ROS and mitochondrial genetic abnormalities as novel indicators to predict anticancer drug efficacy By Tarek Zaidieh School of Pharmacy and Biomedical Sciences University of Portsmouth White Swan Road Portsmouth, United Kingdom April 2020 The thesis is submitted in partial fulfilment of the requirement for the award of the degree of Doctor of Philosophy of the University of Portsmouth Abstract Mitochondria are considered a primary intracellular site of reactive oxygen species (ROS) generation. Mitochondrial genetic abnormalities (mutations and copy number change) have been reported in various cancer types that might be associated with elevated ROS levels in cancer cells compared to normal cells. Since high levels of ROS can trigger apoptosis, treating cancer cells with ROS-stimulating agents may enhance cell death. This study aimed to investigate: 1) how baseline ROS levels might influence cancer cells’ response to ROS-stimulating therapy; 2) the link between mitochondrial genetic abnormalities and baseline ROS levels; 3) how specific mitochondrial DNA mutations might be utilised to predict cancer cells’ response to ROS-stimulating therapy. Sanger sequencing was used to screen for mitochondrial DNA (mtDNA) mutations and qPCR to measure mtDNA copy number (mtDNAcn) in four cancer and one non- cancerous cell lines. Three-dimensional structural mapping and analysis of all the non- synonymous mutations were then performed to assess their functional importance. The overall baseline intracellular ROS levels and mitochondrial superoxide were measured using the 2’,7’-dichlorofluorescin diacetate assay and MitoSOX indicator, respectively. Cells were treated with a conventional drug (cisplatin) and a mitochondria-targeting agent (dequalinium chloride hydrate) separately and jointly. Cell viability was assessed using MTS assays and drug combination synergisms were analysed using the Combination Index method. Apoptosis events were detected and the relative expression levels of the genes and proteins involved in ROS-mediated apoptosis pathways were also investigated. Greater numbers of non-synonymous mutations in the mitochondrial complexes I/III coding regions were detected in cancer cells with higher ROS levels. Our data showed a positive correlation between the baseline ROS levels, number of non-synonymous mutations, mtDNAcn and drug resistance levels in the tested cells. Synergistic effect of both drugs was also observed with ROS being the key contributor in cell death. Our findings assert that cancer cells with low levels of ROS are more sensitive to ROS- stimulating agents, while cells with high levels of ROS are more resistant. Moreover, specific mutations in the complexes I/III coding regions, A10398G, T11120C, C12084T, A13681G, G13708A, C13802T, A13966G and T14798C, could be used to indicate elevated intracellular ROS and therefore to predict drug resistance. Patient-derived i primary cell cultures and tissues, and in vivo experiments will be required to validate the present findings. ii Contents list Abstract ............................................................................................................................. i Contents list .................................................................................................................... iii Declaration ....................................................................................................................... x Word count ...................................................................................................................... x List of tables .................................................................................................................... xi List of figures ................................................................................................................. xii Abbreviations ............................................................................................................... xvi Acknowledgements ..................................................................................................... xxiv Dedication .................................................................................................................... xxv Dissemination ............................................................................................................. xxvi Chapter 1 General introduction .................................................................................... 1 1.1 Mitochondria ...................................................................................................... 2 1.1.1 Mitochondria structure and functions ......................................................... 2 1.1.2 Mitochondrial dynamics.............................................................................. 3 1.1.3 Mitochondrial genome ................................................................................ 4 1.1.3.1 Mitochondrial DNA structure ................................................................. 4 1.1.3.2 Mitochondrial DNA replication and transcription .................................. 6 1.1.4 ATP generation ........................................................................................... 9 1.1.5 Mitochondrial respiratory chain ................................................................ 11 1.1.5.1 Complex I .............................................................................................. 12 1.1.5.2 Complex II ............................................................................................. 14 1.1.5.3 Complex III ........................................................................................... 14 1.1.5.4 Complex IV ........................................................................................... 15 1.1.5.5 Complex V ............................................................................................ 17 1.1.6 Mitochondrial role in apoptosis ................................................................ 18 1.1.7 MtDNA Mutations .................................................................................... 22 1.2 Mitochondria and cancer .................................................................................. 26 1.2.1 The hallmarks of cancer ............................................................................ 26 1.2.2 Cancer as a mitochondrial metabolic disease............................................ 27 1.2.3 Mitochondrial metabolic reprograming in cancers ................................... 29 1.2.4 Apoptosis in cancer ................................................................................... 31 1.2.5 Mitochondrial DNA mutation and epigenetics in tumorigenesis .............. 34 iii 1.2.5.1 D-loop mutations in human cancer........................................................ 35 1.2.5.2 tRNA and rRNA mutations in human cancers ...................................... 36 1.2.5.3 OXPHOS complex I mutations in human cancer .................................. 36 1.2.5.4 OXPHOS complex III mutations in human cancer ............................... 37 1.2.5.5 OXPHOS complex IV mutations in human cancer ............................... 37 1.2.5.6 OXPHOS complex V mutations in human cancer ................................ 38 1.2.5.7 Large mitochondrial deletions in human cancers .................................. 38 1.2.6 MtDNA copy number in human cancers .................................................. 38 1.3 Reactive oxygen species (ROS) ....................................................................... 40 1.3.1 Source of ROS .......................................................................................... 40 1.3.2 Oxidative stress ......................................................................................... 42 1.3.2.1 Effects of oxidative stress on DNA ....................................................... 43 1.3.2.2 Effects of oxidative stress on lipids ....................................................... 44 1.3.2.3 Effects of oxidative stress on proteins ................................................... 44 1.3.2.4 Effects of oxidative stress on signal transduction ................................. 45 1.3.3 Antioxidant defence mechanisms ............................................................. 46 1.3.3.1 Superoxide dismutase ............................................................................ 46 1.3.3.2 Catalase ................................................................................................. 46 1.3.3.3 Peroxiredoxins ....................................................................................... 46 1.3.3.4 Glutathione peroxidase .......................................................................... 47 1.3.3.5 Glutathione ............................................................................................ 47 1.3.4 Antioxidative role of transcriptional factors ............................................. 48 1.3.5 Mitochondrial ROS functions as second messenger molecules ................ 49 1.3.6 ROS regulating apoptosis and cell cycle ................................................... 50 1.3.7 Mitochondrial ROS (mROS) in cancer development ............................... 53 1.3.8 ROS as signalling molecules in cancer survival and metastasis ............... 55 1.3.9 Role of ROS in cancer therapies ............................................................... 57 1.3.9.1 Role of ROS in targeted molecular therapy .......................................... 57 1.3.9.2 Role of ROS in photodynamic