THE EFFECT OF HYPOXIA ON ALTERNATIVE SPLICING IN PROSTATE CANCER CELL LINES ELIZABETH BOWLER Thesis submitted in partial fulfilment of the requirements of the University of the West of England, Bristol for the degree of Doctor of Philosophy Faculty of Applied Sciences, University of the West of England, Bristol 2016 1 ACKNOWLEDGEMENTS First and foremost I would like to thank my primary supervisor Dr. Michael Ladomery. His encouragement and support throughout my PhD has helped me achieve some excellent results and has really built my confidence as a researcher. I am incredibly grateful for all the guidance he has provided through this project. Without Mike’s help and supervision this thesis would not be possible. I would also like to thank my secondary supervisors, Prof. John Hancock and Dr. Ian Wilson. John has been a great support during my time at UWE and has gone above and beyond to offer his help and expertise. Ian’s guidance with statistics was both helpful and insightful. In addition, I would like to acknowledge the collaborators that are linked to this project, Dr. Silvia Pastorekova (Slovak Academy of Sciences, Slovakia) has provided expertise for the hypoxia element of this PhD, and Dr. Roscoe Klick and his team at RNomics Platform Sherbrooke University, Canada) who conducted the high- throughput PCR work. I offer my thanks to the technicians in CRIB laboratory at UWE, especially David Corry who provided assistance with the confocal microscope. Furthermore, I greatly appreciate the encouragement and laughs provided by the Ladomery laboratory team as well as all the other PhD students in the CRIB laboratory. As expected in research, there have been many ups and downs and it has been a pleasure to share them with you all! Next, I would personally like to thank my family. My mum, dad and sister have always been incredibly supportive, encouraging and loving throughout my entire life and I am supremely appreciative for everything that they have done for me. I am exceptionally grateful to my partner Aydin who has provided me with a lot of love, support and reassurance through this period. This project was funded by the UWE RAE2008 QR Fund and the Doctoral Completion Bursary 2015 -2016 (UWE). 2 ABSTRACT Hypoxia is defined as the state in which the availability or delivery of oxygen is insufficient to meet tissue demand. It occurs particularly in aggressive, fast-growing tumours in which the rate of new blood vessel formation (angiogenesis) cannot match the growth rate of tumour cells. Cellular stresses such as hypoxia can cause cells to undergo apoptosis; however some tumour cells adapt to hypoxic conditions and evade apoptosis. Tumour hypoxia has been linked to poor prognosis and to greater resistance to existing cancer therapies. This thesis provides evidence that alterations in alternative splicing patterns of key genes is one method tumour cells adapt to hypoxia. A hypoxic-induced change in the alternative splicing of carbonic anhydrase IX (CA IX) is confirmed. CA IX is one of the best studied hypoxia markers, involved in maintaining an intracellular pH that favours tumour cell growth. Furthermore, evidence is provided here that in the PC3 prostate cancer cell line, the regulation of CA IX splicing involves the scaffold attachment factor B 1 (SAFB1) and pre-mRNA- processing-splicing factor-8 (PRPF8) splice factors. However, SAFB1 expression is shown to decrease in hypoxia. Alternative splicing patterns of previously documented cancer-associated genes are altered in hypoxia in the PC3, VCaP and PNT2 prostate cell lines. There is evidence of significant changes in the alternative splicing of several cancer-associated genes in hypoxia, which have varied roles in the hallmarks of cancer: Apoptosis (APAF1, caspase-9, Bcl-x and survivin); Immune tolerance (BTN2A2); Cellular motility and 3 invasion (CDC42BPA, FGFR1OP and UTRN); Alternative splicing (PUF60); Proliferation (RAP1GDS1) (proliferation); and those with an unknown function but linked to cancers (MBP, PTPN13 and TTC23). Most notably, there was a higher proportion of the pro-oncogenic isoforms of APAF1, Bcl-x, survivin, BTN2A2 and RAP1GDS1 in hypoxia than in normoxia. The mRNA expression of splice factors (SRSF1, SRSF2, SRSF3, SAM68, HuR and hnRNP A1) and SRSF1 protein production were shown to significantly increase in hypoxia. Phosphorylation of SRSF4 and SRSF5 was demonstrated to increase in hypoxia indication that hypoxia may alter alternative splicing patterns. The mRNA expression of the CLK1 and SRPK1 splice factor kinases also increased in hypoxia; however only CLK1 protein production was shown to also increase in hypoxia. There were no significant changes to alternative splicing when SRPK1 was knocked down or inhibited (using SPHINX) suggesting