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TSC2- Deficient Cell Lines New drug therapies to kill TSC2- deficient cell lines Henry Denis McCann 1467575 2019 Thesis submitted to Cardiff University in fulfilment of the of the requirements for the degree of Doctor of Philosophy This thesis is dedicated to Dr. Padraic O’Neill, for all the support given throughout the years. i Publications 1. Johnson, Charlotte E., Dunlop, Elaine A., Seifan, Sara, McCann, Henry D., Hay, Trevor, Parfitt, Geraint J., Jones, Ashley T., Giles, Peter J., Shen, Ming H., Sampson, Julian R., Errington, Rachel J., Davies, David Mark and Tee, Andrew R. 2018. Loss of tuberous sclerosis complex 2 sensitizes tumours to nelfinavir−bortezomib therapy to intensify endoplasmic reticulum stress- induced cell death. Oncogene 37, pp. 5913-5925 2. McCann, Henry, Johnson, Charlotte, Errington, Rachel, Davies, D. Mark, Dunlop, Elaine and Tee, Andrew 2018. Energy stress-mediated cytotoxicity in tuberous sclerosis complex 2-deficient cells with nelfinavir and mefloquine treatment. Cancers 10 (10), 375. ii Contents Publications........................................................................................................... ii Contents ............................................................................................................... iii Figures and Tables .............................................................................................. ix Figures: ............................................................................................................... ix Tables: ................................................................................................................. x Declaration ........................................................................................................... xi Acknowledgements ........................................................................................... xiii Abstract .............................................................................................................. xiv Abbreviations ...................................................................................................... xv Chapter 1: General Introduction .......................................................................... 1 1.1 Tuberous Sclerosis Complex (TSC) .............................................................. 1 1.1.1 History and General Introduction ........................................................... 1 1.1.2 Clinical management of TSC ................................................................. 6 1.2 mTORC1 in tumour growth .......................................................................... 19 1.2.1 mTOR .................................................................................................. 19 1.2.2 mTORC1 and angiogenesis ................................................................ 25 1.2.3 mTORC1 and lipogenesis ................................................................... 26 1.2.4 mTORC1 and autophagy ..................................................................... 27 1.2.5 mTORC1 and energy metabolism ....................................................... 29 1.2.6 mTORC1 and mitochondrial biogenesis .............................................. 30 1.2.7 Potential vulnerabilities of TSC ............................................................ 31 1.3 Nelfinavir ...................................................................................................... 40 1.3.1 General Introduction ............................................................................ 40 1.3.2 Nelfinavir controversy .......................................................................... 41 1.3.3 Nelfinavir and cancer ........................................................................... 41 1.3.4 Nelfinavir and radiotherapy .................................................................. 44 iii 1.3.5 Nelfinavir and TSC .............................................................................. 44 1.4 Aims of Thesis ............................................................................................. 45 Chapter 2: Materials and Methods ..................................................................... 47 2.1 Materials: ..................................................................................................... 47 2.1.1 Cell culture........................................................................................... 47 2.1.2 Drugs ................................................................................................... 47 2.2 Methods: ...................................................................................................... 48 2.2.1 Cell proliferation assay ........................................................................ 48 2.2.2 Flow cytometry .................................................................................... 49 2.2.3 Drug synergy assay ............................................................................. 50 2.2.4 Western blotting ................................................................................... 51 2.2.5 Rescue assays .................................................................................... 53 2.2.6 mRNA extraction and reverse transcription ......................................... 54 2.2.7 XBP-1 splicing ..................................................................................... 54 2.2.8 RNA sequencing .................................................................................. 55 2.2.9 Rhodamine 123 assay ......................................................................... 56 2.2.10 Tumour formation assay .................................................................... 57 2.2.11 Tumour outgrowth assay ................................................................... 57 2.2.12 ROS production analysis ................................................................... 58 2.2.13 Protein synthesis assay ..................................................................... 59 2.2.14 Statistical analysis ............................................................................. 59 Chapter 3: Mefloquine and nelfinavir combination caused selective cell death in mTORC1 hyperactive cells .................................................................. 60 3.1 Introduction: ................................................................................................. 60 3.1.1 Mefloquine ........................................................................................... 60 3.1.2 Hypothesis ........................................................................................... 65 3.2 Results: ........................................................................................................ 66 iv 3.2.1 An optimized combination of mefloquine and nelfinavir synergises to selectively target mTORC1 hyperactive cells and causes caspase- independent cell death. ................................................................................ 66 3.2.2 Mefloquine/nelfinavir combination prevented tumour colony formation and prevented tumour regrowth from treated spheroids. .............................. 72 3.2.3 Mefloquine/nelfinavir combination targeted Tsc2-/- cells in a mTOR and autophagy/lysosomal-independent manner. ................................................. 74 3.2.4 Mefloquine and nelfinavir as single drug agents, as well as in combination, blocked P-glycoprotein activity in Tsc2+/+ and Tsc2-/- MEFS. 77 3.2.5 Mefloquine/nelfinavir combination induced enhanced and prolonged ER stress in Tsc2-/- cells via the PERK pathway. .............................................. 78 3.2.6 Mefloquine/nelfinavir combination affects energy stress levels in Tsc2-/- cells. 86 3.2.7 Mefloquine/nelfinavir combination did not affect the production of reactive oxygen species (ROS). ................................................................... 89 3.3 Discussion: .................................................................................................. 90 Chapter 4: A combination of Bortezomib and Nelfinavir caused selectively cell death in mTORC1 hyperactive cells ........................................................... 97 4.1 Introduction: ................................................................................................. 97 4.1.1 The proteasome .................................................................................. 97 4.1.2 Bortezomib .......................................................................................... 98 4.1.3 Bortezomib and blood cancers ............................................................ 99 4.1.4 Bortezomib and solid cancers ........................................................... 100 4.1.5 Bortezomib combinations .................................................................. 100 4.1.6 Bortezomib limitations ....................................................................... 101 4.1.7 Bortezomib and ER stress ................................................................. 102 4.1.8 Bortezomib and nelfinavir .................................................................. 103 4.1.9 Hypothesis ......................................................................................... 104 4.2 Results: ...................................................................................................... 104 4.2.1 A combination of Bortezomib and nelfinavir selectively targeted Tsc2-/- cells in a caspase-dependent manner. ....................................................... 104 v 4.2.2 Bortezomib/nelfinavir
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