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

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.

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model.

The incidence of both esophageal adenocarcinoma and its precursor, Barrett's Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa. A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa. This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett's Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

Pulsatile exposure to simulated reflux leads to changes in gene expression in a 3D model of oesophageal mucosa.

Oesophageal exposure to duodenogastroesophageal refluxate is implicated in the development of Barrett's metaplasia (BM), with increased risk of progression to oesophageal adenocarcinoma. The literature proposes that reflux exposure activates NF-κB, driving the aberrant expression of intestine-specific caudal-related homeobox (CDX) genes. However, early events in the pathogenesis of BM from normal epithelium are poorly understood. To investigate this, our study subjected a 3D model of the normal human oesophageal mucosa to repeated, pulsatile exposure to specific bile components and examined changes in gene expression. Initial 2D experiments with a range of bile salts observed that taurochenodeoxycholate (TCDC) impacted upon NF-κB activation without causing cell death. Informed by this, the 3D oesophageal model was repeatedly exposed to TCDC in the presence and absence of acid, and the epithelial cells underwent gene expression profiling. We identified ~300 differentially expressed genes following each treatment, with a large and significant overlap between treatments. Enrichment analysis (Broad GSEA, DAVID and Metacore™; GeneGo Inc) identified multiple gene sets related to cell signalling, inflammation, proliferation, differentiation and cell adhesion. Specifically NF-κB activation, Wnt signalling, cell adhesion and targets for the transcription factors PTF1A and HNF4α were highlighted. Our data suggest that HNF4α isoform switching may be an early event in Barrett's pathogenesis. CDX1/2 targets were, however, not enriched, suggesting that although CDX1/2 activation reportedly plays a role in BM development, it may not be an initial event. Our findings highlight new areas for investigation in the earliest stages of BM pathogenesis of oesophageal diseases and new potential therapeutic targets.

Loss of dystroglycan function in oesophageal cancer.

AIMS: Oesophageal cancer is an increasingly common human malignancy, with its incidence in the West rapidly rising. It is associated with a very poor prognosis, and its exact pathogenesis is uncertain. Dystroglycan and E-cadherin are cell adhesion molecules, the loss of which is often related to tumour differentiation, aggressiveness and invasiveness. The aim was therefore to evaluate their roles in oesophageal carcinogenesis. METHODS AND RESULTS: mRNA and protein levels of dystroglycan and E-cadherin were examined in oesophageal normal and tumour tissue samples, and in FLO-1 oesophageal adenocarcinoma cells, using immunohistochemistry, western blotting and reverse transcription polymerase chain reaction. E-cadherin,α-dystroglycan and ß-dystroglycan levels were decreased in the oesophageal primary tumour samples, despite the presence of normal levels of dystroglycan mRNA. In FLO-1 cells, increasing cell density caused a decrease in protein levels of ß-dystroglycan over time, despite the persistent presence of dystroglycan mRNA. Re-expression of dystroglycan in FLO-1 cells reduced the numbers and size of colonies formed in soft agar, indicative of a role for dystroglycan in suppressing the tumour phenotype. CONCLUSIONS: The adenocarcinoma cells mirrored the in vivo situation with respect to dystroglycan function, making this a useful model of oesophageal carcinogenesis; moreover, loss of dystroglycan protein, despite the presence of dystroglycan mRNA, points to a post-translational mechanism of dystroglycan loss.

NF-κB is activated in oesophageal fibroblasts in response to a paracrine signal generated by acid-exposed primary oesophageal squamous cells.

