The pleiotropic effects of vitamin D promoting bowel health MR DANIEL J. LOCK BSC. MRSB. Thesis submitted to the University of East Anglia in requirement for the degree of Doctor of Philosophy (Molecular cell biology with epigenetics) School of Biological Sciences, University of East Anglia, Norwich, UK And Institute of Food Research, Norwich Research Park, Norwich, UK September 2016 Copyright © 2016 Daniel J. Lock This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. 1 Declaration This thesis is submitted to the University of East Anglia for the Degree of Doctor of Philosophy and has not been previously submitted at this, or any other university, for assessment, or for any other degree. Except where stated, and reference or acknowledgement is given, this work is original, and has been carried out by the author alone. 2 Thesis abstract Vitamin D insufficiency is seasonally endemic in populations north of the 40th parallel, and epidemiological data show an association with colorectal cancer risk and prognosis. Molecular mechanisms that underpin the relationship are not well established. Vitamin D status is shown to be associated with age-related silencing of tumour suppressors in colonic stem cells via aberrant DNA methylation, suggesting that insufficiency contributes to transformation. In this text, vitamin D’s ability to promote bowel health via modification of DNA methylation patterns has been investigated. Chronic inflammation drives tumourigenesis, and vitamin D is recognised to promote proper immune function. Data presented here confirm that vitamin D differentiates monocytes to a tissue-resident macrophage phenotype. D-mediated differentiation is associated with hypomethylation of the TNFα promoter and response to LPS. Thus, we suggest that vitamin D attenuates aberrant DNA methylation in colonic stem cells by promoting resolution of systemic inflammation. Mucosal inflammation mediated by PGE2 promotes aberrant DNA methylation. Pericryptal myofibroblasts interact with colonic stem cells via their secretomes, which are a source of PGE2. Supernatants from primary intestinal myofibroblasts were characterised by LC/MS mass spectroscopy in response to vitamin D. Vitamin D attenuated TNFα-induced transcription of COX2 and PGE2 secretion. PGE2 induced hypermethylation of SOX17 and DKK1 in colonic organoids, and myofibroblast supernatants regulated DNA methyltransferase activity in case-matched organoids. Furthermore, vitamin D ameliorated established aberrant DNA methylation in organoids propagated from inflamed mucosa. Thus, we suggest that vitamin D attenuates mucosal inflammation, and the effects of PGE2 driving aberrant DNA methylation in colonic stem cells. Vitamin D status predicts colorectal cancer survival. The effects of vitamin D sufficiency on colorectal cancer cell lines was investigated. Vitamin D-treated cells exhibit a modified methylome, reduced transcription of MAP kinases, reduced phosphorylation of ERK1 and 2, and inhibition of proliferation. Thus we suggest that vitamin D sufficiency improves colorectal cancer prognosis via modification of established aberrant DNA methylation. Taken together, data support vitamin D sufficiency promoting bowel health via attenuation of aberrant age-related DNA methylation in colonic stem cells. 3 Acknowledgements Without question, my sincere thanks are extended first and foremost to Dr Nigel Belshaw, not only for his unwavering support in the face of the challenging circumstances, but also for providing the insightful theoretical framework that forms the basis of this study. Thank you Nigel, for your commitment, lateral support, and championship. Furthermore I appreciate greatly Professor Ian Johnson fighting my corner; your encouragement and objective criticism have enabled me to understand the scientific validity and impact of my work in a broader context. Thanks are also extended to Drs Giles Elliot, Wing Leung, and Ellen Maxwell for their companionship, and initial direction and assistance. I’m indebted to Drs Paul Kroon, Nathalie Juge, and Carmen Pin for their pastoral care keeping me on track during tricky transitional periods. Thanks especially go to my friends Drs Barnabas Shaw, Aimee Parker, and Laura Vaux, for welcoming me to their group(s), and for the daily logistical support and encouragement; it’s by no means an exaggeration to say that I couldn’t have done it without you! I must also sincerely thank UEA and The Institute for the exceptional opportunities afforded to me during the course of the project - practicing science on no fewer than three continents in as many years. Finally I would like to extend warm thanks to my close friends and family, for their (apparent) interest in my research and rallying encouragement, which has proved indispensable - especially Jemma, whose inspiring words have given me the confidence to get the job done on countless occasions. I must also extend a debt of gratitude to my original champions, Professor Richard Ball, Drs Danni Peat, and Peter Cousens, for holding open the doors that have brought me to this juncture; I doubt they’ll ever read this, but it’s true none the less. Cheers all - this text is in honour of, and in return for, your investment in me - I hope to continue doing it justice. 4 Contents Declaration ............................................................................................................................. 2 Thesis abstract ....................................................................................................................... 3 Acknowledgements .............................................................................................................. 4 Contents ................................................................................................................................. 5 List of figures ..................................................................................................................... 8 List of common abbreviations ..................................................................................... 10 Chapter 1 .............................................................................................................................. 12 Abstract ............................................................................................................................ 13 Vitamin D - an evolutionary perspective ................................................................... 14 Physiology and molecular biology of vitamin D ....................................................... 16 Colonic homeostasis and neoplasia ............................................................................ 32 In summary ....................................................................................................................... 48 Research questions; ....................................................................................................... 49 Hypotheses ...................................................................................................................... 50 Aims and objectives ....................................................................................................... 51 Notes ................................................................................................................................. 53 Chapter 2 .............................................................................................................................. 54 Experimental approach .................................................................................................. 55 Readouts ........................................................................................................................... 59 Materials ........................................................................................................................... 60 General methods ............................................................................................................ 60 Chapter 3 .............................................................................................................................. 73 Preface to the work ....................................................................................................... 74 Abstract ............................................................................................................................ 76 Introduction ..................................................................................................................... 77 Methods ............................................................................................................................ 81 Results ............................................................................................................................... 84 Lester’s Oil intervention .......................................................................................... 84 In vitro verification with THP1 leukemic monocytes .......................................... 89 Discussion ........................................................................................................................ 97 In summary ................................................................................................................ 102