The role of the activated form of matrix metalloproteinase-2 in urothelial cancer

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1 BJU International (),, ± The role of the activated form of matrix metalloproteinase- in urothelial cancer K. KANDA, M. TAKAHASHI, Y. MURAKAMI, H. KANAYAMA and S. KAGAWA Department of Urology, School of Medicine, The University of Tokushima, Tokushima, Japan Objective To investigate the expression of matrix metalloproteinase- (MMP-, reportedly associated with cancer cell invasion and metastasis in many human cancers) in urothelial tumours and thus de ne its role in this disease. Materials and methods The expression of both the activated form of MMP- and total MMP- (activated + latent form) was measured using gelatine zymography in tissue obtained surgically from patients with urothelial cancer. The correlation between the level of the activated form of MMP- and clinical and histological variables was assessed. Results The expression of activated and total MMP- were signi cantly higher in invasive tumour tissue and both levels were correlated with histological grade. In particular, the level of activated MMP- was more closely correlated than that of total MMP- in invasive tumour tissue. Moreover, high levels of activated MMP- were strongly associated with shorter disease-speci c survival (P=.). Conclusion These ndings suggest that activated MMP- plays a signi cant role in invasion of urothelial cancer and that the level of activated MMP- expression is a useful prognostic indicator. Keywords Matrix metalloproteinase-, urothelial cancer, gelatine zymography, invasion, prognostic indicator Introduction Cancer cell metastasis to distant organs involves a multistep process, with the acquisition of invasiveness into surrounding tissue being crucial in the malignant progression of cancer. The invasive process involves proteolysis of both basement membrane and stromal extracellular matrix. Matrix metalloproteinases (MMPs) and in particular MMP- (gelatinase A), which degrades type IV collagen, are known to play an important role []. All MMPs, which are zinc-dependent endopeptidases, are produced in a latent form and undergo proteolytic cleavage of an amino-terminal domain during activation [,]. In vivo activation of the latent form of MMP- is thought to take place on the cell surface [] and to be mediated by membrane-type MMPs (MT-MMPs). Recent studies have shown that latent MMP- is recruited to the cell surface by interaction with a tissue inhibitor of MMP- (TIMP-) that is bound to MT--MMP through a tertiary complex. Free MT--MMP close to the tertiary complex in turn activates the latent form of MMP- on the cell surface. Activated MMP- induces the proteolysis of the extracellular matrix required for cancer cell invasion. Previous studies have shown that active MMP- is expressed in a variety of tumour types, including gastric Accepted for publication April carcinoma [,], colorectal carcinoma [], pancreatic carcinoma [], breast cancer [] and bladder cancer [9]. In urothelial cancer, tumour cell in ltration into muscle layers, surrounding tissue or vessels is associated with local recurrence, metastasis and a poor outcome []. However, little is known about the association between activated MMP- and either tumour invasion or patient outcome in urothelial cancer. Gelatine zymography is useful for detecting activated and latent MMP- []. In the present study, we used relative quantitative gelatine zymography, with optical scanning and image analysis, to examine the expression of both forms of MMP- in urothelial cancer; we also evaluated the role of activated MMP- expression in the pathogenesis of the tumour and its impact on prognosis. Materials and methods Tumour tissue samples were obtained during surgery from patients with urothelial cancer; the tumour and patient characteristics are summarized in Table. The tumours were evaluated before surgery and all were diagnosed as localized urothelial cancer (Ta- NM). Disease stage and pathological grade were determined according to the WHO criteria [] or TNM classi cation []. All tissue samples were immediately frozen in liquid nitrogen after surgical removal and stored at xuc until use. # BJU International

