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PEA3/ETV4-related transcription factors coupled with active ERK signalling are associated with poor prognosis in gastric adenocarcinoma

PEA3/ETV4-related transcription factors coupled with active ERK signalling are associated with... Molecular Diagnostics British Journal of Cancer (2011) 105, 124 – 130 & 2011 Cancer Research UK All rights reserved 0007 – 0920/11 www.bjcancer.com PEA3/ETV4-related transcription factors coupled with active ERK signalling are associated with poor prognosis in gastric adenocarcinoma 1,2 1 3 4 3 2 ,1 R Keld , B Guo , P Downey , R Cummins , C Gulmann , YS Ang and AD Sharrocks 1 2 Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK; Faculty of Medicine, University of 3 4 Manchester, Oxford Road, Manchester M13 9PT, UK; Department of Histopathology, Beaumont Hospital, Dublin, Ireland; Department of Pathology, Royal College of Surgeons in Ireland, Dublin, Ireland BACKGROUND: Transcription factors often play important roles in tumourigenesis. Members of the PEA3 subfamily of ETS-domain transcription factors fulfil such a role and have been associated with tumour metastasis in several different cancers. Moreover, the activity of the PEA3 subfamily transcription factors is potentiated by Ras-ERK pathway signalling, which is itself often deregulated in tumour cells. METHODS: Immunohistochemical patterns of PEA3 expression and active ERK signalling were analysed and mRNA expression levels of PEA3, ER81, MMP-1 and MMP-7 were determined in gastric adenocarcinoma samples. RESULTS: Here, we have studied the expression of the PEA3 subfamily members PEA3/ETV4 and ER81/ETV1 in gastric adenocarcinomas. PEA3 is upregulated at the protein level in gastric adenocarcinomas and both PEA3/ETV4 and ER81/ETV1 are upregulated at the mRNA level in gastric adenocarcinoma tissues. This increased expression correlates with the expression of a target gene associated with metastasis, MMP-1. Enhanced ERK signalling is also more prevalent in late-stage gastric adenocarcinomas, and the co-association of ERK signalling and PEA3 expression also occurs in late-stage gastric adenocarcinomas. Furthermore, the co- association of ERK signalling and PEA3 expression correlates with decreased survival rates. CONCLUSIONS: This study shows that members of the PEA3 subfamily of transcription factors are upregulated in gastric adenocarcinomas and that the simultaneous upregulation of PEA3 expression and ERK pathway signalling is indicative of late-stage disease and a poor survival prognosis. British Journal of Cancer (2011) 105, 124–130. doi:10.1038/bjc.2011.187 www.bjcancer.com Published online 14 June 2011 & 2011 Cancer Research UK Keywords: PEA3; ER81; ETV1; ETV4; MMP1; gastric cancer Gastric cancer is the second commonest cause of a cancer death stage and typical 5-year survival is 15%. Cytotoxic chemotherapy worldwide. In Western populations, the incidence has been regimens are largely ineffective in halting the disease (Findlay et al, steadily declining over the past decades (Samalin and Ychou, 1994; Mackay et al, 2001; Tebbutt et al, 2002). Even when surgery 2007; Bosetti et al, 2008). This is thought to be a result of a is possible, neoadjuvant cytotoxic chemotherapy improves 5-year decreasing incidence of Helicobacter pylori by chlorination of survival rates modestly from 20 to 36% (Cunningham et al, 2006). drinking water and policies for antibiotic eradication. Despite New treatments and targets for drug therapies are needed to this reduction, in England gastric cancer remains ranked as the improve outcomes further. sixth commonest cause of a cancer death (Rachet et al, 2009). PEA3/ETV4 is a member of the PEA3 subfamily of ETS-domain This is largely attributable due to the late presentation of the transcription factors. This subfamily also includes ER81/ETV1 and disease and this limits treatment options. Five-year survival is ERM/ETV5. The ETS domain determines DNA-binding specificity, excellent if diagnosed at an early stage and population screening it is highly conserved in all three PEA3 family proteins and this by upper gastrointestinal endoscopy has increased the rates of suggests that they regulate similar promoters (de Launoit et al, early detection and subsequent prognosis in Japanese populations 1997). This has been shown to be the case with other ETS family (Nosho et al, 2005; Tan and Fielding, 2006). In Western members, where both promoter-specific and redundant binding populations, the incidence of gastric cancer is much lower modes are operative (Hollenhorst et al, 2007; Boros et al, 2009). and population screening is not economically viable. As a result, The PEA3 family is important in development, with particular gastric adenocarcinomas are usually diagnosed at an advanced recent emphasis on the nervous system (de Launoit et al, 1997; Oikawa and Yamada, 2003; Sharrocks, 2001, Vrieseling and *Correspondence: Professor AD Sharrocks; Arber, 2006). However, much interest has surrounded PEA3 E-mail: [email protected] proteins because of their association with cancer. The proteins are Received 18 January 2011; revised 27 April 2011; accepted 5 May 2011; generally expressed at low levels in normal adult tissues, however, published online 14 June 2011 in cancer tissues, mRNA and protein expression is often much PEA3/ETV4 and gastric adenocarcinoma R Keld et al higher. PEA3 is associated with a variety of cancers including isolation, PEA3 expression was not associated with poor prognosis colon, breast, ovarian, prostate and oesophageal cancer (de or an advanced disease stage. In adenocarcinoma tissue, PEA3 Launoit et al, 2000; Horiuchi et al, 2003; Cowden Dahl et al, expression in combination with elevated ERK MAP kinase 2007; Tomlins et al, 2007; Keld et al, 2010). ER81 has been signalling is associated with an advanced disease stage and a associated with prostate, oesophageal and gastrointestinal stromal reduced survival compared with specimens with ERK MAP kinase tumours (Cai et al, 2007; Chi et al, 2010; Keld et al, 2010). signalling without PEA3 or in specimens with PEA3 expression in Where present, tumours are generally more aggressive, advanced isolation. This study indicates that the knowledge of transcription and prognosis is worse. It has been established that PEA3 proteins factor expression in combination with the activity of the upstream are normally under auto-inhibitory control (reviewed in signalling pathways may also be important for patient selection to Sharrocks, 2001). For optimal transcriptional activity, PEA3 tailor therapy more effectively to improve treatment response. proteins require activation by post-translational modification from mitogenic signalling pathways. ERK MAP kinase signalling is important for PEA3 and ER81 activation through direct MATERIALS AND METHODS phosphorylation and subsequent SUMOylation (reviewed in de Launoit et al, 2006; Guo et al, 2007; Brown et al, 1998; Janknecht, Tissue collection 2003; Goel and Janknecht, 2004; Guo and Sharrocks, 2009). Various co-factors such as b catenin, c-Jun, p300 and LPP enhance Ethical approval was granted by Wrightington Wigan and Leigh the transcriptional activity of PEA3 in cell line models (Gum et al, Ethics Committee, UK in 2004. Tissue was collected from 40 1996; Crawford et al, 2001; Janknecht, 2003; Liu et al, 2004; Guo patients with gastric adenocarcinomas and 15 healthy controls, et al, 2006; Matsui et al, 2006). This indicates that additional and clinical characteristics recorded as described previously (Keld factors are needed for optimal activity. The poor prognosis et al, 2010). associated with tumours that express PEA3 proteins is thought to be due to the activation of genes encoding matrix metalloproteases (MMP-1, 2, 7, 9, 11, 13 and 14) (Habelhah et al, 1999; Horiuchi RNA isolation and RT–PCR analysis et al, 2003; Cai et al, 2007; Cowden Dahl et al, 2007), cell cycle regulators (Cyclin D3; Jiang et al, 2007) and the production of RNA was extracted, its’ integrity determined and subsequent real- growth factors receptors (HER-2; O’Hagan and Hassell, 1998) and time RT–PCR