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Title: Chemotherapy Response and the Role of the WWOX Tumour Suppressor Name: Haroon Farooq

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Title: Chemotherapy Response and the Role of the WWOX Tumour Suppressor Name: Haroon Farooq Title: Chemotherapy response and the role of the WWOX tumour suppressor Name: Haroon Farooq This is a digitised version of a dissertation submitted to the University of Bedfordshire. It is available to view only. This item is subject to copyright. CHEMOTHERAPY RESPONSE AND THE ROLE OF THE WWOX TUMOUR SUPPRESSOR HAROON FAROOQ PhD 2017 University of Bedfordshire i CHEMOTHERAPY RESPONSE AND THE ROLE OF THE WWOX TUMOUR SUPPRESSOR By HAROON FAROOQ A thesis submitted to the University of Bedfordshire in fulfilment of the requirement of the degree of Doctor of Philosophy August 2017 ii Author’s Declaration I, Haroon Farooq declare that this thesis and the work presented in it are my own and have been generated by me as the result of my own original research. ………………………………………………………………………………………………..………… ……..…………………………………………………………………………………… I confirm that: 1. This work was done wholly or mainly while in candidature for a research degree at this University; 2. Where any part of this thesis has previously been submitted for a degree or any other qualification at this University or any other institution, this has been clearly stated; 3. Where I have cited the published work of others, this is always clearly attributed; 4. Where I have quoted from the work of others, the source is always given. With the exception of such quotations, this thesis is entirely my own work; 5. I have acknowledged all main sources of help; 6. Where the thesis is based on work done by myself jointly with others, I have made clear exactly what was done by others and what I have contributed myself; 7. Either none of this work has been published before submission, or parts of this work have been published as indicated on [insert page number or heading]: “ Name of candidate: Haroon Farooq Signature: Date: 10/08/2017 iii CHEMOTHERAPY RESPONSE AND THE ROLE OF THE WWOX TUMOUR SUPPRESSOR ABSTRACT Paclitaxel is a widely used chemotherapy drug for the treatment of multiple cancers. Paclitaxel works by inhibiting the disassembly of microtubules. Besides the role of paclitaxel in mitotic arrest, many studies suggest that paclitaxel could initiate apoptosis by activating ER-stress pathways. Cisplatin, another chemotherapy drug is also used for the treatment of solid tumours. It works by forming adducts with the structure of DNA leading to cell cycle arrest, DNA damage and apoptosis. These two chemotherapy drugs have different mechanisms of killing tumour cells, but they are often combined to give greater effect. WWOX is a tumour suppressor in many cancers. It has been shown that WWOX promotes apoptosis and suppresses in vivo tumorigenesis, but its function remains unclear. Here in my study I hypothesize that ER-stress is an important part of paclitaxel response in cancer cells, and the WWOX tumour suppressor is important in mediating paclitaxel induced ER- stress response. I also hypothesize that WWOX is important in mediating the apoptotic response to cisplatin. My results show that paclitaxel could not induce ER stress in multiple cancer cells lines, and WWOX did not affect the cytotoxic action of paclitaxel. I show some evidence that ER-stress may influence survival in ovarian cancer patients, but this is independent of the effect of WWOX expression. I show that WWOX is not important for cisplatin response since cisplatin suppresses the expression of WWOX. This effect may relate to its location at a common fragile site since cisplatin also down-regulates another fragile site-associated gene FHIT. However, I also show the apparent involvement of NF-kB2 and PAR2 transcription factors in the down regulation of WWOX. These findings increase our understanding of the unintended effects of chemotherapy treatments. iv Acknowledgments In the name of Allah, the Most Gracious and the most Merciful Alhamdulillah, all praises to Allah for the strengths and His blessings in completing this thesis. First and foremost, I would like to express my gratitude to my supervisor, Dr Adam Paige for his continual encouragement, support and mentoring. This work would not have been possible without his guidance and support. Under his guidance I successfully overcame many difficulties and learned a lot. His unflinching courage and conviction will always inspire me. I would also like to express my gratitude to other members of my supervisory team, Dr Bushra Ahmad and Dr Nicholas Worsfold for their encouragement and support. I would also like to thank Dr Emma Buick and Dr Ariane Standing for the demonstration of several laboratory techniques. This work would not be possible without the help and support of technical staff of the Life Science department. I would also like to