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Genetic and Epigenetic Profiling of Human Prostate Cancer Cell-Subsets

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Genetic and Epigenetic Profiling of Human Prostate Cancer Cell-Subsets Genetic and Epigenetic Profiling of Human Prostate Cancer Cell-Subsets Alberto John Taurozzi PhD University of York Biology September 2016 Abstract Perturbation of androgen signalling drives progression of human prostate cancer (CaP) to castration-resistant prostate cancer (CRPC). Additionally, CaP is initiated and maintained by cancer stem cells (CSC)s which are analogous to normal prostate stem cells (SC)s. This study presents a qPCR assay to detect androgen receptor gene amplification (GAAR), which is the most common mechanism of castration resistance (>30%). Also, the epigenetic regulation and function of two SC-silenced genes with tumour-suppressive activity (Latexin (LXN) and Retinoic Acid Receptor Responder 1 (RARRES1)) were interrogated using micro-ChIP, transcriptional profiling and mass spectrometry. Traditionally, GAAR is detected using FISH which is labour-intensive and semi- quantitative, limiting clinical applicability. The mechanism of action of LXN or RARRES1 in CaP is unknown, and epigenetic regulation by DNA methylation has been ruled-out in primary CaP. The qPCR assay can detect GAAR in minor cell populations (~1%) within a heterogeneous sample and also quantifies X chromosome aneuploidy (XCA) - a predictor of poor- prognosis in CaP. GAAR and XCA were detected in near-patient xenografts derived from CRPC-tissue indicating that these abnormalities are present in cells capable of in vivo tumour-reconstitution. Micro-ChIP analysis of fractionated primary CaP cultures identified bivalent chromatin at LXN and RARRES1 promoters. Transcriptomic profiling failed to reveal significant changes in gene expression after transduction with LXN or RARRES1. However, an interactome for LXN and RARRES1 was successfully generated in PC3 cells. Additionally, confocal microscopy of mVenus-tagged LXN revealed a pan-cellular distribution which is reflected in the interactome. Screening for GAAR and XCA, using a high-throughput qPCR assay, could facilitate a targeted-medicine strategy in the treatment of CaP and CRPC. Further investigation of the LXN and RARRES1 interactomes may identify their mechanism(s) of action and the micro-ChIP assay could be used to identify epigenetic-inducers of LXN and RARRES1 which could provide a CSC-targeted strategy for CaP treatment. 2 List of Contents Content Page Abstract 2 List of Contents 3 List of Figures 8 List of Tables 11 Acknowledgements 12 Author’s declaration 13 1. General Introduction 14 1.1. The Prostate 14 1.1.1. Topographical Anatomy of the Human Prostate 15 1.1.2. Histological Anatomy of the Human Prostate 16 1.1.3. Development and Maturation of the Prostate 19 1.2. Non-Malignant Disorders of the Prostate 23 1.2.1. Prostatitis 23 1.2.2. Benign Prostatic Hyperplasia 23 1.2.3. Prostate Intraepithelial Neoplasia 23 1.3. Prostate Cancer 24 1.3.1. Epidemiology of Prostate Cancer 24 1.3.2. Low Grade-Organ Confined CaP 26 1.3.3. Cancer-Cell Invasion and Metastasis 27 1.3.4. Diagnostics in Prostate Cancer 28 1.3.4.1. PSA Testing 28 1.3.4.2. Gleason Grading 29 1.3.5. Current Treatments for Prostate Cancer 29 1.3.5.1. Active Surveillance 29 1.3.5.2. Radical Prostatectomy 29 1.3.5.3. Radiotherapy 29 1.3.5.4. Androgen Deprivation Therapy 30 1.3.6. Castration-Resistant Prostate Cancer 30 1.4. Cancer Stem Cells 31 1.4.1. Therapeutic Implications of CSCs 33 1.4.2. Prostate Stem cells and Cancer Stem Cells 34 1.5. Introduction to Models 39 1.5.1. Cell Lines 39 1.5.2. Primary Cultures 41 1.5.3. Mouse Models 41 1.6. Regulation of Gene Expression in the Prostate Epithelium 43 1.6.1. Androgen Signalling 47 1.6.2. Retinoic Acid 49 1.7. Epigenetic Regulation 51 1.7.1. Histone Modifications and Chromatin Remodelling 51 1.7.2. MicroRNAs 55 1.7.3. Long Non-Coding RNAs 55 3 1.7.4. DNA Methylation 55 1.8. Research Aims 56 2. Materials and Methods 58 2.1. Mammalian Cell Culture 58 2.1.1. Maintenance of Mammalian Cells 58 2.1.2. Isolation and Maintenance of Primary Cultures 60 2.1.3. Irradiation of Fibroblasts 61 2.1.4. Generation and Maintenance of Xenografts 61 2.1.5. Cryopreservation of Mammalian Cells 61 2.1.6. Determination of Live Cell Number Using a Haemocytometer 61 2.2. Primary Culture Enrichment 62 2.2.1 Enrichment of α2β1-Integrin Expressing Cells 62 2.2.2. Enrichment of CD133 Expressing Cells 62 2.3. Transfection of Cell Lines with Plasmid DNA 63 2.4. Lentivirus Production 63 2.4.1. Bacterial Transformation 64 2.4.2. Bacterial Cultures and