Association of matrilysin-2 (MMP-26) expression with tumor progression and activation of MMP-9 in esophageal squamous cell carcinoma
Hiroyuki Yamamoto4,
Akravit Vinitketkumnuen,
Yasushi Adachi,
Hiroaki Taniguchi,
Tamaki Hirata,
Nobuki Miyamoto,
Katsuhiko Nosho,
Arisa Imsumran,
Masahiro Fujita1,
Masao Hosokawa2,
Yuji Hinoda3 and
Kohzoh Imai
First Department of Internal Medicine, Sapporo Medical University, South-1, West-16, Chuo-ku, Sapporo 060-8543, Japan, 1 Keiyuhkai Institute of Pathology, Sapporo 030-0027, Japan, 2 Department of Surgery, Keiyuhkai Sapporo Hospital, Sapporo 030-0027, Japan and 3 Department of Clinical Laboratory Science, Yamaguchi University School of Medicine, Ube 755-8505, Japan
4 To whom correspondence should be addressed. Tel: +81 11 611 2111; Fax: +81 11 611 2282; Email: h-yama{at}sapmed.ac.jp
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Abstract
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Expression of matrilysin-2, matrix metalloproteinase (MMP)-26, has been implicated in the progression of several types of human cancer. Matrilysin-2 has been reported to be a physiological and pathological activator of pro-MMP-9. The aim of this study was to examine matrilysin-2 expression and determine whether it is correlated with progression of human esophageal squamous cell carcinoma (ESCC). Semi-quantitative reverse transcriptasepolymerase chain reaction, immunohistochemical analysis, zymography and an in vitro invasion assay were performed. Matrilysin-2 mRNA expression was undetectable or only faintly detected in non-tumor tissues, but its overexpression was detected in 24 of the 50 ESCC tissues. Matrilysin-2 overexpression was significantly correlated with depth of invasion, lymph node metastasis and an advance in pathological tumor node metastasis (pTNM) stage. Sections with immunostaining signals in >10% of carcinoma cells at the invasive front, which were observed in 46 of 100 cases, were judged to be positive for matrilysin-2 expression. Matrilysin-2 expression was significantly correlated with depth of invasion, lymph node and distant metastasis, advance in pTNM stage and recurrence. Expression of matrilysin-2 was significantly correlated with nuclear ß-catenin expression and MMP-9 expression. Patients with matrilysin-2-positive cancer had significantly shorter overall and disease-free survival periods than did those with matrilysin-2-negative cancer. Matrilysin-2 expression retained its significant predictive value for overall and disease-free survival in multivariate analysis. Moreover, patients with concomitant expression of matrilysin-2 and MMP-9 had the worst prognosis. Zymography revealed that matrilysin-2 expression was significantly correlated with expression of active MMP-9 in ESCC tissues. Matrilysin-2-transfected TE-1 ESCC cells showed active MMP-9 activity and were more invasive in vitro compared with mock-transfected TE-1 cells. The results of this study suggest that matrilysin-2, the expression of which is closely correlated with nuclear ß-catenin expression and active MMP-9 activity, plays a key role in the progression of ESCC.
Abbreviations: ECM, extracellular matrix; ESCC, esophageal squamous cell carcinoma; MMP, matrix metalloproteinase; PBS, phosphate-buffered saline; RTPCR, reverse transcriptasepolymerase chain reaction; TCF, T cell factor; pTNM, pathological tumor node metastasis
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Introduction
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Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive malignant tumors. Despite recent advances in diagnosis and treatment, the prognosis for patients with ESCC is poor and worse than that for patients with other digestive tract cancers (1,2).
Degradation of the extracellular matrix (ECM) mediated by matrix metalloproteinases (MMPs) is one of the key steps during the invasion and metastasis of ESCC (3,4). Human matrilysin-2, also called MMP-26 or endometase, has been isolated as a matrilysin (MMP-7) homolog and activator of pro-MMP-9 (58). Matrilysin-2 is the smallest MMP identified to date and it has several structural features of MMPs, including the signal sequence, the prodomain involved in enzyme latency and the catalytic domain with the zinc-binding site (58). Matrilysin-2 shares with matrilysin this minimal domain organization required for secretion, latency and activity (58). The protein sequence, minimal modular domain structure, exonintron map and computer modeling demonstrate a relatively close relation of matrilysin-2 with matrilysin (9). However, substrate specificity and transcriptional regulation of matrilysin-2 and those of matrilysin in cancer appear to be distinct (8,1012).