2 K. KANDA et al. Table The characteristics of patients with urothelial cell cancer Characteristics No. of patients Mean (range) age, years. (±9) Male Female Cell type Transitional 9 Squamous Tumour position Bladder 9 Ureter Histological grade Pathological stage pta pt pt pt pt Gelatine zymography was performed according to methods previously described by Heussen and Dowdle [] with slight modi cations. Brie y, each frozen tumour tissue sample was homogenized in % SDS buffer, the extracts then microcentrifuged at 9 g for min and the supernatants collected. The protein concentration of the extracts was determined using an appropriate assay. Gelatine zymography was carried out by electrophoresing equivalent amounts of protein from each tumour tissue sample in % SDS-PAGE containing gelatine at a nal concentration of mg/ml. In each gel, a sample of the UCT- tumour line [] from a renal pelvic tumour (TCC, grade, stage pt pnm) was electrophoresed simultaneously as an internal control to allow a relative quantitative analysis. After electrophoresis the gels were washed twice for min in.% Triton-X- in distilled water, then washed in mmol/l Tris-HCl (ph.) for min. The gels were incubated at uc for h in mmol/l Tris-HCl (ph.) containing. mmol/l CaCl and mmol/l ZnCl, and then stained using.% Coomassie Blue in a solution of % acetic acid and % methanol. After the gels had been de-stained in % acetic acid and % methanol, bands showing gelatinolytic activity were detected as clear zones of lysis against a blue background. The HT- ( brosarcoma) cell line was used as a marker of latent and active MMP- and MMP-9, respectively. The gel image was scanned into a personal computer using a digital camera and the band intensity measured using MacBAS software (version., Fujix, Tokyo, Japan). The expression of activated and latent MMP- in each tumour sample was analysed by measuring the ratio of the intensity of each band to that determined for activated MMP- in the UCT- sample in the same gel. In a preliminary experiment, active MMP- bands were identi ed in parallel with the UCT- sample at different doses of protein (data not shown). Differences in the level of expression were evaluated using Mann±Whitney U-test. Patient outcome was evaluated in relation to activated MMP- expression using the Kaplan-Meier method. The patients were separated into high- and low-expression groups, the threshold value for activated MMP- expression being determined by ROC analysis. The outcome was compared between subgroups using a log-rank test, with statistical signi cance established at P<.. The probabilities of survival and relative hazards are given with 9% CIs. Results Bands of latent and activated MMP- and MMP-9 are shown in Fig. ; the lowest bands are activated MMP-. The expression of activated and total MMP- was plotted for each case (Fig. ); there was a strong correlation between them (r=.9). The correlation of activated and total MMP- with conventional clinicopathological variables (histological grade and pathological stage) was also assessed. The expression of activated MMP- was higher in G than in either G or G tumours (Fig. A), but there was no signi cant difference between G and G tumours (Fig. B). Total MMP- expression levels also increased with increasing grade (Fig. B). The mean (SEM) expression of activated MMP- of pta tumours was. (.), of pt. (.), of pt. (.), of pt. (.) and of pt. (.). There was a signi cant difference in activated MMP- expression between muscle-invasive (opt) tumours and super- Fig.. Gelatine zymography of eight urothelial cancer tissue extract samples (plus a standard in each gel), showing the kda latent form of MMP-. The lowest band is activated MMP- and the 9 kda band latent MMP-9. # BJU International, ±

3 ACTIVATED MATRIX METALLOPROTEINASE- IN UROTHELIAL CANCER Total MMP- levels R =.9 increase with progression in tumour grade and stage, but there were no signi cant differences among the two groups (data not shown). Patient outcome was evaluated for activated MMP- levels using the Kaplan±Meier method. The threshold values for de ning the two groups of patients were. for activated MMP- and. for total MMP- (from ROC analysis, data not shown). The high-expression group for both activated and total MMP- had signi cantly worse cause-speci c survival than the low-expressionr group (P=. and P=., respectively, Fig. ). However, within the groups of patients with either muscle-invasive (opt) or ptax tumours there was no signi cant difference in causespeci c survival between the groups with high and low expression of activated MMP- (data not shown)..... Activated MMP- levels Fig.. The correlation between activated and total MMP- levels in urothelial cancer samples. cial (ptax) tumours (P<., Fig. C). Total MMP- expression in muscle-invasive (opt) tumours was also signi cantly higher than in super cial (ptax) tumours (Fig. D). Any correlation in the expression of MMP-9 with the conventional clinicopathological variables was also assessed. The levels of MMP-9 tended to Discussion Liotta et al. [] reported that the expression of MMPs in cancer cells was correlated with metastatic potential, and that this enzyme plays an important role in both cancer invasion and metastasis. MMP- ( kda Type IV collagenase, gelatinase A) is active against gelatine and basement membrane components (type IV collagen and laminin) [], and is thought to play an important role in invasion and metastasis in a wide range of cancer types. Fig.. The expression of activated (a,c) or total (b,d) MMP- and histological grade (a,b) or pathological stage (c,d; combined pta, pt or o pt) in 9 urothelial cancers (TCC). The limits of the box represent the th and th percentiles, the central horizontal line the sample median, and the central vertical lines the th and 9th percentiles, with outlying points shown as circles. Activated MMP- levels a P <. N.S. P <. G G G Total MMP- levels b P <. N.S. N.S. G G G c d P <. P =. Activated MMP- levels Total MMP- levels pta- pt pta- pt # BJU International, ±

