The effect of the polyadenylation inhibitor Cordycepin on MCF-7 cells Asma Khurshid, MSc (University of Nottingham) Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy July 2015 Declaration Except where acknowledged in the text, I declare that this dissertation is my own work and is based on research that was undertaken by me in the School of Pharmacy, Faculty of Science, University of Nottingham. i Abstract Cordycepin (3′-deoxyadenosine) is a medicinal bioactive component of the caterpillar fungi (Cordyceps and Ophicordyceps). It is reported to have nephroprotective, antiapoptotic, anti-metastatic, hepatoprotective (Yue et al. 2013), inflammatory effects, antioxidant, anti-tumor, immunomodulatory and vasorelaxation activities. Cordycepin is well known to terminate and inhibit polyadenylation, both in vitro and in vivo. Other proposed mechanisms of action of cordycepin include activation of adenosine receptors, activation of AMP dependent kinase (AMPK) and inhibition of PARP1. The purpose of this study is to elucidate the biological and pharmacological effects of cordycepin on cancer cell lines such as MCF-7 cells. In this study I found that cordycepin reduces the cell proliferation in all examined cell lines without always exerting an effect on 4EBP phosphorylation and protein synthesis rates. Therefore, the effects on protein synthesis via inhibition of mTOR, which were previously reported, are not only the sole reason for the effect of cordycepin on cell proliferation. Knockdown of poly (A) polymerases reduces cell proliferation and survival, indicating that poly (A) polymerases are potential targets of cordycepin. I studied different adenosine analogues and found that 8 aminoadenosine, the only one that also consistently inhibits polyadenylation, also reduces levels of P-4EBP. It also inhibits the expression of specific genes indicating that the effects on polyadenylation, mTOR signalling and gene expression are linked. Also consistent with polyadenylation inhibition as the major mode of action is the fact that the effects of cordycepin on gene expression are predominantly post-transcriptional. However, knockdown of poly (A) polymerases did not have the same effects on gene expression or on polyadenylation, indicating that cordycepin may act as a dominant negative rather than as a null mutant. This is consistent with the fact that cordycepin is known to arrest a normally transient polyadenylation complex. We performed microarray analysis of cordycepin treated MCF-7 cells and found that the downregulated mRNAs were predominantly involved in transcriptional regulation, cell proliferation, cell cycle and cell migration. These data show that ii cordycepin is a promising new drug for cancer and indicates that the mode of action it is likely to be through the inhibition of polyadenylation. iii Acknowledgements Firstly, I would like to thank to my supervisor Dr Cornelia H. de Moor for her great support, time, encouragement and co operation throughout my studies. It would not be possible for me to finish off my work without her understanding of my responsibilities being a mother and a student. I do not have enough words to convey my deepest thanks to her. I would also like to thank to Keith Spriggs for his advice and guidance during my studies and especially to Catherine Jopling for helping me to carry out my radiation work safely and efficiently in a friendly manner. I also liked to convey my deepest thanks to Anne Willis and her team (MRC Toxicology unit, Leicester) for her support in performing microarray analysis for this project. I would also like to thank to all of the past and present members of the RNA biology and gene regulation group, for their support and making my time enjoyable during the work particularly Hannah Parker, Hughes Alexandra and Andrew Lewis for their support, enjoyable chats and help during the work. Special thanks to Alexander Kondrashov for not only helping me in experimental work with great experience and expertise over the past three years but also for his conceptual guidance. I would like to thank to my family especially to my father for being supportive, encouraging and for having faith in my capabilities. I would always remain thankful to my late grandmother for her kindness, care and love which will always remain in my heart. Thanks to University of Nottingham for funding support. Lastly, love to my daughters Ayra and Arfa who always reminds me a cheerful and enjoyable face of life with her love, hug and endurable smiles which not only gives me strength but also boost up my energy after many sleepless nights especially during my final stages of research work. iv Table of content Declaration ........................................................................................................... i Abstract ............................................................................................................... ii Acknowledgements ............................................................................................. iv Table of content .................................................................................................. v List of Figures ...................................................................................................... xi List of Tables ...................................................................................................... xiii List of Abbreviations .......................................................................................... xiv 1 Introduction ................................................................................................. 1 1.1 Breast cancer ......................................................................................... 1 1.1.1 Molecular subtypes of breast cancer ..................................................... 1 1.1.2 Role of receptors in Breast cancer ......................................................... 2 1.1.2.1 HER2 ................................................................................................ 2 1.1.2.2 Hormone receptors......................................................................... 3 1.1.3 Breast cancer treatment ........................................................................ 3 1.1.3.1 HER2+ treatment ............................................................................. 4 1.1.3.2 Endocrine therapy .......................................................................... 4 1.1.3.3 Chemotherapy ................................................................................ 5 1.1.4 Molecular mechanisms of breast cancer ............................................... 7 1.1.5 Breast cancer hallmarks ......................................................................... 8 1.1.5.1 Sustaining proliferative signaling .................................................... 8 1.1.5.2 Evading growth suppressors ........................................................... 8 1.1.5.3 Capable of replicative immortality ................................................. 9 1.1.5.4 Activating invasion and metastasis ................................................. 9 1.1.5.5 Inducing angiogenesis ................................................................... 10 1.1.5.6 Resisting cell death ....................................................................... 10 1.1.6 Mutational analysis of human breast cancers: Role of tumour suppressor and oncogenes ................................................................................. 11 1.1.6.1 Breast cancer mutations ............................................................... 12 1.2 Signal transduction by the insulin and growth factor pathways in breast cancer 13 1.2.1 Role of tyrosine kinase receptors ........................................................ 13 1.2.1.1 EGFR signaling in breast cancer .................................................... 14 v 1.2.1.1.1 Types of EGFR ............................................................................ 14 1.2.1.1.2 Types of EGFR ligands ................................................................ 15 1.2.1.1.3 Mode of receptor activation ..................................................... 15 1.2.1.1.4 EGFR mutations in breast cancer .............................................. 16 1.2.1.1.4.1 Role of HER2 ........................................................................ 16 1.2.1.2 Downstream signaling of EGFR ..................................................... 17 1.2.1.2.1 The PI3K/AKT/mTOR pathway ................................................... 19 1.2.1.2.1.1 What is mTOR ..................................................................... 19 1.2.1.2.1.2 Multi-protein complexes of mTOR ..................................... 19 1.2.1.2.1.2.1 mTORC2 ........................................................................ 20 1.2.1.2.1.2.2 mTORC1 ........................................................................ 20 1.2.1.2.1.3 Phosphatidylinositol 2-kinases ........................................... 20 1.2.1.2.1.4 Regulation of mTORC1 by upstream kinases ...................... 21 1.2.1.2.1.4.1 TSC mediated RHEB pathway ....................................... 21 1.2.1.2.1.4.2 AKT: A positive regulator of mTORC1 activity .............. 22 1.2.1.2.1.4.3 AMPK: A negative regulator of mTORC1 activity ........ 23 1.2.1.2.1.5 Downstream signaling of mTOR ......................................... 24 1.2.1.2.1.5.1 eIF4E/4E-BP .................................................................