INCREASED GELATINASE-A AND GELATINASE-B ACTIVITIES IN MALIGNANT VS. BENIGN BREAST TUMORS Roeland HANEMAAIJER 1 , Jan H. VERHEIJEN 1 * , Teresa M. MAGUIRE 3 , Hetty VISSER 1 , Karin TOET 1 , Enda MCDERMOTT 3 , Niall O’HIGGINS 3 and Michael J. DUFFY 2 1 Division of Vascular and Connective Tissue Research, TNO Prevention and Health, Gaubius Laboratory, Leiden, The Netherlands 2 Nuclear Medicine Department, St Vincent’s Hospital, Dublin, Ireland 3 Department of Surgery University College Dublin, Dublin, Ireland Matrix metalloproteinases (MMP) types 2 and 9 (also known as gelatinase A and B) are thought to be causally involved in cancer invasion and metastasis. In normal as well as in malignant tissue, both these MMPs occur in multiple forms such as inactive precursors, active enzymes and en- zyme-inhibitor complexes. Using newly developed quantita- tive activity assays, the levels of active MMP-2, total (active and activatable) MMP-2 and total MMP-9 were found to be significantly higher in breast carcinomas than in fibroadeno- mas. In addition, active MMP-2 and MMP-9 were detected more frequently in malignant than in benign breast carci- noma. These new quantitative activity assays are likely to be of use in studying the mechanism of action of both MMP-2 and -9, assessing their potential prognostic value in different cancers and in the design of MMP inhibitors for preventing cancer metastasis. Int. J. Cancer 86:204 –207, 2000. © 2000 Wiley-Liss, Inc. Matrix metalloproteinases (MMPs) are zinc-dependent en- dopeptidases causally involved in tumor progression (for review, see Woessner, 1991; Chambers et al., 1997). Currently at least 19 different MMPs have been identified in mammalian systems. While some of these MMPs appear to be involved in stimulating tumor cell growth, others are causally involved in invasion and metastasis (Chambers et al., 1997). Two of the MMPs implicated in the spread of cancer are MMP-2 and -9, also known as gelati- nase A and B. These MMPs are thought to mediate invasion and metastasis by catalysing degradation of type IV collagen, the main component of basement membranes. MMP-2 and -9 can however, also degrade other substrates in the extracellular matrix such as types V, VII, IX and X collagen as well as fibronectin and elastin (MacDougal and Matrisian, 1995). MMP-2 and -9, like all other known mammalian MMPs, are initially synthesised as inactive precursors. The mechanism of activation in vivo is largely un- known but is likely to involve proteolytic processes mediated by other MMPs and/or serine proteases (Seiki et al., 1997; Carmeliet et al., 1997). Activation in vitro can be brought about by a variety of agents including organomercurials, alkylating agents, oxidants and other proteinases (Woessner, 1991; Aznavoorian et al., 1993). In biological samples, MMPs can occur in different forms such as inactive pro-enzymes, active enzymes and active enzymes com- plexed with various inhibitors and inactive pro-enzymes com- plexed with inhibitors. Although different types of assay such as immunohistochemistry, ELISA and gelatin zymography are used to detect MMPs (Duffy and McCarthy, 1998; Sier et al., 1996), only the latter can differentiate between precursor and active forms. However, zymography is a tedious procedure and only semi-quantitative. We have described the development of simple, novel and spe- cific activity assays that can quantitatively detect active as well as pro-enzyme forms of both MMP-2 and -9 in biological samples (Verheijen et al., 1997; Hanemaaijer et al., 1998; Capper et al., 1999). These assays are based on a modified urokinase plasmino- gen activator, in which the plasmin activation site has been adapted to an activation site which is only recognised by MMPs. Specific- ity for either MMP-2 or MMP-9 is introduced by a specific capture antibody. As these assays can be performed with microtiter plates, they are particularly convenient when assaying large numbers of samples. In this report, we have used these assays as well as traditional ELISAs to measure MMP-2 and -9 in extracts of benign breast tumors (fibroadenomas) and breast carcinomas. MATERIAL AND METHODS Breast tumors Seventy-one primary breast carcinomas and 17 fibroadenomas were analysed in our study. Following histological examination, the samples were frozen in liquid nitrogen and then transferred to a -80°C freezer. The samples were homogenised in 50 mM Tris-HCl buffer (pH 7.4). The homogenate was centrifuged at 2000g for 10 min and the supernatants used for MMP assays. The main characteristics of the carcinomas used are summarised in Table I. Reagents The MMP-2 ELISA (Biotrak RPN 2617) was purchased from Amersham Pharmacia Biotech (Aylesbury, UK). The urokinase specific substrate L-pyroGlu-Gly-L-Arg-para-nitroanilide (S-2444) was obtained from Chromogenix (Mo ¨lndal, Sweden). Aminophe- nyl-mercuric acetate (APMA) was purchased from Sigma (St. Louis, MO). Modified urokinase was expressed and purified as described by Verheijen et al. (1997). MMP-2 and MMP-9 activity assays Activation of pro-MMP-2 and pro-MMP-9 was carried out by incubation of their pro-forms with APMA (0.5 mM final concen- tration in assay buffer: 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM CaCl 2 ,1 M ZnCl 2 and 0.01% (v/v) Brij-35) at 37°C for 30 min and 2 hr, respectively. At these combinations of activation- time and concentration of APMA, optimum activities were ob- tained (range tested 0 –16 hr and 0 –2 mM APMA). The MMP-9 activity assay was carried out as described by Hanemaaijer et al.. (1998). In brief, MMP-9 was captured from the *Correspondence to: Gaubius Laboratory, TNO-PG, P.O. Box 2215, 2301 CE Leiden, The Netherlands. Fax: +31-71-5181904. E-mail: JH.Verheijen@pg.tno.nl Received 9 July 1999; Revised 28 October 1999 TABLE I – ESTROGEN RECEPTOR (ER) STATUS, NODAL STATUS AND TUMOR SIZE OF BREAST CARCINOMAS ANALYSED n % ER status Negative 14 20 Positive 56 79 Unknown 1 1 Nodal status Negative 28 39 Positive 36 51 Unknown 7 10 Tumor size  2 cm 21 30  2 cm 45 63 Unknown 5 7 Int. J. Cancer: 86, 204 –207 (2000) © 2000 Wiley-Liss, Inc. Publication of the International Union Against Cancer