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The Role of Iron and Haem in Breast Cancer

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The Role of Iron and Haem in Breast Cancer THE ROLE OF IRON AND HAEM IN BREAST CANCER By Thomas Roe A thesis submitted to: The University of Birmingham for the degree of DOCTOR OF MEDICINE School of Cancer Sciences The University of Birmingham January 2015 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties.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 Iron is ubiquitous in the human body, fulfilling many crucial roles. However, accumulating evidence implicates excess iron as a carcinogen. This study aimed to further investigate the role of iron and haem in breast carcinogenesis and the potential utility of chelators in therapy. We demonstrate that the transport machinery for iron and haem is dysregulated in breast cancer, with import being promoted and export down-regulated to allow the accumulation of intra-cellular iron. In vitro studies demonstrate that, in contrast to benign cells, malignant cells are capable of importing haem as well as ionic iron. Both iron and haem stimulate aggressive behaviour in malignant cells, up-regulating viability, proliferation, adhesion, migration and invasion. These changes are abrogated by iron chelation. In addition, the expression profile of iron and haem transporters is shown to favour intra-cellular accumulation even when iron is plentiful and import would be expected to be down-graded. Overall this study suggests that breast cancer may be due to an inappropriate expression profile of iron and haem transporters, leading to excess intra-cellular iron which drives a malignant cell phenotype. In addition the action of chelators to downgrade malignant behaviour implies a potential therapeutic role. ii ACKNOWLEDGEMENTS I woud like to thank Dr Chris Tselepis for his supervisory support throughout the practical side of my work and the subsequent production of this thesis. I would also like to thank Miss Fiona Hoar for her clinical support and assistance in collecting tissue for use in my research. Elisabeth Phillips, Katherine Date and Samuel Ford were all hugely helpful in the laboratory, for which I am extremely grateful. In addition I would like to thank City Hospital, Birmingham for grants to provide consumables for use in this project. iii CONTENTS LIST OF FIGURES ................................................................................................................... x LIST OF TABLES ..................................................................................................................... x ABBREVIATIONS ................................................................................................................ xvii CHAPTER 1: INTRODUCTION AND BACKROUND ................................................... 1 1.1 Breast Cancer ................................................................................................................ 1 1.1.1 Epidemiology ........................................................................................................ 1 1.1.2 Environmental Risk Factors .................................................................................. 2 1.1.2.1 Socio-Economic Status .................................................................................. 2 1.1.2.2 Hormone Replacement Therapy and the Oral Contraceptive Pill .................. 3 1.1.2.3 Reproductive Behaviour and Endogenous Oestrogen .................................... 4 1.1.2.4 Obesity ........................................................................................................... 5 1.1.2.5 Alchohol ......................................................................................................... 5 1.1.3 Genetic Risk Factors .............................................................................................. 6 1.1.4 Staging of Breast Cancer ....................................................................................... 8 1.1.5 Histological Classification and Grading of Breast Cancer .................................... 8 1.2 Iron .............................................................................................................................. 10 1.2.1 Normal Iron Homeostasis .................................................................................... 10 1.2.2 Non-haem Iron Metabolism ................................................................................ 11 1.2.2.1 Duodenal Cytochrome b (Dcytb) ................................................................. 13 1.2.2.2 Divalent Metal Transporter 1 (DMT1)......................................................... 13 1.2.2.3 Ferritin .......................................................................................................... 15 1.2.2.4 Ferroportin .................................................................................................... 16 1.2.2.5 Hephaestin .................................................................................................... 18 iv 1.2.2.6 Hepcidin ....................................................................................................... 19 1.2.2.7 Transferrin Receptor .................................................................................... 23 1.2.2.8 Iron Regulatory Elements and the Iron Response Proteins .......................... 25 1.2.3 Haem Metabolism ............................................................................................... 28 1.2.3.1 Haem Carrier Protein 1 (HCP-1) .................................................................. 30 1.2.3.2 LDL Receptor Related Protein 1 (LRP1) ..................................................... 31 1.2.3.3 Feline Leukaemia Virus Subgroup C Receptor (FLVCR) ........................... 32 1.2.3.4 Breast Cancer Resistance Protein (BCRP) ................................................... 33 1.2.4 Iron and Carcinogenesis ...................................................................................... 35 1.2.4.1 Iron and Redox Chemistry ........................................................................... 35 1.2.4.2 Iron and Cell Cycling ................................................................................... 37 1.2.4.3 Iron and the Immune System ....................................................................... 38 1.2.4.4 Iron and Colon Cancer ................................................................................. 39 1.2.4.5 Iron and Hepatocellular Carcinoma ............................................................. 40 1.2.4.6 Iron and Renal Cell Carcinoma .................................................................... 42 1.2.4.7 Iron and Other (Non-Mammary) Cancers .................................................... 43 1.2.4.8 Iron and Breast Cancer ................................................................................. 43 1.2.5 Iron Chelators ...................................................................................................... 47 1.3 Hypothesis .................................................................................................................. 49 1.4 Aims ............................................................................................................................ 50 CHAPTER 2: MATERIALS AND METHODS .............................................................. 51 2.1 Materials ..................................................................................................................... 51 2.1.1 Laboratory Reagents ............................................................................................ 51 2.1.2 Primary Antibodies .............................................................................................. 54 v 2.1.3 Oligonucleotide Sequences.................................................................................. 56 2.1.4 Cell Lines ............................................................................................................. 57 2.1.5 Fresh Tissue ......................................................................................................... 58 2.2 Methods ...................................................................................................................... 59 2.2.1 Ethical Approval .................................................................................................. 59 2.2.2 Immunohistochemistry ........................................................................................ 59 2.2.2.1 Immunohistochemistry Using W-Cap .......................................................... 59 2.2.2.2 Immunohistochemistry Using Citric Acid ................................................... 60 2.2.2.3 Evaluation of Immunostaining ..................................................................... 60 2.2.3 Cell Culture ......................................................................................................... 61 2.2.3.1 Routine Cell Culture..................................................................................... 61 2.2.3.2 Cell Line Stimulation ................................................................................... 62 2.2.4
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