Expression of ets-related transcriptional factor E1AF is associated with tumor progression and over-expression of matrilysin in human gastric cancer

Hiroyuki Yamamoto1, Shina Horiuchi, Yasushi Adachi, Hiroaki Taniguchi, Katsuhiko Nosho, Yongfen Min and Kohzoh Imai

First Department of Internal Medicine, Sapporo Medical University, South-1, West-16, Chuo-ku, Sapporo 060-8543, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Expression of E1AF/PEA3 (ETV4), an ets family transcriptional factor, has been implicated in tumor progression through induction of matrix metalloproteinase (MMP) expression. The aim of this study was to examine E1AF mRNA expression and to determine whether it is correlated with progression of, and/or MMP expression in, human gastric cancer. Using the semi-quantitative reverse transcriptase–polymerase chain reaction (RT–PCR), we analyzed 100 gastric cancer tissues for E1AF mRNA expression. Expression of ER81 (ETV1) and ERM (ETV5), the other two members of the PEA3 subfamily, and Ets-1 and Ets-2 was also analyzed. The results were correlated with clinicopathological characteristics and MMP expression. Immunohistochemical analysis and an in vitro invasion assay were also performed. E1AF mRNA expression was detected in 64% of the 100 gastric cancer tissues, but was undetectable or only faintly detected in adjacent non-tumor tissues. E1AF expression was significantly correlated with depth of invasion, lymphatic and venous invasion, lymph node and distant metastasis, advance in pathological tumor-node-metastasis stage and recurrence. Patients with E1AF-positive tumors had significantly shorter overall and disease-free survival periods than did those with E1AF-negative tumors (P < 0.0001 and P < 0.0001, respectively). E1AF expression retained its significant predictive value for overall and disease-free survival in multivariate analysis that included conventional clinicopathological factors (P = 0.0082 and P = 0.0096, respectively). Among the MMPs analyzed, expression of matrilysin (MMP-7) was significantly correlated with E1AF expression. Immunohistochemical expression of E1AF was predominantly observed at the invasive front, where the expression of matrilysin was often co-localized. Antisense E1AF-transfected MKN45 gastric cancer cells expressed reduced levels of matrilysin and were less invasive in vitro than mock-transfected MKN45 cells. The results of this study suggest that E1AF, the expression of which is closely correlated with the expression of matrilysin, plays a key role in the progression of gastric cancer.

Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MMP, matrix metalloproteinase; pTNM, pathological tumor-node-metastasis; RT–PCR, reverse transcriptase–polymerase chain reaction; TIMP-1, tissue inhibitor of metalloproteinase-1


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Degradation of the extracellular matrix mediated by matrix metalloproteinases (MMPs) is crucial during tumor invasion and metastasis (1,2). MMPs are over-expressed in a variety of cancer tissues, including gastric cancers (15). Despite the fact that a number of studies have shown that MMPs play important roles in the progression of gastric cancers, the mechanisms underlying the over-expression of MMPs in vivo are poorly understood.

In this regard, E1AF (human PEA3/ETV4) is an engaging target for research. E1AF, a human homolog of mouse PEA3, is an ets family transcriptional factor that binds to the enhancer of the adenovirus E1A gene (6,7). Several lines of evidence suggest that E1AF contributes to the invasion of cancer cells. Over-expression of E1AF has been reported in various cancer cells (819). Previous studies have indicated that E1AF transactivates multiple MMP genes, such as MMP-1, MMP-3 and MMP-9, and plays an important role in tumor invasion (1113). Transfection of a non-invasive human breast cancer cell line, MCF-7, with the E1AF expression vector resulted in induction of invasive activity accompanying an increase in MMP-9 expression level (12). Expression of MMP-1, MMP-9 and E1AF has been found to be correlated with the invasive potential of an oral squamous cell carcinoma cell line (8). Interestingly, E1AF mRNA has been detected at the invasive front of cancer cells seeded on collagen gel (8). Antisense E1AF transfection reportedly restrains oral cancer invasion by reducing the activities of MMP-1, MMP-3 and MMP-9 (13). The expression of E1AF has also been implicated in the invasive potential of cancer cell lines, such as neuroblastoma (10), oral squamous cell carcinoma (13,14) and ovarian cancer (15), through induction of MMP expression. Thus, as an oncogenic transcriptional factor, E1AF appears to play an important role in the enhanced invasive potential of cancer cells by promoting the expression of MMPs. Consistent with the results of in vitro studies, E1AF mRNA has been detected in in vivo cancer tissues, such as oral squamous cell carcinoma (16), breast cancer (17), non-small cell lung cancer (18), hepatocellular carcinoma (19) and colorectal cancer (20).