Matrilysin-2 mRNA is primarily expressed in epithelial cancers, such as lung, breast, endometrial and prostate carcinomas, in their corresponding cell lines (58,1317) and in normal adult tissues, such as the uterus (6,7,18), placenta (5,7) and kidney (8,14). Expression of MMP-26 in normal tissues is higher than that of many other MMPs (19) and thus it may not only be associated with cancer but with normal turnover and repair. The specific expression of matrilysin-2 in malignant tumors and its proteolytic activity against various components of the ECM, including fibronectin, type IV collagen, vitronectin, gelatin and fibrinogen, as well as non-ECM proteins, such as insulin-like growth factor-binding protein 1 and
1-protease inhibitor, suggest that matrilysin-2 plays a key role in tumor progression (14).
Importantly, matrilysin-2 has been reported to be a physiological and pathological activator of pro-MMP-9 (14). Cells transfected with antisense matrilysin-2, showing a significant reduction in matrilysin-2 at the protein level, exhibited a reduction in invasive potential in vitro in addition to a significant diminution in levels of active MMP-9 protein (14). Moreover, matrilysin-2 and MMP-9 proteins were both expressed in the same human prostate carcinoma tissue samples (14). Thus, matrilysin-2, in concert with MMP-9, appears to play an important role in tumor progression.
Since active MMP-9 has been shown to play an important role in the progression of ESCC (20) and its activation mechanism in vivo remains unknown, it seems important to clarify the relevance of matrilysin-2 in ESCC. In an attempt to address this issue, we investigated the expression of matrilysin-2 in 50 ESCC tissues, using semi-quantitative reverse transcriptasepolymerase chain reaction (RTPCR), in relation to clinicopathological characteristics. Immunohistochemical analysis of matrilysin-2 and MMP-9 for 100 ESCC tissues, zymography and an in vitro invasion assay were also performed. Since the promoter of the matrilysin-2 gene has been shown to contain T cell factor (TCF)-binding sites and to be activated by ß-catenin/TCF-4 (9,19), ß-catenin expression was also analyzed.
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Materials and methods
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Patients and tissue samples
Fifty paired surgical specimens of ESCC and adjacent non-tumor tissue and 100 formalin-fixed, paraffin-embedded tumor specimens were obtained from patients who had undergone curative surgical treatment. Tumor tissue samples were taken from the macroscopically visible deepest invading part of the tumor after surgical resection. 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 evaluation of the tumor cell content. Only specimens containing >80% tumor cells were used for analysis. Histopathological features of the specimens were classified according to the pathological tumor node metastasis (pTNM) classification system of the Union International Contre Cancer. Informed consent was obtained from each subject and the institutional review committee approved the experiments. The human ESCC cell lines TE-1TE-15 were provided by the Cell Resource Center of the Biomedical Research Institute on Development, Aging and Cancer, Tohoku University. Cells were cultured in RPMI-1640 containing 10% fetal bovine serum.
Semi-quantitative RTPCR
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 the matrilysin-2 and ß-actin genes in duplex PCRs (21). ß-Actin served as an internal control. Negative control reactions were run without reverse transcriptase. Results were analyzed using a multiimage analyzer (Bio-Rad, Richmond, CA). The levels of gene transcripts were quantified as the ratio of the intensity of the matrilysin-2 gene to the intensity of ß-actin. Overexpression was judged when matrilysin-2 gene expression in the tumor sample was at least three times higher than that in the corresponding normal sample. In cases without matrilysin-2 expression in the corresponding normal sample, overexpression was judged to have occurred when matrilysin-2 gene expression in the tumor sample was at least three times higher than the highest level in the 50 normal samples analyzed. To perform semi-quantitative RTPCR, the ranges of linear amplification for the matrilysin-2 and ß-actin genes were studied using standard curves. Standard curves were drawn using logarithmically serially diluted plasmids containing amplified regions of each target gene. The correlation between the quantity of cDNA before PCR and the band intensities of the target genes was analyzed using a hemi-logarithmic scale. The linear relationships were determined by least squares approximation. Thus, the optimal number of PCR cycles and the optimal 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'-GGGACTTTGTTGAGGGCTAT-3' and 5'-CTGGCGAGATGGAGGTGT-3' for matrilysin-2 (13) and 5'-GTGGGGCGCCCCAGGCACCA-3' and 5'-CTCCTTAATGTCACGGCACGATTT for ß-actin.