Plasmid Purification 64 2.4.3. Gateway™ Cloning 65 2.4.4. Virus Packaging 66 2.4.5. Determining Lentiviral Titres 67 2.5. Transduction of Mammalian Cells 67 2.5.1. Determination of Viral Integration 68 2.5.2. Generation of Stable Lines 68 2.6. Isolation and Analysis of Mammalian Cell DNA 69 2.6.1. DNA Extraction (Qiagen) 69 2.6.2. DNA Extraction (Phenol Chloroform) 69 2.6.3. DNA Extraction from Formalin Fixed Paraffin Embedded Tissue 70 2.6.4. Agarose Gel Electrophoresis 71 2.6.5. PCR Amplification 71 2.6.6. Gel Extraction 72 2.6.7. Quantitative PCR of Genomic DNA 72 2.6.8. Analysis of AR Genomic Copy Number Alterations 73 2.7. Isolation and Analysis of Mammalian Cell RNA 73 2.7.1. RNA Extraction 73 2.7.2. Complementary DNA Synthesis 74 2.7.3. Reverse Transcription PCR 74 2.8. Chromatin Immunoprecipitation 75 2.8.1. Chromatin Preparation 75 2.8.2. Sonication Control DNA Extraction 75 2.8.3. Immunoprecipitation 76 2.8.4. Quantitative PCR Analysis 77 2.8.5. Micro-Chromatin Immunoprecipitation 78 2.9. Analysis of Protein Expression 79 2.9.1. Cell Lysis 79 2.9.2. Bicinchoninic Acid Assay 79 4 2.9.3. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis 79 2.9.4. Western Blot 80 2.9.5. Protein Immunoprecipitation 80 2.10. Protein Localisation Assays 80 2.10.1. Immunofluorescence 80 2.10.2. Subcellular Fractionation 81 3. Development and Validation of a qPCR Test for Simultaneous Detection of AR Amplification and X Chromosome Aneuploidy 83 3.1. Introduction 83 3.2. Premise of the Assay 89 3.3. Assay Work-Flow 92 3.4. Results 94 3.4.1. Semi-Quantitative Analysis of AR Copy Number in Male and Female Genomic DNA by PCR 94 3.4.2. Primer Efficiency and Species Specificity of AR, GAPDH and DMD Primers in qPCR 96 3.4.3. Genomic Copy Number of AR and DMD in Human Prostate Cancer Cell Lines 98 3.4.4. Assay Validation – Sensitivity and Accuracy 100 3.4.5. Genomic Copy Number of AR and DMD in Several Primary Cultures of Human CaP and BPH 103 3.4.6. Genomic Copy Number of AR and DMD in Several Tissue Samples of Human Prostate Cancer 105 3.4.7. Analysis of 10 Blinded Samples – Independently Assessed for AR Amplification Using FISH 107 3.4.8. Analysis of X Chromosome Aneuploidy and AR Amplification in Near-Patient Xenografts 109 3.4.9. Assay Compatibility with Formalin-Fixed Paraffin- Embedded Tissue 113 3.5. Discussion 116 3.5.1. A qPCR Assay Capable of Detecting AR Amplification in a Variety of Heterogeneous Biological Samples has been Successfully Established 117 3.5.2. The Assay also Gives Information Regarding X Chromosome Aneuploidy – a Relatively Common Event in CaP Associated with Poor Prognosis 119 4. Epigenetic Regulation and Investigation of the Mechanism of Action of LXN and RARRES1 in Prostate Epithelial Cells 123 4.1. RARRES1 123 4.2. LXN 125 4.3. Low Expression of RARRES1 or LXN has a Negative Impact on Recurrence-Free Survival in CaP 127 4.4. Co-expression of RARRES1 and LXN in Primary Prostate Cultures 129 4.5. Regulation of LXN and RARRES1 131 4.6. Localisation of LXN in the Prostate Epithelium 131 5 4.7. Overview of Experimental Techniques Used in this Chapter 132 4.7.1. Chromatin Immunoprecipitation 132 4.7.2. Lentiviral Vectors as Gene Delivery Systems 135 4.7.3. Gateway™ Cloning 137 4.7.4. Microarray 140 4.7.5. Mass Spectrometry 141 4.8. Results 143 4.8.1. ChIP Primer Validation 143 4.8.2. ChIP Validation 147 4.8.3. Chromatin Status of RARRES1 and LXN Promoter Sequences in PNT2C2 and LNCaP Cells 149 4.8.4. ChIP Scale Down 151 4.8.5. Establishing a qPCR Protocol for the Estimation of Chromatin Fragmentation After Sonication of Small Quantities of DNA with a View to Micro-ChIP 153 4.8.6. μChIP of Fractionated Primary CaP Samples Interrogated for Relative Enrichment of RARRES1 and LXN Promoter Sequences Using anti- H3K27me3, H3K4me3 and RNApol II Antibodies. 155 4.8.7. Transient Transfection of Primary Prostate Epithelial Cells with Plasmid Vectors is Inefficient and Precludes Their Use 165 4.8.8. Lentiviral Cloning and Virus Production 167 4.8.9. Optimisation of Lentivirus Titres 171 4.8.10. Transduction with Lentivirus Results in Stable Transgene Expression in Primary Human Prostate Epithelial Cells. 173 4.8.11. LXN is a Soluble Pan-Cellular Protein 174 4.8.12. Generation of an Interactome for LXN-HA and RARRES1-HA in PC3 Cells 177 4.8.13. Unsuccessful Attempt to Validated RARRES1 Interactome Through Co-IP of importin 7 in PC3 Cells Transfected with RARRES1-HA Plasmid and Subsequent IP Using anti-HA Antibody. 181 4.8.14. Transcriptome Profiling of Primary Prostate Epithelial Cells – Transduced with LXN, RARRES1 or GUS (Control) Gene Lentiviral Vectors 183 4.9.
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