performed for PEA3, ER81, MMP-1, MMP-7 and 18S mediators of angiogenesis (COX2 and VEGF; Howe et al, 2001; Hua RNA as described previously (Keld et al, 2010). Data are presented et al, 2009; Liu et al, 2004). relative to 18S RNA levels in the same samples. For relative In gastric adenocarcinoma cell line models, PEA3 increases comparison of mRNA levels from tissue specimens, data were MMP-1 and MMP-7 expression and stimulates invasion in vitro further normalised to the level of each gene in a stock standard (Yamamoto et al, 2004). The ERK MAPK kinase pathway has been concentration of RNA isolated from OE33 (for MMP-1 and MMP- shown to activate PEA3 and increase MMP-1 levels by incubating 7), SW480 (for PEA3) and Flo1 (for ER81) cells. This latter gastric cancer cells with H. pylori (Wu et al, 2006). In a recent normalisation enabled experiments performed at different times to study, HER-2 was shown to drive MMP-1 expression and be compared as mRNA concentrations were calculated relative to gastric cancer cell invasion (Bao et al, 2010), but the role of the same standard stock mRNA preparation. The cell lines were PEA3 in this process was not investigated. Our understanding of cultured and lysed as described previously (Keld et al, 2010). the PEA3 family and ERK MAPK signalling in gastric adenocar- cinoma is limited. One previous study looked at PEA3 in this context and demonstrated that PEA3 mRNA expression is associated with Immunohistochemistry poor prognosis and disease recurrence in gastric adenocarcinoma Tissue microarray blocks were constructed from surgical resection in a Japanese population (Yamamoto et al, 2004). The other subfamily members ER81 and ERM did not influence disease stage tumour blocks and biopsies as described previously (Keld et al, or outcome. The role of MAPK signalling was not investigated in 2010). Three arrays were constructed for each case and stained this study. No previous studies have investigated PEA3 or ER81 in with PEA3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; Sc a Western population of gastric adenocarcinomas. Only one study 113) and phosphorylated ERK (P-ERK) antibodies (New England has investigated the status of ERK MAP kinase signalling in gastric Biolabs, Ipswich, MA, USA; #437S) at a 1 : 20 and 1 : 100 dilution, adenocarcinomas. Here, a low occurrence was found but clinical respectively. A negative control slide was tested without the correlations were not made (Feng et al, 2008). A three-way primary antibody to detect any background staining or false- positive results. Three cores for each specimen were constructed association to generate an ERK-PEA3-MMP axis has been and scored by two expert histopathologists blinded to the clinical described in cell lines derived from ovarian, oesophageal and details. A positive score was determined by the presence of positive gastric cancer (Wu et al, 2006; Cowden Dahl et al, 2007; Keld et al, staining in 5% of tumour cells. An intensity score of 1–4 was also 2010). Moreover, both PEA3 and MMP-1 were shown to be determined. Moderate to high expression (intensity score 3 and 4) upregulated in patient-derived early-stage gastric cancer samples was judged to be present if staining was visible easily at  20 from a Japanese population (Wu et al, 2006). This upregulation magnification. The highest score in the triplet of cores was was potentiated in the presence of H. pylori infection, thereby recorded. We took moderate to high expression as positive for suggesting a role for H. pylori-induced ERK pathway activity in PEA3 protein expression and P-ERK occurrence. driving their expression. Here, we revisited this issue and examined more directly whether the ERK-PEA3-MMP axis is operative in gastric adenocarcinoma tissue. Primarily, we investigated the expression of the PEA3 subfamily Statistical analysis members PEA3 and ER81 in gastric adenocarcinomas. Second, we investigated upstream pathways by measuring ERK MAP kinase The software package SPSS 15.0 was utilised to analyse data. The activity and downstream pathways by determining the levels of the t-test was used to compare means of continuous variables. Fisher’s putative targets MMP-1 and MMP-7. We found that increased exact test was used to compare discrete variables. Kaplan–Meier expression of both PEA3 and ER81 are associated with gastric analysis using the Breslow Wilcoxan test was used to calculate adenocarcinomas. Furthermore, PEA3 and ER81 expression are survival curves by univariate analysis. Significance was accepted to associated with elevated MMP-1 and MMP-7 expression. In be present with a P-value o0.05. & 2011 Cancer Research UK British Journal of Cancer (2011) 105(1), 124 – 130 Molecular Diagnostics Molecular Diagnostics PEA3/ETV4 and gastric adenocarcinoma R Keld et al RESULTS associated with gastric adenocarcinomas in a Western population, which is consistent with similar findings in mRNA expression PEA3 and ER81 are associated with gastric levels (Wu et al, 2006) and protein levels in Japanese populations adenocarcinomas (Yamamoto et al, 2004). We recently demonstrated that PEA3 family members are associated with oesophageal adenocarcinomas (Keld et al, 2010) PEA3 and ER81 are associated with MMP-1 and MMP-7 and wanted to know whether they are also associated with gastric expression adenocarcinoma. First, we performed immunohistochemistry to To further extend the immunohistochemistry results, the levels of assess the expression of PEA3 at the protein level using a TMA PEA3 and ER81 mRNA were analysed in a subset of the normal and constructed from 39 gastric adenocarcinoma and 15 non-cancer adenocarcinoma-derived samples from patients. Samples from tissue specimens. Samples were then scored according to PEA3 normal tissue exhibited low-level expression of these two levels and were considered as positive if they had moderate–high transcription factors but a large number of tumour-derived PEA3 protein levels (Figure 1A; bottom panels). Moderate to high samples exhibited elevated expression of PEA3 and/or ER81 PEA3 protein expression was present in 49% of adenocarcinomas (Figures 2A and B; summarised in Figure 2E). The overall median compared with 10% in non-cancer tissue (P-value¼ 0.018) and the distribution of PEA3 and ER81 expression was 11- and (Figure 1B). However, we did not identify a relationship between 12-fold higher, respectively, in gastric cancer tissue compared with PEA3-expressing tumours with H. pylori infection, tumour non-cancer tissue (P-values 0.080 and 0.045, respectively) (Figures differentiation, disease stage or survival, most likely because of 2F and G). Elevated PEA3 and ER81 mRNA expression was seen in the low numbers involved. Thus, PEA3 protein expression is 42 and 79%, respectively, of gastric adenocarcinomas, which was more often than observed in normal tissue. In the case of ER81, the elevated frequency in tumours reached statistical significance × 20 × 40 (P-values 0.063 and 0.008, respectively). PEA3 has previously been shown to regulate MMP-1 and MMP-7 expression in gastric cancer cell lines (Yamamoto et al, 2004; Wu et al, 2006). In Japanese populations, PEA3 expression has been H&E associated with MMP-7 expression in gastric adenocarcinomas (Yamamoto et al, 2004), and high average levels of PEA3 and MMP-1 expression have also been observed (Wu et al, 2006). We Normal therefore tested whether that PEA3 might be involved in regulating MMP-1 and MMP-7 expression in our population. Samples from PEA3 normal tissue contained very low levels of mRNAs encoding these two MMPs (Figures 2C and D; summarised in Figure 2E). In contrast, elevated levels of mRNAs encoding both MMPs were observed in samples from gastric adenocarcinomas, which were particularly marked for MMP-1, where increased expression was observed in 64% of gastric cancers (Figures 2C and D; summarised in Figure 2E). Importantly, in all cases, elevated levels of MMP-1 H&E expression in adenocarcinomas were associated with elevated levels of PEA3 and/or ER81 (Figure 2E). A similar association was seen for elevated MMP-7 expression. These data are therefore Adenocarcinomas