Immunohistochemistry
Five micrometer thick sections of formalin-fixed and paraffin-embedded tissue were dewaxed in xylene and rehydrated in alcohol. The sections were then heated to 105°C in target retrieval solution (DakoCytomation, Carpinteria, CA) in an autoclave for 10 min for antigen retrieval. Endogenous peroxidase activity was suppressed with 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 rabbit anti-human MMP-26 antibody (1/1000 dilution) (Serotec, Oxford, UK), anti-human ß-catenin monoclonal antibody (10 µg/ml) (BD Transduction Laboratories, Lexington, NY) or mouse anti-human MMP-9 monoclonal antibody (5 µg/ml) (Daiichi Fine Chemicals, Takaoka, Japan). 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). Normal rabbit or mouse immunoglobulins were substituted for each primary antibody as negative controls. After washing three times in PBS, the sections were treated with biotinylated anti-rabbit or anti-mouse immunoglobulin (Dako, Glostrup, Denmark) 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 3,3'-diaminobenzidine at room temperature. The sections were counterstained in Mayer's hematoxylin and mounted. The sections were examined microscopically by two pathologists who were blind to the clinicopathological characteristics and patients' outcome. Cytoplasmic expression of matrilysin-2 and MMP-9 and nuclear expression of ß-catenin were defined as positive when immunoreactivity was observed in >10% of cancer cells at the invasive front of the tumor. We defined the cells at the deepest invading part of the tumor as the invasive front.
Gelatin zymography
Gelatin zymography was performed as previously described with some modifications (19). Tissue extracts or cell number-adjusted aliquots of the culture medium from cells grown for 24 h in serum-free medium were electrophoresed on an 8% polyacrylamide gel containing 0.2% gelatin. After electrophoresis, the gels were washed in 2.5% Triton X-100 and incubated for 16 h at 37°C in 50 mM TrisHCl (pH 7.4), 10 mM CaCl2, 1 mM ZnCl2 and 0.02% NaN3, followed by staining with 0.1% Coomassie brilliant blue. Computer-assisted image analysis of the gels was performed.
DNA transfection
A full-length cDNA encoding human matrilysin-2 was cloned into the eukaryotic expression vector pcDNA3.1 (Invitrogen, San Diego, CA) under control of the cytomegalovirus promoter in the 5'
3' orientation. This vector was designated pcDNA mat2. pcDNA mat2 or pcDNA3.1 was transfected into TE-1 cells using SuperFect (Qiagen, Hilden, Germany), following the manufacturer's protocol. After a few weeks of G418 selection, individual colonies were selected and expanded for further analyses. Immunocytochemistry for matrilysin-2 expression in TE-1 cells and their transfectants was done as previously described with some modifications (14).
In vitro invasion assay
Assays were performed by the modified Boyden Chamber method as previously described (22). Aliquots of 2 x 106 cells were seeded onto matrigel-coated filters. After 48 h 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 10 nM MMP-9 inhibitor (23). The results are presented as means ± SD for each sample.
Statistical analysis
Expression of matrilysin-2 was assessed for associations with clinicopathological characteristics using the following statistical tests: Student's t-test for age, the MannWhitney test for histological type, depth of invasion and pTNM stage, and the
2 two-tailed test or Fisher's exact test for the remaining parameters. Cumulative survival rates were calculated by the KaplanMeier method. The difference between survival curves was analyzed by the log rank test. Factors related to survival were analyzed by Cox's proportional hazards regression model. Model selection was performed using a forward stepwise method. A P value of <0.05 was considered significant. For 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.
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Results
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Matrilysin-2 mRNA expression in ESCC tissues
To perform semi-quantitative RTPCR analysis, the ranges of linear amplification for the matrilysin-2 gene and for the control ß-actin gene were examined using standard curves. The optimal number of PCR cycles and the optimal mixing ratio of primers were determined. The expression of matrilysin-2 mRNA was examined in 50 ESCC tissues. Figure 1 shows representative results of RTPCR for matrilysin-2. Matrilysin-2 mRNA was undetectable or only faintly detected in adjacent non-tumor tissues. In 24 of the 50 cases, matrilysin-2 mRNA level was at least three times the level in non-tumor tissue and these cases were regarded as positive for overexpression. The relationship between matrilysin-2 overexpression and clinicopathological characteristics is shown in Table I. Matrilysin-2 mRNA overexpression was significantly correlated with depth of invasion (P = 0.0006), lymph node metastasis (P = 0.0022) and advanced pTNM stage (P < 0.0001). There was no correlation of matrilysin-2 mRNA overexpression with age, gender or tumor location (Table I).

