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Targeting the Splice Factor Kinase CLK1 in Prostate Cancer

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TARGETING THE SPLICE FACTOR KINASE CLK1 IN PROSTATE CANCER CELLS By Simon Ogbonnaya Uzor A thesis submitted in partial fulfilment of the requirements of the University of the West of England, Bristol for the degree of Doctor of Philosophy May, 2019 Supervisors: Dr. Michael Ladomery Dr. Ian Wilson i Author’s Declaration "This thesis is submitted in partial fulfilment of the requirements for the award of PhD in Biomedical Science and except where duly acknowledged or referenced it is entirely my own work. It has not been submitted, either in whole or in part, for any other award at UWE or elsewhere." Signed Date 10/05/2019 Project Supervisor’s Declaration "I confirm that I have read this PhD thesis and that the work it describes was undertaken under my supervision." Signed Date 10/05/2019 Name, Position and Address of Project Supervisor Associate Professor in Biomedical Science, Faculty of Health and Applied Science. University of the West of England, Coldharbour Lane, Frenchay, Bristol, BS16 1QY, United Kingdom ii ABSTRACT Prostate cancer is the most rampant diagnosed cancer and the second leading cause of cancer related death in men between middle age and old age. There is a need to identify the molecular genetic processes that underpin prostate cancer and to search for new treatments. Alternative splicing affects over 94% of human genes and aberrant splicing is implicated in prostate cancer. Splicing is regulated by splice factors and protein kinases (the latter including SRPK1 and CLK1). The CLK1 protein kinase specifically regulates alternative splicing by phosphorylation of SR proteins within the nucleus, particularly in nuclear speckles (splice factor storage sites). CLK1 is also found to be overexpressed during malignant prostate cell transformation-; therefore targeting the splice factor kinase CLK1 by its specific chemical inhibitor (TG003) could be therapeutically useful. We confirm that CLK1 expression is itself regulated through alternative splicing: skipping of exon 4 or retention of intron 4 results in truncated, inactive CLK1. The aims of this project are to study CLK1 alternative splicing and to explore the effects of targeting the splice factor kinase CLK1 in prostate cancer. Prostate cancer cell lines (androgen independent PC3 and DU145 cells) were treated independently for 24, 48 and 72hrs with varying concentrations (10nM -100µM) of the benzothiazole compound (TG003) a specific inhibitor of CLK1. Results suggest that chemical inhibition of CLK1 with TG003 treatment suppressed growth and induced apoptosis in prostate cancer cell lines, as well as causing decreased cell migration and invasion. Similar effects were observed when CLK1 expression was reduced with an siRNA. There was also increased E-cadherin expression with TG003 treatment; in contrast, vimentin expression was reduced suggesting reversal of endothelial- mesenchymal transition following TG003 treatment. RT-PCR analysis revealed that TG003 treatment altered CLK1’s own splicing by altering exon 4 skipping rates in a iii dose dependent manner, suggesting that a feedback loop mechanism contributes to the regulation of CLK1 expression. In vivo mouse work with PC3 cell line xenografts showed a highly significant reduction in tumour growth and volume following intraperitoneal TG003 administration (10 and 50µM). In conclusion, the findings presented in this thesis suggest that targeting CLK1 may bring considerable anticancer benefits. iv ACKNOWLEDGEMENTS I lack words to express my profound gratitude to the Almighty and Sovereign God, the God of all flesh. For in Him I live, move and have my being and by His miraculous work I was able to come to Bristol for studies. I sincerely appreciate the efforts of my Director of studies (Dr. Michael Ladomery and second supervisor Dr. Ian Wilson) whom God has used immensely to bring this work to completion. I cannot thank them enough for reinvigorating my zeal and my stability through their consistent advice and encouragement when I was upset. My sincere gratitude also goes to the Laboratory Technicians of the University of the West of England especially the Technicians at 2G block including Dr. Jef and Dave Corey, for their cheerful assistance in provision of Laboratory materials and technical supports when needed. I earnestly appreciate my research colleagues in the Ladomery group (Layla, Samantha, Chigeru, Marina, Giota, Sean, Tareq and especially Elizabeth Bowler). Great thanks to my professional colleagues. I also appreciate the Ebonyi State University / Tertiary Education Trust Fund of Nigeria for their financial support. Furthermore, I earnestly acknowledge the efforts of my collaborators in Exeter University who assisted me in the in vivo mouse work. Words