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© 2001 American Society for Clinical Oncology Expression of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Human Pancreatic Adenocarcinomas: Clinicopathologic and Prognostic Significance of Matrilysin ExpressionFrom the First Department of Internal Medicine and First Department of Surgery, Sapporo Medical University, Sapporo, Japan. Address reprint requests to Hiroyuki Yamamoto, MD, PhD, First Department of Internal Medicine, Sapporo Medical University, South-1, West-16, Chuo-ku, Sapporo 060-8543, Japan; email: h-yama{at}sapmed.ac.jp
PURPOSE: A disruption in the balance between the matrix metalloproteinases (MMPs) and their natural inhibitors, tissue inhibitors of metalloproteinases (TIMPs), has been implicated in the progression of many types of cancer. The aim of this study was to determine whether a specific MMP or TIMP has clinicopathologic and prognostic significance in pancreatic carcinoma. PATIENTS AND METHODS: Using immunohistochemistry, we analyzed 70 pancreatic ductal adenocarcinoma tissues for expression of MMP-1, MMP-2, MMP-3, MMP-7 (matrilysin), MMP-9, MT1-MMP, TIMP-1, and TIMP-2. The results were matched with clinicopathologic characteristics and patients’ survival. The effects of the suppression of a specific MMP on in vitro invasiveness of pancreatic carcinoma cells were also examined. RESULTS: Expression of MMP-1, MMP-2, MMP-3, matrilysin, MMP-9, MT1-MMP, TIMP-1, and TIMP-2 was detected in either tumor cells or tumor stromal cells, or in both components, at varying frequencies. Among MMPs, matrilysin showed a unique distribution in the tumor nests; its expression was usually most pronounced at the invasive front of the tumors. Sections with immunostaining signals in more than 30% of carcinoma cells at the invasive front, which were observed in 40 cases (57%), were judged to be positive for matrilysin. Matrilysin positivity was significantly correlated with pT, pN, and pM categories and with more advanced pathologic tumor-node-metastasis stages. Patients with matrilysin-positive carcinoma had a significantly shorter overall survival time than did those with matrilysin-negative carcinoma. Matrilysin was a significant independent prognostic factor for overall survival in multivariate analysis. In contrast, there was no correlation between the presence of other MMPs or TIMPs and clinicopathologic characteristics, nor was the presence of individual MMPs or TIMPs related to survival. Antisense matrilysin-transfected CFPAC-1 cells expressed reduced levels of matrilysin and demonstrated a similar growth potential but were less invasive in vitro compared with neotransfected CFPAC-1 cells. CONCLUSION: Our results suggest that matrilysin may play a key role in progression of pancreatic carcinoma and thereby contribute to a poor prognosis. Because different synthetic MMP inhibitors affect different types of MMPs to a different degree, examination of the expression of MMPs, especially that of matrilysin, may serve as an indicator for selecting the most effective MMP inhibitor.
PANCREATIC adenocarcinoma is one of the most lethal malignant tumors. Pancreatic adenocarcinoma is characterized by rapid progression, late clinical presentation, difficulty in early diagnosis, and unresponsiveness to chemotherapy, radiotherapy, and immunotherapy, resulting in low resectability rates after diagnosis, early recurrence after resection, and extremely poor survival rates.1-3 At the time of diagnosis, pancreatic carcinoma usually shows extensive local invasion and/or metastasis, precluding a curative surgical resection. A better understanding of the molecular genetics of pancreatic carcinoma is needed to develop new diagnostic and therapeutic strategies. Several studies have suggested possible roles of growth factors, K-ras oncogene, and tumor suppressor genes such as p16, p53, and DPC4 in pancreatic carcinogenesis4-7; however, the mechanisms underlying the progression of this carcinoma are poorly understood. Although a number of molecular prognostic markers in pancreatic carcinoma have been reported, routine analysis of these markers is not warranted because they do not have any therapeutic implications. Therefore, identification of a molecular prognostic marker, which is susceptible to or modifiable by direct therapeutic intervention, could give increase to therapeutic and prognostic improvements for patients with pancreatic carcinoma. In this regard, the matrix metalloproteinase (MMP) matrilysin is an engaging target for research. Matrilysin has been implicated in tumor invasion and metastasis as well as in tumor initiation or growth in gastrointestinal and other cancers.8-16 As a MMP, matrilysin is unique in its minimum MMP structure, wide spectrum of substrate specificity, potency for starting an activation cascade of MMPs, and, most notably, in its production by cancer cells themselves. The production by cancer cells themselves could be an advantage as a biologic marker of the malignant phenotype. Indeed, we have found that matrilysin expression at the invasive front is correlated with the progression of gastric and colorectal adenocarcinomas as well as esophageal squamous cell carcinoma.17-19 Another advantage is its susceptibility to direct therapeutic intervention. Inhibition of matrilysin by an antisense expression vector or antisense oligonucleotides has been demonstrated to suppress the in vitro invasive potential or in vivo metastatic potential of colon cancer cells.20-22 The crucial roles of MMPs, including matrilysin, in cancers have thus generated considerable interest in the use of synthetic MMP inhibitors as potential therapeutic agents.23-26 Although the frequent expression of matrilysin mRNA or protein has been reported in the limited number of pancreatic carcinoma tissues so far analyzed, it is not clear whether matrilysin plays a role in pancreatic carcinoma.27,28 Thus, it seems promising to assess the clinicopathologic and prognostic significance of matrilysin in pancreatic carcinoma. Nevertheless, it is not sufficient to examine matrilysin expression alone in pancreatic carcinoma because different MMPs, of which each member has different substrate specificities for the extracellular matrix, and tissue inhibitors of metalloproteinases (TIMPs) are expressed at various levels, and complex interactions between tumor and stromal cells influence biologic aggressiveness of many types of cancer.29,30 It is thought that the balance between MMPs and TIMPs determines the proteolysis in vivo. The close ratio of TIMPs to MMPs that is required to neutralize enzymatic activity means that small changes in the levels of either leads to biologically significant changes in net proteolytic activity. If MMP expression increases and/or TIMP expression decreases, the balance favors proteolysis. Indeed, expression of MMP-1, MMP-2, MMP-3, MMP-9, MMP-10, MMP-11, MT1-MMP, MT2-MMP, MT-3-MMP, TIMP-1, and TIMP-2 has been analyzed in pancreatic carcinoma.27,31-35 Gress et al31 have reported that elevated levels of mRNA for MMP-2, MMP-9, TIMP-1, and TIMP-2 in carcinoma tissues are correlated with the degree of the desmoplastic reaction but not with differentiation or tumor stage. Koshiba et al32 have found that the MMP-2 activation ratio in carcinoma tissues is correlated with regional lymph node metastasis, distant metastasis, and postresection recurrence but not with patient survival. Bramhall et al27 analyzed mRNA expression of MMP-1 (positivity in carcinoma tissues: 0%), MMP-2 (93%), MMP-3 (18%), MMP-10 (0%), MMP-11 (41%), TIMP-1 (100%), and TIMP-2 (53%). However, they found no correlation between MMP or TIMP expression and tumor grade, stage, lymph node metastasis, vascular invasion, or perineural invasion.27 Imamura et al33 have suggested the possible involvement of MT1-MMP expression in the desmoplastic reaction. MMP-1 positivity has been reported to be correlated with poor prognosis but not with tumor size, lymph node metastasis, or stage.34 Recently, Ellenrieder et al35 have reported that levels of MT1-MMP and MT2-MMP mRNA and of activated MMP-2 are enhanced in carcinoma tissues and that MT1-MMP expression is correlated with MMP-2 expression and activity. However, no correlation between tumor stage and levels of expression or activation of MMPs was found.35 Although several lines of experimental evidence support the notion that expression of MMP-2 or MMP-9 is correlated with aggressiveness of some pancreatic carcinoma cells in vitro and/or in mice models,36-39 it is not clear which MMP plays a crucial role in the progression of human pancreatic carcinoma. Therefore, we immunohistochemically analyzed expression of matrilysin, MMP-1, MMP-2, MMP-3, MMP-9, MT1-MMP, TIMP-1, and TIMP-2 in 70 primary pancreatic adenocarcinoma tissues. To evaluate the potential for using specific MMP as a therapeutic target, the effect of antisense MMP on in vitro invasive potential of pancreatic carcinoma cells was also examined.