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Fig. 1. RTPCR analysis of mRNA expression for matrilysin-2 in ESCC tissues. N and T, matched samples from non-tumor and tumor tissue, respectively.
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Immunohistochemistry for matrilysin-2 in ESCC tissues
The expression of matrilysin-2 was analyzed immunohistochemically in 100 tumor specimens. In normal esophageal tissues, weak immunoreactivity for matrilysin-2 was only occasionally observed in the cytoplasm of esophageal epithelial cells (Figure 2A). There was strong matrilysin-2 staining of tumor cell cytoplasm and/or membrane in carcinoma tissues in comparison with that in normal epithelial cells (Figure 2B and C). The immunoreactivities at the invasive front were often more intense than those in the superficial layer or in the center of tumors (Figure 2B and C). There was no detectable immunoreactivity with the control non-immune IgG (data not shown). Sections with immunostaining signals in >10% of carcinoma cells at the invasive front were judged to be positive for matrilysin-2, which was thus observed in 46% of cases. Matrilysin-2 expression was significantly correlated with depth of invasion (P = 0.0029), lymph node and distant metastasis (P < 0.0001), advanced pTNM stage (P < 0.0001), recurrence (P < 0.0001) and recurrence within the first post-operative year (P < 0.0001). There was no correlation of matrilysin-2 expression with age, gender or tumor location (Table II). Patients with matrilysin-2-positive carcinoma had significantly shorter overall and disease-free survival periods than did those with matrilysin-2-negative carcinoma (P < 0.0001 and P < 0.0001, respectively) (Figure 3). In the univariate analysis, significant prognostic variables for predicting both overall and disease-free survival were matrilysin-2 expression, depth of invasion, lymph node metastasis, distant metastasis and pTNM stage (Table III). In the multivariate analysis, parameters included matrilysin-2 expression, depth of invasion, lymph node metastasis, distant metastasis and pTNM stage. Only matrilysin-2 and pTNM stage retained significant predictive value for overall and disease-free survival (Table III).

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Fig. 2. Immunohistochemistry in ESCC tissues. (A) Weak cytoplasmic expression of matrilysin-2 in normal esophageal epithelial cells in the parabasal layer. Original magnification x200. (B) Cytoplasmic expression of matrilysin-2 in cancer cells at the invasive front. Original magnification x40. (C) Cytoplasmic expression of matrilysin-2 in cancer cells at the invasive front. In this tumor sample, cells at the periphery showed most intense immunoreactivity. Original magnification x200. (D) Nuclear expression of ß-catenin in cancer cells at the invasive front. Original magnification x40. (E) Cytoplasmic expression of matrilysin-2 in cancer cells in the serial section of (D). Original magnification x40. (F) Nuclear expression of ß-catenin in cancer cells in the lymph node metastatic tissue. Original magnification x100. (G) Cytoplasmic expression of matrilysin-2 in cancer cells in the serial section of (F). Original magnification x100.
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Fig. 3. KaplanMeier overall (A) and disease-free (B) survival curves of patients with ESCC according to expression of matrilysin-2.
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Correlation of matrilysin-2 expression with nuclear ß-catenin and MMP-9 expression
The expression of ß-catenin and MMP-9 was analyzed immunohistochemically in 100 tumor specimens. Nuclear expression of ß-catenin was observed in 32 cases. Matrilysin-2 expression was observed in 24 (75.0%) of the 32 cases positive for ß-catenin expression (Figure 2D and E), while it was positive in 22 (32.4%) of the 68 cases negative for ß-catenin. Thus, matrilysin-2 expression was significantly correlated with nuclear expression of ß-catenin (P < 0.0001). Concomitant expression of nuclear ß-catenin and matrilysin-2 was observed in 7 of the 10 lymph node metastases (Figure 2F and G). Cytoplasmic expression of MMP-9 was observed predominantly in carcinoma cells at the invasive front (Figure 4A) and was positive in 56 cases. Matrilysin-2 expression was positive in 33 (58.9%) of the 56 cases positive for MMP-9 expression (Figure 4A and B), while it was positive in 13 (29.5%) of the 44 cases negative for MMP-9. Thus, expression of matrilysin-2 was significantly correlated with expression of MMP-9 (P = 0.0034). MMP-9 and matrilysin-2 were often co-localized but they were only partially co-localized in some cases. Patients with concomitant expression of matrilysin-2 and MMP-9 at the invasive front had the worst prognosis (Figure 4C and D).