Oesophageal exposure to duodenogastro-oesophageal refluxate leads to reflux oesophagitis and is implicated in the development of Barrett's metaplasia (BM). NF-κB signalling in epithelial cells is associated with the activation of transcription factors believed to be central to BM development, whilst NF-κB activation in fibroblasts plays a critical role in matrix remodelling. Our aim was to study the effects of acid exposure on NF-κB activation in primary human oesophageal fibroblasts (HOFs) and primary and immortalized oesophageal squames and to investigate any epithelial/stromal interactions in the response of these cells to acid. Primary HOFs and primary and immortalized oesophageal epithelial cells were exposed to acid (pH 7 - pH 4 ≤ 120 min) in single or pulsed treatments. Conditioned medium from epithelial cells following acid exposure was also applied to fibroblasts. Cell viability was determined by MTT-ESTA. NF-κB activation was determined by cellular localization of NF-κB/p65 visualized by immunofluorescence. Conditioned medium from oesophageal epithelial cells, subjected to pH 5 pulsatile exposure, activated NF-κB in fibroblasts, with some inter-patient variability, but these conditions did not directly activate NF-κB in the epithelial cells themselves. Significant NF-κB activation was seen in the epithelial cells but only with greater acidity and exposure times (pH 4, 60-120 min). Our findings show that acid exposure can cause indirect activation of stromal cells by epithelial-stromal interactions. This may contribute to the pathogenesis of oesophageal diseases, and the inter-patient variability may go some way to explain why some patients with reflux oesophagitis develop BM and others do not.

Short-chain fatty acid level and field cancerization show opposing associations with enteroendocrine cell number and neuropilin expression in patients with colorectal adenoma.

BACKGROUND: Previous reports have suggested that the VEGF receptor neuropilin-1 (NRP-1) is expressed in a singly dispersed subpopulation of cells in the normal colonic epithelium, but that expression becomes dysregulated during colorectal carcinogenesis, with higher levels in tumour suggestive of a poor prognosis. We noted that the spatial distribution and morphology if NRP-1 expressing cells resembles that of enteroendocrine cells (EEC) which are altered in response to disease state including cancer and irritable bowel syndrome (IBS). We have shown that NRP-1 is down-regulated by butyrate in colon cancer cell lines in vitro and we hypothesized that butyrate produced in the lumen would have an analogous effect on the colon mucosa in vivo. Therefore we sought to investigate whether NRP-1 is expressed in EEC and how NRP-1 and EEC respond to butyrate and other short-chain fatty acids (SCFA - principally acetate and propionate). Additionally we sought to assess whether there is a field effect around adenomas. METHODOLOGY: Biopsies were collected at the mid-sigmoid, at the adenoma and at the contralateral wall (field) of 28 subjects during endoscopy. Samples were fixed for IHC and stained for either NRP-1 or for chromogranin A (CgA), a marker of EEC. Stool sampling was undertaken to assess individuals' butyrate, acetate and propionate levels. RESULT: NRP-1 expression was inversely related to SCFA concentration at the colon landmark (mid-sigmoid), but expression was lower and not related to SCFA concentration at the field. Likewise CgA+ cell number was also inversely related to SCFA at the landmark, but was lower and unresponsive at the field. Crypt cellularity was unaltered by field effect. A colocalisation analysis showed only a small subset of NRP-1 localised with CgA. Adenomas showed extensive, weaker staining for NRP-1 which contrastingly correlated positively with butyrate level. Field effects cause this relationship to be lost. Adenoma tissue shows dissociation of the co-regulation of NRP-1 and EEC. CONCLUSION: NRP-1 is inversely associated with levels of butyrate and other SCFA in vivo and is expressed in a subset of CgA expressing cells. EEC number is related to butyrate level in the same way.

Keratin 8 expression in colon cancer associates with low faecal butyrate levels.

BACKGROUND: Butyrate has been implicated in the mechanistic basis of the prevention of colorectal cancer by dietary fibre. Numerous in vitro studies have shown that butyrate regulates cell cycle and cell death. More recently we have shown that butyrate also regulates the integrity of the intermediate filament (IF) cytoskeleton in vitro. These and other data suggest a link between the role of diet and the implication of a central role for the keratin 8 (K8) as guardian of the colorectal epithelium. METHODS: In this cross-sectional study possible links between butyrate levels, field effects and keratin expression in cancer were addressed directly by analysing how levels of expression of the IF protein K8 in tumours, in adjacent fields and at a distant landmark site may be affected by the level of butyrate in the colon microenvironment. An immunohistochemical scoring protocol for K8 was developed and applied to samples, findings were further tested by immunoblotting. RESULTS: Levels of K8 in colorectal tumours are lower in subjects with higher levels of faecal butyrate. Immunoblotting supported this finding.Although there were no significant relationships with butyrate on the non-tumour tissues, there was a consistent trend in all measures of extent or intensity of staining towards a reduction in expression with elevated butyrate, consistent with the inverse association in tumours. CONCLUSIONS: The data suggest that butyrate may associate with down-regulation of the expression of K8 in the cancerized colon. If further validated these findings may suggest the chemopreventive value of butyrate is limited to early stage carcinogenesis as low K8 expression is associated with a poor prognosis.

