PEA3 and ER81 mRNA and protein expression in oesophageal adenocarcinomas. (A) Examples of TMA staining for PEA3 protein (brown stain in right hand panels) in normal epithelium and adenocarcinoma samples at × 20 magnification. H&E staining of the same samples is shown on the left. (B) Summary of TMA data for moderate-high PEA3 expression in patient samples from different tissue classes. (C) The proportion of patients with T stage or N stage disease, with high PEA3 protein expression. (D and E) Box plots of PEA3 (D) and ER81 (E) mRNA expression in oesophageal tissue taken from healthy controls, Barrett's oesophagus and oesophageal adenocarcinoma patients. Median relative expression levels of PEA3 and ER81 are indicated for each tissue type. mRNA expression is calculated relative to 18S ribosomal RNA. The box plot represents the inter-quartile range and the median value is indicate by the horizontal line. The y axes are split (indicated by dashed lines) and the high outliers are labelled by case number.

Proliferative and invasive properties of oesophageal cells. (A) Relative number of OE33 and Het1A cells invading through a 8 μm matrigel chamber. The data are presented relative to the number of invading OE33 cells. Data are the mean and standard deviations of the relative number of invading cells from 3 experiments. Statistical significance was tested by the t test. (B) Comparative analysis of the relative number of adherent Flo1, OE33 and Het1A, cells grown for 7 days. 2 × 10⁴ cells were seeded at day 0 (indicated as 1). The data are representative of two independent experiments.

Role of ERK MAP kinase signaling in OE33 cell function. (A) Western blot of MMP-1, MMP-7 and phospho-ERK in the indicated oesophageal cell line extracts. ERK2 is used as a loading control. The positions of molecular weight markers are indicated. (B) Invasion assays of OE33 cells in the presence of U0126 or DMSO (-). Assays were performed for 48 hours and the number of invading cells was compared to the average number of invading cells treated with DMSO (taken as 1). Data are the mean and standard deviations of the relative number of invading cells from 2-3 experiments performed in duplicate. (C) Real time RT-PCR analysis of MMP-1 expression in untreated OE33 cells or Flo1 cells treated or untreated with PMA (average of duplicate samples in 2 experiments). (D) Comparative analysis of the relative number of adherent and OE33 cells grown for 96 hours in the presence of U0126 or DMSO (control). 2 × 10⁴ cells were seeded at day 0 (indicated as 1). The data are the mean relative cell numbers and standard deviations from two independent experiments. (E) Western blot of OE33 cell extracts for phospho-ERK. The cells were treated with U0126 at time 0 and allowed to grow for 12 hours. ERK2 was used as a loading control. (F) Real time RT-PCR analysis of PEA3, ER81, MMP1 and VEGF expression in OE33 cells treated with U0126 for the indicated time periods (0-12 hr). Average mRNA levels (from duplicate samples) were calculated relative to the respective mRNA levels of each gene in cells at time zero (taken as 1).

PEA3 expression and MMP-1 regulation. (A) RT-PCR analysis of PEA3, ER81, MMP-1, MMP-7 and osteopontin mRNA expression in the indicated cell lines. GAPDH was used as a loading control. (B-D) Real time RT-PCR analysis of PEA3, ER81, MMP-1 and the indicated putative target genes in untreated Het1A and OE33 cells (B) or OE33 oesophageal adenocarcinoma cells treated with SMARTpool siRNAs directed against PEA3 (C) or ER81 (D). Average mRNA levels (from duplicate samples in 2 experiments) were calculated relative to the respective mRNA levels of each gene in OE33 cells (B) or to cells treated with a non targeting siRNA (C and D) (taken as 1). (E-G) Real time RT-PCR analysis of PEA3 (E), ER81 (F) and MMP-1 (G) expression in OE33 cells treated with SMARTpool (sp) siRNAs directed against PEA3 or ER81 or one of four individual de-convoluted SMARTpool component siRNAs against PEA3, denoted A-D. Average mRNA expression levels (from duplicate samples in 2 experiments) were calculated relative to the respective mRNA levels of each gene in cells treated with a non targeting siRNA (taken as 1). (H) Reporter gene assay of the activity of a MMP-1-luciferase reporter construct in OE33 cells in the presence of the indicated siRNA duplexes. Cells were co-transfected with either empty vector (grey bars) or with a murine PEA3 expression vector (black bars). Data are shown relative to the activity of the reporter in the presence of non-targeting siRNA duplexes in either the presence or absence of mPEA3 (taken as 1 in both cases) and are the average of 2 experiments performed in duplicate (+/- sem).