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Fig. 4. Co-expression of MMP-9 and matrilysin-2 in ESCC tissues. Cytoplasmic expression of MMP-9 (A) and matrilysin-2 (B) in cancer cells at the invasive front. Original magnification x200. KaplanMeier overall (C) and disease-free (D) survival curves of patients with ESCC according to expression of matrilysin-2 and MMP-9 at the invasive front. See online supplementary material for a colour version of this figure.
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Correlation of matrilysin-2 expression with active MMP-9 expression
The gelatinolytic activity of tissue extract was analyzed by gelatin zymography in 50 ESCC tissues. Pro-MMP-9 and pro-MMP-2 were detected in all tumor samples. Representative cases are shown in Figure 5. The active forms of MMP-9 (83 kDa) and MMP-2 (62 kDa) were detected in 28 (56.0%) and 41 (82.0%) of the 50 cases, respectively. The active form of MMP-9 was positive in 20 (83.3%) of the 24 cases positive for matrilysin-2 expression, while it was positive in 8 (30.8%) of the 26 cases negative for matrilysin-2. Thus, expression of the active form of MMP-9 was significantly correlated with matrilysin-2 expression (P = 0.0002). Densitometric analysis showed that the relative activity of MMP-9 (the density of the active form relative to the sum of the densities of the bands of pro-MMP-9 and active MMP-9) was significantly higher in cases positive for matrilysin-2 expression than in those negative for matrilysin-2 (P = 0.0012, MannWhitney test). The relative activity of MMP-2 was not correlated significantly with matrilysin-2 expression.

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Fig. 5. Gelatin zymography of specimen pairs of ESCC and adjacent non-tumor tissue. N and T, matched samples from non-tumor and tumor tissue, respectively.
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In vitro invasion assay
Fifteen esophageal cancer cell lines were examined for matrilysin-2 mRNA expression by semi-quantitative RTPCR analysis. Matrilysin-2 mRNA was detected in TE-2TE-6, TE-9TE-13 and TE-15 cells, but not in the remaining cells (Figure 6A). TE-1 cells were stably transfected with a vector that carried matrilysin-2 cDNA. After G418 selection, 10 different clones were analyzed for matrilysin-2 mRNA expression by RTPCR. A significant amount of matrilysin-2 mRNA was observed in the TE-1-derived clones TE-1-mat-3 and TE-1-mat-6, but not in mock-transfected TE-1 cells or parental cells (Figure 6B). Immunohistochemistry showed matrilysin-2 staining in TE-1-mat-3 and TE1-mat-6, whereas no staining was observed in mock-transfected TE-1 cells and parental cells (data not shown). These clones were then analyzed for gelatinase activity by zymography. Although total activities of MMP-2 and MMP-9 were not affected by transfection, an active form of MMP-9 was induced in matrilysin-2-transfected clones but not in mock-transfectants or parental cells (Figure 6C). In vitro growth rates measured by cell growth kinetics were almost the same among the parental TE-1 cells and the corresponding transfectants (data not shown). The in vitro invasive potential of these cells was then assayed. Both TE-1-mat-3 and TE-1-mat-6 demonstrated more invasive potential than did mock-transfected control cells (P < 0.001) and this difference was partially but significantly diminished by the addition of an MMP-9 inhibitor (Figure 6D).

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Fig. 6. (A) RTPCR analysis of mRNA expression for matrilysin-2 in ESCC cell lines. (B) RTPCR analysis of mRNA expression for matrilysin-2 in TE-1 cells and their transfectants. Lane 1, TE-1; lane 2, mock-transfected TE-1 (TE-1-mock); lane 3, matrilysin-2-transfected TE (TE-1-mat-3); lane 4, TE-1-mat-6. (C) Gelatin zymography of serum-free conditioned medium prepared from TE-1 cells and their transfectants. (D) In vitro invasion assay with or without an MMP-9 inhibitor in TE-1 and their transfectants. Each column indicates means of three experiments; bars, SD. *,**,***P < 0.001.
