Association of Ets-related transcriptional factor E1AF expression with overexpression of matrix metalloproteinases, COX-2 and iNOS in the early stage of colorectal carcinogenesis
Katsuhiko Nosho *,
,
Mio Yoshida
,
Hiroyuki Yamamoto,
Hiroaki Taniguchi,
Yasushi Adachi,
Masashi Mikami,
Yuji Hinoda 1 and
Kohzoh Imai
First Department of Internal Medicine, Sapporo Medical University, S.-1, W.-16, Chuo-ku, Sapporo 0608543, Japan and 1 Department of Clinical Laboratory Science, Yamaguchi University School of Medicine, Ube 7558505, Japan
* To whom correspondence should be addressed. Tel: +81 11 611 2111 (ext.) 3211; Fax: +81 11 611 2282; Email: nosho{at}sapmed.ac.jp
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Abstract
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It is now becoming clear that matrix metalloproteinases (MMPs) play a key role in tumor development and growth. MMPs are overexpressed in a variety of premalignant tumor tissues, including colorectal adenoma. Little is known about the mechanisms underlying the overexpression of MMPs in adenoma tissues. E1AF, an Ets family transcriptional factor, has been shown to play an important role in the expression of MMPs and cyclooxygenase-2 (COX-2) in advanced colorectal cancers. The aim of this study was to examine the E1AF expression and determine whether it is correlated with the expression of MMPs, COX-2 and inducible nitric oxide synthase (iNOS) in human colorectal adenoma and submucosal cancer (pT1). Using the semi-quantitative RTPCR, 90 colorectal tumors, including 63 adenomas and 27 cancers (pT1), were analyzed for the expression of E1AF, MMPs, COX-2 and iNOS. Immunohistochemical analysis and in vitro transfection assays were also performed. E1AF mRNA was detected in 43 (47.8%) of the 90 colorectal tumors. E1AF overexpression was significantly correlated with histopathology. E1AF expression was correlated significantly with the expression of MMP-1 and MMP-7. Overexpression of COX-2 and iNOS mRNA expression was observed in 42.2% and 66.7% of the 90 colorectal tumors, respectively. COX-2 was correlated significantly with size, gender, histopathology and E1AF. iNOS was correlated significantly with size, histopathology, E1AF and COX-2. The correlation of E1AF expression with COX-2 and iNOS expression was also demonstrated by immunohistochemistry. Northern blot analysis of transfectants showed the effect of E1AF on COX-2 expression as well as iNOS on E1AF/COX-2 expression in colon cancer cell lines. The results suggest that E1AF, in conjunction with the expression of MMP-1, MMP-7, COX-2 and iNOS, plays an important role in the early stage of colorectal carcinogenesis.
Abbreviations: COX-2, cyclooxygenase-2; ECM, extracellular matrix; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; iNOS, inducible nitric oxide synthase; MMP, matrix metalloproteinase; NO, nitric oxide; PBS, phosphate-buffered saline; RTPCR, reverse transcriptasepolymerase chain reaction; SMT, S-methylisothiourea sulfate; SSC, standard saline citrate
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Introduction
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Colorectal cancer is one of the most common human malignancies in the world. Although alternative pathways exist, it is accepted that most colorectal cancers arise in pre-existing adenomas (1).
Degradation of the extracellular matrix (ECM) mediated by matrix metalloproteinases (MMPs) is crucial during tumor invasion and metastasis (28). Until recently, the role of MMPs has been associated primarily with tumor invasion and metastasis. It is now becoming clear that MMPs also play a key role in tumor development and growth (28). Several lines of evidence indicate that MMPs regulate cell growth and survival. MMPs directly participate in the generation of signals that induce the proliferation of tumor cells by activating the cell surface growth factor precursors, releasing and activating latent growth factors sequestered in the ECM, and altering the structure of essential ECM components (211). Indeed, MMPs are overexpressed in a variety of premalignant tumor tissues, including colorectal adenoma tissues (1218).
