Iain McCallum

Effects of supplementation with nondigestible carbohydrates on fecal calprotectin and on epigenetic regulation of SFRP1 expression in the large-bowel mucosa of healthy individuals

Background: Hyperactive Wnt signaling is frequently observed in colorectal cancer. Higher intakes of dietary fiber [nondigestible carbohydrates (NDCs)] and the fermentation product butyrate are protective against colorectal cancer and may exert their preventative effects via modulation of the Wnt pathway. Objectives: We investigated the effects of supplementing healthy individuals with 2 NDCs [resistant starch (RS) and polydextrose] on fecal calprotectin concentrations and Wnt pathway–related gene expression. In addition, we determined whether effects on secreted frizzled-related protein 1 (SFRP1) expression are mediated via the epigenetic mechanisms DNA methylation and microRNA expression. Design: In a randomized, double-blind, placebo-controlled trial (the Dietary Intervention, Stem cells and Colorectal Cancer (DISC) Study), 75 healthy participants were supplemented with RS and/or polydextrose or placebo for 50 d in a 2 × 2 factorial design. Pre- and postintervention stool samples and rectal mucosal biopsies were collected and used to quantify calprotectin and expression of 12 Wnt-related genes, respectively. The expression of 10 microRNAs predicted to target SFRP1 was also quantified by quantitative reverse transcriptase-polymerase chain reaction, and DNA methylation was quantified at 7 CpG sites within the SFRP1 promoter region by pyrosequencing. Results: NDC supplementation did not affect fecal calprotectin concentration. SFRP1 mRNA expression was reduced by both RS (P = 0.005) and polydextrose (P = 0.053). RS and polydextrose did not affect SFRP1 methylation or alter the expression of 10 microRNAs predicted to target SFRP1. There were no significant interactions between RS and polydextrose. Conclusions: RS and polydextrose supplementation did not affect fecal calprotectin concentrations. Downregulation of SFRP1 with RS and polydextrose could result in increased Wnt pathway activity. However, effects on Wnt pathway activity and downstream functional effects in the healthy large-bowel mucosa remain to be investigated. The DISC Study was registered at clinicaltrials.gov as NCT01214681.

Non-digestible carbohydrates supplementation increases miR-32 expression in the healthy human colorectal epithelium: a randomized controlled trial

Colorectal cancer (CRC) risk is modulated by diet and there is convincing evidence of reduced risk with higher non‐digestible carbohydrates (NDCs) consumption. Resistant starch (RS), a NDC, positively modulates the expression of oncogenic microRNAs, suggesting that this could be a mechanism through which NDCs protect against CRC. The present study aimed to investigate the effects of supplementation with two NDCs, RS, and polydextrose (PD), on microRNA expression in the macroscopically‐normal human rectal epithelium using samples from the DISC Study, a randomized, double‐blind, placebo‐controlled dietary intervention. We screened 1008 miRNAs in pooled post‐intervention rectal mucosal samples from participants allocated to the double placebo group and those supplemented with both RS and PD. A total of 111 miRNAs were up‐ or down‐regulated by at least twofold in the RS + PD group compared with the control group. From these, eight were selected for quantification in individual participant samples by qPCR, and fold‐change direction was consistent with the array for seven miRNAs. The inconsistency for miR‐133b and the lower fold‐change values observed for the seven miRNAs is probably because qPCR of individual participant samples is a more robust and sensitive method of quantification than the array. miR‐32 expression was increased by approximately threefold (P = 0.033) in the rectal mucosa of participants supplemented with RS + PD compared with placebo. miR‐32 is involved in the regulation of processes such as cell proliferation that are dysregulated in CRC. Furthermore, miR‐32 may affect non‐canonical NF‐κB signaling via regulation of TRAF3 expression and consequently NIK stabilization.

Protocol for a multicentre, prospective, observational cohort study of variation in practice in perioperative analgesic strategies in elective laparoscopic colorectal surgery: the LapCoGesic Study.

Introduction Laparoscopic surgery combined with enhanced recovery programmes has become the gold standard in the elective management of colorectal disease. However, there is no consensus with regard to the optimal perioperative analgesic regime in this cohort of patients, with a number of options available, including thoracic epidural spinal analgesia, patient-controlled analgesia, subcutaneous and/or intraperitoneal local anaesthetics, local anaesthetic wound infiltration catheters and transversus abdominis plane blocks. This study aims to explore any differences in analgesic strategies employed across the North East of England and to assess whether any variation in practice has an impact on clinical outcomes. Methods and analysis All North East Colorectal units will be recruited for participation by the Northern Surgical Trainees Research Association (NoSTRA). Data will be collected over a consecutive 2-month period. Outcome measures will include postoperative pain score, postoperative opioid analgesic use and side effects, length of stay, 30-day complication rates, 30-day reoperative rates and 30-day readmission rates. Ethics and dissemination Ethical approval for this study has been granted by the National Research Ethics Service. The protocol will be disseminated through NoSTRA. Individual unit data will be presented at local meetings. Overall collective data will be published in peer-reviewed journals and presented at relevant surgical meetings.