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Discussion
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The first issue that we addressed in this study was the role of matrilysin-2 expression in the progression of ESCC. Matrilysin-2 mRNA overexpression was found to be significantly correlated with depth of invasion, lymph node and distant metastasis and advanced tumor stage. Moreover, matrilysin-2 overexpression in carcinoma cells at the invasive front was immunohistochemically seen in 46% of patients with ESCC, being associated with depth of invasion, lymph node and distant metastasis and advanced pTNM stage. Thus, the results of mRNA studies were correlated with those of immunohistochemistry. This correlation can be explained, at least in part, by the fact that tissue samples for mRNA studies were taken from the macroscopically visible deepest invading part of the tumor after surgical resection. As shown by immunohistochemistry, weak matrilysin-2 mRNA expression in non-tumor tissues appears to be derived from normal esophageal epithelial cells. Thus, matrilysin-2 may also play a role in normal turnover and/or repair. Matrilysin-2 expression was detected in all 10 lymph node metastases, suggesting that overexpression of matrilysin-2 plays an essential role in the development of lymph node metastasis of ESCC. These results suggest that matrilysin-2 plays an important role in the progression of ESCC. The implication of matrilysin-2 expression at the invasive front was further substantiated by the correlation with disease recurrence and shorter overall and disease-free survival periods. Moreover, only matrilysin-2 expression and pTNM stage provided significant predictive values for overall and disease-free survival in the multivariate analysis, suggesting that matrilysin-2 expression could be a predictor of recurrence and poor prognosis with a significance equaling or surpassing that of other conventional clinicopathological factors. Other MMPs, such as MMP-7 and MMP-13, have also been reported to be correlated with recurrence and/or poor prognosis in patients with ESCC in multivariate analysis (24,25). Therefore, with respect to the prognostic significance, matrilysin-2 is not a unique MMP in ESCC. Nevertheless, unique characteristics of matrilysin-2 shown in this and previous studies (519) suggest that matrilysin-2 plays an important role in the progression of ESCC. Further investigation into the relationship between matrilysin-2 and other MMPs in ESCC is warranted.
The promoter of the matrilysin-2 gene has been shown to contain TCF-binding sites and to be activated by ß-catenin/TCF-4 (9,20). Matrilysin-2 expression was significantly correlated with nuclear expression of ß-catenin in ESCC tissues. These results suggest that ß-catenin directly, or at least cooperatively with other transcription factors or indirectly by transactivating other transcription factors, up-regulates expression of matrilysin-2 in ESCC cells, especially those located at the invasive front, contributing to the progression of ESCC. Nevertheless, because expression of matrilysin-2 at the invasive front in ESCC tissues can not be explained by nuclear ß-catenin expression alone, further analysis is required to clarify the regulation of matrilysin-2 promoter activity in ESCC cells.
The second issue addressed in the present study was the correlation between matrilysin-2 expression and activation of pro-MMP-9. Matrilysin-2 expression determined by immunohistochemistry was significantly correlated with expression of MMP-9. Moreover, matrilysin-2 expression was correlated with expression of active MMP-9 activity determined by zymography. These results suggest that matrilysin-2 plays an important role in the activation of pro-MMP-9 in ESCC tissues. It is notable that patients with concomitant expression of matrilysin-2 and MMP-9 had the worst prognosis. These results, coupled with those of zymography, suggest that tumor-derived matrilysin-2 contributes to tumor progression by degrading the ECM and non-ECM components and by activating pro-MMP-9 in ESCC.
Consistent with the results in tissue samples, matrilysin-2 mRNA was frequently detected in esophageal carcinoma cell lines. To explore the role of matrilysin-2 in invasion of ESCC cells, we introduced matrilysin-2 cDNA into ESCC cells that lack expression of endogenous matrilysin-2 but secrete an inactive form of MMP-9. Interestingly, an active form of MMP-9 was clearly seen in matrilysin-2 transfectants, suggesting that matrilysin-2 plays an important role in the activation of pro-MMP-9 in ESCC cells. Significant enhancement of invasiveness was observed in matrilysin-2 transfectants compared with that of control cells and this difference was partially but significantly diminished by a MMP-9 inhibitor. These results suggest that tumor-derived matrilysin-2 contributes to invasion of ESCC cells by degrading ECM components and by activating pro-MMP-9.
