Verification and unmasking of widely used human esophageal adenocarcinoma cell lines.

For decades, hundreds of different human tumor type-specific cell lines have been used in experimental cancer research as models for their respective tumors. The veracity of experimental results for a specific tumor type relies on the correct derivation of the cell line. In a worldwide effort, we verified the authenticity of all available esophageal adenocarcinoma (EAC) cell lines. We proved that the frequently used cell lines SEG-1 and BIC-1 and the SK-GT-5 cell line are in fact cell lines from other tumor types. Experimental results based on these contaminated cell lines have led to ongoing clinical trials recruiting EAC patients, to more than 100 scientific publications, and to at least three National Institutes of Health cancer research grants and 11 US patents, which emphasizes the importance of our findings. Widespread use of contaminated cell lines threatens the development of treatment strategies for EAC.

Met receptor signaling: a key effector in esophageal adenocarcinoma.

PURPOSE: The incidence of esophageal adenocarcinoma is rising, and survival rates remain poor. The hepatocyte growth factor (HGF) receptor Met has been detected in esophageal cancer. The perturbation of cadherin/catenin complexes has also been shown. We sought to investigate a link among Met expression, cadherin/catenin biology, and cell growth. We assessed the prognostic significance of Met expression in esophageal adenocarcinoma. EXPERIMENTAL DESIGN: Met and HGF expression in esophageal tissues were assessed using immunohistochemistry and ELISA. Met-positive cell lines (OE33 and SEG1) and a Met-negative cell line (TE7) were incubated with HGF. Real-time reverse transcription-PCR and Western blotting were used to assess levels of E-cadherin expression. Nuclear TCF/beta-catenin signaling was assessed following reporter construct transfection. Agar colony formation was used to assess anchorage-independent growth. A panel of 72 resected esophageal adenocarcinomas were assessed for Met expression by immunohistochemistry and correlated to survival data. RESULTS: An increased expression of Met was seen along the metaplasia- adenocarcinoma sequence. Met-positive cells showed reductions in E-cadherin mRNA (37% and 69%) and protein expression following stimulation with HGF (P < 0.01). OE33 and SEG-1 showed up to a 2-fold increase in the levels of beta-catenin nuclear signaling (P < 0.01). TE7 only responded when transfected to express Met; E-cadherin expression decreased by 64% (P < 0.01). HGF stimulation led to increased agar colony formation (P < 0.01). Patients with Met-positive tumors showed lower 6-month survival rates after surgical resection than those with Met-negative tumors (P < 0.05). CONCLUSIONS: Met activation induces changes consistent with early invasion, such as down-regulation of E-cadherin, increased nuclear TCF/beta-catenin signaling, and anchorage-independent growth. This is supported by ex vivo data associating Met with reduced short-term survival. Inhibitors of Met may be effective treatment for esophageal adenocarcinoma.

Tissue inhibitor of metalloproteinase-3 (TIMP-3) gene is methylated in the development of esophageal adenocarcinoma: loss of expression correlates with poor prognosis.

Amongst involvement in diverse physiological and pathological processes, TIMP-3 may have an important role in tumour development, growth and metastasis by interaction with metalloproteases in the extracellular matrix. We studied the role and prognostic effect of TIMP-3 in esophageal adenocarcinoma (EADC). TIMP-3 gene methylation and TIMP-3 mRNA expression were analysed in 5 esophageal cell lines and 24 resected EADCs. TIMP-3 protein expression was examined in the 5 cell lines and 79 resected EADCs with known clinicopathological features. TIMP-3 methylation signal was only detected in the OE33 EADC cell line. In tissues, 0% of case-matched normal, 72% of BE and 90% of EADC were positive for methylation. TIMP-3 mRNA was detected in all the cell lines and normal, metaplastic and tumour tissues. TIMP-3 protein was localised to the cytoplasm in cell lines and tissues. Demethylating treatment of OE33 increased protein expression. At the invading edge of tumours, protein staining was equal to, or reduced, compared to normal tissues. Reduction of protein expression was associated with disease stage (p = 0.046) and poor patient survival (OR 2.1, 95% CI 1.2-3.5, p = 0.007). Mean survival time was halved in patients with reduced tumour TIMP-3 expression, from 49 to 24 months. These studies have demonstrated association between methylation of the TIMP-3 gene and BE and EADC. Reduced expression of TIMP-3 protein in EADC is associated with increased tumour invasiveness and reduced patient survival.

p16 expression in Barrett's esophagus and esophageal adenocarcinoma: association with genetic and epigenetic alterations.