consistent with a role for PEA3 and/or ER81 in regulating MMP expression, although overexpression of the transcription factors PEA3 alone appears insufficient for inducing MMP expression, as there are cases where PEA3 and/or ER81 are expressed but either MMP-1 and/or MMP-7 are not overexpressed (Figure 2E). Because of the small number of tumours that do not express elevated expression of PEA3 and/or ER81, it is not possible to determine whether the lack of MMP expression in these tumours is statistically significant. Similarly, we do not have any tumour samples that 40 express MMP1 and/or MMP7 and lack PEA3 and/or ER81, which further exacerbates the inability to determine statistical signifi- cance of the observed associations. Nevertheless, our results show a strong association between the expression of PEA3 and/or ER81 and downstream MMPs. Thus, PEA3 and ER81 are often upregulated at the mRNA level in gastric adenocarcinomas, which is consistent with the results of TMA analysis demonstrating overexpression of PEA3 protein in this cancer type. Further, correlations with MMP expression suggest that PEA3 family members may play an important role in controlling these important mediators of tumour metastasis in this context. Figure 1 PEA3 protein expression in gastric adenocarcinomas. (A) Examples of typical TMA staining (brown stain) for PEA3 protein in ERK MAPK signalling in combination with PEA3 normal gastric epithelium and adenocarcinoma (top panels H&E, bottom expression is a marker of poor prognosis panels PEA3 staining) at  20 and  40 magnification. (B) Summary of The influence of ERK MAP kinase signalling on gastric cancer is TMA data for moderate – high PEA3 protein expression (scored as intensity 3 or 4) in patient samples from normal and adenocarcinoma tissue classes. poorly understood. As ERK MAP kinase signalling is an important British Journal of Cancer (2011) 105(1), 124 – 130 & 2011 Cancer Research UK Normal Adenocarcinoma % High PEA3 protein PEA3/ETV4 and gastric adenocarcinoma R Keld et al PEA3 ER81 1.2 0.25 0.2 0.8 0.15 0.6 0.1 0.4 0.05 0.2 0 0 MMP-1 MMP-7 0.09 0.014 0.08 0.012 0.07 0.01 0.06 0.008 0.05 0.04 0.006 0.03 0.004 0.02 0.002 0.01 0 0 PEA3 0.25 0.20 0.15 ER81 0.10 2.0 0.04 1.5 0.03 1.0 0.02 0.5 0.01 0 0 Basal Normal Low Metaplasia Medium Carcinomas High Figure 2 Correlative expression of PEA3 and ER81 with MMP-1 and MMP-7 in gastric tissue specimens. (A–D) mRNA levels of PEA3 (A), ER81 (B), MMP-1 (C) and MMP-7 (D) relative to 18S mRNA in tissue specimens are presented. All samples were standardised to mRNAs isolated from SW480 (for PEA3), Flo1 (for ER81) or OE33 (for MMPs) cell lines. The average relative mRNA levels and standard deviations derived from at least two readings from one sample are shown. The individual tissue specimens are numbered. The samples are grouped according to the gastric tissue sub-types of normal gastric epithelium (N), adenomatous polyp (P), intestinal metaplasia (IM) and adenocarcinomas (T). The average mRNA levels of the normal samples is shown in red and indicated by the horizontal dotted line. (E) Heat map summary of the relative mRNA levels of PEA3, ER81, MMP1 and MMP7 in the gastric tissue samples. Samples are categorised as ‘normal’ (light grey dots), ‘metaplasia (dark grey dots) or from patients with oesophageal adenocarcinomas (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; 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. Groups of samples showing increased expression of MMP-1 and either PEA3 or ER81 relative to basal levels are bracketed. (F and G) Box plots of PEA3 (F) and ER81 (G) mRNA expression in oesophageal tissue taken from normal healthy controls and gastric adenocarcinoma patients. Median relative expression levels of PEA3 and ER81 are indicated for each tissue type. The box plot represents the inter-quartile range and the median value is indicated by the horizontal line. The y axes in (F) is split and the high outliers are labelled by case number. upstream activator of members of the PEA3 subfamily, we specimens and 15 non-cancer tissue specimens that we examined therefore tested whether ERK signalling was activated in gastric for PEA3 expression. Active ERK signalling was observed in both adenocarcinoma samples and whether this signalling was asso- non-cancer tissue and cancer tissue, 79% compared with 54% ciated with PEA3 subfamily member expression and subsequent (Figure 3B) reflecting ERK signalling as a normal physiological disease progression. An antibody raised against the activated event in this tissue. However, subgroup analysis performed on the P-ERK was used to stain TMAs derived from gastric adenocarci- 39 gastric cancer specimens demonstrated that there were clear noma samples to test for active ERK signalling. Examples of the differences in the levels of ERK signalling among cancer samples nuclear staining patterns of P-ERK in different classes of tissue are (Figures 3A and C). Specimens with active ERK MAP kinase illustrated in Figure 3A. We tested the same 39 gastric cancer signalling were more frequently seen in tumours with metastases & 2011 Cancer Research UK British Journal of Cancer (2011) 105(1), 124 – 130 Normal Normal Adenocarcinoma Adenocarcinoma PEA3 ER81 MMP1 MMP7 87T 87T 87T 87T 61P 82N 194N 61N 81N 103N 61P 82N 194N 61N 81N 103N 61P 82N 194N 61N 81N 103N 61P 82N 194N 103N 61N 81N Mean 148T 152T 187T 144T 145T 146T 147T 173T Mean 181T 154T 185T 196T 148T 197T 152T 187T 144T 145T 146T 147T 173T 181T 154T 185T Mean 196T 197T 148T 152T 187T 144T 145T 146T 147T Mean 173T 181T 154T 185T 196T 148T 197T 152T 187T 144T 145T 146T 147T 173T 181T 154T 185T 196T 197T 73IM 73IM 73IM 73IM Relative MMP-1 mRNA levels Relative PEA3 mRNA levels Relative PEA3 mRNA levels Relative MMP-7 mRNA levels Relative ER81 mRNA levels Relative ER81 mRNA levels Molecular Diagnostics Molecular Diagnostics PEA3/ETV4 and gastric adenocarcinoma R Keld et al ×20 ×40 H&E Normal 40 P-ERK H&E Adenocarcinomas (P-ERK negative) T&N stage P-ERK 80 M stage H&E Adenocarcinomas (P-ERK positive) PEA3 protein: – – + + P-ERK P-ERK: – + – + -PEA3 / -P-ERK (n=6) -PEA3 / +P-ERK (n=13) +PEA3 / -P-ERK (n=11) +PEA3 / +P-ERK (n=9) 0 20 40 60 80 100 120 Months Figure 3 Correlative expression of PEA3 with high levels of ERK MAPK signalling in gastric adenocarcinoma specimens. (A) Examples of typical TMA staining (brown stain) for phosphorylated active ERK (P-ERK) levels in normal gastric epithelium, negative adenocarcinoma and positive adenocarcinoma (top panels H&E, bottom panels P-ERK staining) at  20 and  40 magnification. (B) Summary of TMA data for moderate –high P-ERK signalling in patient samples from normal and adenocarcinoma tissue classes. Positive ERK MAP kinase signalling is defined as more than 5% tumour cells staining positive for P-ERK at intensity 3–4 at  20 magnification. (C) Histograms correlating the percentage of patients with TNM stage and phospho-ERK (P-ERK) levels (as defined in B). (D) Patients with a combination of higher than basal P-ERK levels and above basal PEA3 protein expression are correlated with metastatic M-stage disease. Positive P-ERK signals and PEA3 protein presence are defined as in (B). Data are presented as the percentage of patients containing the indicated combinations of P-ERK and PEA3 levels, which have T and N (white bars) or M-stage (black bars) tumours. (E) Patients with the indicated combinations of basal level (), and above basal P-ERK levels and PEA3 protein expression (þ ) are correlated with % survival. The numbers of cases with each combination of markers are shown in brackets. (Figure 3C; P-value¼ 0.040) and median survival was significantly metastases (M stage) than be at the T&N stage. Indeed, this worse, 10 months compared with 27 months in samples with lower association with M stage was much stronger with this dual ERK MAP kinase signalling activity (P-value¼ 0.050)(data not shown). combination compared with tumours with low expression or Next, we investigated if active ERK MAP kinase signalling activation of one factor in isolation (Figure 3D; w P-value¼ 0.038). influenced the clinical outcomes