 (Core-binding factor, runt domain, alpha subunit 2; translocated to 3) 22 1.3.1.2.3 – CYBA (Cytochrome b-245, alpha polypeptide) 23 1.3.1.3 - Does 16q24.3 harbour multiple breast cancer tumour suppressor genes? 24 1.3.1.3.1 - ANKRD11 (Ankyrin repeat domain 11) 25 CHAPTER 2: GENERAL MATERIALS AND METHODS 26 2.1 - Cell lines and transient transfections 26 2.2 – Antibodies 27 2.3 - Immunofluorescence 28 2.4 - Reverse transcription real time-PCR (RT-PCR) 28 2.5 - Western blot analysis 29 III 2.6 – Oligo-primers and DNA constructs 30 2.7 – DNA sequence analysis 30 2.8 – In silico analysis 30 2.9 – Cell Culture Synchronisation 31 CHAPTER 3: FUNCTIONAL CHARACTERISATION OF ANKRD11 43 3.1 – Identification of ANKRD11 as a novel p53 coactivator involved in the rescue of mutant p53 43 3.1.1 – Prelude 43 3.1.1.1 – Selection of ANKRD11 as a candidate 16q24.3 breast cancer tumour suppressor gene 43 3.1.1.2 – Characterisation of the ANKRD11 gene 43 3.1.2 – Introduction 45 3.1.3 – Materials and Methods 48 3.1.3.1 – Cell lines and antibodies 48 3.1.3.2 – Plasmids 48 3.1.3.3 – Immunofluorescence 48 3.1.3.4 – Glycerol Gradient Fractionation 48 3.1.3.5 – In silico analysis 49 3.1.3.6 – Co-immunoprecipitation assays 49 3.1.3.7 – In vitro binding assays 49 3.1.3.8 – Promoter binding assays 50 3.1.3.9 – Reporter assays 50 3.1.3.10 – Reverse transcription real time-PCR (RT-PCR) 51 3.1.3.11 – Cell-based assays 51 3.1.3.12 – Yeast two-hybrid screen 51 3.1.3.13 − β-Galactosidase assay 52 3.1.3.14 – Statement of work 52 3.1.4 – Results 53 3.1.4.1 – ANKRD11 associates with p53, p14ARF and PML in extranucleolar inclusions 53 3.1.4.2 – ANKRD11 interacts with p53 in vivo and in vitro through the ankyrin repeat domain 57 3.1.4.3 – ANKRD11 enhances p53-mediated transcription 61 3.1.4.4 – ANKRD11 expression in breast cell lines 61 3.1.4.5 – Restoration of ANKRD11 enhances p53-dependent expression of p21waf1 64 3.1.4.6 – Restoration of ANKRD11 expression suppresses the growth characteristics of breast cancer cell lines 65 3.1.4.7 – Silencing of ANKRD11 reduces p53 transcriptional activity 68 3.1.4.8 – ANKRD11 associates with hADA3 and P/CAF and increases p53 acetylation 68 3.1.4.9 – ANKRD11 enhances DNA-binding of the p53R273H mutant 69 3.1.4.10 – ANKRD11 is a p53 target gene 72 3.1.5 – Discussion 75 3.2 – Identification of a putative p53-interaction motif located within the ANK domain 80 3.2.1 – Prelude 80 3.2.2 – Introduction 80 3.2.2.1 – The stuctural architecture of the ANK domain 80 3.2.2.2 – Ankyrin proteins: a novel family of p53 regulators? 81 3.2.2.2.1 – BARD1: a regulator of the apoptotic function of p53 83 3.2.2.2.2 – 53bp2: a regulator of the transcriptional function of p53 83 IV 3.2.2.2.3 – IκBα: a regulator of p53 localisation 84 3.2.2.3 – The ANK domain is frequently involved in p53 interaction 84 3.2.3 – Materials and Methods 85 3.2.3.1 – In silico analysis 85 3.2.4 – Results 86 3.2.4.1 – Homology between ANK domains of ANKRD11, BARD1, IκBα and 53bp2 86 3.2.4.2 – Identification of a putative ANK-based p53-interaction motif 86 3.2.5 – Discussion 89 3.3 – Characterisation of a role for ANKRD11 as a putative cell cycle checkpoint protein 92 3.3.1 – Introduction 92 3.3.1.1 – The cell cycle 92 3.3.1.2 – Regulation of the cell cycle 92 3.3.1.3 – The Ubiquitin Proteolytic Pathway 93 3.3.1.3.1 – Polyubiquitination 93 3.3.1.3.2 – E3 ubiquitin ligases 95 3.3.1.3.2.1 – E3 ubiquitin ligases: the SCF complex 96 3.3.1.3.2.2 – E3 ubiquitin ligases: the APC/C complex 96 3.3.1.3.3 – Cell cycle checkpoints 101 3.3.1.3.3.1 – The G1/S phase