Taken together, the findings in this study suggest that expression of matrilysin-2, in conjunction with expression of MMP-9, leads to coordinated up-regulation of protease activity in ESCC cells, especially those located at the invasive front, contributing to the progression of ESCC. Identification of matrilysin-2 as a molecular marker that is correlated with disease recurrence and poor prognosis would provide new insights into disease management by making it possible to define a high risk of recurrence, thus providing a more accurate estimation of the prognosis of patients with ESCC. Consequently, early post-operative screening and/or intense post-operative therapy should be performed for patients with matrilysin-2-positive carcinoma, especially those with concomitant overexpression of matrilysin-2 and MMP-9. Immunohistochemical analysis is a technique that is feasible in daily clinical practice. Although other MMPs, such as MMP-7 and MMP-13, have also been reported to be correlated with poor prognosis (24,25), the combination of matrilysin-2 and MMP-9 appears to surpass previously reported MMPs. Therefore, analysis of expression of matrilysin-2 and MMP-9 could be an important routine part of the management of patients with ESCC. Use of the diagnostic strategy examined in this study and advances in therapeutic approaches, including the use of MMP inhibitors, should improve the prognosis of patients with ESCC.
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Acknowledgments
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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.).
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References
|
---|
- Messmann,H. (2001) Squamous cell cancer of the oesophagus. Best Pract. Res. Clin. Gastroenterol., 15, 249265.[CrossRef][ISI][Medline]
- Kuwano,H., Nakajima,M., Miyazaki,T. and Kato,H. (2003) Distinctive clinicopathological characteristics in esophageal squamous cell carcinoma. Ann. Thorac. Cardiovasc. Surg., 9, 613.[Medline]
- DeClerck,Y.A. (2000) Interactions between tumour cells and stromal cells and proteolytic modification of the extracellular matrix by metalloproteinases in cancer. Eur. J. Cancer, 36, 12581268.[CrossRef][ISI][Medline]
- Lynch,C.C. and Matrisian,L.M. (2002) Matrix metalloproteinases in tumorhost cell communication. Differentiation, 70, 561573.[CrossRef][ISI][Medline]
- de Coignac,A.B., Elson,G., Delneste,Y. et al. (2000) Cloning of MMP-26. A novel matrilysin-like proteinase. Eur. J. Biochem., 267, 33233329.[Abstract/Free Full Text]
- Park,H.I., Ni,J., Gerkema,F.E., Liu,D., Belozerov,V.E. and Sang,Q.X. (2000) Identification and characterization of human endometase (matrix metalloproteinase-26) from endometrial tumor. J. Biol. Chem., 275, 2054020544.[Abstract/Free Full Text]
- Uria,J.A. and Lopez-Otin,C. (2000) Matrilysin-2, a new matrix metalloproteinase expressed in human tumors and showing the minimal domain organization required for secretion, latency and activity. Cancer Res., 60, 47454751.[Abstract/Free Full Text]
- Marchenko,G.N., Ratnikov,B.I., Rozanov,D.V., Godzik,A., Deryugina,E.I. and Strongin,A.Y. (2001) Characterization of matrix metalloproteinase-26, a novel metalloproteinase widely expressed in cancer cells of epithelial origin. Biochem. J., 356, 705718.[CrossRef][ISI][Medline]
- Marchenko,G.N., Marchenko,N.D., Leng,J. and Strongin,A.Y. (2002) Promoter characterization of the novel human matrix metalloproteinase-26 gene: regulation by the T-cell factor-4 implies specific expression of the gene in cancer cells of epithelial origin. Biochem. J., 363, 253262.[CrossRef][ISI][Medline]
- Marchenko,N.D., Marchenko,G.N. and Strongin,A.Y. (2002) Unconventional activation mechanisms of MMP-26, a human matrix metalloproteinase with a unique PHCGXXD cysteine-switch motif. J. Biol. Chem., 277, 1896718972.[Abstract/Free Full Text]