in relation to PEA3 expression. In Furthermore, a similar pattern is observed in patient survival cultured gastric cancer cells, it has been shown that ERK MAP where the Kapplan–Meier graph shows a clear relationship kinase signalling enhances PEA3 and MMP-1 expression (Wu et al, between PEA3 levels, P-ERK levels and patient survival 2006). In gastric adenocarcinoma tissue, we have identified an (Figure 3E). Patient survival is worse in the presence of elevated association between PEA3/ER81 and MMP-1 expression. We levels of either PEA3 protein or P-ERK and even worse when both therefore postulated that ERK signalling might influence clinical levels are elevated together. However, in comparison with the features of gastric adenocarcinoma patients in relation to PEA3 double-negative population neither P-ERK alone (P-value¼ 0.072) expression either directly via influencing MMP expression or or PEA3 expression alone (P-value¼ 0.199) reached statistical through some other mechanism. The expression of elevated levels significance compared with the significant association seen of PEA3 protein and P-ERK signal was therefore compared among with patients double positive for P-ERK and PEA3 expres- the 39 tumour samples (Figure 3D). Tumours with little PEA3 sion (P-value¼ 0.019). Conversely, in comparison with the protein expression and low P-ERK levels were all of the T&N stage double-positive population neither alone (P-value¼ 0.345) class. Conversely, tumours with a dual combination of elevated nor PEA3 expression alone (P-value¼ 0.053) reached statistical PEA3 protein and P-ERK levels were more likely to have distant significance. Thus, only the presence of elevated levels of both British Journal of Cancer (2011) 105(1), 124 – 130 & 2011 Cancer Research UK Normal Adenocarcinoma T stage N stage M stage % Survival % High P-ERK % Tumours samples % P-ERK positive PEA3/ETV4 and gastric adenocarcinoma R Keld et al PEA3 and P-ERK provides a reliable indicator of worse patient studies suggest that similar mechanisms might also operate in prognosis. gastric adenocarcinomas as tumours with combinations of high Together these results indicate that although the occurrence of PEA3 and P-ERK levels fare worse than those with low levels of one elevated P-ERK in tumours is associated with late-stage disease, or both of these molecular events. However, we do not see an the combination of elevated P-ERK levels and PEA3 expression is obvious correlation between P-ERK and levels of PEA3 protein more strongly associated with advanced-stage metastatic disease expression in cancer samples, but this would likely only be and shorter survival. revealed by more quantitative western analysis rather than the more subjective TMA analysis. Our data suggest that an axis of ERK-PEA3/ER81-MMP-1/7 exists in these tumours as observed in DISCUSSION oesophageal adenocarcinomas (Keld et al, 2010) but this is not confirmed in the current study, which is limited by design and Members of the PEA3 subfamily are associated with a variety of patient number. However, the information from the current study cancers including colon, breast, ovarian, prostate and oesophageal may be important in tailoring cancer therapy in the future. We cancer (de Launoit et al, 2000; Horiuchi et al, 2003; Cowden Dahl have shown that PEA3 upregulation in isolation does not predict et al, 2007; Tomlins et al, 2007; Chi et al, 2010; Keld et al, 2010). prognosis in any stage of gastric cancer but that simultaneous Here, we have demonstrated that increased expression of PEA3 upregulation of PEA3 expression and ERK pathway signalling is and ER81 is associated with gastric adenocarcinoma in a Western indicative of late-stage disease. Our data suggest a role for population and this is consistent with previous studies on an Asian targeting cancer therapies to tumours not only with an active population (Yamamoto et al, 2004). Moreover, we have shown a signalling pathway but also ones with a highly expressed relationship between PEA3 and ER81 expression with the transcription factors known to regulate key carcinogenic genes. expression of MMP-1 and, to a lesser extent, MMP-7 in gastric Indeed, there are clearly two distinct gastric cancer populations adenocarcinomas. This mirrors the findings in gastric cancer cell with high and low levels of active ERK, but importantly, normal lines and in gastric cancer biopsies derived from Japanese gastric tissue samples have high levels of active ERK, implying that populations where PEA3 and MMP-1 overexpression was also downregulation of ERK signalling occurs during tumourigenesis observed (Wu et al, 2006) or where an association between PEA3 with subsequent re-activation at some point, which associates with and MMP-7 expression was thought to be more important for late-stage disease. Clinical drug trials that inhibit the ERK pathway cancer cell invasion (Yamamoto et al, 2004). In addition, we have at the growth factor receptor level (e.g., EGFR) or at the signal presented data for PEA3 expression in all stages of gastric transducer protein level (e.g., MEK) have yielded limited results adenocarcinoma in our population whereas these previous findings (Rinehart et al, 2004; Rojo et al, 2006). The identification of were mainly focussed on early gastric cancer. Our data suggest that additional signal-dependant transcription factors that influence both PEA3 and ER81 are related to MMP-1 and MMP-7 expression. prognosis may improve future outcomes. Although the RNA analysis is underpowered and larger studies are needed, the findings indicate that an inter-play between PEA3 and ER81 may exist in relation to expression of MMP-1. Other ETS ACKNOWLEDGEMENTS proteins including ETS1, GABPa and Elk-1 are known to bind and regulate the same gene promoters (Hollenhorst et al, 2007; Boros We thank Karren Palmer for excellent technical assistance; et al, 2009). It is plausible that this mechanism may also be members of our laboratory for stimulating discussions; Ian operative with PEA3 and ER81 in different gastric adenocarcinomas. Donaldson for help with data visualisation. We thank Catherine Importantly, our results indicate that PEA3 mRNA or protein West and Helen Valentine for their assistance with RNA tissue expression in isolation is insufficient to specify cancer progression, specimens, Susan Pritchard (UHSM FT, Manchester, UK), David as no relationship was demonstrated with disease stage or Harrison, Craig Rodgers (WWL FT, Wigan, UK) and Stephen prognosis. Mechanisms derived from cell lines inform us that Hayes (SRH FT, Salford, UK) for their help with tissue dissection PEA3 needs activation for optimal promoter transactivation and location of archived paraffin tissue specimens, Ian Welch, activity. A model of PEA3 activation by H. Pylori through ERK Simon Galloway (UHSM FT, Manchester, UK), Laura Formela and MAP kinase signalling has been suggested in gastric cancer cells John Vickers (SRH FT, Salford, UK) for their assistance in and a similar mechanism of ERK activation appears to be retrieving surgical specimens. This work was supported by grants necessary for PEA3 activity in oesophageal and ovarian cancer from Research and Development (ABJ0026) and the Cancer cell lines (Wu et al, 2006; Cowden Dahl et al, 2007; Keld et al, Therapy and Research Fund from the WWL NHS foundation trust 2010). 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Biochem Biophys Res Commun 358: 53–58 Attribution-NonCommercial-Share Alike 3.0 Unported Keld R, Guo B, Downey P, Gulmann C, Ang YS, Sharrocks AD (2010) The License. To view a copy of this license, visit http://creativecommons. ERK MAP kinase-PEA3/ETV4-MMP-1 axis is operative in oesophageal adenocarcinoma. Molecular Cancer 9: 313 org/licenses/by-nc-sa/3.0/ British Journal of Cancer (2011) 105(1), 124 – 130 & 2011 Cancer Research UK http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png British Journal of Cancer Springer Journals

PEA3/ETV4-related transcription factors coupled with active ERK signalling are associated with poor prognosis in gastric adenocarcinoma

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Springer Journals