that SRPK1 was not involved in the alteration of alternative splicing of the cancer-associated genes studied. However, siRNA knockdown and chemical inhibition of CLK1 (using TG003) suggested a shift in FGFR1OP splicing that mirrored the effect of hypoxia on FGFR1OP splicing. This suggests that CLK1 activity is inhibited in hypoxia. This conflicts the finding that CLK1 production increases in hypoxia and suggests that there are more mechanisms concerned in the regulation of CLK1 during hypoxia. This work has provided an insight into mechanisms that are involved in alternative splicing changes in hypoxia in mammalian cell lines. These novel research findings may aid in the understanding of how cells adapt to hypoxia especially in regards to alternative splicing and may offer future therapeutic targets in hypoxic tumours. 4 CONTENTS 1.0 Introduction……………………………………………………………………………….. 15 1.1 Prostate Cancer…………………………………………………………………………….…… 15 1.1.1 Incidence…………………………………………………………………………... 15 1.1.2 Risk factors for prostate cancer…………………………………..…….. 15 1.1.3 Current prostate cancer therapies……………………………………… 16 1.2 Hypoxia…………………………………………………………………………...………………… 23 1.2.1 Hypoxia and cancer therapy…………………………………………..….. 23 1.2.2 Hypoxic tumour microenvironment……………………………..……. 25 1.2.3 The hypoxia inducible factor (HIF) pathway……………………….. 27 1.2.4 Genes expressed as a results of hypoxia inducible factor (HIF) activation…………..………………………………………………..……. 29 1.2.5 HIF-α subunits………………………………………………………..…………. 32 1.3 Change in post transcriptional regulation in hypoxia……………………….... 34 1.3.1 Translation – Internal Ribosome Entry Sites (IRES)………..…… 34 1.3.2 Pre-mRNA splicing……………………………………………………………… 35 1.3.3 Alternative splicing……………………………………………………..…….. 39 1.3.4 Regulation of alternative splicing………………………………………. 41 1.3.5 Alternative splicing during cellular stress…………………………… 47 1.4 Hypotheses, aims and objectives……………………………………………………….. 54 1.4.1 Hypothesis 1………………………………………………………………………. 54 1.4.2 Hypothesis 2………………………………………………………………………. 55 1.4.3 Hypothesis 3………………………………………………………………………. 56 2.0 Methods and Materials………………………………………………………………. 57 2.1 Cell lines…………………………………………………………………………………………….. 57 2.2 Trypsinisation of adherent cells…………………………………………………………. 57 2.3 Cryopreserving cells…………………………………………………………………………… 58 2.4 Thawing cryo-preserved cells……………………………………………………..……… 59 2.5 Hypoxia treatment……………………………………………………………………..……… 59 2.6 Chemical inhibition of splice factor kinases……………………………………….. 61 2.7 Knockdowns by RNA interference (RNAi)…………………………………………... 62 2.8 RNA extraction………………………………………………………………………………….. 63 2.9 cDNA synthesis………………………………………………………………………………….. 64 2.10 Standard PCR……………………………………………………………………..……………. 64 2.10.1 Calculating % exon inclusion for alternatively spliced genes (%ψ)………….............................……………………………………………. 65 2.11 Qualitative (real-time) PCR………………………………………………………………. 65 5 2.12 High-throughput PCR……………………………………………………………………….. 66 2.12.1 High-throughput PCR of cassette exon inclusion in cancer- associated genes in hypoxic PC3 cells………………………………… 66 2.12.2 High-throughput PCR analysis of the effect of splice factor knockdowns on CA IX alternative splicing……………….…………. 67 2.13 Protein extraction and quantification………………………………………………. 68 2.14 Western blot analysis………………………………………………………………………. 69 2.14.1 Acrylamide gels…………………………..…………………………………….. 69 2.14.2 SDS PAGE…………………………………………..………………………………. 70 2.14.3 Transfer of proteins to a membrane………………………………….. 70 2.14.4 Detection of antigens……………………………………..…………………. 71 2.14.5 Image acquisition…………………………………………..………………….. 71 2.14.6 Scaffold attachment factor B1 (SAFB1) normalisation to β-actin……………………………………………………..………………………… 72 2.15 Cellular localisation of CLK1 protein.………………………………………………… 72 2.15.1 Nuclear and nuclear-free cellular fractions………………………… 72 2.15.2 Immunofluorescence analysis of CLK1 splice factor kinase subcellular localisation to the cytoplasm…………………………… 74 2.16 Statistical Analysis.............................................................................. 76 3.0 Examining Alternative Splicing Changes in Hypoxia in a Prostate Cancer Cell Line Model………………..……………………………………………… 82 3.1 Background………………………………………………………………………………………… 83 3.2 Confirming that hypoxia treatment was successful through use of the hypoxia marker carbonic anhydrase 9 (CA IX).…………………………………………. 85 3.3 Examining the effect of hypoxia on alternative splicing in genes involved in apoptosis…..………………………………………………………………………….. 91 3.4 High-throughput PCR of exon inclusion in cancer-associated genes………………………………………………………………………………………………… 101 3.5 Verification of high-throughput PCR results……………………………………….. 104 3.6 Discussion………………………………………………………………………………………….. 135 3.6.1 CA IX 135 3.6.2 Alternative splicing of