The Effect of Vitamin D on Gene Expression in Colorectal Tumours and Normal Colon Francesca M. Munro A thesis submitted for the degree of Masters of Biochemistry, University of Otago, New Zealand Supervisors: John L. McCall and Michael A. Black August 2015 ABSTRACT Background Colorectal cancer (CRC) is the third most frequently diagnosed malignancy and the fourth leading cause of death from cancer worldwide. In New Zealand, incidence rates of CRC are projected to decline in all age groups. The overall burden is projected to continue increasing as a result of population growth and an aging population. Epidemiological studies have reported an inverse association between vitamin D status and incidence of CRC. Higher serum vitamin D levels at the time of diagnosis or post- surgery have been associated with improved long-term outcome in CRC, but there is no proven causal link. It has been postulated that vitamin D levels should be maintained at >80 nmol/L for both bone and overall health. Vitamin D deficiency is common in NZ particularly in the winter months. The primary aim of this study was to examine whether a single large dose of vitamin D administered to CRC patients in the window between diagnosis and surgery could have a measurable effect on vitamin D responsive genes in the tumour and corresponding normal colon. Methods The study was a randomised, double-blind, placebo-controlled trial of vitamin D supplementation in patients undergoing elective surgery for CRC at Dunedin Hospital. For the primary outcome, RNA from resected normal and tumour tissue was profiled on microarray gene-chips. Array gene expression data were analysed using single gene analysis to identify differentially expressed individual genes and over-representation and gene-set analysis (GSA) to identify differences in pathway expression. Secondary outcomes including complication rate, length of hospitalisation, post-operative recovery and survival were measured. Results There were no baseline differences between the treatment and control groups. Pre- incision vitamin D concentrations were higher in the treatment than in the placebo group (mean 87 +/- 22 vs 49 +/- 19 nmol/L; p= >0.001). The complication rate and length of hospitalisation were within the expected range for this cohort. No differences in post-operative recovery, cancer recurrence or survival were observed. There were no i significant differences in single gene expression between the treatment and control groups. There were also no significant differences in gene expression of vitamin D modulated genes between the groups. Differences in pathway expression were identified between the treatment arm and placebo arm. In the normal tissue, patients randomised to receive vitamin D had down-regulation of a number of pathways compared to those randomised to the placebo group including Fatty Acid Metabolism, Drug Metabolism - Cytochrome P450, Metabolism of Xenobiotics by Cytochrome P450, Vitamin Digestion and Absorption and Negative Regulation of Growth pathways. In the tumour tissue the treated group also had down-regulation of several pathways compared to the placebo group including the Fatty Acid Metabolism, Fatty Acid beta- Oxidation and Oxidative Phosphorylation pathways. In the paired analysis the expression of the Ribosome and Translational Termination pathways were enhanced by vitamin D in the tumour tissue of a subgroup of patients and reduced in another subgroup, compared to the normal tissue. Over-representation analyses identified a number of other pathways that may be different between the study groups. Conclusion In this randomised controlled trial potentially significant biological differences between the vitamin D and placebo groups were identified. The Fatty Acid Metabolism and Fatty Acid beta-Oxidation pathways were down-regulated in the tumour tissue of the treated patients compared with the untreated patients. Down-regulation of fatty acid metabolism in the tumour may lead to the slowing of tumour growth. Un-metabolised butyrate (short-chain fatty acid utilised by colon epithelia) may also precipitate reduced cell proliferation and enhanced differentiation and apoptosis. Further work is required to develop our understanding of the impact of vitamin D on the biology of colorectal cancer. ii ACKNOWLEDGEMENTS This study was designed by the candidate’s supervisors, John McCall and Michael Black. The candidate had no input into the study design. The candidate explained the study to the patients, obtained written consent, collected and stored tissue and collated clinical data. RNA extraction and purity assessment was performed by Mrs Victoria Phillips of the Department of Surgical Sciences of the University of Otago Medical School. RNA quality assessment was carried out by Dr