However, little is known about E1AF expression and its relationship with MMP expression in gastric cancer tissues. Because various MMPs have been shown to play important roles in the progression of gastric cancer (3,2124), it seems important to clarify the relevance of E1AF in gastric cancer tissues. In an attempt to address these issues, we investigated the expression of E1AF in 100 gastric cancer tissues, using the semi-quantitative reverse transcription– polymerase chain reaction (RT–PCR), in relation to clinicopathological characteristics and MMP expression. We also analyzed the expression of ER81 (ETV1) and ERM (ETV5), the other two members of the PEA3 subgroup, and Ets-1 and Ets-2.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
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Patients and tissue samples
100 paired surgical specimens of gastric cancer and adjacent non-tumor tissue and 30 formalin-fixed, paraffin-embedded tumor specimens were obtained from patients who had undergone curative surgical treatment. Each tissue specimen was divided into two pieces after resection. For total RNA extraction, one sample was immediately frozen in liquid nitrogen at the time of surgery and stored at -80°C until extraction. The other sample was processed for pathological examination using hematoxylin and eosin staining for the evaluation of the tumor cell content. Only specimens containing >80% tumor cells were used for analysis. All of the tumors were adenocarcinoma and the histopathological features of the specimens were classified according to the TNM classification system of the Union International Centre Cancer. Informed consent was obtained from each subject and the institutional review committee approved the experiments. The human gastric cancer cell line MKN45 was purchased from the Japanese Cancer Research Resources Bank (Osaka, Japan). Cells were cultured in DMEM containing 10% fetal bovine serum.

Semi-quantitative RT–PCR
Total RNA was extracted from specimens using the acid guanidinium thiocyanate–phenol–chloroform extraction method and treated with DNase I. cDNA was synthesized from 1 µg of total RNA using SuperScript II reverse transcriptase (Invitrogen, San Diego, CA) with random hexamers. PCR was performed using primers specific for each target gene and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene in duplex PCRs (25). GAPDH served as an internal control of the reaction. All reactions were controlled without reverse transcriptase. Results were analyzed using a multi-image analyzer (Bio-Rad, Richmond, CA). The levels of gene transcripts were quantified as the ratio of the intensity of the target gene to the intensity of GAPDH. Over-expression was judged when target gene expression in the tumor sample was at least three times higher than that in the corresponding normal sample. To perform semi-quantitative RT–PCR, the ranges of linear amplification for the target gene and for the GAPDH gene were studied by using standard curves. Standard curves were drawn by using logarithmically serially diluted plasmids containing amplified region of each target gene. The correlation between the quantity of cDNA before PCR and the band intensities of target genes was analyzed by using a hemilogarithmic scale. The linear relationships were determined by the least squares approximation. Thus, the optimal number of PCR cycles and the mixing ratio of primers were determined. After confirming the specificity of each primer set, we used those primers published previously. The primers used were 5'-GCCCATTTCATTGCCTGGAC-3' and 5'-GACTTGCCATTTCTCCACTTTCC-3' for E1AF, 5'-AGCAGCATGGATGGATTTTAT-3' and 5'-CTCCTGCTTAAAGCCTTGTGGTGG-3' for ER81 (9), 5'-CCATGGTCCCAGGGAAATCTCGAT-3' and 5'-TGGCAGGGTTCAGACAGTTGTCTC-3' for ERM (9), 5'-GGGTAGCGACTTCTTGTTTG-3' and 5'-GTTAATGGAGTCAACCCAGC-3' for Ets-1, 5'-GCCTCAATAAGCCAACCATGTC-3' and 5'-TCAATCCTGCCTTTCCTGGGTC-3' for Ets-2, 5'-AGATGTGGAGTGCCTGATGT-3' and 5'-AGCTAGGGTACATCAAAGCC-3' for MMP-1, 5'-CACTGAGGGCCGCACGGAT-3' and 5'-CTTGATGTCATCCTGGGACA-3' for MMP-2, 5'-AGAGGTGACTCCACTCACAT-3' and 5'-GGTCTGTGAGTGAGTGATAG-3' for MMP-3, 5'-TCTTTGGCCTACCTATAACTGG-3' and 5'-CTAGACTGCTACCATCCGTC-3' for matrilysin, 5'-ACGGGCTCCTGGCACACG-3' and 5'-CGTCCCGGGTGTAGAGTC-3' for MMP-9, 5'-GTGGTGTGGGAAGTATCATCA-3' and 5'-GCATCTGGAGTAACCGTATTG-3' for MMP-13, 5'-CCCTATGCCTACATCCGTGA-3' and 5'-TCCATCCATCACTTGGTTAT-3' for MMP-14.