Alteration of the p16 tumor suppressor gene has been implicated as a critical lesion in the molecular pathogenesis of esophageal adenocarcinoma. The aim of this study was to characterize the spectrum of p16 alterations in surgically resected esophageal tissues, comprising histologically normal esophageal squamous and gastric epithelia, premalignant Barrett's epithelia, and associated esophageal adenocarcinomas, and to explore associations between p16 mRNA expression and p16 mutations, deletions, promoter hypermethylation, p16 protein expression, and clinico-pathologic features for the same tissues. We have shown that while p16 mutations are uncommon (2%; 1/54), hypermethylation of the p16 promoter is detected in 43% (9/21) of histologically normal epithelia, in 77% (14/18) of associated Barrett's epithelia, and in 85% (18/21) of esophageal adenocarcinomas. However, p16 mRNA levels (relative to matched normal epithelia) were variable in Barrett's epithelia and adenocarcinomas, having no clear correlation with methylation status or other molecular and clinico-pathological parameters. These findings are consistent with a role for the p16 tumor suppressor gene early in the molecular progression of Barrett's epithelium to invasive esophageal adenocarcinoma, but do not support the notion that the detection of hypermethylation is systematically associated with low levels of expression.

Clinical implications of p53 tumor suppressor gene mutation and protein expression in esophageal adenocarcinomas: results of a ten-year prospective study.

OBJECTIVE: This study was undertaken to characterize the spectrum of p53 alterations (mutations and protein expression) in surgically resected esophageal adenocarcinomas, and to correlate molecular alterations with clinicopathologic findings and outcome. METHODS: Between 1991 and 2001, 91 consecutive patients with esophageal adenocarcinomas underwent subtotal esophagectomy. No patient received induction therapy. Strict clinicopathologic criteria were used to define primary esophageal adenocarcinomas. Genomic DNA was extracted from esophageal tumors, each matched with histologically normal esophageal epithelium (internal control) from the resection margin. Polymerase chain reaction was used to amplify p53 exons 4 through 10. Mutations were studied by single-strand conformation polymorphism analysis and direct DNA sequencing. Immunohistochemical testing (monoclonal antibody DO7) was used to evaluate p53 protein distribution. RESULTS: Five-year overall survival was 27.3%. No p53 alterations (mutations and/or protein overexpression) were found in normal esophageal epithelium. A total of 57.1% (n = 52) of tumors had p53 alterations (mutations and/or protein overexpression), which on univariate analysis were associated with poor tumor differentiation (P =.001), advanced pTNM stage (P =.009), and number of involved lymph nodes (0, 1-3, >3; P =.04). Patients with p53 alterations had significantly reduced 5-year overall survival relative to patients with wild-type p53 (15% vs 46%; P =.004). The p53 mutations were predominantly G:C to A:T transitions at CpG dinucleotides (52.2%, 24/46) CONCLUSIONS: We conclude that p53 alterations (mutations and/or protein overexpression) are a predictor of reduced postoperative survival after surgical resection of esophageal adenocarcinomas and that p53 may be a clinically useful molecular marker for stratifying patients in future clinical trials. Patterns of p53 mutations suggest endogenous mutational mechanisms.

Tumour necrosis factor-alpha in Barrett's oesophagus: a potential novel mechanism of action.

Barrett's metaplasia (BM) is an early lesion in the progression from oesophageal inflammation through dysplasia to the development of Barrett's adenocarcinoma (BA). Previous work indicates that BM and BA are associated with reduced E-cadherin expression and increased cytoplasmic/nuclear pools of its associated protein beta-catenin. beta-catenin participates in Wnt signalling and activates oncogene transcription by complexing with T-cells factors (TCF). One such oncogene is c-myc. We have previously shown that TNF-alpha can down-regulate E-cadherin expression. Here, we assess TNF-alpha expression in Barrett's metaplasia and examine if TNF-alpha can promote beta-catenin mediated transcription of oncogenes in a gastrointestinal model system. Employing immunohistochemistry and Western blot analysis of oesophageal tissue, epithelial expression of TNF-alpha increases with progression along the metaplasia-dysplasia-carcinoma sequence (P<0.001). beta-catenin mediated transcription was then assessed in TNF-alpha stimulated cell lines using the TOPFLASH reporter system whilst c-myc expression was assessed by real time PCR. In a columnar intestinal cell model, TNF-alpha induces c-myc expression which is induced via beta-catenin mediated transcription (P<0.05). This beta-catenin mediated transcription is independent of NF-kappaB activation. Thus, TNF-alpha is up-regulated in the progression of Barrett's oesophagus and beta-catenin mediated transcription of c-myc is a novel pathway whereby elevated levels of TNF-alpha may lead to oncogene transcription and altered biology in gastrointestinal epithelia and metaplasia.