Matrix metalloproteinases and bladder cancer Hiro-omi Kanayama Department of Urology, The University of Tokushima School of Medicine, Tokushima, Japan Abstract: Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes which degrade the extracellular matrix or components of the basement membrane. They have essential roles in tumor invasion and metastasis. In bladder cancer, elevated MMP-2 and MMP-9 expression in tumor tissues, correlated with tumor stage, grade or prognosis, were reported in several studies. Moreover, high levels of serum or urine MMP and TIMP were observed in patients with bladder cancer especially in advanced cases. However, the true roles of MMPs and TIMPs in bladder cancer progression are not yet clarified. Here, we discuss the roles and clinical implications of MMPs in bladder cancer. J. Med. Invest. 48:31-43, 2001 Keywords:matrix metalloproteinase, tumor invasion, metastasis, progression, bladder cancer INTRODUCTION The process of cancer progression consists of multisteps, which can be rate limiting since a failure or an insufficiency at any of the steps aborts the process (1-3). The outcome of the process is dependent on both the intrinsic properties of the tumor cells and the responses of the host. The steps or events required for the formation of tumor invasion and metastasis are the same in all tumors (Fig. 1). The major steps in tumor progression are as follows : 1) After the initial transformation, tumor cells grow at the primary site. 2) Neovascularization must occur when the tumor mass forms 2 mm more in diameter (4). Several angiogenic factors play key roles to establish neovascularization (5-7). 3) Local invasion of the basement membrane and degradation of the stroma are necessary for migration from the primary site (8-11). Matrix metalloproteinases may play the most important role in these steps. 4) Thin-walled venules, like lymphatic channels or small capillaries, must be penetrated by tumor cells for tumor cell entry into the circulation. Some carcinomas metastasize and grow via the lymphatic system, and others spread via the hematogeneous route. 5) After the circulating tumor cells attach to the epithelium of venules, extravasation occurs via a similar mechanism as the initial invasion. 6) Tumor cells grow at distant sites with neovascularization, similar to primary site, and metastatic tumors can be established. Then the metastatic process can be completed. To produce detectable lesions, the metastases must develop neovascularization, evade the host immune system (12), and respond to organ-specific factors that influence their growth (13-17). Many factors have essential roles in this metastatic process, and the matrix metalloproteinases (MMPs) must be one of the most important factors in several steps. MATRIX METALLOPROTEINASES (MMPs) : STRUCTURE, FUNCTION AND REGULATION In many physiological states or processes, degradation of extracellular matrix is very important and essential, for example, during development, growth, and repair or remodeling of organ tissues (18-21). However, excessive degradation of tissues or proteolysis causes several pathological conditions, for example, rheumatoid arthritis, osteoarthritis, autoimmune disorders of skin, and others (18, 22, 23). In addition, in tumor invasion, metastasis and angiogenesis, degradation of the extracellular matrix is an essential steps and increased expression levels of matrix metalloproteinases (MMPs) is associated with tumor invasion and metastasis with different histogenetic origin (19, 24). (1) Structure MMPs are a family of at least 20 human zinc-dependent endopeptidases, which, collectively capable of degrading extracellular matrix components (19, 22, 23, 25-27). These members of the MMP gene family can be classified into subgroups of collagenases, stromelysins, gelatinases, membrane-type MMPs, and other MMPs according to their substrate specificity and structure (Table 1). In general, MMPs contain a signal/propeptide domain, a catalytic domain with the highly conserved zinc binding site, hinge region, and a hemopexin-like domain (Fig. 2). In addition, gelatinase-A (MMP-2) and gelatinase-B (MMP-9) contain a gelatin-binding site as fibronectin type II inserts within the catalytic domain, and MT-MMPs contain a transmembrane domain in the c-terminal of the hemopexin-like domain. A hemopexin-like domain is absent in matrilysin (MMP-7), the smallest MMP (18, 27). The substrate specificity of MMPs has been determined by their ability to degrade different components of extracellular matrix in vitro, however, no direct evidence was obtained in vivo. (2) Function Collagenase, including interstitial collagenase (MMP-1), neutrophil collagenase (MMP-8), and collagenase-3 (MMP-13) are the secreted neutral proteinases capable of degradation of native fibrillar collagens of types I, II, III (18, 23). These collagenases play a crucial role in degradation of collagenous extracellular matrix in various physiological and pathological situations (28-30). Gelatinase-A (MMP-2, 72 kDa Type IV collagenase) is expressed by a variety of normal and transformed cells. Gelatinase-B (MMP-9, 92 kDa Type IV collagenase) is produced by monocytes, alveolar macrophages, and various malignant cells (18, 23). In addition, MMP-2 and MMP-9 can degrade gelatin, laminin, and MMP-2 has also been reported to degrade native type I collagen and activate MMP-9 and MMP-13 (24, 31). The stromelysin subgroup contains stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), and stromelysin-3 (MMP-11) (18, 23). MMP-3 and MMP-10 are expressed by fibroblastic cells and by normal and transformed squamous epithelial cells (28, 30). Stromelysins degrade basement membrane components, type IV collagen, and fibronectin (18, 23). The cDNA of stromelysin-3 (MMP-11) was cloned from invasive breast cancer tissue (32). The predicted structure of MMP-11 resembles that of other stromelysins and collagenases (18, 23). The smallest MMP-7, matrilysin can degrade gelatin I, III, IV, V and some other extracellular matrix including fibronectin. Moreover, both matrilysin and macrophage metalloelastase have the ability to degrade elastin (18, 22, 23). The first membrane-type MMP (MT1-MMP, MMP-14) was cloned from invasive lung cancer cells (33) and revealed a typical five-domain modular structure resembling collagenases and stromelysins;it also contains an additional short carboxyl-terminal transmembrane domain. Three other MT-MMPs ; MT2-MMP (MMP-15), MT3-MMP (MMP-16), and MT4-MMP (MMP-17) were cloned. Active MT1-MMP serves as a cell membrane receptor for the complex formed of latent MMP-2 (proMMP-2) and tissue inhibitor of metalloproteinases-2 (TIMP-2). The complex of MT1-MMP and MT2-MMP works at the cell surface as an activator for proMMP-2. Most MMPs are secreted as latent proenzyme that are proteolytically activated in the extracellular space, with the exception of MMP-11 and MT1-MMP, which are activated prior to secretion intracellularly by furin-like proteases (18, 22, 23, 31). The activity of MMPs in the extracellular space is specifically inhibited by tissue inhibitors of metalloproteinases (TIMPs), which bind to the highly conserved zinc binding site of active MMPs at molar equivalence. The TIMP gene family consists of four structurally related members, TIMP-1, -2, -3, and -4, which show 30 to 40% identity at the amino acid level and possess 12 conserved cysteine residues (34). TIMP-1, -2, and -4 are secreted in soluble form whereas TIMP-3 is associated with extracellular matrix. TIMPs have biological effects that extend beyond their role as inhibitors of MMP activity (35). They induce changes in cell morphology, stimulate growth of several cell types, and TIMP-2 is also involved in activation of MMP-2 (31). (3) Regulation Regulation of the MMPs is exerted at many levels and involves both transcriptional and post-transcriptional mechanisms (25, 36). Many different factors have been shown to influence the transcription of MMPs, including hormones, growth factors, oncogenes, and cytokines (19, 37, 38). The mechanism of transcriptional activation has been extensively studied and the AP-1 binding site has been the focus of much recent research (36). The AP-1 binding site is located upstream from the transcriptional activation site and has been considered to play an important role in the transcriptional activation of the MMP promoters. From a recent study, the interaction of this site with other cis-acting elements is necessary for basal transcription and induction by cytokines and growth factors (39, 40). The AP-1 site is also involved in the down-regulation of MMPs by transforming growth factor beta, retinoids and glucocorticoids. Several findings suggested that the MMP subgroup was independently regulated (41, 42). In a recent study, a single nucleotide polymorphism in the MMP-1 promoter increased the transcription rate of the MMP-1 gene (43). The increased frequency of the GG polymorphism in several tumor-derived cell lines may be associated with increased matrix degradation (43). The soluble MMPs, such as collagenases, gelatinases, and stromelysins, are secreted as proenzymes, requiring activation. Acquisition of proteolytic activity is associated with the loss of the amino-terminal pro-domain (18, 23). It is becoming apparent that the MMPs interact with other proteases, for example, plasmin and MT1-MMP (44). THE ROLE OF MMPs IN TUMOR INVASION AND METASTASIS (1) MMPs expression in malignant tumors Liotta et al. described the role of MMPs in cancer in1980 (45). They identified a type IV collagenase involved in melanoma invasion and metastasis, and suggested that proteolysis was essential step in tumor invasion. From the cloning of the type IV collagenase, this activity has been considered to be attributed to MMP-2 or MMP-9 (46, 47). Although it was initially suggested that the tumor cells produced these MMPs, it has become clear that the interaction between host stromal cells and tumor cells was important