Patients and Tissue Samples Ten fresh paired surgical specimens of pancreatic carcinoma and adjacent nontumorous tissue were obtained from patients receiving surgical treatment. Each resected specimen was evaluated for its tumor cell content in a hematoxylin and eosin–stained section. Only specimens containing more than 75% of tumor cells were used for zymography. Paraffin-embedded tumor specimens from 70 patients with pancreatic carcinoma who had undergone surgical resection in our university hospital or related hospitals were used for immunohistochemical analysis. All of the tumors were adenocarcinoma, and the histopathologic and clinical features of the specimens were classified according to the guidelines of the International Union Against Cancer.40 Informed consent in writing was obtained from each patient and the experiments were approved by the institutional review committee. The human pancreatic adenocarcinoma cell lines BxPC-3, Capan-1, CFPAC-1, Hs 766T, MIA PaCa-2, and PANC-1 were purchased from the American Type Culture Collection (Rockville, MD). Cells were cultured in DMEM or RPMI 1640 containing 10% fetal bovine serum.
Zymography
Immunohistochemistry
Northern Blot Analysis
DNA Transfection
In Vitro Invasion Assay
Statistical Analysis
Casein and Gelatin Zymography in Pancreatic Carcinoma Tissues Using casein zymography, the levels of secreted matrilysin were examined in 10 paired specimens of pancreatic carcinoma and nontumorous tissues. Representative results are shown in Fig 1A. Both latent (28 kDa) and activated (19 kDa) forms of matrilysin were detected in six of 10 carcinoma tissues, but matrilysin was undetectable or only the inactive form was faintly detected in normal tissues. Figure 1B shows representative results of gelatin zymography. The latent forms of both MMP-9 (92 kDa) and MMP-2 (72 kDa) were detected in all of the carcinoma and nontumorous tissues. The activated form of MMP-9 (82 kDa) was detected in three of 10 carcinoma tissues and in two of 10 nontumorous tissues. The activated form of MMP-2 (66 kDa) was detected in nine of 10 carcinoma tissues and in four of 10 nontumorous tissues.
Matrilysin Expression in Pancreatic Carcinoma Tissues Figure 2 shows representative results of immunohistochemistry for matrilysin in pancreatic carcinoma tissues. Weak expression of matrilysin was identified in some islet cells of normal pancreas tissue but not in normal duct cells or acinar cells (Fig 2A). Stromal cells, other than some monocytes, were not stained. Matrilysin immunoreactivity in carcinoma cells was heterogeneous both between and within cases. The immunoreactivity was present in the cytoplasm of carcinoma cells and was usually most pronounced at the invasive front of the tumors (Fig 2A and 2B). In addition to the cytoplasmic staining, the lumenal surface of neoplastic glands was apically stained in some cases (Fig 2C). There was no detectable immunoreactivity with the control AI-206 antibody (data not shown). Sections with cytoplasmic immunostaining signals in more than 30% of carcinoma cells at the invasive front, which were observed in 40 cases (56%), were judged to be positive for matrilysin. All of the six carcinomas that showed matrilysin activity in casein zymography were positive in immunohistochemistry, but the remaining four were negative. The relationship between matrilysin positivity and clinicopathologic characteristics is summarized in Table 1. Matrilysin positivity was significantly correlated with pT (P < .0001), pN (P = .008), and pM categories (P = .020), and with advanced pTNM stage (P < .0001). On the other hand, there were no significant relationships between matrilysin positivity and age, sex, size of tumor, or tumor differentiation (Table 1 and data not shown).