PEA3 controls the proliferation and invasion of OE33 oesophageal adenocarcinoma cells. (A-C) Invasion assays of Het1A (A) and OE33 (B and C) cells in the presence of siRNA directed against PEA3, MMP-1 or a non targeting siRNA (control). Assays were performed for 24 hours and the number of invading cells was compared to the average number of invading cells treated with a non targeting control (taken as 1). Data are the mean and standard deviations of the relative number of invading cells from duplicate samples in 2-3 independent experiments. Statistical significance was tested by the t test. (D and E) Comparative analysis of the relative number of adherent Het1A (D) and OE33 (E) cells grown for 96 hours in the presence of the indicated targeting or non-targeting (control) siRNA duplexes. SMARTpool siRNAs (D and E) and individual siRNA contructs A and B (E) against PEA3 were used. 2 × 10⁴ cells were seeded at day 0 (indicated as 1). The data are representative of three independent experiments, and in (D) show the mean relative cell numbers and standard deviations from two experiments.

Correlative expression of PEA3, ER81, MMP-1 with high levels of ERK signaling in oesophageal tissue specimens. (A) Heat map of the relative mRNA levels of PEA3, ER81, MMP1 and MMP7 in the oesophageal tissue samples. Samples are categorized as "normal" (light grey dots) or from patients with Barrett's disease (dark grey dots) or oesophageal adnenocarcinomas (black dots). Expression was defined as mRNA levels more than one standard deviation above the mean for the normal samples for each gene. Basal levels are anything below this value (turquoise rectangles). Expression was then divided into three categories relative to the mean for the normal samples; high expression was anything more than 100 fold (red rectangles), medium was between 25-100 fold (orange rectangles) and low was up to 25 fold (yellow rectangles) over the mean. The primary data for individual samples is shown in additional file 1: figures S1 and S2. Groups of samples showing increased expression of MMP-1 and either PEA3 or ER81 relative to basal levels are bracketed. (B and C) Histograms correlating the number of patients with AJCC stage 1, 2, 3 and 4 disease and phospho-ERK (P-ERK) and PEA3 protein levels. (B) The number of patient samples showing the presence or absence of P-ERK is correlated with each disease stage. Positive ERK MAP kinase signalling is defined as more than 5% tumour cells staining positive for P-ERK at intensity 3-4. (C) Patients with a combination of high and low P-ERK levels and high and low PEA3 protein expression are correlated with either early stage disease (1&2; grey bars) or late stage disease (3&4; black bars). High PEA3 protein is defined as more than 5% tumour cells staining positive at intensity 4. Data are presented as the percentage of patients within each group representing the indicated combinations of P-ERK and PEA3 levels. (D) Box plots of MMP1 mRNA expression in oesophageal tissue taken from oesophageal adenocarcinoma patients. The data are grouped according to the presence or absence of high P-ERK levels and/or increased levels of PEA3 and/or ER81 (defined as mRNA levels more than two standard deviations above the mean for the normal samples for each gene). Median relative expression levels of MMP-1 are indicated for each combination of P-ERK and PEA3/ER81. mRNA expression is calculated relative to 18S ribosomal RNA. The box plot represents the inter-quartile range and the median value is indicated by the horizontal line. The y axes are split (indicated by dashed lines) and the high outliers are labelled by case number.