Riboflavin depletion impairs cell proliferation in adult human duodenum: identification of potential effectors.

BACKGROUND AND AIMS: Riboflavin (vitamin B2) is an essential dietary component with a known function in oxidative metabolism. Our previous data using a rat model of riboflavin deficiency suggested that riboflavin also functions as a luminal signaling molecule regulating crypt development and cell turnover. Riboflavin deficiency is prevalent in both high- and low-income countries across the globe. This study aims to establish whether riboflavin deficiency has consequences for gastrointestinal (GI) morphology in adults and what the effects and effectors of any such alteration may be. METHODS: Duodenal biopsies and blood samples were collected from a cross-section of gastroscopy patients. Crypt morphology and cell division were studied by immunohistochemistry, and biochemical riboflavin status was determined. Additionally a cell culture model of riboflavin deficiency was developed and analyzed using a combination of flow cytometry, and microarray and clonogenic assays. RESULT: Duodenal crypts from subjects in the lowest quartile of riboflavin status were significantly shorter (P=0.023), less cellular (P=0.007), and had fewer cell divisions (P=0.034) than the crypts of subjects in the top quartile of riboflavin status. Following riboflavin depletion of colon cells in culture, cell cycle slowed. Microscopy revealed impaired mitosis and accumulation of aneuploid cells. Alterations in gene expression profiles reflected this alteration, with several mitosis-related genes altered, including AspM, cyclin B1, and Birc5 downregulated and Kif23 upregulated. Riboflavin depletion in vitro caused irreversible loss of proliferative potential of cells. CONCLUSIONS: Riboflavin depletion in adult humans impairs proliferation and proliferative potential of intestinal cells, which may have implications for gastrointestinal function.

The development and characterization of an organotypic tissue-engineered human esophageal mucosal model.

There is a demand for a reliable three-dimensional tissue-engineered model of the esophageal mucosa for use as an experimental platform for investigating esophageal epithelial biology and the pathogenesis of esophageal neoplasia and precursor lesions such as Barrett's metaplasia. A number of models have been described, but there has been little systematic assessment of the different approaches, making selection of a preferred platform difficult. This study assesses the properties of organotypic cultures using four different scaffolds (human esophageal matrix, porcine esophageal matrix, human dermal matrix, and collagen) and two different epithelial cell types (primary human esophageal squamous cells and the Het-1A esophageal squamous cell line). Human esophageal matrix and dermis did not give consistent results, but porcine esophageal matrix and collagen proved more reliable and were studied in greater detail. Both matrices supported the formation of a mature stratified epithelium that was similar to that of the normal human esophagus, demonstrated by Ki67, CK4, CK14, and involucrin staining. However, collagen showed reduced epithelial adherence, while fibroblast penetration into the porcine matrix was poor. Composite cultures using Het-1A cells formed a hyperproliferative epithelium with no evidence of differentiation. We propose human esophageal squamous cells seeded onto porcine esophageal matrix as the preferred model of the normal human esophagus.

A study protocol to investigate the relationship between dietary fibre intake and fermentation, colon cell turnover, global protein acetylation and early carcinogenesis: the FACT study.