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Copyright © 2011 by The Author(s)
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Biomedicine; Biomedicine, general; Cancer Research; Epidemiology; Molecular Medicine; Oncology; Drug Resistance
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10.1038/bjc.2011.187
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Abstract

Molecular Diagnostics British Journal of Cancer (2011) 105, 124 – 130 & 2011 Cancer Research UK All rights reserved 0007 – 0920/11 www.bjcancer.com PEA3/ETV4-related transcription factors coupled with active ERK signalling are associated with poor prognosis in gastric adenocarcinoma 1,2 1 3 4 3 2 ,1 R Keld , B Guo , P Downey , R Cummins , C Gulmann , YS Ang and AD Sharrocks 1 2 Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK; Faculty of Medicine, University of 3 4 Manchester, Oxford Road, Manchester M13 9PT, UK; Department of Histopathology, Beaumont Hospital, Dublin, Ireland; Department of Pathology, Royal College of Surgeons in Ireland, Dublin, Ireland BACKGROUND: Transcription factors often play important roles in tumourigenesis. Members of the PEA3 subfamily of ETS-domain transcription factors fulfil such a role and have been associated with tumour metastasis in several different cancers. Moreover, the activity of the PEA3 subfamily transcription factors is potentiated by Ras-ERK pathway signalling, which is itself often deregulated in tumour cells. METHODS: Immunohistochemical patterns of PEA3 expression and active ERK signalling were analysed and mRNA expression levels of PEA3, ER81, MMP-1 and MMP-7 were determined in gastric adenocarcinoma samples. RESULTS: Here, we have studied the expression of the PEA3 subfamily members PEA3/ETV4 and ER81/ETV1 in gastric adenocarcinomas. PEA3 is upregulated at the protein level in gastric adenocarcinomas and both PEA3/ETV4 and ER81/ETV1 are upregulated at the mRNA level in gastric adenocarcinoma tissues. This increased expression correlates with the expression of a target gene associated with metastasis, MMP-1. Enhanced ERK signalling is also more prevalent in late-stage gastric adenocarcinomas, and the co-association of ERK signalling and PEA3 expression also occurs in late-stage gastric adenocarcinomas. Furthermore, the co- association of ERK signalling and PEA3 expression correlates with decreased survival rates. CONCLUSIONS: This study shows that members of the PEA3 subfamily of transcription factors are upregulated in gastric adenocarcinomas and that the simultaneous upregulation of PEA3 expression and ERK pathway signalling is indicative of late-stage disease and a poor survival prognosis. British Journal of Cancer (2011) 105, 124–130. doi:10.1038/bjc.2011.187 www.bjcancer.com Published online 14 June 2011 & 2011 Cancer Research UK Keywords: PEA3; ER81; ETV1; ETV4; MMP1; gastric cancer Gastric cancer is the second commonest cause of a cancer death stage and typical 5-year survival is 15%. Cytotoxic chemotherapy worldwide. In Western populations, the incidence has been regimens are largely ineffective in halting the disease (Findlay et al, steadily declining over the past decades (Samalin and Ychou, 1994; Mackay et al, 2001; Tebbutt et al, 2002). Even when surgery 2007; Bosetti et al, 2008). This is thought to be a result of a is possible, neoadjuvant cytotoxic chemotherapy improves 5-year decreasing incidence of Helicobacter pylori by chlorination of survival rates modestly from 20 to 36% (Cunningham et al, 2006). drinking water and policies for antibiotic eradication. Despite New treatments and targets for drug therapies are needed to this reduction, in England gastric cancer remains ranked as the improve outcomes further. sixth commonest cause of a cancer death (Rachet et al, 2009). PEA3/ETV4 is a member of the PEA3 subfamily of ETS-domain This is largely attributable due to the late presentation of the transcription factors. This subfamily also includes ER81/ETV1 and disease and this limits treatment options. Five-year survival is ERM/ETV5. The ETS domain determines DNA-binding specificity, excellent if diagnosed at an early stage and population screening it is highly conserved in all three PEA3 family proteins and this by upper gastrointestinal endoscopy has increased the rates of suggests that they regulate similar promoters (de Launoit et al, early detection and subsequent prognosis in Japanese populations 1997). This has been shown to be the case with other ETS family (Nosho et al, 2005; Tan and Fielding, 2006). In Western members, where both promoter-specific and redundant binding populations, the incidence of gastric cancer is much lower modes are operative (Hollenhorst et al, 2007; Boros et al, 2009). and population screening is not economically viable. As a result, The PEA3 family is important in development, with particular gastric adenocarcinomas are usually diagnosed at an advanced recent emphasis on the nervous system (de Launoit et al, 1997; Oikawa and Yamada, 2003; Sharrocks, 2001, Vrieseling and *Correspondence: Professor AD Sharrocks; Arber, 2006). However, much interest has surrounded PEA3 E-mail: [email protected] proteins because of their association with cancer. The proteins are Received 18 January 2011; revised 27 April 2011; accepted 5 May 2011; generally expressed at low levels in normal adult tissues, however, published online 14 June 2011 in cancer tissues, mRNA and protein expression is often much PEA3/ETV4 and gastric adenocarcinoma R Keld et al higher. PEA3 is associated with a variety of cancers including isolation, PEA3 expression was not associated with poor prognosis colon, breast, ovarian, prostate and oesophageal cancer (de or an advanced disease stage. In adenocarcinoma tissue, PEA3 Launoit et al, 2000; Horiuchi et al, 2003; Cowden Dahl et al, expression in combination with elevated ERK MAP kinase 2007; Tomlins et al, 2007; Keld et al, 2010). ER81 has been signalling is associated with an advanced disease stage and a associated with prostate, oesophageal and gastrointestinal stromal reduced survival compared with specimens with ERK MAP kinase tumours (Cai et al, 2007; Chi et al, 2010; Keld et al, 2010). signalling without PEA3 or in specimens with PEA3 expression in Where present, tumours are generally more aggressive, advanced isolation. This study indicates that the knowledge of transcription and prognosis is worse. It has been established that PEA3 proteins factor expression in combination with the activity of the upstream are normally under auto-inhibitory control (reviewed in signalling pathways may also be important for patient selection to Sharrocks, 2001). For optimal transcriptional activity, PEA3 tailor therapy more effectively to improve treatment response. proteins require activation by post-translational modification from mitogenic signalling pathways. ERK MAP kinase signalling is important for PEA3 and ER81 activation through direct MATERIALS AND METHODS phosphorylation and subsequent SUMOylation (reviewed in de Launoit et al, 2006; Guo et al, 2007; Brown et al, 1998; Janknecht, Tissue collection 2003; Goel and Janknecht, 2004; Guo and Sharrocks, 2009). Various co-factors such as b catenin, c-Jun, p300 and LPP enhance Ethical approval was granted by Wrightington Wigan and Leigh the transcriptional activity of PEA3 in cell line models (Gum et al, Ethics Committee, UK in 2004. Tissue was collected from 40 1996; Crawford et al, 2001; Janknecht, 2003; Liu et al, 2004; Guo patients with gastric adenocarcinomas and 15 healthy controls, et al, 2006; Matsui et al, 2006). This indicates that additional and clinical characteristics recorded as described previously (Keld factors are needed for optimal activity. The poor prognosis et al, 2010). associated with tumours that express PEA3 proteins is thought to be due to the activation of genes encoding matrix metalloproteases (MMP-1, 2, 7, 9, 11, 13 and 14) (Habelhah et al, 1999; Horiuchi RNA isolation and RT–PCR analysis et al, 2003; Cai et al, 2007; Cowden Dahl et al, 2007), cell cycle regulators (Cyclin D3; Jiang et al, 2007) and the production of RNA was extracted, its’ integrity determined and subsequent real- growth factors receptors (HER-2; O’Hagan and Hassell, 1998) and time RT–PCR performed for PEA3, ER81, MMP-1, MMP-7 and 18S mediators of angiogenesis (COX2 and VEGF; Howe et al, 2001; Hua RNA as described previously (Keld et al, 2010). Data are presented et al, 2009; Liu et al, 2004). relative to 18S RNA levels in the same samples. For relative In gastric adenocarcinoma cell line models, PEA3 increases comparison of mRNA levels from tissue specimens, data were MMP-1 and MMP-7 expression and stimulates invasion in vitro further normalised to the level of each gene in a stock standard (Yamamoto et al, 2004). The ERK MAPK kinase pathway has been concentration of RNA isolated from OE33 (for MMP-1 and MMP- shown to activate PEA3 and increase MMP-1 levels by incubating 7), SW480 (for PEA3) and Flo1 (for ER81) cells. This latter gastric cancer cells with H. pylori (Wu et al, 2006). In a recent normalisation enabled experiments performed at different times to study, HER-2 was shown to drive MMP-1 expression and be compared as mRNA concentrations were calculated relative to gastric cancer cell invasion (Bao et al, 2010), but the role of the same standard stock mRNA preparation. The cell lines were PEA3 in this process was not investigated. Our understanding of cultured and lysed as described previously (Keld et al, 2010). the PEA3 family and ERK MAPK signalling in gastric adenocar- cinoma is limited. One previous study looked at PEA3 in this context and demonstrated that PEA3 mRNA expression is associated with Immunohistochemistry poor prognosis and disease recurrence in gastric adenocarcinoma Tissue microarray blocks were constructed from surgical resection in a Japanese population (Yamamoto et al, 2004). The other subfamily members ER81 and ERM did not influence disease stage tumour blocks and biopsies as described previously (Keld et al, or outcome. The role of MAPK signalling was not investigated in 2010). Three arrays were constructed for each case and stained this study. No previous studies have investigated PEA3 or ER81 in with PEA3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; Sc a Western population of gastric adenocarcinomas. Only one study 113) and phosphorylated ERK (P-ERK) antibodies (New England has investigated the status of ERK MAP kinase signalling in gastric Biolabs, Ipswich, MA, USA; #437S) at a 1 : 20 and 1 : 100 dilution, adenocarcinomas. Here, a low occurrence was found but clinical respectively. A negative control slide was tested without the correlations were not made (Feng et al, 2008). A three-way primary antibody to detect any background staining or false- positive results. Three cores for each specimen were constructed association to generate an ERK-PEA3-MMP axis has been and scored by two expert histopathologists blinded to the clinical described in cell lines derived from ovarian, oesophageal and details. A positive score was determined by the presence of positive gastric cancer (Wu et al, 2006; Cowden Dahl et al, 2007; Keld et al, staining in 5% of tumour cells. An intensity score of 1–4 was also 2010). Moreover, both PEA3 and MMP-1 were shown to be determined. Moderate to high expression (intensity score 3 and 4) upregulated in patient-derived early-stage gastric cancer samples was judged to be present if staining was visible easily at  20 from a Japanese population (Wu et al, 2006). This upregulation magnification. The highest score in the triplet of cores was was potentiated in the presence of H. pylori infection, thereby recorded. We took moderate to high expression as positive for suggesting a role for H. pylori-induced ERK pathway activity in PEA3 protein expression and P-ERK occurrence. driving their expression. Here, we revisited this issue and examined more directly whether the ERK-PEA3-MMP axis is operative in gastric adenocarcinoma tissue. Primarily, we investigated the expression of the PEA3 subfamily Statistical analysis members PEA3 and ER81 in gastric adenocarcinomas. Second, we investigated upstream pathways by measuring ERK MAP kinase The software package SPSS 15.0 was utilised to analyse data. The activity and downstream pathways by determining the levels of the t-test was used to compare means of continuous variables. Fisher’s putative targets MMP-1 and MMP-7. We found that increased exact test was used to compare discrete variables. Kaplan–Meier expression of both PEA3 and ER81 are associated with gastric analysis using the Breslow Wilcoxan test was used to calculate adenocarcinomas. Furthermore, PEA3 and ER81 expression are survival curves by univariate analysis. Significance was accepted to associated with elevated MMP-1 and MMP-7 expression. In be present with a P-value o0.05. & 2011 Cancer Research UK British Journal of Cancer (2011) 105(1), 124 – 130 Molecular Diagnostics Molecular Diagnostics PEA3/ETV4 and gastric adenocarcinoma R Keld et al RESULTS associated with gastric adenocarcinomas in a Western population, which is consistent with similar findings in mRNA expression PEA3 and ER81 are associated with gastric levels (Wu et al, 2006) and protein levels in Japanese populations adenocarcinomas (Yamamoto et al, 2004). We recently demonstrated that PEA3 family members are associated with oesophageal adenocarcinomas (Keld et al, 2010) PEA3 and ER81 are associated with MMP-1 and MMP-7 and wanted to know whether they are also associated with gastric expression adenocarcinoma. First, we performed immunohistochemistry to To further extend the immunohistochemistry results, the levels of assess the expression of PEA3 at the protein level using a TMA PEA3 and ER81 mRNA were analysed in a subset of the normal and constructed from 39 gastric adenocarcinoma and 15 non-cancer adenocarcinoma-derived samples from patients. Samples from tissue specimens. Samples were then scored according to PEA3 normal tissue exhibited low-level expression of these two levels and were considered as positive if they had moderate–high transcription factors but a large number of tumour-derived PEA3 protein levels (Figure 1A; bottom panels). Moderate to high samples exhibited elevated expression of PEA3 and/or ER81 PEA3 protein expression was present in 49% of adenocarcinomas (Figures 2A and B; summarised in Figure 2E). The overall median compared with 10% in non-cancer tissue (P-value¼ 0.018) and the distribution of PEA3 and ER81 expression was 11- and (Figure 1B). However, we did not identify a relationship between 12-fold higher, respectively, in gastric cancer tissue compared with PEA3-expressing tumours with H. pylori infection, tumour non-cancer tissue (P-values 0.080 and 0.045, respectively) (Figures differentiation, disease stage or survival, most likely because of 2F and G). Elevated PEA3 and ER81 mRNA expression was seen in the low numbers involved. Thus, PEA3 protein expression is 42 and 79%, respectively, of gastric adenocarcinomas, which was more often than observed in normal tissue. In the case of ER81, the elevated frequency in tumours reached statistical significance × 20 × 40 (P-values 0.063 and 0.008, respectively). PEA3 has previously been shown to regulate MMP-1 and MMP-7 expression in gastric cancer cell lines (Yamamoto et al, 2004; Wu et al, 2006). In Japanese populations, PEA3 expression has been H&E associated with MMP-7 expression in gastric adenocarcinomas (Yamamoto et al, 2004), and high average levels of PEA3 and MMP-1 expression have also been observed (Wu et al, 2006). We Normal therefore tested whether that PEA3 might be involved in regulating MMP-1 and MMP-7 expression in our population. Samples from PEA3 normal tissue contained very low levels of mRNAs encoding these two MMPs (Figures 2C and D; summarised in Figure 2E). In contrast, elevated levels of mRNAs encoding both MMPs were observed in samples from gastric adenocarcinomas, which were particularly marked for MMP-1, where increased expression was observed in 64% of gastric cancers (Figures 2C and D; summarised in Figure 2E). Importantly, in all cases, elevated levels of MMP-1 H&E expression in adenocarcinomas were associated with elevated levels of PEA3 and/or