Immunohistochemistry
Formalin-fixed, paraffin-embedded specimens of 5 µm in thickness were dewaxed in xylene and rehydrated in alcohol, and then heated to 105°C in an autoclave for 10 min. Endogenous peroxidase activity was suppressed by a solution of 3% hydrogen peroxide in methanol for 5 min. After being rinsed twice in phosphate-buffered saline (PBS), the sections were treated for 18 h with an anti-human PEA3 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), an anti-human ß-catenin monoclonal antibody (Transduction Laboratories, Lexington, NY), or an anti-human matrilysin monoclonal antibody (141-7B2, Daiichi Fine Chemicals, Takaoka, Japan) at a concentration of 10 µg/ml. Non-immune mouse IgG was used as a negative control. After washing three times in PBS, the sections were treated with biotinylated goat anti-mouse immunoglobulin (Dako, Glostrup, Denmark) for 10 min and then with horseradish peroxidase–avidin complex, diluted as recommended by the manufacturer, for 10 min. The slides were then washed in PBS and developed in 0.05 M Tris–HCl (pH 7.5) containing 0.6 mg/ml 3,3'-diaminobenzidine at room temperature. The sections were counterstained in Mayer's hematoxylin and mounted. The sections were examined microscopically by two well-trained pathologists who were blinded to the clinicopathological characteristics and patients' outcome. Nuclear expression of E1AF and ß-catenin and cytoplasmic expression of matrilysin were defined as positive when immunoreactivity was observed in >10% of cancer cells at the invasive front of the tumor.

DNA transfection
A full-length cDNA encoding human E1AF was cloned into a eukaryotic expression vector, pcDNA3 neo (Invitrogen) under the control of the cytomegalovirus (CMV) promoter in 3'–5' orientation and the vector was designated pcDNA E1AF-as. pcDNA E1AF-as or pcDNA3 neo was transfected into MKN45 cells using Effectene (Qiagen, Hilden, Germany), following the manufacturer's protocol. After a few weeks of G418 selection, individual colonies were selected and expanded for further analyses. Transfectants containing the selection plasmid pcDNA3 neo alone were used as controls.

Northern blot analysis
Total RNA was prepared from cells using the acid guanidinium thiocyanate–phenol–chloroform extraction method, followed by treatment with deoxyribonuclease I. Ten micrograms of total RNA was electrophoresed on a 1% denaturing agarose gel and transferred to a nitrocellulose membrane. The membrane was hybridized with a complementary DNA for E1AF, ER81, ERM, MMP-1, MMP-2, MMP-3, matrilysin, MMP-9, MMP-13 or MMP-14 labeled using the random primer method in 50% formamide/5x Denhardt's solution/3x standard saline citrate (SSC)/100 µg/ml salmon sperm DNA/1% SDS at 42°C overnight. The membrane was then washed twice in 2x SSC/0.1% SDS at room temperature for 10 min and three times in 0.1x SSC/0.1% SDS at 55°C for 15 min. After washing, the membrane was exposed to X-ray films at -70°C. The membrane was then stripped and reprobed with a ß-actin complementary DNA probe to control for the quantity of loading and the integrity of total RNA in each lane.

