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Functional Analysis of the F-Box Protein Fbxl17

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Functional Analysis of the F-Box Protein Fbxl17 Functional analysis of the F-box protein Fbxl17 Bethany Jane Mason Clare College December 2019 This dissertation is submitted for the degree of Doctor of Philosophy Declaration This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except where specified in the text. It is not substantially the same as any work that has already been submitted before for any degree or other qualification at the University of Cambridge or any other University or similar institution. This dissertation does not exceed the prescribed word limit of 60,000 words. Bethany Jane Mason 1 Abstract Advances in DNA sequencing technology have allowed detailed characterisation of cancer genomes and has highlighted the contribution of somatic structural variations to the mutational landscape of epithelial tumours. However, our understanding of the functional consequences of such genome rearrangements remains rudimentary. By surveying the METABRIC dataset, consisting of segmented array-CGH copy number data, and paired-end whole-genome DNA and RNA sequencing data from primary breast tumours, we found that the F-box protein encoded by FBXL17 is frequently rearranged in breast cancer. F-box proteins are the substrate-recognition components of Skp1-cullin 1-F-box protein (SCF) E3 ligases. As essential components of the ubiquitin proteasome system (UPS) they are responsible for directing target proteins for ubiquitination. Fbxl17 is a relatively understudied member of the FBXL family of F-box proteins and, in breast cancers, is disrupted in the region of the gene that encodes its substrate-binding leucine rich repeat (LRR) domain. Truncating Fbxl17 LRRs impaired its association with the other SCF holoenzyme subunits Skp1, Cul1 and Rbx1, and decreased its ubiquitination activity. Loss of the LRRs also affected Fbxl17 binding to its targets. Thus, genomic rearrangements in FBXL17 are likely to disrupt SCFFbxl17-regulated networks in cancer cells. To investigate the functional effect of these rearrangements, we performed a yeast two-hybrid screen to identify Fbxl17-interacting proteins. Among the 37 binding partners Uap1, an enzyme involved in O-GlcNAcylation of proteins was identified most frequently. We demonstrate that Fbxl17 binds to UAP1 directly and inhibits its phosphorylation, which we propose regulates UAP1 activity. Knockdown of Fbxl17 expression elevated O-GlcNAcylation in breast cancer cells, arguing for a functional role for Fbxl17 in this metabolic pathway. To identify further interacting partners of Fbxl17, we performed a mass spectrometry analysis of purified Fbxl17 SCF E3 ubiquitin ligases. Co-immunoprecipitates were enriched for DNA damage/ DNA repair proteins suggesting a novel role for Fbxl17 in the DNA damage response (DDR). We have demonstrated that Fbxl17 is recruited to DNA damage sites rapidly upon double-stand break (DSB) induction and knockdown of Fbxl17 protein expression sensitises cells to the DNA damaging agent Camptothecin. Furthermore, Fbxl17 can ubiquitinate the tandem BRCT domain of the well-known DDR protein 53BP1, which we propose targets 53BP1 for proteasomal degradation. In conclusion, we have identified two regulatory networks of Fbxl17 which provide an insight into the role of Fbxl17 in breast cancer pathogenesis. These pathways may be amenable to therapeutic targeting in the future for the treatment of breast cancers rearranged in FBXL17. 2 Acknowledgements Thanks must first go to my supervisor Dr Heike Laman, for her unwavering support and guidance throughout my PhD. You are a true champion of women in science and it has been an absolute joy working in the lab these last four years. To the past and present members of the Laman lab, thank you for always being on hand to answer questions and offer support when I needed it. It would have been a lonely journey without you all there to make the failed experiments seem that little less disappointing. A special thanks to my colleague and friend Dr Suzie Randle, still offering her support and answering my questions even after leaving the lab for pastures new. I would like to thank Dr Paul Edwards for his continued encouragement throughout this PhD and for his bioinformatics expertise. Thank you for all your help in extracting the FBXL17 primary tumour data and of course to Professor Carlos Caldas and his lab for access to the primary tumour data before it has been published. In addition, thank you to Dr Edwards and Dr Susanne Flach for their initial work on Fbxl17 and their contributions to our published manuscript. Many thanks to Dr Yaron Galanty and the Jackson lab, at the Gurdon Institute, Cambridge, for their help with all things DNA damage. I am hugely grateful for your assistance with experiments and generous donation of reagents. I would also like to acknowledge and thank Dr Robin Antrobus, for performing the mass spectrometry analysis of Fbxl17, Dr Christian Frezza and lab members for their LC-MS expertise and Drs Vincenzo D’Angiolella and Svetlana Khoronenkova for reagents. To my friends in Cambridge, thank you for four fun-filled years. Adam, you are my still point in an ever-turning world. Thanks for looking after me and always knowing when I need food. Finally, I would like to thank my amazing parents, you believe in me even when I doubt myself and I hope I have made you both proud. Not bad for a Yorkshire lass in the South. 3 Table of Contents Declaration ................................................................................................................................. 1 Abstract ...................................................................................................................................... 2 Acknowledgements .................................................................................................................... 3 Table of Contents ....................................................................................................................... 4 List of Figures .............................................................................................................................. 8 List of Tables ............................................................................................................................. 11 Abbreviations ........................................................................................................................... 12 CHAPTER 1 - Introduction ........................................................................................................ 16 1.1 Breast Cancer .......................................................................................................... 16 1.1.1 Stratification of breast cancers .................................................................... 16 1.1.2 Genomic rearrangements in breast cancer ................................................. 17 1.2 The Hexosamine Biosynthetic Pathway .................................................................. 20 1.2.1 O-GlcNAcylation in cancer ............................................................................ 23 1.2.2 Uap1 ............................................................................................................. 24 1.2.2.1 Uap1 in cancer ..................................................................................... 26 1.3 DNA double-strand break repair ............................................................................ 28 1.3.1 DNA end resection ....................................................................................... 29 1.3.2 Non-homologous end-joining ...................................................................... 32 1.3.3 53BP1 ............................................................................................................ 33 1.3.4 BRCA1 ........................................................................................................... 36 1.3.5 BRCA1 antagonises 53BP1............................................................................ 36 1.4 The Ubiquitin Proteasome System (UPS) ............................................................... 40 1.4.1 SCF-type E3 ubiquitin ligases ........................................................................ 41 1.4.2 F-box proteins .............................................................................................. 43 1.4.3 F-box proteins in cancer ............................................................................... 43 1.4.4 Fbxl17 ........................................................................................................... 45 1.4.5 Fbxl17 is recurrently rearranged in breast cancer ....................................... 48 1.5 Aims of this thesis ................................................................................................... 50 1.5.1 Investigating the functional consequences of FBXL17 rearrangements ..... 50 1.5.2 Identifying and verifying new target substrates of Fbxl17 .......................... 50 CHAPTER 2- Materials and methods ........................................................................................ 52 4 2.1 CELL CULTURE AND ANALYSIS ................................................................................ 52 2.1.1 Cell line maintenance ................................................................................... 52 2.1.2 Transfection of adherent cells...................................................................... 52 2.1.3 Retroviral infection of adherent cells..........................................................
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