4 K. KANDA et al. Cause specific survival rate (%) a 9 P =. Follow-up period (months) Cause specific survival rate (%) b 9 P =. Follow-up period (months) Fig.. Kaplan±Meier cause-speci c survival curves for groups of patients categorized by activated (a) or total (b) MMP- expression, as high (red, patients in a and 9 in b) or low (green, patients in a and in b). We previously evaluated the mrna expression of MMP-, TIMP- and MT-MMP in bladder cancer tissue specimens using semi-quantiatative RT-PCR []. Levels of MMP- and TIMP- expression were higher in muscle-invasive bladder cancer tissue than in low-stage ptax tumours. Talvensaari-Mattila et al. [] used immunohistochemistry to show that MMP- positivity was associated with a shorter survival independent of major prognostic indicators in patients with breast cancer. However, mrna expression and immunohistochemical analysis are ineffective for resolving latent and activated MMP-. The relative expression of activated MMP- can be differentiated using gelatine zymography. Davies et al. [9] reported that the expression of MMP-9 and the activated form of MMP- were correlated with tumour grade and stage in bladder cancer, using quantitative gelatine zymography. The same result was obtained for activated MMP- in the present study, but although the expression of MMP-9 increased with tumour grade and stage, the differences were not signi cant. The present expression of activated and total MMP- in invasive tumours were signi cantly higher than they were in super cial tumours, indicating that MMP- expression in urothelial tumours is highly correlated with tumour invasion. There was also a strong correlation between the level of activated MMP- and those of total MMP-. Both expression levels were associated with tumour invasion, but the results suggested that activated MMP- expression is a better indicator of tumour invasion. Moreover, patients with tumours that had a high expression of activated and total MMP- had a signi cantly worse cause-speci c survival than those with low expression. A high expression of activated MMP- was more closely associated with an unfavourable prognosis than was total MMP- expression. As far as we are aware, this study represents the rst report of an association between activated MMP- expression and shorter survival in patients with urothelial cancer. In summary, activated and total MMP- both play an important role in urothelial cancer invasion and progression. In particular, activated MMP- is a more effective protease than total MMP-. Furthermore, activated MMP- is particularly associated with patient outcome, suggesting that the level of activated MMP- is an important prognostic indicator in urothelial cancer. Acknowledgements This work was supported in part by a grant-in aid ()() for Scienti c Research from the Ministry of Education, Science and Culture of Japan. References Stetler-Stevenson WG. Type IV collagenase in tumor invasion and metastasis. Cancer Metast Rev 99; 9: 9± Atkinson SJ, Ward RV, Reynolds JJ, Murphy G. Cellmediated degradation of type IV collagen and gelatin lms is dependent on the activation of matrix metalloproteinases. Biochem J 99; : ± Kieiner DE Jr, Stetler-Stevenson WG. Structural biochemistry and activation of matrix metalloproteinases. Curr Opin Cell Biol 99; : 9± Sato H, Takino T, Okade Y et al. A matrix metalloproteinase expressed on the surface of invasive tumor cells. Nature 99; : ± Mori M, Minori K, Shiraishi T et al. Analysis of MT-MMP and MMP- expression in human gastric cancers. Int J Cancer 99; : ± Yamagata S, Yoshii Y, Suh JG, Tanaka R, Shimizu S. Occurrence of an active form of gelatinase in human gastric and colorectal carcinoma tissues. Cancer Lett 99; 9: ± Koshiba T, Hosotani R, Wada M et al. Involvement of matrix metalloproteinase- activity in invasion and metastasis of pancreatic carcinoma. Cancer 99; : ± Lee KS, Rha SY, Kim SJ et al. Sequential activation and production of matrix metalloproteinase- during breast cancer progression. Clin Exp Metastasis 99; : ±9 # BJU International, ±

5 ACTIVATED MATRIX METALLOPROTEINASE- IN UROTHELIAL CANCER 9 Davies B, Waxman J, Wasan H et al. Levels of matrix metalloproteinase in bladder cancer correlate with tumor grade and invasion. Cancer Res 99; : ±9 Lipponen PK, Eskelinen MJ, Kiviranta J, Pesonen E. Prognosis of transitional cell bladder cancer: a multivariate prognostic score for improved prediction. J Urol 99; : ± Heussen C, Dowdle EB. Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Ann Biochem 9; : 9± Mosto FK, Sobin LH, Torloni H. Histological typing of urinary bladder tumors. Geneva: World Health Organization, 9 Hermanek P, Sobin H, eds. International Union against Cancer (UICC). TNM Classi cation of Malignant Tumors. th edn. Geneva: Springer-Verlag 9 Furukawa A, Tsuji M, Nishitani M et al. Role of the matrix metalloproteinase and tissue inhibitors of metalloproteinase families in noninvasive and invasive tumors transplanted in mice with severe combined immunode ciency. Urology 99; : 9± Liotta LA, Tryggvason K, Garbisa S, Hart I, Folz CM, Sha e S. Metastatic potential correlates with enzymatic degradation of basement membrane collagen. Nature 9; : ± Liotta LA, Stetler-Stevenson WG. Metalloproteinases and cancer invasion. Semin Cancer Biol 99; : 99± Kanayama H, Yokota K, Kurokawa Y, Murakami Y, Nishitani M, Kagawa S. Prognostic values of matrix metalloproteinase- and tissue inhibitor of metalloproteinase- expression in bladder cancer. Cancer 99; : 9± Talvensaari-Mattila A, Paakko P, Hoyhtya M, Blanco- Sequeiros G, Turpeenniemi-Hujanen T. Matrix metalloproteinase- immunoreactive protein: a marker of aggressiveness in breast carcinoma. Cancer 99; : ± Authors K. Kanda, MD, Urologist. M. Takahashi, MD, Urologist. Y. Murakami, MD, Urologist. H. Kanayama, MD, Associate Professor of Urology. S. Kagawa, MD, Professor of Urology. Correspondence: Kazuya Kanda, Department of Urology, School of Medicine, The University of Tokushima, ±± Kuramoto, Tokushima ±, Japan. kkanda@clin.med.tokushima-u.ac.jp # BJU International, ±