In vitro invasion assay
Assays were performed by the modified Boyden Chamber method as described previously (26). 2 x 106 cells were seeded onto matrigel-coated filters. After 48 h of incubation, cells on the upper surface of the filters were completely removed by wiping with a cotton swab, as monitored visually under high magnification. The filters were fixed with methanol and stained with hematoxylin and eosin. Cells that had invaded the lower surface of the filters were counted under a microscope at a magnification of x200. Assays were also performed with a 10 µg/ml of tissue inhibitor of metalloproteinase-1 (TIMP-1) (27). The results were presented as means ± SD for each sample.

Statistical analysis
The expression of each target gene was assessed for associations with clinicopathological characteristics using the following statistical tests: Student's t-test for age; the Mann–Whitney test for histological type, depth of invasion and pathological tumor-node-metastasis (pTNM) stage; and the {chi}2 two-tailed test or Fisher's exact test for the remaining parameters. Cumulative survival rates were calculated by the Kaplan–Meier method. In the analysis of disease-free survival, patients who died of a disease other than gastric cancer recurrence were censored at the date of death. The difference between the survival curves was analyzed by the log-rank test. Factors related to survival were analyzed by Cox's proportional hazards regression model. P values <0.05 were considered significant. For the invasion assay, all of the data were first analyzed by one-way analysis of variance. When a significant difference was found by analysis of variance, the data were analyzed using the Bonferroni (Dunn) multiple-comparison method.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
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E1AF mRNA expression in gastric cancer tissues
To perform semi-quantitative RT–PCR analysis, the ranges of linear amplification for each target gene and for the control GAPDH gene were examined by using standard curves. The optimal number of PCR cycles and the mixing ratios of primers were determined. The expression of E1AF mRNA was examined in 100 gastric cancer tissues. Figure 1 shows representative results of RT–PCR for E1AF. E1AF mRNA was detected in 64% of the 100 gastric cancer tissues, but was undetectable or only faintly detected in adjacent non-tumor tissues. E1AF mRNA was detected in all 10 liver metastases from gastric cancers (data not shown). The relationship between E1AF expression and clinicopathological characteristics is shown in Table I. E1AF mRNA expression was significantly correlated with depth of invasion (P < 0.0001), lymphatic and venous invasion (P = 0.0006 and P = 0.0142, respectively), lymph node and distant metastasis (P < 0.0001 and P = 0.0088, respectively), advanced pTNM stage (P < 0.0001) and recurrence (P = 0.0016). There was no correlation of E1AF mRNA expression with age, gender, size or histological type (Table I). Expression of ER81 and ERM was occasionally detected in non-tumor tissues at low levels (Figure 1). Over-expression of ER81 and ERM mRNA was observed in 31 and 26% of the 100 gastric cancer tissues, respectively, but the expressions were not significantly correlated with clinicopathological characteristics (data not shown). The expression of the three related genes, E1AF, ER81 and ERM, was not significantly correlated with each other. For comparison, we also analyzed expression of Ets-1 and Ets-2 in gastric cancer tissues (Figure 1). Over-expression of Ets1 mRNA was detected in 42% of the 100 gastric cancer tissues, and the expression was significantly correlated with lymph node metastasis. On the other hand, over-expression of Ets2 mRNA was detected in 39% of the 100 gastric cancer tissues, but the expression was not significantly correlated with clinicopathological characteristics.



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Fig. 1. RT–PCR analysis of mRNA expression for E1AF, ER81, ERM, Ets-1 and Ets-2 in gastric cancer tissues. N and T, matched samples from non-tumor and tumor tissue, respectively.

 

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Table I. Expression of E1AF and clinicopathological characteristics in patients with gastric cancer

 
Patients whose tumors were E1AF-positive had significantly shorter overall and disease-free survival periods than did those with E1AF-negative tumors (P < 0.0001 and P < 0.0001, respectively, Figure 2). In univariate analysis, significant prognostic variables for predicting both overall and disease-free survival were E1AF expression, age, size, differentiation, depth of invasion, lymph node metastasis, distant metastasis, pTNM stage, lymphatic invasion and vascular invasion (Table II). In multivariate analysis, E1AF retained its significant predictive value for overall and disease-free survival (Table II). In contrast to E1AF expression, expression of ER81, ERM, Ets-1 or Ets-2 was not correlated with survival (data not shown).