are not enough to express my gratitude to my lovely wife (Uzor Glory Ogbonneya) and my little kids (Uzor Praise Nneoma, Uzor Bright Chukwuemerie and Uzor Divinegrace Ebubechi) whom by their permission I am here. I also thank Pastor and Pastor Mrs Favour Obasi, Pastor Yemi Ladipo, my lovely mother and my siblings for their prayer support. Silver and gold are not enough to appreciate Pastor Tony and the entire members of Filton Community Church, Bristol for their agape love, consistent prayers and other supports. I sadly missed my dear father and my lovely sister Ijeoma who passed to the great beyond when I am highly in need of them. v Contents …………………………………………………………………......vi Title page ……………………………………………………………………………….......i Declaration ………………………………………………………………………………….ii Abstract …………………………………………………………………………………….iii Acknowledgement …………………………………………………………………………v List of table …………………………………………………………………………………xii List of figures ...........................................................................................................xiii List of Abbreviations and units ………………………………………………………….xiv Chapter 1: Introduction ……………………………………………………………………………….1 1.1 Prostate cancer ………………………………………………………………………..2 1.1.1 Structure and function of the prostate gland ……………………………7 1.1.2 Benign prostate hyperplaxia (BPH) and prostate cancer ……………..9 1.1.3 Epidemiology and risk factors in prostate cancer ……………………..11 1.1.4 Prostate cancer risk factors ……………………………………………..15 1.1.5 Genetic and other factors in prostate cancer predisposition …………18 1.1.6 Diagnosis of prostate cancer …………………………………………....21 1.1.7 Established treatments of prostate cancer …………………………….35 1.2 Alternative (differential) splicing and prostate cancer ……………………………41 1.2.1 Pre-mRNA splicing ……………………………………………………….41 1.2.2 Pre-mRNA splicing mechanism ………………………………………...42 vi 1.2.3 Functional role of spliceosome in pre-mRNA splicing ……………...46 1.2.4 Assembly of spliceosomes via intron and exon definition …………..48 1.2.5 Types of alternative splicing ………………………..………………….49 1.2.6 Alternative splicing mutation and disease development …………….51 1.2.7 Regulation of alternative splicing by splice factors …………………..57 1.2.8 SR protein kinases ………………………………………………………60 1.2.9 Regulation of splice factors (SR proteins) by splice factor kinases (SRPK and CLK) ………………………………………………………………………….61 1.2.10 Alternative splicing and epithelial mesenchymal transition (EMT) …67 1.2.11 Identification of mutations causing splicing defects ………………….68 1.3 Targeting the splicing machinery in prostate cancer ……………………………69 1.3.1 Therapeutic manipulation of alternative splicing ……………………..69 1.3.2 CLK inhibition in prostate cancer ……………………………………...71 1.4 Aims and Objectives ………………………………………………………………..73 1.4.1 Hypothesis ……………………………………………………………….73 1.4.2 Aims ………………………………………………………………………73 1.4.3 Objectives ………………………………………………………………..73 Chapter 2: 2.0 Materials and Methods ……………………………………………………………75 2.1 Cell culture …………………………………………………………………………..76 2.2 Cell treatment ……………………………………………………………………….76 2.3 Adherent cell trypsinisation ………………………………………………………..76 2.4 Cell cryopreservation ……………………………………………………………...77 vii 2.5 Thawing cells from liquid nitrogen ………………………………………………..77 2.6 Cell proliferation by trypan blue assay …………………………………………...78 2.7 Cell proliferation by Ki67 assay …………………………………………………...78 2.8 Spectrophotometric measurement of colour changes and media depletion following TG003 treatment (indirect proliferation assay) …………………...79 2.9 Acridine orange assay ……………………………………………………………..79 2.10 Caspase 3/7 apoptosis assay ……………………………………………………80 2.11 RNA extraction …………………………………………………………………….80 2.12 RNA quality (nano drop) ………………………………………………………….81 2.13 cDNA synthesis ……………………………………………………………………81 2.14 DNA quantitation …………………………………………………………………..83 2.15 CLK1 primer design ……………………………………………………………….83 2.16 Gel extraction (DNA purification) ………………………………………………...84 2.17 DNA sequencing …………………………………………………………………..84 2.18 RT-PCR …………………………………………………………………………….85 2.19 Protein isolation ……………………………………………………………………85 2.20 Determination of protein concentration using the Bradford assay …………..85 2.21 SDS- PAGE ……………………………………………………………………….85 2.22 Buffer preparation …………………………………………………………………87 2.23 Western blotting by wet transfer …………………………………………………87 viii 2.24 Antibodies ……… …………………………………………………………………87 2.25 Film development. .……………………………………………………………….88 2.26 Wound healing-scratch assay …………………………………………………..88 2.27 Cell migration and invasion assay ………………………………………………89 2.28 Heat shock assay …………………………………………………………………90 2.29 Osmotic stress induction and harmine treatment ……………………………..90 2.30 CLK1 siRNA knockdown …………………………………………………………90 2.31 EMT marker expression ………………………………………………………….91 2.32 PC3 xenografts ……………………………………………………………………92 2.33 Statistical analysis ………………………………………………………………..92
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