BACKGROUND: A number of studies, notably EPIC, have shown a descrease in colorectal cancer risk associated with increased fibre consumption. Whilst the underlying mechanisms are likely to be multifactorial, production of the short-chain fatty-acid butyrate fro butyratye is frequently cited as a major potential contributor to the effect. Butyrate inhibits histone deacetylases, which work on a wide range of proteins over and above histones. We therefore hypothesized that alterations in the acetylated proteome may be associated with a cancer risk phenotype in the colorectal mucosa, and that such alterations are candidate biomarkers for effectiveness of fibre interventions in cancer prevention. METHODS AN DESIGN: There are two principal arms to this study: (i) a cross-sectional study (FACT OBS) of 90 subjects recruited from gastroenterology clinics and; (ii) an intervention trial in 40 subjects with an 8 week high fibre intervention. In both studies the principal goal is to investigate a link between fibre intake, SCFA production and global protein acetylation. The primary measure is level of faecal butyrate, which it is hoped will be elevated by moving subjects to a high fibre diet. Fibre intakes will be estimated in the cross-sectional group using the EPIC Food Frequency Questionnaire. Subsidiary measures of the effect of butyrate on colon mucosal function and pre-cancerous phenotype will include measures of apoptosis, apoptotic regulators cell cycle and cell division. DISCUSSION: This study will provide a new level of mechanistic data on alterations in the functional proteome in response to the colon microenvironment which may underwrite the observed cancer preventive effect of fibre. The study may yield novel candidate biomarkers of fibre fermentation and colon mucosal function. TRIAL REGISTRATION NUMBER: ISRCTN90852168.

Commonly used bowel preparations have significant and different effects upon cell proliferation in the colon: a pilot study.

BACKGROUND: Markers of crypt cell proliferation are frequently employed in studies of the impact of genetic and exogenous factors on human colonic physiology. Human studies often rely on the assessment of tissue acquired at endoscopy. Modulation of cell proliferation by bowel preparation with oral laxatives may confound the findings of such studies, but there is little data on the impact of commonly used bowel preparations on markers of cell proliferation. METHODS: Crypt length, crypt cellularity and crypt cell proliferation were assessed in biopsies acquired after preparation with either Klean-Prep or Picolax. Crypt cell proliferation was assessed by whole-mount mitotic figure count, and by two different immunohistochemical (IHC) labelling methods (Ki-67 and pHH3). Subsequent biopsies were obtained from the same patients without bowel preparation and similarly assessed. Parameters were compared between groups using analysis of variance and paired t-tests. RESULTS: There were significant differences in labelling indices (LI) between biopsies taken after Klean-prep and those taken after Picolax preparation, for both Ki67 (p = 0.019) and pHH3 (p = 0.017). A similar trend was seen for whole-mount mitotic figure counts. Suppression or elevation of proliferation parameters by bowel preparation may mask any effect due to an intervention or disease. CONCLUSION: Commonly used bowel preparations may have significant and different effects on crypt cell proliferation. This should be taken into account when designing studies and when considering the findings of existing studies.

Overexpression of cellular iron import proteins is associated with malignant progression of esophageal adenocarcinoma.

PURPOSE: There is growing evidence that iron is important in esophageal adenocarcinoma, a cancer whose incidence is rising faster than any other in the Western world. However, how iron mediates carcinogenesis at the molecular level remains unclear. In this study, we investigated the expression of iron transport proteins involved in cellular iron import, export, and storage in the premalignant lesion Barrett's metaplasia and esophageal adenocarcinoma. EXPERIMENTAL DESIGN: Perls' staining was used to examine iron deposition in tissue. mRNA expression in samples of Barrett's metaplasia matched with esophageal adenocarcinoma and samples of Barrett's metaplasia without evidence of adenocarcinoma were examined by real-time PCR. Semiquantitative immunohistochemistry was used to examine cellular localization and protein levels. The effect of iron loading on cellular proliferation and iron transporter expression was determined in esophageal cell lines OE33 and SEG-1 using a bromodeoxyuridine assay and real-time PCR, respectively. RESULTS: In the progression of Barrett's metaplasia to adenocarcinoma, there was overexpression of divalent metal transporter 1 (DMT1), transferrin receptor 1, duodenal cytochrome b, ferroportin, and H-ferritin, and these changes were associated with increased iron deposition. Overexpression of DMT1 was further associated with metastatic adenocarcinoma. Iron loading OE33 and SEG-1 cells caused increased cellular proliferation, which was associated with increased H-ferritin and decreased transferrin receptor 1 and DMT1 expression. CONCLUSIONS: Progression to adenocarcinoma is associated with increased expression of iron import proteins. These events culminate in increased intracellular iron and cellular proliferation. This may represent a novel mechanism of esophageal carcinogenesis.