However, despite the fact that several studies have shown that MMPs play important roles in the early stage of colorectal carcinogenesis, little is known about the mechanisms underlying the overexpression of MMPs in adenoma tissues. We recently reported that E1AF (human PEA3/ETV4), an Ets family transcriptional factor, plays a key role in the progression of colorectal cancer (19). Interestingly, the expression of E1AF was closely correlated with the expression of MMP-1 and MMP-7 in advanced colorectal cancer tissues.
Interestingly, it has been reported that E1AF is one of the potent activators of cyclooxygenase-2 (COX-2) transcription (20). Increasing mounting evidence indicates that COX-2 plays an important role in the early and late stages of colorectal carcinogenesis (2126). COX-2 is overexpressed in 8090% of colorectal cancer tissues and in 4050% of premalignant adenoma tissues (16,17).
Inducible nitric oxide synthase (iNOS) has been reported to play a crucial role in the development of cancer by promoting angiogenesis (27). Cianchi et al. (28,29) reported that COX-2 and iNOS upregulated VEGF, which is one of the most important proangiogenic factors, in human colorectal cancer. Moreover, nitric oxide (NO), generated by iNOS, reportedly stimulates E1AF to increase the COX-2 expression in colorectal cancer (30). In addition, NO has been shown to augment the synergistic interaction between E1AF and its transcription coactivator CBP/p300, resulting in the facilitation of COX-2 induction (30).
Thus, it seems important to clarify whether E1AF, MMPs, COX-2, and iNOS are overexpressed concomitantly in the early stage of colorectal carcinogenesis. If this is the case, as a transcriptional factor, E1AF may play an important role in the regulation of multiple genes involved in tumour promotion. In an attempt to address these issues, we investigated the expression of E1AF and other Ets-related transcriptional factors in 90 colorectal tumor tissues, including 63 adenoma tissues and 27 cancer tissues with submucosal invasion (pT1), by using the semi-quantitative RTPCR, with respect to clinicopathological characteristics and the expression of MMPs, COX-2 and iNOS. Considering the limits of semi-quantitative RTPCR analysis, we further analyzed the expression of E1AF, COX-2, and iNOS immunohistochemically. In vitro transfection assays were also performed.
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Materials and methods
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Patients and tissue samples
Ninety paired specimens of colorectal tumor and non-tumor tissues were obtained by polypectomy or surgical treatment. These tumor samples consisted of 63 adenomas and 27 adenocarcinomas with submucosal invasion (pT1 in the TNM classification of the Union Internationale Contre le Cancer). Each tissue specimen was divided into two pieces. For total RNA extraction, one sample was immediately frozen in liquid nitrogen at the time of endoscopy or 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. The histopathological features of the specimens were classified according to the TNM classification system. Locations of the colorectal tumors were divided into proximal colon (cecum, ascending and transverse colon) and distal colon (descending and sigmoid colon and rectum). Macroscopic types were divided into protruded type (height of tumor
3 mm) and flat type (height of tumor <3 mm). The clinicopathological characteristics of colorectal tumors are shown in Table I. An informed consent was obtained from each subject and the institutional review committee approved this study.
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Table I. Clinicopathological characteristics and mRNA expression profiles in 90 colorectal tumor tissues. Each row is a colorectal adenoma (n = 63) or pT1 cancer (n = 27). Black rectangles indicate each mRNA expression as positive. Gender (M, male; F, female), location (D, distal; P, proximal), macroscopic type (P, protruded; F, flat).