Expression of other MMPs and TIMPs in Pancreatic Carcinoma Tissues Figure 3 shows representative results of immunohistochemistry for MMP-2, MMP-3, matrilysin, MMP-9, MT1-MMP, and TIMP-2 in pancreatic carcinoma tissues. There was no detectable immunoreactivity with the control AI-206 antibody (data not shown). These MMPs and TIMPs had no special distribution patterns of the immunoreactive carcinoma cells in the tumor-cell nests. Expression of MMP-1, MMP-2, MMP-3, MMP-9, MT1-MMP, TIMP-1, and TIMP-2 was observed in 70%, 94%, 20%, 93%, 67%, 87%, and 71% of the 70 cases of tumor cells, respectively, and in 57%, 74%, 17%, 60%, 61%, 46%, and 40% of the tumor stromal cells, respectively. There was no correlation between the presence of individual MMPs or TIMPs in either tumor cells or tumor stromal cells, or in both components, and clinicopathologic characteristics, including age, sex, size of tumor, tumor differentiation, and pT, pN, pM, or pTNM stage (data not shown). Expression of MMP-1, MMP-2, MMP-3, MMP-9, MT1-MMP, TIMP-1, and TIMP-2 was stronger in tumor cells than in stromal cells in 34%, 81%, 9%, 72%, 51%, 74%, and 60% of the 70 cases, respectively. However, enhanced expression of these MMPs or TIMPs in tumor cells was not correlated with clinicopathologic characteristics (data not shown).
Prognostic Significance of Matrilysin and Other MMPs and TIMPs Patients whose tumors were matrilysin-positive in immunohistochemistry had a significantly shorter overall survival time than did those with matrilysin-negative tumors (P = .0018, Fig 4). Size of tumor (P = .009), pT (P = .0001), pN (P = .0002), and pM categories (P = .0001), and pTNM stage (P = .0001) were also significantly correlated with poor survival in univariate analysis. In multivariate analysis of these variables, only matrilysin remained a significant prognostic variable for predicting poor survival (hazard ratio, 4.85; 95% confidence interval, 1.22 to 10.8; P = .022). In contrast, none of the other MMPs and TIMPs analyzed in this study demonstrated prognostic significance (data not shown).
In Vitro Invasion Assay Six pancreatic carcinoma cell lines were analyzed for matrilysin mRNA expression by Northern blot analysis. Matrilysin mRNA was detected in Capan-1 and CFPAC-1 cells but not in the remaining cells ( Fig 5A). In an attempt to suppress matrilysin in pancreatic carcinoma cells, we used the antisense technique.20 CFPAC-1 or Capan-1 cells were stably transfected with the cytomegalovirus-based vector which carried the matrilysin cDNA in antisense orientation.20 After G418 selection, 10 different clones were analyzed for matrilysin mRNA by Northern blot analysis. A considerable reduction in the amount of matrilysin mRNA was observed in CFPAC-1-derived clones AS 5 and AS 8 (Fig 5B). The expression of other related genes, such as MMP-2, MMP-9, TIMP-1, and TIMP-2, was not affected by the transfection (data not shown). The enzyme activity of matrilysin in the conditioned culture media of these clones was hardly detectable by casein zymography (data not shown). In vitro growth rates measured by cell growth kinetics were almost the same among the parental CFPAC-1 and the corresponding transfectants (data not shown). In vitro invasive potential of these cells was then assayed. Both AS 5 and AS 8 demonstrated about one third the invasive potential of control cells (P < .01), and this difference was eliminated by TIMP-1 (Fig 5C). Suppression of in vitro invasive potential was also observed in the antisense matrilysin-transfected Capan-1 cells (data not shown).
