In contrast, samples from oesophageal adenocarcinomas generally showed higher levels of either PEA3, ER81 or both transcription factors. Indeed of the 38 adenocarcinomas analysed, 29 showed levels of either PEA3 or ER81, or both, that were higher than found in samples from normal tissue. Together these data therefore provide strong evidence which this associates PEA3 and ER81 expression with Inhibitors,Modulators,Libraries adeno carcinomas, and association with patient parameters suggests that PEA3 expression is associated with meta static disease. The expression of PEA3 family members and their target genes in oesophageal cell lines Next we investigated whether oesophageal cell lines showed similar characteristics to the tumour samples.

Two cell lines derived from oesophageal adenocarcino mas, Flo 1 and OE33 cells were tested alongside OE21 oesophageal squamous cancer cells, and Het1A, a cell line derived from normal oesophageal epithelial tissue. SW480 and 293T cells were used as controls Inhibitors,Modulators,Libraries as these have previously been shown to be positive Inhibitors,Modulators,Libraries and negative respectively for PEA3 expression. Both of the adenocarcinoma cell lines showed detectable PEA3 mRNA expression whereas normal Het1A cells showed little expression. Low levels of ER81 mRNA were seen in all cell lines, except OE21 where it was barely detectable and Flo1 cells where high level expression was observed. These results were confirmed in OE33 and Het1A cells by real time PCR, where PEA3 levels are clearly greatly elevated in OE33 cells. OE33 and Het1A cells therefore represent reasonable models in which to study PEA3 function as PEA3 expression mirrors that seen in tissue samples, being high in adeno carcinomas and low in normal oesophageal cells.

PEA3 has been shown to control the expression of several matrix Inhibitors,Modulators,Libraries metalloproteases, including MMP 1 and MMP 7, and other genes such as osteo pontin and VEGF. We therefore examined whether PEA3 presence correlated with expression of any of these potential targets in the cell line models. MMP 1 was expressed in both OE21 and OE33 cell lines, alongside PEA3 suggesting a causal relationship. These results were confirmed in OE33 and Het1A cells by real time PCR, where MMP 1 levels are clearly greatly elevated in OE33 cells. In contrast MMP 7 was only expressed to high levels in OE33 cells and reciprocally, osteopontin was only expressed to high levels in OE21 cells.

Flo1 cells showed little MMP expression despite the presence of PEA3 and ER81, indicating that these transcription factors are not sufficient to activate MMP expression. To further investigate the potential links between PEA3 and ER81 and putative target gene expression, we performed siRNA mediated depletion Inhibitors,Modulators,Libraries MEK162 ARRY-162 experiments in OE33 cells using SMARTpools and measured target gene expression. Depletion of PEA3 had little effect on GAPDH and VEGF levels, but caused a 75% reduction in MMP 1 mRNA expression. A moderate 1.