for the induction of MMPs (48). The concept of stromal cell expression of MMPs has been acceptable by the identification of stromelysin-3 as a stromal metalloproteinase associated with breast cancer (32). In situ hybridization for MMP revealed that the expression of MMPs in stromal cell is more common than in tumor cells. Many MMPs are induced in connective tissue cells, including fibroblasts and inflammatory cells. There is some evidence that the mRNA of MMP-2 is produced by stroma cells, but the protein is located in tumor cells, especially in the invasive front of tumor tissue (49). In addition, matrilysin is commonly expressed in the epithelial component of adenocarcinomas (50). There is a general correlation between the stage of tumor progression and the level of MMPs expression (26, 27). In a murine system of squamous cell carcinomas, high levels of stromelysin-1 in highly metastatic spindle-cell carcinomas and very low levels in benign papillomas were observed (51). The expression of MMP-9 is associated with melanoma growth and subsequent metastasis (52), and high expression of MMP-2 is associated with high tumor grade (53). MMP-2 is widely expressed in breast cancers, however, the ratio of the active form of MMP-2 is increased in advanced disease. In addition, malignant tumors tend to express various MMPs rather than benign tissues (54, 55). Colon adenocarcinomas express matrilysin, stromelysin-1, stromelysin-3, MMP-2, and collagenase-1 (56). From these findings, there is a general positive correlation between tumor aggressiveness and the expression of MMPs. The ability of the diagnostic or prognostic value of the expression of the MMPs or TIMPs were observed in some studies (26). Expression of stromelysin-3 has been associated only in malignant breast tumors, and it is not expressed in benign tissues (57). Although some other studies have reported associations between stromelysin-3 expression and lymph node metastasis, and, thus, shorter survival in patients with infiltrating ductal carcinoma of the breast, larger studies are necessary to clarify the true value (58-61). In addition, high levels of serum MMP-2 were observed in the patients with prostate cancer (62). Another study mentioned that tissue levels of active MMP-2 were associated with Gleason score and lymph node metastases, and high levels of serum MMP-2 were also found in patients with prostate cancer (63). Similar findings about plasma TIMP-1 were also reported in prostate cancer (64, 65). In colon cancer, immunohistochemical detection of interstitial collagenase is associated with a poor prognosis (66). Matrilysin expression, measured using reverse transcriptase polymerase chain reaction (RT-PCR), has also been suggested to have prognostic value in colon and esophageal cancer (67). In a study of esophageal carcinoma, patients with tumors that demonstrated no matrilysin expression had a better disease-free and overall survival (68). (2) The role of MMPs and TIMPs in tumor progression The increased expression of MMPs in advanced tumors and the ability of these enzymes to degrade extracellular matrix barriers suggested that these enzymes have important roles in tumor invasion and metastasis (9, 10, 27). This hypothesis was supported by the findings from experimental and spontaneous metastasis models (69). Recombinant TIMP-1 decreased the number of lung nodules of B16-F10 melanoma cells (70). Increased establishment of lung metastases after intravenous injection of several cancer cells were associated with MMP-2, MMP-9 and MT1-MMP (71-73). It has also been shown that MMP-2, MMP-9, and matrilysin metastasize to appropriate target organs from their primary sites in orthotopic models of bladder, fibrosarcoma, and colon cancer (71, 72, 74). In addition, B16-F10 melanoma cells transfected with TIMP-1 produced significantly fewer metastatic nodules than control cells (75). These findings suggest that MMPs and TIMPs have some roles for extravasation to target organs from vessels. However, the role of MMPs in tumor cell intravasation, or entry into the vessels, appears to be better established. TIMPs have been shown to inhibit tumor cell invasion in in vitro invasion assay (69). Matrilysin was observed to promote invasion of DU145 prostate cells into the diaphragm of nude mice (76). Recently, an in vivo model of tumor cell intravasation using the chick chorioallantoic membrane demonstrated that the intravasation of human epidermoid carcinoma cells was associated with the production of MMP-9 (77). The ability of tumor cells to penetrate the epithelial basement membrane, migrate through stroma, and enter vessels appears to be dependent on matrix-degrading proteases, including the MMPs. However, the process of tumor cell invasion and metastasis is tightly coupled to neovascularization, and MMPs have also been implicated in the process of angiogenesis (78). Based on recent studies with MMP-knock-out mice, MMP-2 or MMP-9 may be associated with neovascularization (79, 