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Characterising Mechanisms of Aberrant Mutant Androgen Receptor Signalling in Advanced Prostate Cancer

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Characterising Mechanisms of Aberrant Mutant Androgen Receptor Signalling in Advanced Prostate Cancer Characterising mechanisms of aberrant mutant androgen receptor signalling in advanced prostate cancer Wenrui Guo 120444241 Supervisors: Dr. Luke Gaughan Prof. Craig Robson Surgical Oncology Research Group Northern Institute for Cancer Research, Newcastle. September 2018 1 Abstract Prostate cancer (PC) is the most commonly diagnosed disease in the UK which causes approximately 10,000 deaths annually. Although an initially effective response to androgen deprivation therapy (ADT) occurs in most patients, the tumour normally recurs in a more aggressive form of the disease termed castrate resistant PC (CRPC) and is largely untreatable at this stage. In many cases, disease is driven by inappropriate androgen receptor (AR) signalling. It is therefore vital to have better understanding of mechanisms that re-activate AR and promote ADT resistance in the clinic and hence better treatments for advanced tumour. Activation of AR by testosterone is crucial for prostate growth and transformation. Anti- androgens, the second most common PC therapy after surgery, antagonise ligand binding to the receptor and hence deactivate AR signalling. In 2012, enzalutamide, a more potent agents in terms of availability to block AR was approved by the FDA and ENA as a second-generation anti- androgen for clinical usage. Although it demonstrated several advantages over its pervious counterpart bicalutamide, response rates of just 50% in CRPC patients and subsequent resistance observed in responders has limited its effectiveness. Critically, several lines of evidence from pre- clinical models and patient samples indicate that one particular resistant mechanism is the emergence of AR mutant(s), in part, driven by a specific AR mutation F876L that enables the compound to act as an agonist. Importantly, the same mutant was later detected in metastatic PC patient had been treated with apalutamide. Evidently, novel therapies emerging into clinical treatment of advance disease have the added challenge of being efficacious in the background of mutant AR and thus developing model systems to test this is of paramount importance. In order to enable more physiological modelling of aberrant ARF876L activity that would highlight potentially distinct mechanisms that could be exploited in future therapies, this project aimed to generate CRISPR-edited LNCaP and CWR22Rv1 cell lines expressing the enzalutamide-activated ARF876L mutant. Meanwhile, a part of project has also focused on generation of a physiologically relevant AR rescue/replacement in vitro cell line model (LNCaP- ARF876L cells) which permits ability for studying ARF876L directly regulated gene expression profiles by effectively knockdown endogenous AR without impacting on the ectopically expressed mutant. Furthermore, by using 2 Illumina Human HT-12 arrays analysing LNCaP-ARF876L cells revealed a comprehensive transcriptomic data-set to provide an insight into how an enzalutamide-activated AR mutant can drive a distinct gene-set in advanced PC. This is important as it may enable distinct biomarker discovery in enzalutamide-resistance disease and has highlighted interplay between the ARF876L mutation and the glucocorticoid receptor. Lastly, the LNCaP- ARF876L cell lines was utilised to demonstrate that aberrantly-functioning receptor is sensitive to BET inhibitors. In all, the work has shown that the ARF876L mutant drives a distinct transcriptional programme to the endogenous AR in LNCaP cells and the model can be utilised effectively to indicate sensitivities of the receptor to clinically-relevant compounds. 3 Acknowledgments At the beginning of this thesis, I would like to express my deepest appreciation to all those who provided me the possibility to complete this research project. Firstly, I would like to thank my supervisors Dr. Luke Gaughan and Pro. Craig Robson, for their continuous guidance, motivation, patience and engagement during all my PhD study. Also, I want to thank all the team both present and past members of my group; thanks Dr. Dominic Jones, Dr. Mark Wade, Lewis Chaytor and Evangelia Kounatidou for their technical assistance and helpful discussion., My deep gratitude for my parents Rongfu Guo and Kelin Chen, the most generosity and loveable person. Thanks for their unconditional love, support, encouragement and understanding that hold me up through all the ups and downs. I wouldn’t be where I am without you. I would also like to thank all my friends, specially, my dearest mate Yun Wei and her husband. I also want to dedicate this thesis to Mr. Xu Zhao who has been by my side snice day one we met in High school. Thanks, for his love, support and understanding that help me through the toughest time. I would like to thank Dr. Daniel O’Neill from AstraZeneca, for his suggestion on the generation of CRISPR PC cell model; Finally, thanks to all members of staff at the Northern Institute for Cancer Research for creating such an amazing welcoming atmosphere within the institute and for their continuous help. 4 5 Contents List of Abbreviations ..................................................................................................................... 11 List of Figures ................................................................................................................................ 16 List of Tables ................................................................................................................................. 18 General introduction ................................................................................................... 19 1.1. Prostate cancer and treatment. ......................................................................................... 20 1.2. Androgen sensitivity in prostate cancer. ........................................................................... 21 1.3. The androgen-receptor signalling cascade in prostate cancer and castration-resistant prostate cancer ......................................................................................................................... 23 1.4. The structure of the androgen receptor ............................................................................ 26 1.4.1. N-terminal transactivation domain (NTD) .................................................................. 26 1.4.2. The DNA-binding Domain (DBD) and hinge region (H) ............................................... 27 1.4.3. The ligand-binding domain (LBD) ............................................................................... 28 1.5. Direct AR-targeting anti-androgens. .................................................................................. 28 1.6. Acquisition of AR mutation as a mechanism of therapy evasion. ..................................... 31 1.7. ARF876L ................................................................................................................................. 34 1.8. Alternative AR targeting agents ......................................................................................... 35 1.9. Cross Talk between the AR and Glucocorticoid Receptor (GR) ......................................... 36 1.10. The bromodomain and extra terminal domain (BET) family proteins involves in AR- mediated transcriptional programme. ..................................................................................... 38 1.11. P300/CBP histone acetylation activity associates with progression of prostate cancer. 42 1.12. The novel gene-editing technology is advancing current study tools. ............................ 45 1.13. ZINC finger and TALEN ..................................................................................................... 46 1.14. Mechanism and development of CRISPR......................................................................... 48 1.15. Application of CRISPR....................................................................................................... 51 6 Aims ............................................................................................................................. 53 Methods and Materials ............................................................................................... 54 3.1 General expression plasmids and primers, ......................................................................... 55 3.2 Bacterial transformations and plasmid preparation .......................................................... 55 3.3 Site- directed mutagenesis ................................................................................................. 55 3.4 Primers ................................................................................................................................ 56 3.5 Mammalian cell culture and passage ................................................................................. 56 3.6 Transient plasmid DNA transfection ................................................................................... 57 3.7 Construction of lentiviral expression vectors ..................................................................... 57 3.8 Virus production and titre determination .......................................................................... 58 3.9 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting ...................... 58 3.10 Cytoplasmic and nuclear extract preparation .................................................................. 59 3.11 Immunofluorescence ........................................................................................................ 60 3.12
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