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Semi-quantitative RTPCR
Total RNA was extracted from specimens using the acid guanidinum thiocyanatephenolchloroform 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) genes in duplex PCR (12). GAPDH served as an internal control of the reaction. The 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. Overexpression was judged when the target gene expression in the tumor samples was at least three times higher than that in the corresponding normal sample. To perform semi-quantitative RTPCR, the ranges of linear amplification for the target gene and for the GAPDH genes were studied. The optimal number of PCR cycles and the mixing ratio of primers were determined. The primers used were 5'-GCCCATTTCATTGCCTGGAC-3' and 5'-GACTTGCCATTTCTCCACTTTCC-3' for E1AF, 5'-AGCAGCATGGATGGATTTTAT-3' and 5'-CTCCTGCTTAAAGCCTTGTGGTGG-3' for ER81, 5'-TTATGGTCCCAGGGAAATCTCGAT-3' and 5'-TGGCAGGGTTCAGACAGTTGTCTC-3' for ERM, 5'-GGGTAGCGACTTCTTGTTTG-3' and 5'-GTTAATGGAGTCAACCCAGC-3' for Ets-1, 5'-GCCTCAATAAGCCAACCATGTC-3' 5'-TCAATCCTGCCTTTCCTGGGTC-3' for Ets-2, AGATGTGGAGTGCCTGATGT-3' and 5'-AGCTAGGGTACATCAAAGCC-3' for MMP-1, 5'-AGAGGTGAC TCCACTCACAT-3' and 5'-GGTCTGTGAGTGAGTGATAG-3' for MMP-3, 5'-TCTTTGGCCTACCTATAACTGG-3' and 5'-CTAGACTGCTACCATCCGTC-3' for MMP-7, 5'-A CGGGCTCCTGGCACACG-3' and 5'-CGTCCCGGGTGTAGAGTC-3' for MMP-9, 5'-TTCAAATGAGATTGTGGGAAAAT-3' and 5'-AGATCATCTCTGCCTGAGTATCTT-3' for COX-2, 5'-GCCTCGCTCTGGAAAGA-3' and 5'-TCCATGCAGACAACCTT-3' for iNOS, 5'-GGCGTCTTCACCACCATGGAG-3' and 5'-AAGTTGTCATGGATGACCTTGGC-3' for GAPDH.
Immunohistochemistry
Sixty formalin-fixed, paraffin-embedded colorectal tumor specimens were obtained from patients who had undergone polypectomy or surgical treatment. These tumor samples consisted of 42 adenomas and 18 adenocarcinomas with submucosal invasion. Sections of formalin-fixed and paraffin-embedded tissue of 5 µm thickness were de-waxed in xylene and rehydrated in alcohol. The sections were then heated to 105°C in a target retrieval solution (DakoCytomation, Carpinteria, CA) in an autoclave for 10 min, for antigen retrieval. 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 incubated for 18 h at 4°C with an anti-human PEA3 mouse monoclonal antibody (10 µg/ml, Santa Cruz Biotechnology, Santa Cruz, CA), anti-human COX-2 mouse monoclonal antibody (10 µg/ml, ZYMED Laboratories, San Francisco, CA) or anti-human iNOS mouse monoclonal antibody (10 µg/ml, Transduction Laboratories, Lexington, NY). The antibodies were diluted in antibody diluent with background reducing components (0.05 mol/l TrisHCl buffer containing 0.1% Tween and 0.015 mol/l sodium azide) (DakoCytomation, Glostrup, Denmark). Normal mouse immunoglobulins were substituted for each primary antibody as negative controls. After washing three times in PBS, the sections were treated with biotinylated anti-mouse immunoglobulin (DakoCytomation) for 10 min and then with horseradish peroxidaseavidin complex, diluted as recommended by the manufacturer, for 10 min. The slides were then washed in PBS and developed in 0.05 M TrisHCl (pH 7.5) containing 0.6 mg/ml of 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. Nuclear expression of E1AF and cytoplasmic expression of COX-2 and iNOS were defined as positive when an immunoreactivity was observed in >10% of tumor cells.
DNA transfection
The human colon cancer cell line RCM-1 was purchased from Japanese Collection of Research Bioresources (Tokyo). Cells were cultured in RPMI-1640 containing 10% fetal bovine serum. A full-length cDNA encoding human iNOS was cloned into a eukaryotic expression vector, pcDNA3.1 (+) (Invitrogen, San Diego, CA) under the control of the cytomegalovirus promoter in a sense orientation and the vector was designated as pcDNA3.1/iNOS. pcDNA3.1/iNOS or pcDNA3.1 was transfected into RCM-1 cells using SuperFect transfection reagent (Quiagen, 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 vector plasmid pcDNA3.1 alone were used as controls. iNOS inhibitor S-methylisothiourea sulfate (SMT) was purchased from Calbiochem (San Diego, CA). Some transfectants were treated with 50 µM SMT for 14 h.
