Background and aims: Approximately 10% of adults experience gastro-oesophageal reflux symptoms with a variable oesophageal response. A total of 60% have no endoscopic abnormality, 30% have oesophagitis, and 10% have Barrett's oesophagus. We investigated whether the inflammatory cell infiltrate and cytokine profiles of these clinical phenotypes merely vary in severity or are fundamentally different. Methods: Patients with reflux symptoms and a normal oesophagus (n=18), oesophagitis (n=26), and Barrett's oesophagus (n=22 newly diagnosed, n=28 surveillance) were recruited. Endoscopic and histopathological degrees of inflammation were scored. Cytokine expression was determined by competitive reverse transcriptase-polymerase chain reaction and immunohistochemistry. Results: In oesophagitis, endoscopic and histopathological grades of inflammation correlated highly. mRNA expression of proinflammatory interleukin (IL)-1β, IL-8, and interferon γ (IFN-γ) were increased 3-10-fold compared with non-inflamed squamous or Barrett's oesophageal samples. There was a modest increase in anti-inflammatory IL-10 but no increase in IL-4. In Barrett's oesophagus, 29/50 had no endoscopic evidence of inflammation and histopathological inflammation was mild in 17/50 and moderate in 24/50, independent of acid suppressants. Expression of IL-1β, IL-8, and IFN-γ was similar to non-inflamed squamous mucosa. IL-10 was increased 1.6-fold similar to oesophagitis. IL-4 was increased fourfold, with 100-fold increase in IL-4/T cell receptor expression, compared with squamous oesophagus or oesophagitis. Conclusions: Barrett's oesophagus is characterised by a distinct Th-2 predominant cytokine profile compared with the proinflammatory nature of oesophagitis. The specific oesophageal immune responses may influence disease development and progression.

There is increasing evidence that epithelial to mesenchymal transition (EMT) is involved in cancer progression. Because local invasion and metastasis occurs early in the pathogenesis of esophageal adenocarcinoma, we hypothesized that EMT may be important in this disease. Using immunohistochemistry in a well-characterized set of adenocarcinoma tissues, we showed down-regulation of epithelial markers (E-cadherin and cytokeratin 18) and up-regulation of mesenchymal markers (vimentin and A-smooth muscle actin) with concomitant transforming growth factor-B1 (TGF-B1) expression at the invasive margin compared with the central tumor. A panel of esophageal cell lines was examined for the ability of TGF-B1 to induce EMT in vitro. TE7 cells were selected as a model because TGF-B1 (0-5 ng/mL) treatment induced morphologic and molecular expression changes suggestive of EMT. In TE7 cells, these TGF-B1-induced changes were reversed by 100 ng/ mL of bone morphogenetic protein 7 (BMP7), another member of the TGF-B1 superfamily. EMT was mediated via canonical TGF-B1 signaling with concomitant up-regulation of SMAD-interacting protein 1. Alterations in functional variables (aggregation, wounding, motility, and invasion) following TGF-B1 treatment were consistent with a more invasive phenotype. These functional changes were reversed by BMP7 and SMAD4 RNA interference in vitro. These data suggest that TGF-B1-mediated EMT may be relevant in esophageal carcinogenesis. (Cancer Res 2006; 66(19): 9583-90)

Background and aims: Transforming growth factor b (TGF-b) is frequently involved in gastrointestinal carcinogenesis although its contribution to oesophageal adenocarcinoma (AC) and its precursor Barrett's oesophageal epithelium (BE) metaplasia are unclear. Methods: Expression of TGF-b signalling components was assessed by reverse transcription-polymerase chain reaction (PCR), western blot, and immunohistochemistry in oesophageal endoscopic biopsies and cell lines. Genomic alterations in SMAD4 were characterised by fluorescence in situ hybridisation, methylation specific PCR, and sequencing. Functional integrity of TGF-b signalling was assessed by characterisation of p21 and proliferation status. Smad4 negative BIC-1 cells were transiently transfected with smad4 and TGF-b responsiveness evaluated. Results: smad4 mRNA expression was progressively reduced in the metaplasia-dysplasia-adenocarcinoma sequence (p,0.01). A quarter of AC samples displayed an abnormal Smad4 protein isoform, with no corresponding changes in gene sequence or organisation. Methylation of smad4 has not been described previously but we found promoter methylation in 70% of primary AC samples. In 6/8 oesophageal cell lines, chromosomal rearrangements affected the smad4 locus. Lack of smad4 expression in BIC-1 cells occurred secondary to loss of one copy and extensive deletion of the second allele's promoter region. TGF-b dependent induction of p21 and downregulation of minichromosome maintenance protein 2 was lost in .80% of BE and AC. TGF-b failed to inhibit proliferation in 5/8 oesophageal cell lines. In BIC-1, the antiproliferative response was restored following transient transfection of smad4 cDNA. Conclusions: In BE carcinogenesis, downregulation of Smad4 occurs due to several different mechanisms, including methylation, deletion, and protein modification. Frequent alterations in TGF-b signalling lead to a functionally significant impairment of TGF-b mediated growth suppression.

In cancer, Transforming Growth Factor β (TGFβ) increases proliferation and promotes invasion via selective loss of signalling pathways. Oesophageal adenocarcinoma arises from Barrett's oesophagus, progresses rapidly and is usually fatal. The contribution of perturbed TGFβ signalling in the promotion of metastasis in this disease has not been elucidated. We therefore investigated the role of TGFβ in Barrett's associated oesophageal adenocarcinoma using a panel of cell lines (OE33, TE7, SEG, BIC, FLO). 4/5 adenocarcinoma cell lines failed to cell cycle arrest, down-regulate c-Myc or induce p21 in response to TGFβ, and modulation of a Smad3/4 specific promoter was inhibited. These hyperproliferative adenocarcinoma cell lines displayed a TGFβ induced increase in the expression of the extracellular matrix degrading proteinases, urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1), which correlated with an invasive cell phenotype as measured by in vitro migration, invasion and cell scattering assays. Inhibiting ERK and JNK pathways significantly reduced PAI and uPA induction and inhibited the invasive cell phenotype. These results suggest that TGFβ Smad-dependent signalling is perturbed in Barrett's carcinogenesis, resulting in failure of growth-arrest. However, TGFβ can promote PAI and uPA expression and invasion through MAPK pathways. These data would support a dual role for TGFβ in oesophageal adenocarcinoma.