Regulation of BTRC by gain-of-function TP53 mutation(s) in cancer cells By Peter Xu A thesis submitted in conformity with the requirements for the degree of Masters of Science (M.Sc) Graduate Department of Molecular Genetics University of Toronto © Copyright by Peter Xu (2015) Regulation of BTRC by gain-of-function TP53 mutation(s) in cancer cells Peter Xu Masters of Science Department of Molecular Genetics University of Toronto 2015 ABSTRACT Regulation of BTRC by gain-of-function TP53 mutation Peter Xu, Masters of Science (2015), Department of Molecular Genetics, University of Toronto Mutation or loss of TP53 has been detected in more than 50% of all human tumours. Gain-of-function (GOF) TP53 mutations have been linked to metastasis, altered metabolism, and drug resistance. Cancer patients carrying GOF TP53 mutations in their tumours respond poorly to standard of care treatments and have a worse prognosis. In my Master’s thesis, I have focused on understanding the relationship between a frequently observed mutation of TP53 (TP53 R248W ), and BTRC , an E3 ubiquitin ligase with functions impinging on cell cycle, morphology and metabolism. I observed a correlation between BTRC protein expression and TP53 genotype across a panel cancer cell lines. Additionally, I observed that TP53 R248W -mediated regulation of BTRC expression is dependent on a post-transcriptional mechanism, likely involving more than one micro-RNA. In conclusion, I present a model describing the regulation of BTRC by TP53 , which may have implications for targeted strategies in cancers harboring GOF TP53 mutations. ii ACKNOWLEDGEMENTS Foremost, I would like to express my sincere gratitude to my advisor Prof. Jason Moffat for his continuous support of my M.Sc study and research. In particular, I want to thank him for his patience, motivation, and immense knowledge during both high and especially low stages of my graduate career. In addition, his guidance propelled me to persevere through my research and thesis writing. Besides my advisor, I want to take this opportunity to thank the rest of my committee members: Prof. Leonardo Salmena, Prof. Mark Minden and Prof. Freda Miller for their time, encouragement, and insightful comments. I want to thank Prof. Leonardo Salmena especially for his time and our lively and fun discussion both research and non-research related. I want to thank all my fellow Moffat labmates for their guidance and support throughout my graduate studies. In particular, I want to thank my fellow friend and labmate, Sachin Kumar for the endless nights of research and stimulating discussions. I want to thank Dr. Taras Makhnevych setting up an example of research vigor and scientific standard for me to follow closely. I want to thank my friends in the Moffat lab past and present, Hayoung Yoo, Eun Jee Koh, Megha Chandrashekhar, Rashida Williams, Esther Lau, Satra Nim, Andrea Uetrecht, Hong Han, and Michael Aregger. Last but not least, I would like to thank my family and especially my mother and father for their endless support for the past quarter century. iii TABLE OF CONTENTS Chapter Title Page Number ABSTRACT ............................................................................................................................................. ii ACKNOWLEDGEMENTS................................................................................................................... iii TABLE OF CONTENTS....................................................................................................................... iv LIST OF TABLES .................................................................................................................................. v LIST OF FIGURES ................................................................................................................................ v LIST OF ABBREVIATIONS ............................................................................................................... vi Chapter 1 – Introduction ........................................................................................................................ 1 1.1 – TP53 Mutation and Cancer .......................................................................................................... 1 1.2 – Molecular Role of TP53 as a Tumour Suppressor ....................................................................... 2 1.3 – Metabolic Role of TP53 in Cancer .............................................................................................. 2 1.4 - Molecular Role of BTRC .............................................................................................................. 3 1.5 - TP53 and BTRC ............................................................................................................................ 6 1.6 – Hypothesis .................................................................................................................................... 6 Chapter 2 – Experimental Methods ...................................................................................................... 7 2.1 - Cell Culture ................................................................................................................................... 7 2.2 - Western Blot ................................................................................................................................. 7 2.3 - Preparation of protein lysates ....................................................................................................... 8 2.4 - Lentiviral Infection ....................................................................................................................... 8 2.5 - Cellular RNA Isolation ................................................................................................................. 9 2.6 - nCounter Nanostring Analysis ..................................................................................................... 9 2.7 – CRISPR sgRNA Constructs ......................................................................................................... 9 2.9 – HCT116 Cell Transfections ....................................................................................................... 10 2.10 - Intracellular Flow Cytometry ................................................................................................... 10 Chapter 3 -Results ................................................................................................................................. 11 3.1 – Gain-of-function TP53 mutant cells express low levels of BTRC ............................................ 11 3.2 – Survey of TP53 and BTRC protein levels across a panel of cancer cell lines ........................... 12 3.3 – An inducible system to examine relationship between TP53 R248W and BTRC ........................... 14 3.4 – BTRC levels are not regulated by the proteasome, cytoskeleton remodeling, or cell cycle ...... 15 3.5 - An inducible system to examine relationship between TP53 R248W , BTRC , and DICER1 ........... 16 3.6 – Examining BTRC in miR-195 knockout cells ............................................................................ 18 3.7 – A model for TP53 regulation of BTRC levels ........................................................................... 20 Chapter 4 – Conclusion and Future Direction ................................................................................... 22 4.1 – Conclusion ................................................................................................................................. 22 4.2 - Future Directions ........................................................................................................................ 23 Chapter 5 - References ......................................................................................................................... 26 iv LIST OF TABLES Chapter 2.1 - Table 1. Culture method of cancer cell lines Chapter 2.7 - Table 2. Sequences of sgRNA against hsa-mir-195 and BTRC LIST OF FIGURES Chapter 1.1 - Fig 1. The distribution of missense mutations in TP53 : the hotspot region. Chapter 1.4 - Fig 2. Schematic of basic ubiquitin pathway. Chapter 1.4 - Fig 3. List of proteins regulated by ubiquitin through BTRC . Chapter 1.4 - Fig 4. Regulation of AKT signaling by BTRC . Chapter 1.4 - Fig 5. TP53 R248W genotype influences AKT phosphorylation. Chapter 3.1 - Fig 6. Gain-of-function TP53 mutant cells express low levels of BTRC. Chapter 3.2 - Fig 7. BTRC and GOF TP53 protein levels across a panel of cancer cell lines. Chapter 3.3 - Fig 8. Induction of wild-type and GOF TP53 reduces BTRC expression. Chapter 3.4 - Fig 9. BTRC protein levels were not dependent on proteasome, cytoskeleton remodeling or cell cycle. Chapter 3.5 - Fig 10. Induction of wild-type and TP53 R248W in DICER1 deficient cells rescues BTRC expression. Chapter 3.6 - Fig 11. Schematic of sgRNA targets locations at the hsa-mir-195 genomic locus. Chapter 3.6 - Fig 12. miR-195 knockout modestly induces BTRC expression. Chapter 3.7 - Fig 13. Overexpressed miRNAs in TP53 GOF cancer cells. Chapter 3.7 - Fig 14. Schematic model for the metabolic TP53 mediated regulation of BTRC . v LIST OF ABBREVIATIONS TERM MEANING AKT Protein kinase B AMPK 5' Adenosine monophosphate-activated protein kinase ATP Adenosine triphosphate BTRC Beta-transducin repeat containing E3 ubiquitin protein ligase CRISPR Clustered regularly interspaced short palindromic repeats Cas9 CRISPR associated protein 9 CTNNB1 Catenin