Northern blot analysis
Total RNA was prepared from cells using the acid guanidinium thiocyanatephenolchloroform extraction method, followed by a treatment with deoxyribonuclease I. Ten micrograms of total RNA were electrophoresed on a 1% denaturing agarose gel and transferred onto a nitrocellulose membrane. The membrane was hybridized with a complementary DNA for E1AF, COX-2 or iNOS 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 integrity of total RNA in each lane.
Statistical analysis
Expression of each target gene was assessed for associations with clinicopathological characteristics using the following statistical tests: MannWhitney U-test for age, size and average tumornormal expression ratios, and the chi-square two-tailed test or Fisher's exact test for the remaining parameters.
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Results
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E1AF mRNA expression in colorectal tumor tissues
To perform semi-quantitative RTPCR 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 optimal mixing ratios of primers were determined. The expression of E1AF mRNA in 90 colorectal tumor tissues was examined. Figure 1 shows the representative results of RTPCR for E1AF. E1AF mRNA expression was detected in 43 (47.8%) of the 90 colorectal tumor tissues but was undetectable in adjacent non-tumor tissues. The relationships between E1AF expression and clinicopathological characteristics are shown in Table I. E1AF mRNA expression was correlated significantly with histopathology (P = 0.0188). There was no correlation of E1AF mRNA expression with age, size, gender, location or macroscopic type. The average tumornormal expression ratio was significantly higher in pT1 cancer than in adenoma (P = 0.0006). Expression of ER81 and ERM was faintly detected in non-tumor tissues (Figure 1). Overexpression of ER81 and ERM mRNA was observed in 20.0% and 16.7% of the 90 colorectal tumor tissues, respectively, but the expression was not correlated significantly with any of the clinicopathological characteristics (data not shown). The expression patterns of the three related genes, E1AF, ER81 and ERM were not significantly correlated with each other. For comparison, we also analyzed the expression of Ets-1 and Ets-2 in colorectal tumor tissues (Figure 1). Overexpression of Ets-1 and Ets-2 mRNA was observed in 38.9% and 60.0% of the 90 colorectal tumor tissues, respectively, but the expression was not correlated significantly with any of the clinicopathological characteristics (data not shown).

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Fig. 1. RTPCR analysis of mRNA expression for E1AF, ERM, ER81, Ets-1 and Ets-2 in colorectal tumor tissues. T and N, matched samples from tumor and non-tumor tissue, respectively. Cases 14 are colorectal adenomas and cases 58 are colorectal carcinomas (pT1).
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Expression of MMPs and their relationships with E1AF expression
The expression of MMPs mRNA was examined in 90 colorectal tumor tissues. Figure 2 shows the representative results of RTPCR for E1AF, MMP-1, MMP-3, MMP-7 and MMP-9. Expression of MMP-7 was undetectable or only faintly detected in adjacent non-tumor tissues. Overexpression of MMP-1, MMP-3, MMP-7 and MMP-9 mRNA was observed in 37.8%, 0%, 77.8% and 16.7% of the 90 colorectal tumor tissues, respectively.

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Fig. 2. RTPCR analysis of mRNA expression for E1AF, MMP-1, MMP-3, MMP-7 and MMP-9 in colorectal tumor tissues. T and N, matched samples from tumor and non-tumor tissue, respectively. Cases 14 are colorectal adenomas and cases 58 are colorectal carcinomas (pT1).