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Fig. 2. Kaplan–Meier overall (A) and disease-free (B) survival curves of patients with gastric cancer according to the expression of E1AF.

 

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Table II. Univariate and multivariate analyses of overall and disease-free survival in patients with gastric cancer

 
Expression of MMPs and their relationship with E1AF expression
Forty randomly selected paired samples of tumor and non-tumor tissues were subjected to semi-quantitative RT–PCR analysis for MMP-1, MMP-2, MMP-3, matrilysin, MMP-9, MMP-13 and MMP-14. To perform semi-quantitative RT–PCR analysis, the ranges of linear amplification for each target gene and for the control GAPDH gene were examined. The optimal number of PCR cycles and the mixing ratios of primers were determined. Representative results are shown in Figure 3. Among the genes analyzed, expression of matrilysin was significantly correlated with E1AF expression (21 matrilysin-positive/26 E1AF-positive versus four matrilysin-positive/14 E1AF-negative, P = 0.0018). On the other hand, over-expression of ER81, ERM, Ets-1 or Ets-2 was not correlated with the expression of MMPs (data not shown).



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Fig. 3. RT–PCR analysis of mRNA expression for matrilysin, MMP-1, MMP-2, MMP-3, MMP-9, MMP-13 and MMP-14 in gastric cancer tissues. N and T, matched samples from non-tumor and tumor tissue, respectively.

 
Immunohistochemistry
The expression of E1AF, ß-catenin and matrilysin was analyzed immunohistochemically in 30 tumor specimens. In the normal gastric tissues, immunoreactivity for E1AF was only occasionally observed in the nuclei of epithelial cells. There was stronger E1AF staining of tumor cell nuclei in carcinoma tissues by comparison with normal epithelial cells. The immunoreactivities at the invasive front were often more intense than those at the superficial layer or in the center of tumors (Figure 4A). There was no detectable immunoreactivity with the control non-immune IgG (data not shown). As we and others have reported previously, cytoplasmic expression of matrilysin was observed predominantly in cancer cells at the invasive front (Figure 4B). Consistent with the results in previous reports, nuclear and/or cytoplasmic expression of ß-catenin was observed predominantly at the invasive front in a few cases (Figure 4C). Among the 30 cases examined, matrilysin was positive in 15 of the 19 cases positive for E1AF, while it was positive in only one of 11 cases negative for E1AF (P = 0.0003). Matrilysin was positive in all of the six cases positive for both E1AF and ß-catenin, but matrilysin was negative in eight cases negative for both E1AF and ß-catenin. Matrilysin was positive in nine of the 13 cases positive for E1AF alone, while it was positive in one of three cases positive for ß-catenin alone.



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Fig. 4. Immunohistochemical analysis for E1AF (A) and matrilysin (B) in serial sections of gastric cancer tissues and for ß-catenin (C) in the near section. (A) Expression of E1AF in cancer tissues. Nuclear expression can be seen in cancer cells at the invasive front (arrow). (B) Expression of matrilysin in cancer tissues. Cytoplasmic expression can be seen in cancer cells at the invasive front (arrow). (C) Expression of ß-catenin in cancer tissues. Nuclear and/or cytoplasmic expression can be seen in cancer cells. Original magnification x200 (A–C). See Supplementary material for colour version of this figure.