Each member of the MMP family is secreted as a latent proenzyme requiring the removal of an amino-terminal domain to attain enzyme activity.29 Although immunohistochemical techniques provide useful information concerning the overall expression of enzymes, inactive and active forms can not be distinguished. On the other hand, zymography can not provide information concerning the cellular distribution of enzymes in tissues. Because fresh tumor specimens are required for zymography, it is not practical for routine assessment of MMPs in diagnostic practice. Therefore, before detailed immunohistochemical analysis, we decided to analyze all of the frozen tissues available for the levels of casein or gelatin degrading MMP by zymography and to compare the results with those of immunohistochemistry. Although only 10 paired frozen tissues were available in this study, the results of casein and gelatin (MMP-2) zymography paralleled those of immunohistochemistry, suggesting that immunohistochemistry is an appropriate technique, at least, for analyzing matrilysin and MMP-2 expression in pancreatic carcinomas. In contrast, immnuoreactivity of MMP-9 may not necessarily be correlated with the expression of the activated form of MMP-9. Further study using a large number of fresh tumor specimens is required to clarify these issues. As regards the activation of matrilysin and MMP-2, it is notable that the activated forms of both matrilysin and MMP-2 were detected in almost all of the carcinoma tissues that showed matrilysin or MMP-2 activity, suggesting that matrilysin and MMP-2 could be efficiently activated in pancreatic carcinomas. MT1-MMP is likely to be involved in the activation of MMP-2.35 The activation mechanism of matrilysin in vitro seems to be similar to that of the other MMPs, which could be activated by activator proteinases such as serine proteinases.46,47 It is of interest that matrilysin-positive cell lines, CFPAC-1 and Capan-1 cells, have been reported to produce pancreatic trypsinogen.48 Trypsin is also one of the promising candidate activators of matrilysin in vivo.49 It is notable that trypsin(ogen) was expressed in 75% of 23 pancreatic carcinoma tissues and was more pronounced at the infiltrative margins of tumors.50 Thus, trypsin may play an important role in the activation of matrilysin in pancreatic carcinoma and this possibility warrants further study. Matrilysin-positive carcinoma cells showed a unique distribution in the tumor nests. First, polarized immunoreactivity was observed on the lumenal surfaces of neoplastic glands in some cases. This staining pattern is similar to that reported in several exocrine glands9,51 and supports the notion that matrilysin may play a role in exocrine functions. MMPs reportedly activate lumenal or membrane-bound cytokines or growth factors, such as tumor necrosis factor alpha and heparin-binding epidermal growth factor, to locally perturb the growth of responsive cells.52,53 Therefore, it is tempting to speculate that matrilysin plays a role in early pancreatic carcinogenesis,15,54 and this possibility deserves further analysis. In contrast, most tumor cells showed depolarized diffuse cytoplasmic staining, which was usually most pronounced at the invasive front of the tumors. With respect to the mechanism(s) underlying the depolarized matrilysin expression in tumor cells at the invasive front, genetic alterations in tumor cells and/or tumor-host interactions may play a key role.55,56 Matrilysin positivity was significantly correlated with the extent of tumor invasion, lymph node and distant metastasis, and advanced tumor stage, suggesting that matrilysin may play an important role in the progression of pancreatic carcinoma. We have previously reported that the immunohistochemically detected matrilysin expression was correlated with the progression of gastric and colorectal adenocarcinomas as well as esophageal squamous cell carcinomas.17-19 These findings suggest that upregulation of matrilysin expression may be a common mechanism for the progression of tumor cells of an epithelial origin. Nevertheless, it is clear that the aggressive phenotype of pancreatic carcinomas can not be determined by matrilysin expression alone. It is well known that different MMPs and TIMPs are expressed at various levels and that complex interactions between tumor and stromal cells influence biologic aggressiveness of many types of cancer.29,30 Therefore, we immunohistochemically analyzed the expression of MMP-1, MMP-2, MMP-3, MMP-9, MT1-MMP, TIMP-1, and TIMP-2 in pancreatic adenocarcinoma tissues. Overall, localization and frequency of the expression of MMP-1, MMP-2, MMP-3, MMP-9, MT1-MMP, TIMP-1, and TIMP-2 were almost consistent with the results of previous reports on pancreatic cancer.27,31-35 Our results, in part, confirm previous findings and provide further evidence through comparative analysis that both tumor cells and tumor stromal cells are the major sources of various members of MMP and TIMP families in pancreatic cancer tissues. Also, in agreement with the results of previous reports, none of the expressions of MMPs or TIMPs showed significant correlation with pathologic factors, such as tumor size, lymph node metastasis, distant metastasis, or tumor stage. Therefore, they may be involved in processes such as the desmoplastic reaction in the stroma and remodeling of the pancreas in chronic pancreatitis31,35 