ER81 (Figure 2E). A similar association was seen for elevated MMP-7 expression. These data are therefore Adenocarcinomas consistent with a role for PEA3 and/or ER81 in regulating MMP expression, although overexpression of the transcription factors PEA3 alone appears insufficient for inducing MMP expression, as there are cases where PEA3 and/or ER81 are expressed but either MMP-1 and/or MMP-7 are not overexpressed (Figure 2E). Because of the small number of tumours that do not express elevated expression of PEA3 and/or ER81, it is not possible to determine whether the lack of MMP expression in these tumours is statistically significant. Similarly, we do not have any tumour samples that 40 express MMP1 and/or MMP7 and lack PEA3 and/or ER81, which further exacerbates the inability to determine statistical signifi- cance of the observed associations. Nevertheless, our results show a strong association between the expression of PEA3 and/or ER81 and downstream MMPs. Thus, PEA3 and ER81 are often upregulated at the mRNA level in gastric adenocarcinomas, which is consistent with the results of TMA analysis demonstrating overexpression of PEA3 protein in this cancer type. Further, correlations with MMP expression suggest that PEA3 family members may play an important role in controlling these important mediators of tumour metastasis in this context. Figure 1 PEA3 protein expression in gastric adenocarcinomas. (A) Examples of typical TMA staining (brown stain) for PEA3 protein in ERK MAPK signalling in combination with PEA3 normal gastric epithelium and adenocarcinoma (top panels H&E, bottom expression is a marker of poor prognosis panels PEA3 staining) at  20 and  40 magnification. (B) Summary of The influence of ERK MAP kinase signalling on gastric cancer is TMA data for moderate – high PEA3 protein expression (scored as intensity 3 or 4) in patient samples from normal and adenocarcinoma tissue classes. poorly understood. As ERK MAP kinase signalling is an important British Journal of Cancer (2011) 105(1), 124 – 130 & 2011 Cancer Research UK Normal Adenocarcinoma % High PEA3 protein PEA3/ETV4 and gastric adenocarcinoma R Keld et al PEA3 ER81 1.2 0.25 0.2 0.8 0.15 0.6 0.1 0.4 0.05 0.2 0 0 MMP-1 MMP-7 0.09 0.014 0.08 0.012 0.07 0.01 0.06 0.008 0.05 0.04 0.006 0.03 0.004 0.02 0.002 0.01 0 0 PEA3 0.25 0.20 0.15 ER81 0.10 2.0 0.04 1.5 0.03 1.0 0.02 0.5 0.01 0 0 Basal Normal Low Metaplasia Medium Carcinomas High Figure 2 Correlative expression of PEA3 and ER81 with MMP-1 and MMP-7 in gastric tissue specimens. (A–D) mRNA levels of PEA3 (A), ER81 (B), MMP-1 (C) and MMP-7 (D) relative to 18S mRNA in tissue specimens are presented. All samples were standardised to mRNAs isolated from SW480 (for PEA3), Flo1 (for ER81) or OE33 (for MMPs) cell lines. The average relative mRNA levels and standard deviations derived from at least two readings from one sample are shown. The individual tissue specimens are numbered. The samples are grouped according to the gastric tissue sub-types of normal gastric epithelium (N), adenomatous polyp (P), intestinal metaplasia (IM) and adenocarcinomas (T). The average mRNA levels of the normal samples is shown in red and indicated by the horizontal dotted line. (E) Heat map summary of the relative mRNA levels of PEA3, ER81, MMP1 and MMP7 in the gastric tissue samples. Samples are categorised as ‘normal’ (light grey dots), ‘metaplasia (dark grey dots) or from patients with oesophageal adenocarcinomas (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; 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. Groups of samples showing increased expression of MMP-1 and either PEA3 or ER81 relative to basal levels are bracketed. (F and G) Box plots of PEA3 (F) and ER81 (G) mRNA expression in oesophageal tissue taken from normal healthy controls and gastric adenocarcinoma patients. Median relative expression levels of PEA3 and ER81 are indicated for each tissue type. The box plot represents the inter-quartile range and the median value is indicated by the horizontal line. The y axes in (F) is split and the high outliers are labelled by case number. upstream activator of members of the PEA3 subfamily, we specimens and 15 non-cancer tissue specimens that we examined therefore tested whether ERK signalling was activated in gastric for PEA3 expression. Active ERK signalling was observed in both adenocarcinoma samples and whether this signalling was asso- non-cancer tissue and cancer tissue, 79% compared with 54% ciated with PEA3 subfamily member expression and subsequent (Figure 3B) reflecting ERK signalling as a normal physiological disease progression. An antibody raised against the activated event in this tissue. However, subgroup analysis performed on the P-ERK was used to stain TMAs derived from gastric adenocarci- 39 gastric cancer specimens demonstrated that there were clear noma samples to test for active ERK signalling. Examples of the differences in the levels of ERK signalling among cancer samples nuclear staining patterns of P-ERK in different classes of tissue are (Figures 3A and C). Specimens with active ERK MAP kinase illustrated in Figure 3A. We tested the same 39 gastric cancer signalling were more frequently seen in tumours with metastases & 2011 Cancer Research UK British Journal of Cancer (2011) 105(1), 124 – 130 Normal Normal Adenocarcinoma Adenocarcinoma PEA3 ER81 MMP1 MMP7 87T 87T 87T 87T 61P 82N 194N 61N 81N 103N 61P 82N 194N 61N 81N 103N 61P 82N 194N 61N 81N 103N 61P 82N 194N 103N 61N 81N Mean 148T 152T 187T 144T 145T 146T 147T 173T Mean 181T 154T 185T 196T 148T 197T 152T 187T 144T 145T 146T 147T 173T 181T 154T 185T Mean 196T 197T 148T 152T 187T 144T 145T 146T 147T Mean 173T 181T 154T 185T 196T 148T 197T 152T 187T 144T 145T 146T 147T 173T 181T 154T 185T 196T 197T 73IM 73IM 73IM 73IM Relative MMP-1 mRNA levels Relative PEA3 mRNA levels Relative PEA3 mRNA levels Relative MMP-7 mRNA levels Relative ER81 mRNA levels Relative ER81 mRNA levels Molecular Diagnostics Molecular Diagnostics PEA3/ETV4 and gastric adenocarcinoma R Keld et al ×20 ×40 H&E Normal 40 P-ERK H&E Adenocarcinomas (P-ERK negative) T&N stage P-ERK 80 M stage H&E Adenocarcinomas (P-ERK positive) PEA3 protein: – – + + P-ERK P-ERK: – + – + -PEA3 / -P-ERK (n=6) -PEA3 / +P-ERK (n=13) +PEA3 / -P-ERK (n=11) +PEA3 / +P-ERK (n=9) 0 20 40 60 80 100 120 Months Figure 3 Correlative expression of PEA3 with high levels of ERK MAPK signalling in gastric adenocarcinoma specimens. (A) Examples of typical TMA staining (brown stain) for phosphorylated active ERK (P-ERK) levels in normal gastric epithelium, negative adenocarcinoma and positive adenocarcinoma (top panels H&E, bottom panels P-ERK staining) at  20 and  40 magnification. (B) Summary of TMA data for moderate –high P-ERK signalling in patient samples from normal and adenocarcinoma tissue classes. Positive ERK MAP kinase signalling is defined as more than 5% tumour cells staining positive for P-ERK at intensity 3–4 at  20 magnification. (C) Histograms correlating the percentage of patients with TNM stage and phospho-ERK (P-ERK) levels (as defined in B). (D) Patients with a combination of higher than basal P-ERK levels and above basal PEA3 protein expression are correlated with metastatic M-stage disease. Positive P-ERK signals and PEA3 protein presence are defined as in (B). Data are presented as the percentage of patients containing the indicated combinations of P-ERK and PEA3 levels, which have T and N (white bars) or M-stage (black bars) tumours. (E) Patients with the indicated combinations of basal level (), and above basal P-ERK levels and PEA3 protein expression (þ ) are correlated with % survival. The numbers of cases with each combination of markers are shown in brackets. (Figure 3C; P-value¼ 0.040) and median survival was significantly metastases (M stage) than be at the T&N stage. Indeed, this worse, 10 months compared with 27 months in samples with lower association with M stage was much stronger with this dual ERK MAP kinase signalling activity (P-value¼ 0.050)(data not shown). combination compared with tumours with low expression or Next, we investigated if active ERK MAP