Aaron Jeffs of the Department of Biochemistry of the University of Otago. RNA labeling, RNA hybridisation to the genechips and profiling and scanning of the genechips was conducted by Mr Leslie McNoe and Dr Luxmanan Selvanesan of the Department of Biochemistry of the University of Otago. The candidate analysed the microarray gene expression data and measured the secondary outcomes of the study. iii THANKS Firstly, I’d like to thank John McCall for giving me the opportunity to undertake further study (further thanks to John are in order and can be found below). I’d like to acknowledge the University of Otago for encouraging its staff to continue their education and having processes in place to enable this. There are many employees of the university, Dunedin Hospital and Southern Community Laboratories who provided help and support for this project. I won’t name them all individually but their involvement was very much appreciated, especially the banter ;) . These fabulous people included staff from the Department of Surgery, Wards 4A and 4B (and occasionally staff from other wards), Pre-admission Clinics, the Radiology Department, the Oncology Department and any other department that allowed me to follow their patients around to discuss the study, obtain consent and blood and administer the study treatment. The Southern Community Laboratories’ phlebotomists are always so helpful (nothing’s ever a problem) including at the out-of- town clinics. Staff from other hospital departments were indispensable, including the Pharmacy Department. Theatre staff were amazing (and still are), always happy to provide me with a bucket of fresh flesh. The Southern Community Laboratories’ histology prep staff, pathologists and especially the pathology registrars were stunning! Thanks so much for providing tissue samples from fresh specimens which usually meant lots of mucky brown stuff. Thanks for not barfing on my shoes. Vicky Phillips is the lab manager from heaven. Not only does she provide copious amounts of cake, but she recruits patients and extracts RNA as well. Quite a woman! I’d like to say, “Les, where’s my cake?” But at least you are a master with microarrays. Cheers to you too, Lux. And Aaron. You all make working with little things look fun! One of my fellow students became an unpaid ‘go-to’ for my myriad ‘R’ questions. Poor guy has been promoted to friend and adopted son! Thanx Tom! I rate ya! Big ups to my various work roomies over the duration of this project. You were almost bearable…nah I love you all, especially when you’re quiet Lesley! x iv Arohanui to all my fantastic and supportive friends including you Bea. Xxx Thanx Lesley B. for reading this thesis for me. Hopefully you don’t find too many errors! Projects like this one are not possible without patients consenting to be involved. I’m in awe of how much these wonderful people wanted to do something to help others even though many of these people had only just received their diagnosis. I hope your generosity combined with our efforts make for better outcomes for patients in the future. Being grateful for one’s supervisors is to be expected but mine have had to put up with a distracted, hormonal and tired student in me (we won’t mention retarded- though my kids would enjoy that!). I am really grateful guys that you managed to stick with me through this and provide me with ‘extra support’ in various ways. Hopefully that ‘extra support’ pays off in my role for the department and the CTRG. Thanks John. Thanks Mik. J And of course the obvious gratefulness towards one’s family because they’ve had to put up with various inconveniences while you’ve been ‘tripping’. Unfortunately my family has had to endure a major loss while I’ve been preoccupied with study. I think we’ve all done an amazing job of getting through what could have been a very messy time. I want to say a heartfelt thank you to you Bruce for helping to make this an amicable process and for your support during this project. Big love to all my kids, Bea, Zac, Elliot, Josi and Gianni. You’re all fantabulous & I love you guys truckloads!! v TABLE OF CONTENTS Chapter 1. INTRODUCTION……………………………………………………………………………………1 1.1 Colorectal Cancer…………………………………………………………………………………………1 1.1.1 Colorectal cancer worldwide………………………………………………………………………1 1.1.2 Colorectal cancer in New Zealand……………………………………………………………….1 1.1.3 Presentation, treatment and prognosis for colorectal cancer……...………………..2 1.2 Health Effects of Vitamin D ………………………………..………………………………………..4 1.2.1 Vitamin D – basic biology…………………………………………………………………………...4 1.2.2 Mechanism of action of calcitriol………………………………………………………………...6 1.2.3 Vitamin D – role in calcium/phosphorous homeostasis and bone health………9 1.2.4 Vitamin D – other health effects………………………………………………………………….9