Differences in the expression of microRNAs implicated in colorectal carcinogenesis and involved in the WNT signalling pathway in the macroscopically-normal epithelium of people at higher-risk of colorectal cancer

People with ulcerative colitis (UC) or adenomatous polyps (adenomas) are at increased risk of colorectal cancer (CRC). Altered expression of microRNAs (miRNAs), small non-coding RNAs that regulate gene expression post-transcriptionally, has been observed in those with UC and adenomas. Importantly, abnormally-expressed miRNAs contribute to the initiation and progression of CRC and are potential biomarkers for diagnosis and prognosis of this cancer. This study aimed to investigate differences in the expression of a panel of miRNAs in the macroscopically-normal mucosa of people at higher-risk of CRC. We quantified expression of 8 miRNAs that are (i) implicated in CRC, (ii) regulators of WNT signalling, a pathway frequently aberrantly activated in CRC, and/or (iii) whose expression is altered by butyrate treatment in healthy participants (n= 56) and in patients at higher CRC risk with quiescent UC (n= 26) or history of adenomatous polyps (n= 12). RNA was isolated from mucosal biopsies of macroscopically-normal tissue collected at 10 cm from the anal verge. cDNA was synthesised by reverse transcription and used to quantify the expression of miR-17, miR-19a, miR-19b, miR-20a, miR-25, miR-93, miR-106b and miR-424 by quantitative PCR. For normally distributed data, the ANOVA General Linear Model was used to compare miRNA expression between the 3 risk groups, adjusting for age, sex and endoscopy procedure as covariates. Where data were not normally distributed, the nonparametric Kruskal-Wallis test was used.

Anti-neoplastic effects of non-digestible carbohydrates on WNT signalling in the bowel: a randomised controlled dietary intervention

Colorectal cancer (CRC) is the third most common cancer in the UK. The majority of CRC cases develop sporadically, and the WNT signalling pathway is implicated in the aetiology of both sporadic and inherited forms. Environmental factors, including diet, modify CRC risk and non-digestible carbohydrates (NDCs), such as dietary fibre and resistant starch (RS), appear to be protective. These beneficial effects are believed to result from the production of butyrate, a short-chain fatty acid, by colonic bacteria and evidence exists for the modulation of WNT signalling by butyrate. This study aimed to investigate the effects of supplementing healthy participants with NDCs on WNT-related gene expression and its functional outcomes in the bowel. It is hypothesised that increasing NDC intake will increase colonic butyrate concentrations and modulate WNT signalling. 75 healthy participants were recruited as part of the DISC Study and supplemented with RS and/or polydextrose or placebo for 7 weeks in a 2*2 factorial design. Rectal mucosal biopsies were collected preand post-intervention. RNA was extracted from OCT-embedded biopsies and reverse transcribed to cDNA for the quantification of CCND1, MYC and SFRP1 expression in duplicate by quantitative PCR.

Abstract SY01-01: Genetic and epigenetic mechanisms linking diet and bowel cancer

