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Investigation of the Role of HMGB2 in Endocrine Resistant Breast Cancer

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Investigation of the Role of HMGB2 in Endocrine Resistant Breast Cancer Investigation of the role of HMGB2 in endocrine resistant breast cancer Katie O’ Brien B.Sc., M.Sc. Waterford Institute of Technology Supervisors: Dr. Leonie Young (RCSI), Dr. Orla O’Donovan (W.I.T.) Thesis submitted to Waterford Institute of Technology for the award of M.Sc. June 2012 I declare that this thesis, which I submit to RCSI and W.I.T for consideration of the award of M.Sc. is my own personal effort. Where any of the content presented is the result of input from any other source other than my own work this is duly acknowledged in the text. Furthermore, I took reasonable care to ensure that the work is original, and, to the best of my knowledge, does not breach copyright law, and has not been taken from other sources except where such work has been cited and acknowledged within the text. Signed Student Number: W20042831 Date: 24 June 2012 Acknowledgements There are many people to whom I am incredibly grateful to for their guidance, support and their friendship during the course of this research M.Sc. This project would never have been possible without the funding provided by the Council of Directors, under the Strand III Technological Sector Research Funding, which is gratefully acknowledged. I would also like to thank Dr. Leonie Young in the Royal College of Surgeons, Ireland. Dr. Young has shared with me her extensive scientific knowledge and has provided me with constant mentorship throughout the course of this research. I must sincerely thank Dr. Aisling Redmond who has patiently given her time in assisting me with the research, making a significant contribution to the bulk of this work. Her patience in explaining experimental techniques and her corrections for this thesis are very much appreciated. Dr. Damian McCartan also made a significant contribution to this research and is acknowledged in the results chapters accordingly. I was fortunate to be surrounded by an amazing research team in the Royal College of Surgeons (endocrine oncology lab). I am grateful to Fiona Bane for her support and her friendship during my time in RCSI. Damir Varešlija and Sinead Cocchiglia have both been a great support and good friends to me in the lab. Also Eamon Hughes has to be mentioned for making me laugh every single day; which is often needed during research. I am grateful to Paul Tibbitts for assisting me with the immunhistochemistry. Dr. Christopher Byrne was a constant source of scientific knowledge and provided me with many entertaining stories. I also have to thank Dr. Marie McIlroy and Dr. Jean McBryan for their advice throughout the research project; two women I admire very much. Finally, I am sincerely thankful for the support and encouragement of my parents, Marie and Michael O’ Brien and Martin Collier. Table of Contents Table of Contents i Table of Figures iv List of Tables v Abbreviations vi Abstract ix 1. General Introduction 1 1.1 Introduction to breast cancer 1.1.1 Breast cancer epidemiology 2 1.1.2 Genetic susceptibility to breast cancer 3 1.1.3 Molecular classification of breast cancer 4 1.2 Hormone signalling in breast cancer 1.2.1 The Estrogen Receptor α 6 1.2.2 Endocrine therapies for breast cancer 10 1.2.3 Resistance to endocrine therapies 