checkpoint 101 3.3.1.3.3.2 – The intra-S phase checkpoint 102 3.3.1.3.3.3 – The G2/M phase checkpoint 103 3.3.1.3.3.4 – The spindle assembly checkpoint 103 3.3.1.3.4 – Aberrant regulation of cell cycle proteins and checkpoints during breast tumorigenesis 104 3.3.2 – Materials and Methods 105 3.3.2.1 – Cell lines and antibodies 105 3.3.2.2 – Plasmids 105 3.3.2.3 – Immunofluroescence 105 3.3.2.4 – Cell culture synchronization 105 3.3.2.5 – Subcellular biochemical fractionation 105 3.3.3 – Results 107 3.3.3.1 – ANKRD11 exhibits partial localisation with PML bodies 107 3.3.3.2 – ANKRD11 localises with PML isoforms 109 3.3.3.3 – ANKRD11 associates with chromatin during mitosis and is rapidly degraded during mitotic progression 109 3.3.3.4 – ANKRD11 associates with histone H2A 115 3.3.3.5 – ANKRD11 is a cell cycle regulated protein 117 3.3.3.6 – ANKRD11 translocates to the nucleolus following proteasomal inhibition 118 3.3.3.7 – ANKRD11 contains a consensus destruction box and multiple PEST sequences 119 3.3.4 – Discussion 121 3.3.4.1 – The dynamic regulation of ANKRD11 during mitotis 121 3.3.4.2 – Association of ANKRD11 with chromatin-rich regions 122 3.3.4.2.1 – Is ANKRD11 recruited to chromatin via interaction with nucleosomes? 122 3.3.4.2.2 – ANKRD11-histone association: direct or indirect? 123 3.3.4.3 – Proteasomal-mediated degradation of ANKRD11 124 3.4 – Investigation into a role for ANKRD11 in the modulation of estrogen receptor activity 126 3.4.1 – Prelude 126 V 3.4.2 – Introduction 126 3.4.2.1 – Steroid nuclear receptors and transcriptional regulation 126 3.4.2.2 – The p160 family of steroid recetor coactivators 127 3.4.2.2.1 – Structural and function domains of the SRC family – the bHLH-PAS domain 127 3.4.2.2.2 – Structural and function domains of the SRC family – the LXXLL motif 127 3.4.2.2.3 – Structural and function domains of the SRC family – the AD domain 128 3.4.2.3 – AIB1 and breast cancer 129 3.4.2.4 – ANKRD11: a novel antagonist of the p160 coactivators 129 3.4.3 – Materials and Methods 131 3.4.3.1 – Cell lines and antibodies 131 3.4.3.2 – Plasmids 131 3.4.3.3 – Estrogen receptor transactivation assays 131 3.4.3.4 – Co-immunoprecipitation assays 131 3.4.4 – Results 132 3.4.4.1 – ANKRD11 enhances estrogen receptor-mediated transcriptional activity 132 3.4.4.2 – ANKRD11 interacts with ERBP in vivo via the amino-terminal ankyrin region 136 3.4.5 – Discussion 138 CHAPTER 4: FBXO31 IS THE CHROMOSOME 16Q24.3 SENESCENCE GENE, A CANDIDATE BREAST TUMOUR SUPPRESSOR, AND A COMPONENT OF AN SCF COMPLEX 142 4.1 - Prelude 142 4.2 – Introduction 142 4.3 – Materials and Methods 144 4.3.1 - Cell lines and antibodies 144 4.3.2 – Plasmids 144 4.3.3 – Human tissues 144 4.3.4 – Northern blot 144 4.3.5 – Real-time reverse transcription-PCR 145 4.3.6 – Cell-based assays 145 4.3.7 – Cell synchronization 146 4.3.8 – Cell cycle analysis 146 4.3.9 – Western blot analysis 146 4.4 – Results 149 4.4.1 – Analysis of 16q24.3 senescence region 149 4.4.2 – Northern blot and in silico analysis 153 4.4.3 – Ectopic expression of FBXO31 induces cellular senescence in MCF-7 153 4.4.4 – Alteration of FBXO31 expression affects growth of breast cell lines 156 4.4.5 – FBXO31 expression and mutation screening 159 4.4.6 – FBXO31 is cell cycle regulated 161 4.4.7 – FBXO31 exists as a component of SCFFBXO31 complex 164 4.5 – Discussion 167 REFERENCES 172 VI Abstract Loss of heterozygosity (LOH) on the long arm of chromosome 16 is frequently observed during the onset of breast cancer.
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