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. 2015 Apr 17;2(1):e000022.
doi: 10.1136/bmjgast-2014-000022. eCollection 2015.

Reduced keratin expression in colorectal neoplasia and associated fields is reversible by diet and resection

Caroline A Evans  1 , Ria Rosser  2 , Jennifer S Waby  3 , Josselin Noirel  4 , Daphne Lai  5 , Phillip C Wright  1 , Elizabeth A Williams  6 , Stuart A Riley  7 , Jonathan P Bury  8 , Bernard M Corfe  9
Affiliations

Reduced keratin expression in colorectal neoplasia and associated fields is reversible by diet and resection

Caroline A Evans et al. BMJ Open Gastroenterol. .

Abstract

Background: Patients with adenomatous colonic polyps are at increased risk of developing further polyps suggesting field-wide alterations in cancer predisposition. The current study aimed to identify molecular alterations in the normal mucosa in the proximity of adenomatous polyps and to assess the modulating effect of butyrate, a chemopreventive compound produced by fermentation of dietary residues.

Methods: A cross-sectional study was undertaken in patients with adenomatous polyps: biopsy samples were taken from the adenoma, and from macroscopically normal mucosa on the contralateral wall to the adenoma and from the mid-sigmoid colon. In normal subjects biopsies were taken from the mid-sigmoid colon. Biopsies were frozen for proteomic analysis or formalin-fixed for immunohistochemistry. Proteomic analysis was undertaken using iTRAQ workflows followed by bioinformatics analyses. A second dietary fibre intervention study arm used the same endpoints and sampling strategy at the beginning and end of a high-fibre intervention.

Results: Key findings were that keratins 8, 18 and 19 were reduced in expression level with progressive proximity to the lesion. Lesional tissue exhibited multiple K8 immunoreactive bands and overall reduced levels of keratin. Biopsies from normal subjects with low faecal butyrate also showed depressed keratin expression. Resection of the lesion and elevation of dietary fibre intake both appeared to restore keratin expression level.

Conclusion: Changes in keratin expression associate with progression towards neoplasia, but remain modifiable risk factors. Dietary strategies may improve secondary chemoprevention.

Trial registration number: ISRCTN90852168.

Keywords: ADENOMA; BUTYRATE; CYTOKERATINS; DIETARY FIBRE.

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Figures

Figure 1
Figure 1
Experimental workflow. The figure summarises the workflow used in this study. Participants recruited had biopsies taken and provided stool (for faecal butyrate) and diet information. The frozen biopsies were lysed to yield soluble and insoluble fractions. The former was pooled according to butyrate and subject to immunoprecipitation for acetyl proteins, to yield an acetyl-enriched and a soluble fraction. The former was analysed by 2DGE whereas the latter was analysed by iTRAQ. The insoluble fraction was processed to yield IF and then analysed by iTRAQ. iTRAQ, isobaric tags for relative and absolute quantification; 2DGE, two dimensional gel electrophoresis; IF, intermediate filament.
Figure 2
Figure 2
iTRAQ analysis of the insoluble proteome shows effects of lesional proximity and butyrate level. Samples were separated and relatively quantified by 8-plex iTRAQ. Global analysis of the data was undertaken by principal component analysis (PCA) (panel A) and hierarchical clustering analysis (HCA) (panel B). PCA showed clustering by butyrate level with low butyrate samples in the blue oval and high butyrate in the orange oval. HCA showed the high-butyrate samples were more alike than the other samples. A protein interaction network was generated from the whole dataset (orphan nodes not shown) indicating proteins interlinked with clusters around extracellular matrix (solid line), keratins (dashed line) and metabolism (dotted line). Significant differences between samples according to lesional proximity and controlling for butyrate were computed and are shown in panel C. Proteins listed in red are significantly downregulated, while those in green are upregulated. iTRAQ, isobaric tags for relative and absolute quantification.
Figure 3
Figure 3
iTRAQ analysis of the global soluble proteome shows effects of butyrate and presence of an adenoma. Samples were separated and relatively quantified by 8-plex iTRAQ. Global analysis of the data was undertaken by principal component analysis (PCA) (panel Ai) and hierarchical clustering analysis (HCA) (panel Aii). PCA showed macroscopically normal samples could be separated by a single factor to distinguish high from low butyrate (dashed axis) and a straight line (function of two factors) could separate normal from lesion-associated samples. Panel B hierarchical clustering was used to group the data based on the degree of similarity between the samples analysed using the complete iTRAQ data set. Quantitative data were used to identify significant differences between the soluble proteome samples according to lesional proximity and controlling for butyrate. Panel C shows comparisons of lesion proximity in high and low butyrate samples and panel D shows changes associated with butyrate level, controlling for lesional proximity. Proteins listed in red are significantly down-regulated, while those in green are up-regulated. iTRAQ, isobaric tags for relative and absolute quantification.
Figure 4
Figure 4
Integrative analysis of changes in keratin. Data on relative expression of keratins 8, 18 and 19 are extracted from the isobaric tags for relative and absolute quantification (iTRAQ) analyses and presented in panel A to allow comparison of trends across samples. Panel Bi shows immunoblot for keratin 8 and keratin 19 of different soluble fraction pools with varying mean butyrate at the mid-sigmoid (MS), contralateral (CL) and adenoma (AD) biopsy sites. Bands were quantified by densitometry and are represented in Bii, MS is used as the reference sample, levels at the CL wall and lesion AD are shown in the white and grey bars respectively. Panel C shows immunoblot analysis of keratin 8 and 18 immunoreactivity in the insoluble fractions at varying butyrate level (high and low). Comparative analysis of trend in change in the iTRAQ data for soluble and insoluble fractions is shown in Panel D. FFPE sections were stained and scored for keratin 8. Panels Ei and Eii show representative sections for high and how scores. Three aspects of keratin organisation were scored: surface intensity, crypt intensity and crypt depth. Box-and-whiskers plots show distributions of each data for each end point between three different sample sets—mid-sigmoid (normal and adenoma) and contralateral to adenoma. All end points the data showed significant differences (Jonkheere-Terpstra). When the normal group were separated into new cases (sporadic—spor) or surveillance cases free from pathology but with a history of adenoma (surveillance) there were no significant differences between end points.
Figure 5
Figure 5
A high-fibre intervention elevates keratin level. Panel A graphical protocol: participants were recruited to an 8-week fibre intervention. Biopsies, food diary and faecal samples were collected at baseline and at exit from the intervention. Non-starch polysaccharide intake was significantly increased (panel B), however, there were no significant effects on faecal acetate or butyrate (Ci, Ciii) whereas propionate (Cii) approached a significant reduction. Levels of keratins 8 and 19 were measured by immunoblot, in samples preintervention and postintervention. Owing to effects of SCFAs and diet on ‘housekeeping’ markers (not shown), Coomassie staining of duplicate gels was used to show controlled loading of gels.
Figure 6
Figure 6
Integrative model of impact of microenvironment on keratin and functional consequences.
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