thank Dr Mona El-Bahrawy (Imperial College, London) for immunohistochemistry work. At this time I think of my parents whose selfless sacrificial life and their great efforts and prayers enabled me to reach the present position in my life. I would like to thank my wife for her unconditional support and help. Finally I would like to dedicate all my work to my Late Father, who was a great role model for me in my life. The greatest gift that my father gave me was, he believed in me. I will always miss his guidance and support in my life. v List of abbreviations 5-FU - Fluorouracil ASK1- Apoptotic signalling kinase 1 ATF6 - Activating transcription factor 6 ATP - Adenosine triphonspahte ATR - Ataxia-telangiectasia and Rad3 Related Bcl - B-cell lymphoma BUB - Budding uninhibited by benzimidazoles cDNA - Complementary deoxyribonucleic acid CFS - Common Fragile site DMF - Dimethylformamide DMSO - Dimethyl sulfoxide EGFP - Enhanced green fluorescent protein eIF2α - Eukaryotic translation initiation factor 2 on the alpha subunit ERAD - Endoplasmic reticulum associated ERK - Extracellular signal regulated kinase ER Stress - Endoplasmic reticulum-stress FBS - Fetal bovine serum FDA - Food and drug administration FHIT - Fragile histidine triad FIGO - The international federation of gynaecology and obstetrics GRP-78 - Glucose regulated protein IHC - Immunohistochemistry IRE1 - Inositol requiring enzyme 1 JNK - Jun amino terminal kinases kDa - Kilo Dalton KO - Knockout LOH - Loss of heterozygosity MAP4 - Microtubule associated protein 4 vi MAPK - Mitogen activated protein kinases MDM2 - MOUSE DOUBLE MUTANT 2 MFSD2A - Major facilitator superfamily domain containing protein 2 mRNA - Messenger RNA ND - No drug NF-Kb - Nuclear factor kappa B NLS - Nuclear localisation sequence OS - overall survival PBS-T - Phosphate buffer saline-tween PERK - Protein kinase like ER kinase PFS - Progression free survival QRT-PCR - Quantitative real time polymerase chain reaction SDR - Short-chain dehydrogenase/reductase SEM - Standard error mean SRB - Sulforhodamine B TBP - TATA binding protein TCA - Trichloro acetic acid TCGA - The cancer genome atlas TNF - Tumour necrosis factor TRAF2 - TNF receptor-associated protein TVS - Transvaginal ultrasonography UPR - Unfolded protein response UV - Ultraviolet VCP - Valosin containing protein WT - Wild type WWOX - WW Domain Containing Oxidoreductase XBP-1 - X-box binding protein vii List of contents Chapter 1 Introduction ........................................................................................................... 1 1.1 FRA16D and WWOX ................................................................................................... 1 1.1.1 WWOX and spliced variants .................................................................................. 2 1.1.2 Subcellular and tissue distribution of WWOX ............................................................ 3 1.1.3 WWOX: Tyr33 phosphorylation, death signalling and tumour suppression ............... 8 1.1.4 WWOX binding proteins ........................................................................................... 9 1.1.5 Murine models of WWOX ....................................................................................... 11 1.2 Ovarian Cancer ............................................................................................................. 13 1.2.1 Epidemiology .......................................................................................................... 13 1.2.2 Molecular pathology of ovarian cancers .............................................................. 13 1.2.2.1 Hereditary ovarian carcinomas ......................................................................... 13 1.2.2.2 Sporadic ovarian cancer ................................................................................... 14 1.2.2.3 Borderline epithelial ovarian cancer .................................................................. 14 1.2.4 The dualistic model of epithelial ovarian carcinogenesis ............................................ 16 1.2.4.1 Type I ovarian tumours ........................................................................................ 16 1.2.4.1.1 Endometrioid Carcinoma and Clear Cell Carcinoma ...................................... 17 1.2.4.1.2 Mucinous Carcinoma ..................................................................................... 18 1.2.4.1.3 Low grade serous Carcinoma ........................................................................ 19 1.2.4.2 Type II ovarian tumours ....................................................................................... 20 1.2.4.2.1 High grade serous carcinomas ...................................................................... 20 1.2.5 Stages of Ovarian cancer ....................................................................................... 21 1.2.5.1
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