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The relationships between the expression of MMPs and clinicopathological characteristics are shown in Table I. The expression of MMP-1 was correlated significantly with size (P = 0.0035). MMP-7 mRNA expression was correlated significantly with size (P = 0.0008), age (P = 0.0150), location (P = 0.0394) and histopathology (P = 0.0057). The expression of MMP-9 was correlated significantly with size (P = 0.0003) and histopathology (P < 0.0001). When only adenoma tissues were considered, the correlation of MMP-1 with size (P = 0.0013) and that of MMP-7 expression with size (P = 0.0170) and age (P = 0.0354) were still significant. Average tumornormal expression ratios of MMP-1, MMP-7 and MMP-9 were significantly higher in pT1 cancer than in adenoma (P = 0.0490, P = 0.0310 and P < 0.0001, respectively). Among the expression of MMPs analyzed, E1AF expression was correlated significantly with the expression of MMP-1 and MMP-7 (P = 0.0385 and P = 0.0048, respectively, Table II). When only adenoma tissues were considered, the correlation between E1AF and MMP-7 expression was still significant (P = 0.0109).
COX-2 and iNOS mRNA expression in colorectal tumor tissues
Figure 3 shows the representative results of RTPCR for COX-2 and iNOS. COX-2 mRNA expression was detected in 38 (42.2%) of the 90 colorectal tumor tissues but was undetectable or only faintly detected in adjacent non-tumor tissues. The relationships between the COX-2 overexpression and clinicopathological characteristics are shown in Table I. COX-2 expression was correlated significantly with size (P = 0.0002), gender (P = 0.0042), histopathology (P = 0.0004) and E1AF expression (P < 0.0001; Table III). Average tumornormal expression ratios were significantly higher in pT1 cancer than in adenoma (P < 0.0001). When only adenoma tissues were considered, the correlation of COX-2 expression with size, gender and E1AF expression was still significant (P = 0.0443, P = 0.0118 and P = 0.0003, respectively).

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Fig. 3. RTPCR analysis of mRNA expression for E1AF, COX-2 and iNOS in colorectal tumor tissues. T and N, matched samples from tumor and non-tumor tissue, respectively. Cases 14 are colorectal adenomas and cases 58 are colorectal carcinomas (pT1).
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iNOS mRNA expression was detected in 60 (66.7%) of the 90 colorectal tumor tissues but was undetectable or only faintly detected in adjacent non-tumor tissues. The relationships between iNOS overexpression and clinicopathological characteristics are shown in Table I. iNOS expression was correlated significantly with size (P < 0.0001), histopathology (P = 0.0006), COX-2 expression (P < 0.0001) and E1AF expression (P = 0.0002; Table III). Average tumornormal expression ratios were significantly higher in pT1 cancer than in adenoma (P < 0.0001). When only adenoma tissues were considered, the correlation of iNOS expression with size, COX-2 and E1AF expression was still significant (P = 0.0047, P = 0.0004 and P = 0.0081, respectively).
Immunohistochemical expression of E1AF, COX-2 and iNOS in colorectal tumor tissues
Figure 4 shows the representative the results of immunohistochemical expression of E1AF, COX-2 and iNOS in a patient (Case no. 3 in cancer group). Immunohistochemical expression of E1AF, COX-2 and iNOS was positive in 24 (40.0%), 28 (46.7%) and 37 (61.7%) of the 60 tumors, respectively. E1AF expression was correlated significantly with COX-2 expression and iNOS expression (18 COX-2-positive/24 E1AF-positive versus 10 COX-2-positive/36 E1AF-negative, P = 0.0003 and 19 iNOS-positive/24 E1AF-positive versus 18 iNOS-positive/36 E1AF-negative, P = 0.0013). COX-2 expression was correlated significantly with iNOS expression (20 iNOS-positive/28 COX-2-positive versus 17 iNOS-positive/32 COX-2-negative, P = 0.0023).

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Fig. 4. Hematoxylineosin staining (A) and immunohistochemical analysis for E1AF (B), COX-2 (C) and iNOS (D) in serial sections of colon cancer tissues (Case no. 3 in cancer group). (A) Hematoxylineosin stained section. (B) Nuclear expression of E1AF in cancer cells. (C) Cytoplasmic expression of COX-2 in cancer cells. (D) Cytoplasmic expression of iNOS in cancer cells. (AD) Original magnification x200.