 
In vitro invasion assay
The antisense technique was used in an attempt to suppress E1AF expression in gastric cancer cells (26). MKN45 cells were stably transfected with a CMV-based vector that carried the E1AF cDNA in an antisense orientation. After G418 selection, 10 different clones were analyzed for E1AF mRNA by northern blot analysis. A considerable reduction in the amount of E1AF mRNA was observed in the MKN45-derived clones MKN AS-5 and MKN AS-8, and the expression of matrilysin was significantly down-regulated in these clones (Figure 5A). MKN45 cells were negative for expression of ER81, ERM, MMP-2, MMP-3, MMP-9, MMP-13 and MMP-14 and expression was not affected by the transfection (data not shown). In vitro growth rates measured by cell growth kinetics were almost the same among the parental MKN45 cells and the corresponding transfectants. The in vitro invasive potential of these cells was then assayed. Both MKN AS-5 and MKN AS-8 demonstrated about one-third the invasive potential of mock-transfected control cells (P < 0.01), and this difference was eliminated by the addition of an MMP inhibitor, TIMP-1 (Figure 5B).



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Fig. 5. (A) Northern blot analysis of MKN45 and transfectants. Lane 1, MKN45; lane 2, mock-transfected MKN45 (MKN-neo); lane 3, antisense E1AF-transfected MKN45 (MKN AS-5); lane 4, MKN AS-8. (B) In vitro invasion assay with or without TIMP-1 (10 µg/ml) in MKN45 and their transfectants. Each column indicates the means of three experiments; bars, SD. *P < 0.01.

 

    Discussion
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 Materials and methods
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The first issue that we addressed in this study was the role of E1AF mRNA expression in the progression of gastric cancer. E1AF mRNA expression was found to be significantly correlated with depth of invasion, lymphatic and venous invasion, lymph node and distant metastasis and advanced tumor stage. These results suggest that E1AF plays an important role in the progression of gastric cancer. It is thought that E1AF induces invasion of cancer cells through its target genes and that cancer cells infiltrate successively to lymph ducts and veins, reaching lymph nodes and/or distant organs. Moreover, E1AF mRNA was detected in all 10 liver metastases, suggesting that over-expression of E1AF also plays an essential role in the development of liver metastasis in gastric cancer. Thus, our results suggest that E1AF contributes to the aggressive phenotypes of gastric cancer cells. The significance of E1AF mRNA expression was further substantiated by its correlation with recurrence and poor prognosis. Therefore, an analysis of E1AF expression could be an important part of the management of patients with gastric cancer.

The second issue addressed in the present study was the correlation between E1AF mRNA expression and expression of matrilysin. This result is important because the prognostic significance of matrilysin expression in gastric cancer has been reported (24). The promoter regions of the MMP genes often contain binding sites for Ets and AP-1 transcriptional factors, mediating oncogene- and growth factor-induced transcription of the genes. Indeed, the ets-binding sequence is found in the promoter of matrilysin gene (28). Our results suggest that E1AF directly, or at least co-operatively with other transcription factors or indirectly by transactivating other transcription factors, up-regulates the expression of matrilysin in gastric cancer. On the other hand, E1AF expression was not correlated with expression of other MMPs, including MMP-1, MMP-3 or MMP-9. E1AF has been implicated in the progression of oral squamous cell carcinoma cell lines through induction of expression of these MMPs (11,13,14). It is possible that different types of cancer may use different pathways to enhance expression of various MMPs (19). Many factors, including various transcriptional factors, are involved in the regulation of MMP promoter. In gastric cancer cells, the promoter of matrilysin may be easily up-regulated by E1AF. Further analyses are needed to clarify the link between E1AF and MMPs in various types of cancer.

Immunohistochemical expression of E1AF was predominantly observed at the invasive front, where the expression of matrilysin was often co-localized. E1AF expression was significantly correlated with matrilysin expression, further supporting the notion that E1AF plays an important role in the induction of matrilysin expression. Although the frequency of expression was lower than those of E1AF and matrilysin, nuclear ß-catenin expression was occasionally co-localized. The promoter of the matrilysin gene has been shown to contain T cell factor (TCF)-binding sites and has been reported to be activated by ß-catenin/TCF-4 (29,30). Concomitant expression of ß-catenin and matrilysin has been shown in human colorectal cancer tissues (30,31). However, the frequencies (at most 30%) of nuclear over-expression of ß-catenin in gastric cancer tissues are too low to explain matrilysin expression (3234). Moreover, predominant expression of matrilysin at the invasive front in gastric cancer tissues cannot be explained by nuclear ß-catenin expression alone. Indeed, it has been reported that over-expression of ß-catenin alone was not sufficient to induce the expression of matrilysin (35). Thus, nuclear expression of ß-catenin may play a less important role in the induction of matrilysin expression in gastric cancer. We and others reported previously that matrilysin plays an important role in the progression of gastric cancer (2224). Taken together, these findings suggest that the expression of E1AF, occasionally in conjunction with the nuclear accumulation of ß-catenin, leads to up-regulation of matrilysin gene transcription in gastric cancer cells, especially those located at the invasive front, contributing to the progression of gastric cancer.