rather than in tumor progression. Nevertheless, because each tumor displays varying expression patterns for MMPs and TIMPs, it is possible that they also influence the invasive and metastatic potential of the respective tumor. If MMP expression increases and/or TIMP expression decreases, the balance favors proteolysis. However, both TIMP-1 and TIMP-2 were rather overexpressed in pancreatic carcinoma tissues. These results may be explained by coordinate regulation of proteases and their corresponding inhibitors or, alternatively, the synthesis of inhibitors may be a cellular reaction to the presence of proteases.57 It should be noted that TIMPs are multifunctional proteins. Both TIMP-1 and TIMP-2 have erythroid-potentiating activity and have been shown to stimulate the mitogenesis of several cell types in vitro.58 The results of mutagenesis experiments have suggested that the erythroid-potentiating activity and the MMP-inhibitory property of TIMP-1 are independent and separable activities.59 Indeed, high levels of TIMP-1 mRNA have been correlated with aggressiveness in several types of tumor.60 Moreover, high levels of TIMP-1 or TIMP-2 protein have been associated with poor prognosis in breast cancer57 or in both breast and bladder cancers, respectively.61,62 It has also been reported that the growth-stimulatory activity of TIMP-2 is concentration and cell type–dependent and that lower concentrations of TIMP-2 promote the activity, whereas higher doses have either no effect or are growth-inhibitory in vitro.63 These results suggest that MMPs and TIMPs, and the balance between MMPs and TIMPs, play complex roles in tumor progression.64 Further analyses are needed to clarify these issues in pancreatic cancer. The significance of matrilysin expression was further substantiated by its correlation with a shorter overall survival time. Moreover, only matrilysin provided a significant predictive value for overall survival in multivariate analysis, suggesting that matrilysin expression could be a powerful predictor of poor prognosis with a significance equaling or surpassing that of other conventional clinicopathologic factors. Thus, our results suggest that matrilysin contributes to the aggressive behavior of tumors and thereby to poor prognosis. Matrilysin could be a new prognostic marker that would allow us to identify patients with a poor prognosis who might benefit from more aggressive treatments. Immunohistochemical analysis is a technique available in daily clinical settings, and analysis of matrilysin expression could, therefore, be an important routine part of the management of patients with pancreatic carcinoma. Nevertheless, the effect of conventional therapy is limited for patients with pancreatic carcinoma, especially for patients with matrilysin-positive carcinoma. Another important clinical implication of our results, beyond the prognostic significance, is that matrilysin could be a potentially useful target for therapeutic intervention either by the use of an antisense strategy or synthetic MMP inhibitors. To evaluate the potential for using matrilysin as a therapeutic target, the effect of antisense matrilysin on in vitro invasive potential of pancreatic carcinoma cells was examined. Downregulation of matrilysin by the antisense vector markedly reduced the invasive potential of the metastasis-derived pancreatic carcinoma cell lines CFPAC-1 and Capan-1. In vitro invasion assay thus provided experimental evidence that matrilysin contributes to the aggressive tumor behavior of pancreatic carcinomas and could be a therapeutic target. Although considerable optimization is necessary in many aspects, the matrilysin antisense strategy offers a feasible possibility as an adjuvant therapy for pancreatic carcinoma in the future. The use of synthetic MMP inhibitors is the currently used strategy in clinical settings. Some inhibitors have been proven to be of therapeutic significance in clinical trials.23-26 Considering our results, matrilysin could be a primary target of such broad-type MMP inhibitors in a subset of pancreatic carcinomas. However, it is clear that matrilysin expression does not determine the aggressive phenotype of all pancreatic carcinomas. For a specific selection of patients who would benefit from those therapies, examination of MMP expression will be necessary. Because different synthetic MMP inhibitors affect different types of MMPs to a different degree, examination of expression of not only matrilysin but also other MMPs and TIMPs would be ideal for selecting the most effective MMP inhibitor. The diagnostic strategy presented here and advances in therapeutic approaches, including anti-MMP therapy, are expected to improve the prognosis of patients with pancreatic carcinoma.
Supported by grants-in-aid from the Ministry of Education, Science, Sports and Culture (F.I. and K.I.) and from the Ministry of Health and Welfare (F.I. and K.I.), Japan.
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Copyright © 2001 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 two-tailed test for the remaining parameters. Survival analysis was carried out for all patients except those who died of causes other than carcinoma. Cumulative survival rates were calculated by the Kaplan-Meier method. 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. A P value of less than .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. 
