Understanding the molecular interplay between senescence, rejuvenation, and healthy ageing. Eleanor Tyler A thesis presented for the degree of Doctor of Philosophy 2016 Supervisors: Dr Cleo L. Bishop Prof. Mike P. Philpott Centre for Cell Biology and Cutaneous Research The Blizard Institute Barts and the London School of Medicine and Dentistry Queen Mary University of London Statement of originality I, Eleanor Tyler, confirm that the research included within this thesis is my own work or that where it has been carried out in collaboration with, or supported by others, that this is duly acknowledged below and my contribution indicated. I attest that I have exercised reasonable care to ensure that the work is original, and does not to the best of my knowledge break any UK law, infringe any third party’s copyright or other Intellectual Property Right, or contain any confidential material. I accept that the College has the right to use plagiarism detection software to check the electronic version of the thesis. I confirm that this thesis has not been previously submitted for the award of a degree by this or any other university. The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author. Signature: Eleanor Tyler Date: 19.12.16 1 Abstract Senescence is classically defined as an irreversible cell cycle arrest. There is now convincing evidence that senescent cells accumulate during human ageing, potentially driving age-related dysfunction through depletion of mitotically active cells and stimulation of chronic inflammation. Recently, a landmark paper demonstrated that removal of p16INK4A (p16)-positive senescent cells in mice prolonged healthy lifespan, suggesting a direct link between senescence and age-related dysfunction. As such, restraining the senescent pool or slowing their rate of accumulation presents an attractive therapeutic strategy for extending healthspan. Previously, our laboratory has demonstrated that siRNA transfection can reverse deep senescence in p16-positive primary adult human mammary epithelial cells using a panel of senescence markers. Subsequent siRNA screening revealed 28 hits which strongly induced reversal in the deeply senescent HMECs. In this project, siRNA knockdown of p16 combined with p21WAF1/CIP1 (p21) was found to reverse deep replicative senescence in primary adult human mammary fibroblasts, as defined by a panel of senescence markers. This discovery provided the opportunity to screen for novel siRNAs which induce reversal in both cell types. Screening in the deeply senescent HMFs of the 28 shortlisted candidates and 33 protein interactors identified using bioinformatics revealed 45 siRNAs which significantly increased cell number compared to the negative siRNA control. Subsequent immunofluorescence staining and high content analysis of the top 14 candidates identified 10 hit siRNAs which induced senescence reversal as defined by a panel of markers. Interestingly, these 10 hits were enriched for cytoskeletal and cell adhesion processes, suggesting an interplay between external forces and senescence induction. The top siRNA hit, early growth response 2 or EGR2, a transcription factor, was validated and selected for further exploration as a novel driver of senescence. Bioinformatics analysis revealed an enrichment for EGR2 binding sites within genes dynamically expressed in HMEC senescence, including p16, p21, and nine hits identified to reverse senescence in HMFs and HMECs. Furthermore, deeply senescent HMFs and HMECs were found to have a significantly increased nuclear EGR2 foci number compared to proliferating cells, and this was significantly decreased in reversed HMFs. In conclusion, it is proposed that EGR2 may represent a novel driver of both HMF and HMEC senescence. 2 Acknowledgements This work was supported by a Medical Research Council PhD Studentship (Grant number: MR/K501372/1). First and foremost, I would like to thank my supervisor, Dr Cleo Bishop, for her invaluable, unwavering support and guidance throughout my PhD. Your relentless optimism has been an inspiration. Thank you. I am also grateful for the support from my second supervisor, Prof. Mike Philpott, who has been there for me throughout all the PhD milestones. To Deb, the members of the Bishop lab., and everyone in the Blizard (Anke, Heather, Hannah, to name but a few), thank you for making our institute such a fun place to work. In particular, I would like to thank Dr Maddi Moore. We grew up together in the lab. and I will always look to you as my academic big sister. Special thanks also to Mat for sharing his seemingly endless wealth of knowledge. A big thank you goes to my support at home, Mike, who made sure I was always fed and watered. You are my rock. Also, to my friends and family, Kim, Sylvie, Alex, Gemma, Mum and Dad, who were always there for me and always okay when I couldn’t be there. Thank you all. 3 Contents Chapter 1 Introduction .................................................................................................... 20 1.1 The cell cycle .......................................................................................................... 21 1.1.1 Cell cycle regulation ........................................................................................ 21 1.1.2 The INK4/ARF locus ......................................................................................... 24 1.1.3 p21 .................................................................................................................. 26 1.2 Fibroblast replicative senescence ......................................................................... 28 1.3 Other types of senescence .................................................................................... 31 1.3.1 Epithelial cellular senescence ......................................................................... 31 1.3.2 Premature senescence ................................................................................... 32 1.3.3 Senescence in other cell types ........................................................................ 33 1.4 Markers of senescence .......................................................................................... 34 1.4.1 Commonly used markers of senescence ........................................................ 34 1.4.2 Senescence-associated secretory phenotype (SASP) ..................................... 35 1.5 Senescence in vivo ................................................................................................. 38 1.5.1 Senescence, tissue dysfunction, and ageing .................................................. 38 1.5.2 Other roles of senescence in vivo ................................................................... 42 1.6 Senescence as a therapeutic target ...................................................................... 44 1.7 Reversal of senescence.......................................................................................... 47 1.8 Early growth response 2 (EGR2) ............................................................................ 49 1.8.1 EGR2 gene locus, isoforms, and family ........................................................... 49 1.8.2 EGR2 and development .................................................................................. 50 1.8.3 EGR2 and the peripheral nervous system ...................................................... 51 1.8.4 EGR2 and the immune system ........................................................................ 52 1.8.5 EGR2, cancer, and senescence ....................................................................... 54 1.9 Small interfering RNA (siRNA) technology ............................................................ 57 1.10 Project aims ......................................................................................................... 59 Chapter 2 Materials and Methods .................................................................................. 60 4 2.1 Mammalian cell culture .................................................................................... 61 2.1.1 Culture of normal human mammary fibroblasts ...................................... 61 2.1.2 Culture of wildtype and p21 double knockout human fibroblasts ........... 62 2.1.3 Culture of normal human mammary epithelial cells ................................ 62 2.2 UV irradiation-induced senescence ................................................................. 62 2.3 Paracrine senescence ....................................................................................... 63 2.4 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blotting ......................................................................................................... 63 2.4.1 Protein lysate preparation ........................................................................ 63 2.4.2 SDS-PAGE .................................................................................................. 64 2.4.3 Immunoblot analysis ................................................................................. 64 2.4.4 Densitometry............................................................................................. 66 2.4.5 Membrane stripping ................................................................................. 67 2.5 siRNA forward-transfection optimisation ........................................................ 67