13 1.2.4 SRC1 role in endocrine resistance 14 1.3 High Mobility Group Box 2 protein 16 1.4 Breast Cancer Amplified Sequence 3 gene 17 1.5 Hypothesis 19 1.6 Aims 20 2. Materials and Methods 21 2.1 Cell Culture 2.1.1 Breast cancer cell lines 22 2.1.2 Cell culture environment 23 2.1.3 Passaging of cells 23 2.1.4 Culturing of cells from cryo-storage ` 23 2.1.5 Cell treatments 23 2.1.6 Cell counting 24 2.2 Protein Biochemistry 2.2.1 Protein extraction 24 2.2.2 Protein quantification 25 2.2.3 Coimmunoprecipitation 25 2.2.4 SDS-PAGE and western blotting 26 i 2.2.5 Mass Spectrometry 29 2.2.6 Chromatin Immunoprecipitation 30 2.3 Nucleic Acid Biochemistry 2.3.1 PCR 32 2.3.2 Real-Time PCR 34 2.4 Transfections 2.4.1 Gene silencing 36 2.4.2 Protein over-expression 37 2.5 Immunohistochemistry 2.5.1 Biological samples 38 2.5.2 Staining protocol 38 2.5.3 Immunohistochemistry scoring system 40 2.6 3D Mammosphere Assay 40 3. Investigation of HMGB2 in the ER/SRC1 complex in endocrine resistant breast cancer 42 3.1 Introduction 43 3.2 Results 3.2.1 ER α knockdown in MCF7 and LY2 cells prior to 3D culture assay 45 3.2.2 3D Culture Assay in MCF7 and LY2 cells with siRNA ER α 46 3.2.3 Mass Spectrometry 47 3.2.4 HMGB2 expression in the MCF7 and LY2 cell lines 49 3.2.5 Tamoxifen driven HMGB2 regulates the expression of SRC1 in LY2 cells 50 3.2.6 ER α/ SRC1 interactions following HMGB2 knock down in LY2 cells 51 3.2.7 HMGB2 Immunohistochemistry 53 3.3 Discussion 55 4. HMGB2 regulates the expression of estrogen responsive genes in endocrine resistant breast cancer 57 4.1 Introduction 58 4.2 Results 4.2.1 HMGB2 regulates the expression of cMyc in LY2 cell line 60 4.2.2 SRC1 ChIP-Sequencing 61 4.2.3 SRC1 regulates the expression of BCAS3 in LY2 cell line 62 ii 4.2.4 HMGB2 regulates the expression of BCAS3 in LY2 cell line 63 4.2.5 ER α regulates the expression of BCAS3 in LY2 cell line 64 4.2.6 HMGB2 recruitment to the BCAS3 promoter via PCR in the LY2 cell line 65 4.2.7 HMGB2 recruitment to the BCAS3 promoter via Real-Time PCR in the LY2 cell line 66 4.2.8 BCAS3 regulates the expression of ER α in LY2 cell line 67 4.3 Discussion 68 5. GENERAL DISCUSSION 71 5.1 HMGB2 acts as a facilitator in ER α/SRC1 interactions 73 5.2 HMGB2 regulates protein expression of BCAS3 75 5.3 Conclusion 78 6. REFERENCES 79 7. APPENDIX 89 iii Table of Figures 1.1 The average number of newly diagnosed breast cancer cases from 2004-2007 3 1.2 Location of luminal epithelial cells on the inner lining of the mammary duct 5 1.3 Basic structure of ER α 7 1.4 Classical and non-classical regulation of estrogen responsive genes 9 1.5 Endocrine therapies for breast cancer 12 1.6 Cruciform structures in chromosomal region of 17q23 18 2.1 Schematic representation of the method of 3D cell culture on Matrigel 41 3.1 ER α knock-down in MCF7 and LY2 cell lines 45 3.2 3D culture assay following ER α knock-down in MCF7 and LY2 cell lines 46 3.3 SDS Page gel from which proteins were selected for mass spectrometry 48 3.4 Expression of HMGB2 in MCF7 and LY2 cell lines 49 3.5 Expression of SRC1 in LY2 cell line following HMGB2 knock-down 50 3.6 ER α/SRC1 interaction following HMGB2 knock-down in LY2 cell line 52 3.7 HMGB2 immunohistochemistry and Kaplan-Meier survival curve 53 4.1 c-Myc expression following HMGB2 knock-down in LY2 cell line 60 4.2 SRC1 ChIP sequencing in LY2 cell line 61 4.3 BCAS3 expression following SRC1 knock-down and over-expression in LY2 62 cell line 4.4 BCAS3 