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Effect of E1AF on COX-2 expression as well as iNOS on E1AF/COX-2 expression in colon cancer cell lines
We previously cloned antisense E1AF-transfected HT-29 cells and confirmed a considerable reduction in the amount of E1AF mRNA in the HT-29-derived clones HT AS-3 and HT AS-7 (19). Therefore, we analyzed the COX-2 expression by northern blot analysis in parental HT-29 cells, mock-transfected HT-29 cells, and HT AS-3 and HT AS-7 clones. A considerable reduction in the amount of COX-2 mRNA was observed in the HT AS-3 and HT AS-7 clones (Figure 5A).

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Fig. 5. The effect of E1AF on COX-2 expression as well as iNOS on E1AF/COX-2 expression in colon cancer cell lines. (A) Northern blot analysis of parental HT-29 cells and transfectants. Lane 1, parental HT-29 cells; 2, mock-transfected HT-29; 3, antisense E1AF-transfected HT-29 (HT-AS-3); 4, HT-AS-7. (B) Northern blot analysis of parental RCM-1 cells and transfectants. Lane 1, parental RCM-1 cells; 2, mock-transfected RCM-1; 3, iNOS-transfected RCM-1 (RCM/iNOS-2); 4, RCM/iNOS-6. (C) Northern blot analysis of iNOS-transfectants without or with treatment of iNOS inhibitor SMT. Lane 1, RCM/iNOS-2; 2, RCM/iNOS-6; 3, RCM/iNOS-2 treated with SMT; 4, RCM/iNOS-6 treated with SMT.
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RCM-1 cells were stably transfected with a cytomegalovirus-based vector that carried the iNOS cDNA in a sense orientation. After G418 selection, 10 different clones were analyzed for iNOS mRNA by northern blot analysis. A considerable amount of iNOS mRNA was observed in the RCM-1-derived clones RCM/iNOS-2 and RCM/iNOS-6, and the expression of E1AF and COX-2 was significantly upregulated in these clones (Figure 5B). iNOS inhibitor SMT suppressed the effect of iNOS in augmenting the expression of E1AF and COX-2 (Figure 5C).
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Discussion
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The issue that we addressed in this study was the expression of E1AF and its relationship with the expression of MMPs, COX-2 and iNOS in the early stage of colorectal carcinogenesis. The reason why we chose pT1 cancer is that it represents the early stage of colorectal cancer.
E1AF mRNA was detected in 47.8% of the 90 colorectal tumor tissues but not in adjacent non-tumor tissues, suggesting that the tumor-associated expression of E1AF occurs in the early stage of colorectal carcinogenesis. The frequency of E1AF mRNA expression was significantly higher in pT1 cancer (66.7%) than in adenoma (39.7%). It has been shown that an increase in both the amount and activity of E1AF is needed to assure a high E1AF target gene expression (31). In this aspect, it is interesting that the average tumornormal expression ratio of E1AF was significantly higher in pT1 cancer than in adenoma. It is possible that an initiating genetic event leads to the upregulation 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 (31).
As for the expression of MMPs, overexpression of MMP-1, MMP-3, MMP-7 and MMP-9 mRNA was observed in 37.8%, 0%, 77.8% and 16.7% of the 90 colorectal tumor tissues, respectively. The expression of MMP-1 was correlated significantly with size. MMP-7 mRNA expression was correlated significantly with size and histopathology. The expression of MMP-9 was correlated significantly with size and histopathology. Average tumornormal expression ratios of MMP-1, MMP-7 and MMP-9 were significantly higher in pT1 cancer than in adenoma. These results suggest that MMP-1, MMP-7 and MMP-9 are involved in tumor growth and/or early invasion of tumor cells.
The expression of E1AF mRNA was correlated significantly with expression of MMP-1 and MMP-7 in the early stage of colorectal carcinogenesis. Interestingly, a concomitant expression of E1AF and MMP-7 mRNA has been shown in intestinal adenoma tissues in Min mice (32). These results further support the notion that E1AF plays an important role in the induction of MMP-7 expression in colorectal adenoma tissues.