It has been shown previously that all three PEA3 subfamily members are expressed in several colon cancer cell lines (35). However, the extent of expression of each gene differed considerably among the cell lines (35). The expression pattern of ER81 has been found to be distinct from that of the other two PEA3 subfamily members in human breast cancer cell lines and Min mice tumors (9,35). Therefore, it is not surprising that the expression of the three related genes, E1AF, ER81 and ERM, was not significantly correlated with each other in human gastric cancer tissues. In contrast to E1AF expression, the expression of ER81, ERM, Ets-1 or Ets-2 was not correlated with survival or the expression of MMPs analyzed in this study. These results suggest that E1AF is the specific Ets gene that activates the target matrilysin gene in gastric cancer.

Consistent with the result of the previous study, Ets-1 expression was significantly correlated with lymph node metastasis (36). However, Ets-1 expression was not correlated with MMP expression analyzed in this study. It has been suggested that different Ets transcriptional factors have different effects on target gene expression by cooperating with or opposing such expression (19,37). Further analyses are needed to clarify target genes of Ets-1 in the progression of gastric cancer. A significant correlation of Ets-1 expression with matrilysin expression has been reported in human hepatocellular carcinoma tissues (19). Thus, several regulatory mechanisms may be involved in the over-expression of matrilysin in cancers and different types of cancer may use different pathways to enhance matrilysin expression (19).

Regarding the regulation of E1AF expression, the promoter of the E1AF gene contains a TCF-binding site and E1AF itself has been suggested to be a direct target of ß-catenin (38). However, the frequencies (at most 30%) of nuclear over-expression of ß-catenin in gastric cancer tissues are too low to explain E1AF expression (3234). It has been reported that E1AF positively regulates its own transcription (17). It is possible that an initiating event leads to increased ß-catenin/TCF activity or to increased other equivalent signals, resulting in up-regulation of the transcriptional activity of E1AF, and that transcriptionally activated E1AF in turn stimulates the expression of its target genes, including the E1AF gene itself (17). It has also been shown that an increase in both the amount and the activity of E1AF is needed to ensure high E1AF target gene expression (17). Thus, the mechanisms underlying E1AF over-expression appear to be complicated and further analysis is required to elucidate them.

Antisense E1AF-transfected MKN45 gastric cancer cells expressed reduced levels of matrilysin and were less invasive in vitro than mock-transfected MKN45 cells. However, down-regulation of E1AF could affect other target genes, including those involved in tumor invasion, in MKN45 cells. Nevertheless, the difference between invasiveness in antisense E1AF-transfected cells and that in control cells was eliminated by the addition of TIMP-1. Moreover, the expression of other MMPs was not affected by the transfection. Therefore, these results suggest that E1AF contributes to the invasive potential of gastric cancer cells, at least in part, through induction of the expression of matrilysin.

The E1AF and matrilysin axis could be a potent therapeutic target in gastric cancer patients. Therapeutic agents that inhibit the expression or function of E1AF may prove efficacious or might complement agents that directly compromise MMP activities in the treatment of gastric cancer and other cancers characterized by E1AF over-expression.


    Supplementary material
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 Supplementary material
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Supplementary material can be found at http://www.carcin.oupjournals.org


    Notes
 
1 To whom correspondence should be addressed Email: h-yama{at}sapmed.ac.jp Back


    Acknowledgments
 
This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (H.Y. and K.I.) and from the Ministry of Health, Labor and Welfare of Japan (H.Y. and K.I.).


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 

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Received July 24, 2003; revised September 16, 2003; accepted October 28, 2003.