expression following HMGB2 knock-down in LY2 cell line 63 4.5 BCAS3 expression following ER α knock-down in LY2 cell line 64 4.6 HMGB2 recruitment to the BCAS3 promoter in the LY2 cell line 65 4.7 Q-PCR of HMGB2 ChIP to BCAS3 promoter in the LY2 cell line 66 4.8 ER α expression following BCAS3 knock-down in the LY2 cell line 67 iv List of Tables 2.1 Gel preparation for SDS-PAGE 27 2.2 Conditions used for western blotting for proteins studied 28 2.3 PCR reaction reagents and composition 32 2.4 Primers used 33 2.5 Cycling conditions for PCR reactions used 34 2.6 Calculation of fold enrichment of ChIP samples on qPCR 35 2.7 Run conditions used for qPCR reactions 36 2.8 Conditions used for immunohistochemistry 39 v Abbreviations ABC Avidin-biotin complex AD Activation domain AF-1 and AF-2 Activation function domains AI Aromatase inhibitor AIB1 Amplified-in-breast cancer 1 AP-1 Activating protein BCA Bichinchonic Assay bFGF Basic fibroblast growth factor bp Base pairs BCAS3 Breast Cancer Amplified Sequence 3 gene BRCA Breast cancer susceptibility gene BSA Bovine serum albumin CARM1 Coactivator-associated arginine methyltransferase 1 CBP CREB-binding protein Cdk1 Cyclin-dependent kinase 1 cDNA Complementary DNA CDS-FCS Charcoal dextran-stripped fetal calf serum ChIP Chromatin Immunoprecipitation Co-IP Co-Immunoprecipitation DAB 3,3-diaminobenzidine tetrahydrochloride DAPI 4’,6-diamidino-2-phenylindole dihydrochloride DBD DNA binding domain DCIS Ductal carcinoma in situ dH 2O Distilled water DFS Disease-free survival DMEM Dulbecco’s modified Eagle’s medium DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid dNTP Deoxyribonucleotide triphosphate E2 17 β-estradiol ECL Enhanced chemiluminescent reagent vi EGF Epidermal growth factor EGFR Epidermal growth factor receptor ER Estrogen receptor Erb Erythroblastic Leukemia Viral Oncogene ERE Estrogen response elements ERK Extracellular signal-regulated kinase HAT Histone acetyltransferases HDAC Histone deacetylases HER-2 Human epidermal growth factor receptor 2 HMGB2 High Mobility Group Box 2 gene HRP Horseradish peroxidase IGF-1 Insulin-like growth factor 1 IMS Industrial methylated spirits JNK C-Jun NH2-terminal kinase LiCl Lithium chloride MAPK Mitogen-activated protein kinase MEM Minimal essential medium MgCl 2 Magnesium chloride MMP Matrix metalloproteinases MTA1 Metastasis associated protein 1 NCoA Nuclear co-activator NCoR Nuclear co-repressor PCR Polymerase Chain Reaction PELP1 Proline glutamic acid leucine rich protein 1 PI3K Phosphatidylinositol-3 kinase PR Progesterone receptor qPCR Quantitative PCR RIPA Radioimmunoprecipitation RNA Ribonucleic acid RT-PCR Reverse transcriptase polymerase chain reaction SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis Ser Serine SERM Selective estrogen receptor modulator SMRT Silencing mediator of retinoic acid and thyroid hormone receptor vii SNP Single nucleotide polymorphisms SRC Steroid receptor coactivators TAE Tris acetate-EDTA Taq Taq polymerase (Thermophilus aquaticus ) TBS Tris buffered saline TBS-T Tris buffered saline plus 1% Tween TMA Tissue microarray TNF α Tumour necrosis factor-α UV Ultraviolet v/v Volume/volume w/v Weight/volume viii Investigation of the role of HMGB2 in endocrine resistant breast cancer Abstract The high mobility group box 2 protein (HMGB2) is an abundant chromatin remodelling protein with an affinity for unusual DNA structures.
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