The expression of E1AF mRNA was also correlated significantly with the expression of COX-2 in colorectal tumor tissues. The association was further substantiated by immunohistochemistry. Moreover, a northern blot analysis of antisense E1AF transfectants also showed the effect of E1AF on COX-2 expression in colon cancer cell lines. Although COX-2 expression is regulated by both transcriptional and post-transcriptional mechanisms, transcriptional regulation may play a more decisive role in COX-2 expression in human colon carcinoma cells (26,33,34). It has been shown that while ß-catenin only weakly activates the COX-2 promoter, E1AF is a potent activator of COX-2 transcription (20). It has been shown that the expression of MMP-7 and COX-2 is responsive to Wnt signaling and that their expression is regulated by E1AF in colorectal cancer cell lines (20). Thus, our results suggest that E1AF plays an important role in the induction of MMP-1, MMP-7 and COX-2 in the early stage of colorectal carcinogenesis. The overall frequency of nodal metastasis of pT1 colorectal cancer has been reported to be
10%. Liver metastasis is also rare. To clarify whether the COX-2 expression is associated with a metastasis of early invasive colorectal cancer, studies on a large number of pT1 cancer tissues are needed. Because transcriptional and translational regulation of COX-2 is complex (20,26,33), additional studies are required to clarify the complexity of COX-2 regulation in the early stage of colorectal carcinogenesis.
As for the expression of iNOS, Yagihashi et al. (35) previously reported a 5-fold increase in the mRNA level in 50% of six colorectal cancer tissues. In the current study, iNOS mRNA expression was detected in 66.7% of the 90 colorectal tumor tissues but was undetectable or only faintly detected in adjacent non-tumor tissues. iNOS mRNA overexpression was correlated significantly with size and histopathology. Average tumor-normal expression ratio was significantly higher in pT1 cancer than in adenoma. These results suggest that iNOS contributes to the tumor growth and early invasion of tumor cells. Interestingly, the expression of iNOS mRNA was correlated significantly with the expression of E1AF and COX-2. The associations were further substantiated by immunohistochemistry. Moreover, northen blot analysis of iNOS transfectants with or without iNOS inhibitor also showed the effect of iNOS on E1AF/COX-2 expression in colon cancer cell lines. It has been thought that NO, generated by iNOS, stimulates the activity of MMPs, which in turn, causes the degradation of E-cadherin and the relocalization of ß-catenin to the cytoplasm and nucleus, and activates the ß-cateninTCF/LEF signaling pathway (30). ß-cateninTCF/LEF reportedly activates E1AF, which stimulates the COX-2 activity (30). In addition, NO has been shown to augment the synergistic interaction between E1AF and its transcription coactivator CBP/p300, resulting in the facilitation of COX-2 induction (30). Our results support those of the previous in vitro study (30) and further suggest that iNOS, in conjunction with E1AF and COX-2, plays an important role in the early colorectal carcinogenesis.
Thus, the most important aspect of this study is the early appearance of gene products of the E1AF, MMPs, COX-2 and iNOS in colorectal tumor tissue samples. As a tumor-associated transcriptional factor, E1AF appears to play an important role in the regulation of multiple genes involved in tumour promotion. The results of in vitro studies also support this notion.
All the biopsy samples were obtained from the surface of the tumor in this study. Therefore, E1AF mRNA expression is derived from the tumor cells in the lamina propria mucosae. In addition, using the immunohistochemical method, staining of E1AF was observed not only at the invasive front but also in the upper part of the muscularis mucosae (19). Accordingly, it is thought that a tumor in which E1AF is overexpressed, already has malignant potential before it invades the submucosa. Therefore, analysis of the E1AF expression in colorectal adenoma tissues obtained by biopsy could be useful for the prediction of malignant potential, preoperatively. The E1AF-MMP1-MMP-7-COX-2-iNOS axis could be a potent therapeutic target in patients with a colorectal tumor. Therapeutic agents that inhibit the expression or function of E1AF or its target genes may prove efficacious or might complement agents that compromise MMP, COX-2 and iNOS activities in the treatment of colorectal tumors and other tumors characterized by an E1AF overexpression (36).
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Notes
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The first two authors contributed equally to this work. 
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Acknowledgments
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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.).
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Received August 3, 2004;
revised January 12, 2005;
accepted January 17, 2005.