Genetic basis of colorectal cancer Tumours in the large bowel arise though unrepaired genomic damage in individual stem cells located at the base of the crypts within this columnar epithelium. In the hereditary forms of bowel cancer, familial adenomatous polyposis (FAP) and Lynch Syndrome (LS), risk is increased greatly through germ-line defects in tumour suppressor genes notably the APC gene and in genes encoding the DNA mismatch repair consortium, respectively. Despite these potent genetic influences on bowel cancer development in FAP and LS, there is now good evidence that cancer risk in such patients can be reduced or delayed significantly by chemopreventive agents 1-3. Defects in the same genes are acquired through genomic damage in the much more common sporadic bowel cancer. Diet and colorectal cancer risk There is compelling evidence that lifestyle factors, especially diet, are major modulators of bowel cancer risk. For example, a recent meta-analysis demonstrated a dose-dependent reduction in bowel cancer risk with increasing intake of dietary fiber, in particular cereal fiber and whole grains.4 In addition, bowel cancer risk increases with increasing adiposity (obesity). Whilst there is evidence for cancer-related phenotypic changes in response to some dietary interventions5, the molecular mechanisms through which dietary factors modulate the acquisition or repair of the genomic damage which is fundamental for tumorigenesis remains poorly understood. Epigenetic mechanisms We have hypothesised that poor dietary choices and/or increased adiposity may enhance bowel cancer risk through increased inflammation in the colorectal epithelium. This inflammatory load, together with other stressors e.g. altered redox state, may be responsible for aberrant gene expression though epigenetic mechanisms. Epigenetics describes the nature and application of the information overlaid on the primary genetic sequence which is used to regulate gene expression under specific circumstances. The constellation of marks and molecules which is encompassed by epigenetics includes DNA methylation, histone modifications, DNA/histone binding proteins and small (non-coding) RNAs. Importantly, epigenetic marks and molecules are plastic in response to a wide range of environmental exposures, including dietary factors,6 and provide a clear mechanistic link between such exposures and the aberrant gene expression which is a cardinal feature of tumours. Diet-responsive biomarkers of bowel cancer risk Using both proteomic and sensitive gene methylation approaches, we have demonstrated that the macroscopically normal mucosa of those at higher bowel cancer risk (i.e. patients with adenomatous polyps or carcinoma) can be distinguished from that of normal (low risk) individuals.7,8 The genes showing differential patterns of methylation in those at greater bowel cancer risk included genes in the WNT signalling pathway e.g. APC, SFRP4 and WIF1 and other tumour suppressor (or putative tumour suppressor genes) i.e. HPP1, P16 and ESR1.8 Such observations suggest that there may be field changes in the vulnerable large bowel epithelium which are i) detectable long in advance of any evidence of colorectal mucosal pathology and ii) potential biomarkers of risk which could be used as surrogate endpoints in dietary intervention studies. Developing diet-related epigenetic biomarkers of bowel cancer risk We have undertaken a randomised controlled trial (the DISC Study) in which 75 healthy volunteers were given food supplements of resistant starch (RS) and polydextrose (PD) for 50 days in a 2*2 factorial design. RS and PD are non-digestible carbohydrates which show bioactivity in the large bowel where they appear to have beneficial effects on gut physiology and immune function including anti-inflammatory effects. These beneficial effects may be mediated by short-chain fatty acids (SCFA) which are produced by bacterial fermentation of the ingested RS and PD. One of the SCFA, butyrate, has potent antineoplastic actions which may be attributed to epigenetically-mediated changes in gene expression since butyrate is a powerful histone deacetylase inhibitor.9 We are assessing systemic and local inflammation in the colon using the biomarkers hsCRP in blood and calprotectin in stool, respectively. Bioactivity of the RS and PD in the colon will be monitored by measurement of SCFAs in stool and urine. We collected colo-rectal mucosal biopsies before and after the intervention for biomarker assessment. These biomarker studies include measurement of the methylation status of genes (those involved in WNT signalling and other tumour suppressor genes) known to be important in the early stages of cancer development and which may be modifiable by diet. In addition we will assess functional outcomes including expression of marker proteins identified though our earlier proteomic studies7 and mucosal cell kinetics.5 This work is on-going and recent data will be reported. Summary A wealth of epidemiological evidence provides assurance that much bowel cancer can be attributed adverse environmental exposures and this is a firm basis for the development of dietary (and other lifestyle-based) interventions which aim to reduce or delay colorectal cancer development. However, the lack of robust, validated biomarkers of bowel cancer risk is a significant impediment to the conduct of such interventions since studies with cancer as the endpoint3 are costly in both time and other resources. Epigenetic markers, especially patterns of DNA methylation, are an attractive target for development of surrogate endpoints for nutritional intervention studies since they are plastic in response to dietary exposures and provide a clear mechanistic link with the aberrant gene expression which is a cardinal feature of cancer development. Acknowledgement The DISC Study is funded by the Biotechnology and Biological Sciences Research Council (BBSRC) (Grant no. BH090948). References 1. Burn J et al. (2008) N Engl J Med 359, 2567-2578. Effect of Aspirin or Resistant Starch on colorectal neoplasia in the Lynch Syndrome. 2. Burn J et al. (2011) Cancer Prevention Research 4, 655-665. A Randomized Placebo-Controlled Prevention Trial of Aspirin and/or Resistant Starch in Young People with Familial Adenomatous Polyposis. 3. Burn J et al. (2011) Lancet 378, 2081-2087. Long-term effect of aspirin on cancer risk in carriers of hereditary colorectal cancer: an analysis from the CAPP2 randomised controlled trial. 4. Aune D et al. (2011) BMJ 343; d6617. Dietary fibre, whole grains, and risk of colorectal cancer: systematic review and dose-response meta-analysis of prospective studies. 5. Dronamraju SS et al. (2008) Gut 58, 413-420. Cell kinetics and gene expression changes in colorectal cancer patients given resistant starch - A randomised controlled trial. 6. Mathers JC et al. (2010) Adv. Genet. 71, 3-39. Induction of epigenetic alterations by dietary and other environmental factors. 7. Polley AC et al. (2006) Cancer Res. 66, 6553-6562. Proteomic analysis reveals field-wide changes in protein expression in the morphologically normal mucosa of patients with colorectal neoplasia. 8. Belshaw NJ et al. (2008) Br J Cancer 99, 136-42. Profiling CpG island field methylation in both morphologically normal and neoplastic human colonic mucosa. 9. Williams EA et al. (2003) Proc Nutr Soc 62, 107-115. Anti-cancer effects of butyrate: use of micro-array technology to investigate mechanisms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY01-01. doi:1538-7445.AM2012-SY01-01