kinase signalling activation of one factor in isolation (Figure 3D; w P-value¼ 0.038). influenced the clinical outcomes in relation to PEA3 expression. In Furthermore, a similar pattern is observed in patient survival cultured gastric cancer cells, it has been shown that ERK MAP where the Kapplan–Meier graph shows a clear relationship kinase signalling enhances PEA3 and MMP-1 expression (Wu et al, between PEA3 levels, P-ERK levels and patient survival 2006). In gastric adenocarcinoma tissue, we have identified an (Figure 3E). Patient survival is worse in the presence of elevated association between PEA3/ER81 and MMP-1 expression. We levels of either PEA3 protein or P-ERK and even worse when both therefore postulated that ERK signalling might influence clinical levels are elevated together. However, in comparison with the features of gastric adenocarcinoma patients in relation to PEA3 double-negative population neither P-ERK alone (P-value¼ 0.072) expression either directly via influencing MMP expression or or PEA3 expression alone (P-value¼ 0.199) reached statistical through some other mechanism. The expression of elevated levels significance compared with the significant association seen of PEA3 protein and P-ERK signal was therefore compared among with patients double positive for P-ERK and PEA3 expres- the 39 tumour samples (Figure 3D). Tumours with little PEA3 sion (P-value¼ 0.019). Conversely, in comparison with the protein expression and low P-ERK levels were all of the T&N stage double-positive population neither alone (P-value¼ 0.345) class. Conversely, tumours with a dual combination of elevated nor PEA3 expression alone (P-value¼ 0.053) reached statistical PEA3 protein and P-ERK levels were more likely to have distant significance. Thus, only the presence of elevated levels of both British Journal of Cancer (2011) 105(1), 124 – 130 & 2011 Cancer Research UK Normal Adenocarcinoma T stage N stage M stage % Survival % High P-ERK % Tumours samples % P-ERK positive PEA3/ETV4 and gastric adenocarcinoma R Keld et al PEA3 and P-ERK provides a reliable indicator of worse patient studies suggest that similar mechanisms might also operate in prognosis. gastric adenocarcinomas as tumours with combinations of high Together these results indicate that although the occurrence of PEA3 and P-ERK levels fare worse than those with low levels of one elevated P-ERK in tumours is associated with late-stage disease, or both of these molecular events. However, we do not see an the combination of elevated P-ERK levels and PEA3 expression is obvious correlation between P-ERK and levels of PEA3 protein more strongly associated with advanced-stage metastatic disease expression in cancer samples, but this would likely only be and shorter survival. revealed by more quantitative western analysis rather than the more subjective TMA analysis. Our data suggest that an axis of ERK-PEA3/ER81-MMP-1/7 exists in these tumours as observed in DISCUSSION oesophageal adenocarcinomas (Keld et al, 2010) but this is not confirmed in the current study, which is limited by design and Members of the PEA3 subfamily are associated with a variety of patient number. However, the information from the current study cancers including colon, breast, ovarian, prostate and oesophageal may be important in tailoring cancer therapy in the future. We cancer (de Launoit et al, 2000; Horiuchi et al, 2003; Cowden Dahl have shown that PEA3 upregulation in isolation does not predict et al, 2007; Tomlins et al, 2007; Chi et al, 2010; Keld et al, 2010). prognosis in any stage of gastric cancer but that simultaneous Here, we have demonstrated that increased expression of PEA3 upregulation of PEA3 expression and ERK pathway signalling is and ER81 is associated with gastric adenocarcinoma in a Western indicative of late-stage disease. Our data suggest a role for population and this is consistent with previous studies on an Asian targeting cancer therapies to tumours not only with an active population (Yamamoto et al, 2004). Moreover, we have shown a signalling pathway but also ones with a highly expressed relationship between PEA3 and ER81 expression with the transcription factors known to regulate key carcinogenic genes. expression of MMP-1 and, to a lesser extent, MMP-7 in gastric Indeed, there are clearly two distinct gastric cancer populations adenocarcinomas. This mirrors the findings in gastric cancer cell with high and low levels of active ERK, but importantly, normal lines and in gastric cancer biopsies derived from Japanese gastric tissue samples have high levels of active ERK, implying that populations where PEA3 and MMP-1 overexpression was also downregulation of ERK signalling occurs during tumourigenesis observed (Wu et al, 2006) or where an association between PEA3 with subsequent re-activation at some point, which associates with and MMP-7 expression was thought to be more important for late-stage disease. Clinical drug trials that inhibit the ERK pathway cancer cell invasion (Yamamoto et al, 2004). In addition, we have at the growth factor receptor level (e.g., EGFR) or at the signal presented data for PEA3 expression in all stages of gastric transducer protein level (e.g., MEK) have yielded limited results adenocarcinoma in our population whereas these previous findings (Rinehart et al, 2004; Rojo et al, 2006). The identification of were mainly focussed on early gastric cancer. Our data suggest that additional signal-dependant transcription factors that influence both PEA3 and ER81 are related to MMP-1 and MMP-7 expression. prognosis may improve future outcomes. Although the RNA analysis is underpowered and larger studies are needed, the findings indicate that an inter-play between PEA3 and ER81 may exist in relation to expression of MMP-1. Other ETS ACKNOWLEDGEMENTS proteins including ETS1, GABPa and Elk-1 are known to bind and regulate the same gene promoters (Hollenhorst et al, 2007; Boros We thank Karren Palmer for excellent technical assistance; et al, 2009). It is plausible that this mechanism may also be members of our laboratory for stimulating discussions; Ian operative with PEA3 and ER81 in different gastric adenocarcinomas. Donaldson for help with data visualisation. We thank Catherine Importantly, our results indicate that PEA3 mRNA or protein West and Helen Valentine for their assistance with RNA tissue expression in isolation is insufficient to specify cancer progression, specimens, Susan Pritchard (UHSM FT, Manchester, UK), David as no relationship was demonstrated with disease stage or Harrison, Craig Rodgers (WWL FT, Wigan, UK) and Stephen prognosis. Mechanisms derived from cell lines inform us that Hayes (SRH FT, Salford, UK) for their help with tissue dissection PEA3 needs activation for optimal promoter transactivation and location of archived paraffin tissue specimens, Ian Welch, activity. A model of PEA3 activation by H. Pylori through ERK Simon Galloway (UHSM FT, Manchester, UK), Laura Formela and MAP kinase signalling has been suggested in gastric cancer cells John Vickers (SRH FT, Salford, UK) for their assistance in and a similar mechanism of ERK activation appears to be retrieving surgical specimens. This work was supported by grants necessary for PEA3 activity in oesophageal and ovarian cancer from Research and Development (ABJ0026) and the Cancer cell lines (Wu et al, 2006; Cowden Dahl et al, 2007; Keld et al, Therapy and Research Fund from the WWL NHS foundation trust 2010). 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Biochem Biophys Res Commun 358: 53–58 Attribution-NonCommercial-Share Alike 3.0 Unported Keld R, Guo B, Downey P, Gulmann C, Ang YS, Sharrocks AD (2010) The License. To view a copy of this license, visit http://creativecommons. ERK MAP kinase-PEA3/ETV4-MMP-1 axis is operative in oesophageal adenocarcinoma. Molecular Cancer 9: 313 org/licenses/by-nc-sa/3.0/ British Journal of Cancer (2011) 105(1), 124 – 130 & 2011 Cancer Research UK

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British Journal of Cancer – Springer Journals

Published: Jun 14, 2011

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