- Park,H.I., Turk,B.E., Gerkema,F.E., Cantley,L.C. and Sang,Q.X. (2002) Peptide substrate specificities and protein cleavage sites of human endometase/matrilysin-2/matrix metalloproteinase-26. J. Biol. Chem., 277, 3516835175.[Abstract/Free Full Text]
- Park,H.I., Jin,Y., Hurst,D.R., Monroe,C.A., Lee,S., Schwartz,M.A. and Sang,Q.X. (2003) The intermediate S1' pocket of the endometase/matrilysin-2 active site revealed by enzyme inhibition kinetic studies, protein sequence analyses and homology modeling. J. Biol. Chem., 278, 5164651653.[Abstract/Free Full Text]
- Zhang,J., Cao,Y.J., Zhao,Y.G., Sang,Q.X. and Duan,E.K. (2002) Expression of matrix metalloproteinase-26 and tissue inhibitor of metalloproteinase-4 in human normal cytotrophoblast cells and a choriocarcinoma cell line, JEG-3. Mol. Hum. Reprod., 8, 659666.[Abstract/Free Full Text]
- Zhao,Y.G., Xiao,A.Z., Newcomer,R.G. et al. (2003) Activation of pro-gelatinase B by endometase/matrilysin-2 promotes invasion of human prostate cancer cells. J. Biol. Chem., 278, 1505615064.[Abstract/Free Full Text]
- Tunuguntla,R., Ripley,D., Sang,Q.X. and Chegini,N. (2003) Expression of matrix metalloproteinase-26 and tissue inhibitors of metalloproteinases TIMP-3 and -4 in benign endometrium and endometrial cancer. Gynecol. Oncol., 89, 453459.[CrossRef][ISI][Medline]
- Impola,U., Uitto,V.J., Hietanen,J., Hakkinen,L., Zhang,L., Larjava,H., Isaka,K. and Saarialho-Kere,U. (2004) Differential expression of matrilysin-1 (MMP-7), 92 kD gelatinase (MMP-9) and metalloelastase (MMP-12) in oral verrucous and squamous cell cancer. J. Pathol., 202, 1422.[CrossRef][ISI][Medline]
- Zhao,Y.G., Xiao,A.Z., Park,H.I., Newcomer,R.G., Yan,M., Man,Y.G., Heffelfinger,S.C. and Sang,Q.X. (2004) Endometase/matrilysin-2 in human breast ductal carcinoma in situ and its inhibition by tissue inhibitors of metalloproteinases-2 and -4: a putative role in the initiation of breast cancer invasion. Cancer Res., 64, 590598.[Abstract/Free Full Text]
- Isaka,K., Nishi,H., Nakai,H., Nakada,T., Li,Y.F., Ebihara,Y. and Takayama,M. (2003) Matrix metalloproteinase-26 is expressed in human endometrium but not in endometrial carcinoma. Cancer, 97, 7989.[CrossRef][ISI][Medline]
- Marchenko,N.D., Marchenko,G.N., Weinreb,R.N., Lindsey,J.D., Kyshtoobayeva,A., Crawford,H.C. and Strongin,A.Y. (2004) Beta-catenin regulates the gene of MMP-26, a novel metalloproteinase expressed both in carcinomas and normal epithelial cells. Int. J. Biochem. Cell Biol., 36, 942956.[CrossRef][ISI][Medline]
- Koyama,H., Iwata,H., Kuwabara,Y., Iwase,H., Kobayashi,S. and Fujii,Y. (2000) Gelatinolytic activity of matrix metalloproteinase-2 and -9 in oesophageal carcinoma; a study using in situ zymography. Eur. J. Cancer, 36, 21642170.[CrossRef][ISI][Medline]
- Yamamoto,H., Horiuchi,S., Adachi,Y., Taniguchi,H., Nosho,K., Min,Y. and Imai,K. (2004) Expression of ets-related transcriptional factor E1AF is associated with tumor progression and over-expression of matrilysin in human gastric cancer. Carcinogenesis, 25, 325332.[Abstract/Free Full Text]
- Horiuchi,S., Yamamoto,H., Min,Y., Adachi,Y., Itoh,F. and Imai,K. (2003) Association of ets-related transcriptional factor E1AF expression with tumour progression and overexpression of MMP-1 and matrilysin in human colorectal cancer. J. Pathol., 200, 568576.[CrossRef][ISI][Medline]
- Levin,J.I., Chen,J., Du,M. et al. (2001) The discovery of anthranilic acid-based MMP inhibitors. Part 2: SAR of the 5-position and P1(1) groups. Bioorg. Med. Chem. Lett., 11, 21892192.[CrossRef][ISI][Medline]
- Yamashita,K., Mori,M., Shiraishi,T., Shibuta,K. and Sugimachi,K. (2000) Clinical significance of matrix metalloproteinase-7 expression in esophageal carcinoma. Clin. Cancer Res., 6, 11691174.[Abstract/Free Full Text]
- Etoh,T., Inoue,H., Yoshikawa,Y., Barnard,G.F., Kitano,S. and Mori,M. (2000) Increased expression of collagenase-3 (MMP-13) and MT1-MMP in oesophageal cancer is related to cancer aggressiveness. Gut, 47, 5056.[Abstract/Free Full Text]
Received June 10, 2004;
revised July 21, 2004;
accepted August 10, 2004.