80). One of the primary effects of MMPs on tumor progression also appears to be the ability to create the space for tumor growth (19). The effect of TIMP on the establishment of distant metastases was its ability to inhibit the growth of tumor cells at metastatic sites (81). In addition, MMPs also appear to contribute to the establishment and growth of primary tumors (82-84). Thus, MMPs appear to be able to alter the extracellular environment and induce tumor cell establishment and growth. MMPs IN BLADDER CANCER (1) Expression of MMPs in tumor tissues In bladder cancer patients, the presence of deep muscle invasion, infiltrative proliferation, or infiltration to vessels is associated with a high recurrence rate and poor prognosis (85). We evaluated the expression of MMP-2, MMP-9 and its inhibitors TIMP-1 and TIMP-2 in 22 bladder cancer tissues by Northern blot and slot blot analysis and High MMP-2, TIMP-1, and TIMP-2 expression levels were observed in advanced tumor tissues (86). Davies et al. reported that MMP-2 and MMP-9 activities quantitated by gelatin zymography correlated with tumor grade and invasion in bladder cancer (87). Grignon et al. showed that high levels of TIMP-2 expression were associated with poor outcome in bladder cancer patients undergoing radical cystectomy (88). We also evaluated MMP-2, TIMP-2 and MT1-MMP expression in bladder cancer tissues using RT-PCR analysis (89). MMP-2 and TIMP-2 expression levels were strongly associated with tumor stage and prognosis. High levels of expression of MMP-2 and TIMP-2 were observed in muscle invasive ≥pT2 bladder cancer tissues compared with low stage pTa-1 tumors. However, MT1-MMP expression was not correlated with tumor invasion. Although, the levels of MT1-MMP was not associated with tumor invasion, it was associated with patient outcome. Other studies have shown high expression of MT1-MMP in cancer tissues and association with invasiveness of cervical cancer cells (90) or lymph node metastases in lung cancers (91). Thus, there may be some tissue specificity regarding the involvement of MT1-MMP in tumor invasion or metastasis. In bladder cancer, MT1-MMP may involve distant metastasis directly but not tumor invasion. The relatively activated MMP-2 expression can be measured by gelatin zymography which is capable of highly sensitive differentiation of latent and activated forms of gelatinases. Therefore, we determined activation of MMP-2 using gelatin zymography (92) (Fig. 3). The expression of activated MMP-2 and the expression of total MMP-2 in invasive tumors (≥pT2) were both significantly higher than in superficial tumors (pTa-1). These findings indicated that MMP-2 expression in urothelial tumors was highly correlated with tumor invasion, and showed a strong correlation between the levels of activated MMP-2 and those of total MMP-2. Both expression levels were associated in tumor invasion, but the findings suggested that the activated form of MMP-2 expression was a better indicator of tumor invasion. Moreover, we observed that the high expression groups of the activated form of MMP-2 and total MMP-2 showed significantly worse cause-specific survival than did the low expression groups. High expressions of activated MMP-2 were more strongly linked with an unfavorable prognosis than was total MMP-2 expression. We also reported MMP-2 and MT1-MMP expression in invasive urothelial tumor tissues transplanted in SCID mice (93). In bladder cancer, the pathologic stage or tumor grade is associated with patient survival. However, even within patients of the same stage or grade, there are some differences in patient survival (85). Therefore, there is a need to identify other predictors for bladder cancer patients. We examined the survival of patients according to the levels of expression of MMP-2, TIMP-2, and MT1-MMP to evaluate the usefulness of these proteins as predictors. Patients with high expression levels of MMP-2, TIMP-2, or MT1-MMP showed worse cause-specific survival, even within the group of patients with muscle invasive ≥pT2 tumors resected radically (89). However, differences in the MMP-2/TIMP-2 ratios did not affect survival. In our study, the expression levels of MMP-2 was correlated with those of TIMP-2. Therefore, the ratio of MMP-2/TIMP-2 might not affect patient outcome. Grignon et al. showed that high levels of TIMP-2, as assessed by immunohistochemical staining, were associated with poor outcome in patients with invasive bladder cancer treated by cystectomy but that MMP-2 expression did not affect survival (88). However, it is complicated to evaluate MMP-2 or TIMP-2 expression by immunohistochemical staining methods. Moreover, evaluation of expression by gelatin zymography or Northern blot analysis is also difficult to use in clinical settings. Our results demonstrated that levels of MMP-2, TIMP-2, or MT1-MMP in tumor tissues obtained by operation or biopsy may be useful for prognosis, even in patients with muscle invasive tumors (89, 92). It is