Identification of signalling pathways involved in the oxidative stress response triggered by Low Temperature Plasma in prostate epithelial cells and the assessment of tumour-associated allelic expression in Prostate Cancer John Packer BSc. (Hons) Ph.D University of York Biology June 2018 Abstract Pairing of cancer genome and transcriptome data has revealed that heterozygous mutations aren’t always expressed in cells. The potential for point mutation or genomic rearrangement to alter tumour allelic expression has implications for understanding cellular heterogeneity and application of treatments. Mutation of SPOP, PTEN and IDH-1 was assessed in 51 primary prostate cancer cultures to establish allelic heterozygosity and ascertain whether oncogenic change to coding regions altered allelic expression. No mutations were detected in the three genes, although 18% of tested cultures had loss of heterozygosity in PTEN. The TMPRSS2-ERG fusion, present in half of all prostate cancers, is selectively expressed at an allelic level by cancer stem cells. Monoallelic expression didn’t correlate with TMPRSS2 promoter hypermethylation. Prostate cultures expressed fusion transcript, however epigenetic features of monoallelically expressed genes were not investigated in the epithelial subpopulations. Understanding of allelic chromatin states may inform treatment strategies that permit tumour suppressor expression or oncogenic protein repression. Inability to predict indolent or aggressive progression of organ-confined prostate cancers has created the problem of surgical overtreatment. Focal therapies targeting the tumour core are being met with increasing rates of recurrence, necessitating development of novel treatments. The anti-cancer properties of Low Temperature Plasma (LTP) are being explored in prostate models where it produces autophagy and necrosis through generation of reactive species. Initial gene expression response to LTP and the activation of upstream transcription factors were analysed. LTP activated Nrf2, AP-1 and Notch signalling in patient matched prostate normal and cancer cultures. The progenitor-containing cell fraction was more responsive to LTP than differentiated epithelial cells in both transcription of response genes and nuclear accumulation of active Notch1. When linked to cell-fate outcomes, these immediate molecular responses of prostate cancer to LTP could be used as hallmarks of resistance or treatment efficacy in patients. 2 Thesis Contents Abstract ............................................................................................................................................... 2 Thesis Contents ................................................................................................................................... 3 List of Tables ....................................................................................................................................... 8 List of Figures ...................................................................................................................................... 9 Acknowledgements ........................................................................................................................... 13 Author’s Declaration ......................................................................................................................... 14 1.1 - The Prostate; Anatomy and Cellular Hierarchy ........................................................................ 15 1.1.1 – Prostate Anatomy ............................................................................................................. 15 1.1.2 – Prostate Development ...................................................................................................... 15 1.1.3 - Cells of the Prostate ........................................................................................................... 18 1.1.4 - Prostate Epithelial Stem Cells ............................................................................................ 21 1.2 - Disorders of the Prostate .......................................................................................................... 26 1.2.1 - Benign Prostatic Hyperplasia ............................................................................................. 26 1.2.2 - Prostatitis ........................................................................................................................... 26 1.2.3 - Inflammatory Aetiology of Prostate Cancer ...................................................................... 26 1.2.4 - Proliferative Inflammatory Atrophy .................................................................................. 30 1.2.5 - Prostatic Intraepithelial Neoplasia .................................................................................... 31 1.2.6 - General Attributes of Prostate Cancer .............................................................................. 33 1.2.7 - Prostate Cancer Epidemiology ........................................................................................... 33 1.3 - Tumour Heterogeneity and Cancer Stem Cells ......................................................................... 36 1.3.1 - Clonal Evolution Model ...................................................................................................... 36 1.3.2 - Linear step carcinogenesis ................................................................................................. 39 1.3.3 - Field Cancerisation ............................................................................................................. 39 1.3.4 - Cancer Stem Cell Model ..................................................................................................... 40 1.3.5 - Pre-tumour development .................................................................................................. 44 1.3.6 - The stem cell niche ............................................................................................................ 45 1.3.7 - The immortal strand hypothesis ........................................................................................ 45 1.3.8 - Cancer relapse; a stem cell triggered event? .................................................................... 46 3 1.3.9 - Prostate Cancer Stem Cells ................................................................................................ 47 1.4 - Prostate Cancer Diagnosis ........................................................................................................ 50 1.4.1 - Prostate Specific Antigen Testing ...................................................................................... 50 1.4.2 - The Gleason Grading System ............................................................................................. 51 1.4.3 – Treatment of Low Grade Prostate Cancer ........................................................................ 54 1.4.4 - Treatment of Advanced Prostate Cancer .......................................................................... 57 1.5 – The Genetic Background of Primary Prostate Cancer .............................................................. 61 1.6 - Castrate Resistant Prostate Cancer (CRPC) ............................................................................... 65 1.6.1 - Androgen signalling in end-stage disease .......................................................................... 65 1.6.2 - Involvement of Basal Cancer Stem Cells in Metastatic disease ......................................... 67 1.6.3 - Neuroendocrine Prostate Cancer ...................................................................................... 68 1.6.4 - Genetic heterogeneity in Castrate Resistant Prostate Cancer .......................................... 69 1.6.5 - Prostate Cancer Invasion and Metastasis .......................................................................... 70 1.7 - Prostate Cancer Models ............................................................................................................ 75 1.7.1 - Cell Lines and Primary Cultures ......................................................................................... 75 1.7.2 - Mouse Models; Xenograft and Transgenic lines ................................................................ 79 1.7.3 - Cancer Stem Cells in Mice .................................................................................................. 82 1.8 - Alterations of Allelic expression in Prostate Cancer ................................................................. 83 1.8.1 - Classical monoallelic expression ........................................................................................ 83 1.8.2 - Random monoallelic expression; involvement in cancer? ................................................ 87 1.8.3 - Epigenetic regulation of random monoallelic expression and perturbation in cancer ..... 89 1.8.4 - Epigenetic modifiers as cancer treatments ....................................................................... 94 1.8.5 - PTEN and Prostate Cancer ................................................................................................. 95 1.8.6 - SPOP and Prostate Cancer ............................................................................................... 100 1.8.7 - Isocitrate Dehydrogenase-1 and Prostate Cancer ........................................................... 106 1.8.8 - TMPRSS2-ERG; a defining rearrangement in Prostate Cancer .......................................