possible that adjuvant chemotherapy can be avoided in patients with low levels of MMP-2 or TIMP-2whose muscle invasive bladder cancers have been radically resected. Recently, several studies reported MMP-2 and MMP-9 in bladder cancer. Papathoma et al. reported that zymographical analysis of the levels of MMP-9 and active MMP-2 showed a significant increase with tumor grade and invasiveness, however, the correlation between the levels of both gelatinases with recurrence in superficial tumors or progression in invasive tumors was not significant (94). We evaluated MMP-9 expression in bladder tumor tissues using gelatin zymography (92), however, the correlation between MMP-9 expression levels and tumor invasion was weak and not significant. The background of patients might have been different in those two studies. Ozdemir et al. reported a strong correlation of basement membrane degradation with p53 inactivation and/or MDM2 overexpression in superficial urothelial carcinomas, and they suggested that MMP-9 plays a key role in the invasion step of superficial urothelial carcinomas (95, 96). Kitagawa et al. reported that MT1-MMP and MT2-MMP may play an important role in the development and multifocal occurrence of urothelial cancer (97). (2) Serum MMPs and TIMPs Recently, some studies evaluated serum levels of MMPs and TIMPs, and their clinical usefulness. Gohji et al. reported the prognostic significance of serum MMPs and TIMPs, and the imbalance between serum matrix metalloproteinase-2 and its inhibitor as a predictor of recurrence of urothelial cancer (98-100). Measurement of plasma/serum MMP and TIMP levels may provide important information for selecting and following patients considered for treatment with drugs that interfere with MMP activity (101). We also observed high levels of serum TIMP-1 in patients with advanced bladder cancers (102). (3) Urinary MMPs and TIMPs Moses et al. evaluated the incidence of matrix metalloproteinases in urine of cancer patients (103). They detected three molecular weight classes of urinary MMPs, MMP-2, MMP-9, and high molecular weight (Mr≥150,000) species, correlated with disease status. The presence of biologically active MMP-2 or MMP-9 was an independent predictor of organ-confined cancer, and the high molecular weight species was an independent predictor of metastatic cancer. Monier et al. detected 72 kDa proMMP-2 and its activated 68 kDa form, a 92 kDa proMMP-9, and a higher molecular weight complex (115 kDa) which was identified as proMMP-9 (104). MMPs in urine can be used as predictors of bladder cancer diagnosis and prognosis. Sier et al. also reported that urinary MMP-2 and MMP-9 activity levels were significantly correlated with each other, and they concluded that enhanced urinary MMP activity or especially in combination with other markers, might be useful as a marker for superficial bladder carcinoma (105). Detection of proMMP-9 in bladder washes also may be a novel approach for the identification of patients with more aggressive forms of bladder cancer (106). (4) Regulation of MMP-2 and MMP-9 in bladder cancer cells by cytokines Shin et al. observed that MMP-2 and MMP-9 expression was up-regulated by tumor necrosis-alpha or interferon-gamma, and they suggested BCG immunotherapy may enhance the invasiveness of bladder cancer in certain conditions with induction of MMPs (107).Kageyama et al. also suggested the possibility that BCG promotes invasion of bladder cancer cells because of the induced secretion of MMP-9 from peripheral mononuclear cells by BCG (108). Basic fibroblast growth factor also induces MMP-2 and MMP-9 secretion from bladder cancer cells (109). However, Slaton et al. observed interferon-alpha down regulated the production of basic fibroblast growth factor and MMP-9 from invasive bladder cancer cells (110). From these findings, certain cytokines may be useful for treatment of invasive bladder cancer by regulating MMPs. CONCLUSIONS It has been clarified that MMPs have important roles in tumor invasion and metastasis. Large numbers of studies have examined the presence of individual MMPs in different types of cancer, using various methods. In bladder cancer, although these studies suggested the clinical usefulness of MMP-2 and MMP-9 in tissues, serum and urine for further diagnosis of tumor biological activities or patient status, further examinations of large numbers of patients are necessary. In the near future, it may be possible to use MMPs or TIMPs for clinical applications as MMPs expression levels in biopsied cancer tissues, serum or urine MMPs and TIMPs obtained from patients with bladder cancer. The development of antibodies that distinguish between pro-enzymes and activated enzymes, and between free MMP and MMP complexed with TIMP may provide new approaches for bladder cancer diagnosis or treatment. Moreover, MMP-2 or MMP-9 would be the target for therapy in especially advanced bladder cancer patients. 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