[CANCER RESEARCH 62, 283–289, January 1, 2002] Cancer Cell-associated Fibronectin Induces Release of Matrix Metalloproteinase-2 from Normal Fibroblasts 1 Sonia Saad, David J. Gottlieb, Kenneth F. Bradstock, Christopher M. Overall, and Linda J. Bendall 2 Westmead Institute for Cancer Research, Westmead Millennium Institute [S. S., L. J. B.], Department of Medicine, University of Sydney [D. J. G.], Department of Hematology, Westmead Hospital [K. F. B.], Westmead. NSW. 2145, Australia, and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver V6T 123, Canada [C. M. O.] ABSTRACT The bone and bone marrow are the most common sites of metastasis in breast cancer. Matrix metalloproteinases (MMPs), particularly MMP-2, produced by cancer cells or, more typically, induced in the adjacent normal stroma are necessary for the degradation of extracellular matrix essential for cancer metastasis. Here we describe a mechanism by which breast cancer cells can rapidly use MMP-2 produced by bone marrow fibroblasts (BMFs). MMP-2 is stored in an inactive conformation in association with the cell surface or extracellular matrix of BMFs. Cocul- tures of BMFs and the human breast cancer cell line MDA-MB-231 induce release of MMP-2 into the culture supernatant without up-regulation of MMP-2 synthesis in either cell. MMP-2 is present on the surface of BMFs and is displaced by MDA-MB-231 cells or by fibronectin or fragments of fibronectin containing the fibronectin type II modules. Moreover, when fibronectin is eluted from the surface of MDA-MB-231 cells, they lose the ability to induce the release of MMP-2 from BMFs. These data are consistent with the displacement of inactive MMP-2 bound to normal fibroblasts via its collagen-binding domain by fibronectin type II modules of cancer cell-associated fibronectin. Cancer cells can then use the pro- teinase to facilitate tissue invasion. Because an identical mechanism can be demonstrated using fibroblasts from different sources, it is likely to be important for the rapid movement of malignant cells into a variety of normal tissues. INTRODUCTION Breast cancer is one of the most common forms of cancer in women, but despite being responsive to hormonal manipulation and chemotherapy, relapse after treatment is common, particularly in patients presenting with metastatic disease (1). One of the most common sites of metastasis is bone and bone marrow, which may provide a favorable microenvironment for the growth of tumor cells and act as a reservoir of disease, allowing further hematogenous spread (2, 3). The process of tumor cell invasion and metastasis requires the degradation of connective tissue associated with vascular basement membranes and interstitial connective tissue (4, 5). Several lines of evidence strongly implicate MMPs, 3 particularly MMP-2, in this process. These include a positive correlation between MMP-2 expres- sion and invasive potential and the inhibition of metastasis formation in vivo by MMP inhibitors (4, 6, 7). Interestingly, MMP-2 is often associated with adjacent normal tissues rather than the tumor cells themselves, suggesting that neoplastic cells can use MMPs produced by normal cells to facilitate their egress from the tumor mass and potentially their entry into new sites (8). MDA-MB-231 and T47D cells have been shown to bind MMPs on their cell membranes by specific receptors, optimally locating these enzymes to maximize invasive potential (9). MMPs are a family of zinc-dependent enzymes consisting of a propeptide, catalytic, hinge, and COOH-terminal (hemopexin like) domains. All MMPs are produced in latent forms requiring catalytic removal of the propeptide domain for function. MMP-2 and MMP-9 are unique because of the inclusion of three fibronectin type II repeats within their catalytic domain (10). These repeats, referred to as the CBD, facilitate adhesion to collagen (11–13). MT-MMPs are a sub- family of MMPs which contain a transmembrane domain (14). MT1- MMP was the first physiological activator of MMP-2 discovered and is still likely to be the major activator of MMP-2 on the cell surface (15). Whereas all active MMPs can be inhibited by TIMPs, TIMP-2 has a biphasic effect on MMP-2 activation, potentiating at low con- centrations and inhibiting when present at high concentrations (15–17). We have demonstrated previously the importance of MMP-2 in the invasion of BMF monolayers by the invasive breast cancer cell lines MDA-MB-231 and BT-549 (18). Using a short-term coculture sys- tem, we have also shown that contact between the breast cancer cell line MDA-MB-231 and bone marrow-derived fibroblasts results in a rapid increase in the concentration of MMP-2 in the culture superna- tant (18). Here we demonstrate that the MMP-2 observed in the supernatant is primarily derived from MMP-2 bound to collagen associated with the BMF monolayer. MMP-2 is displaced from the collagen by fibronectin bound to the breast cancer cells. Once dis- placed from the BMF-associated collagen, the MMP-2 can be acti- vated by MT1-MMP/TIMP-2 complexes expressed by the breast cancer cells. This mechanism provides a rapid method by which neoplastic cells can use MMP-2 produced by normal cells to facilitate their invasion of normal tissues. MATERIALS AND METHODS Materials. The following antibodies were purchased: 4B4 (anti-1) from Coulter Clones, Hialeah, FL; 5D11 (anti-MMP-2) from Oncogene Research Products Cambridge, MA; 36006.211 (anti-hMMP-2 for immunofluorescent staining) from R&D Biomedicals, Minneapolis, MN; 556000 (antihuman fibronectin) from Cortex Biochem, San Leandro, CA; collagen antibody (MAB1334) from Chemicon International, Temecula, CA; SAMF from Sile- nus, Hawthorne, Vic, Australia; and goat antimouse immunoglobulins biotin or horseradish peroxidase conjugated from DAKO Corp., Carpinteria, CA. Marimistat was a kind gift from British Biotech Pharmaceuticals, Oxford, United Kingdom. Human recombinant MMP-9 was purchased from Chemicon International; human plasma fibronectin and laminin was from Life Technol- ogies, Inc., Rockville, MD; and hyaluronic acid, actinomycin-D, cyclohexam- ide, Triton X-100, phenylmethylsulfonyl fluoride, leupeptin, and EDTA was from Sigma Chemical Co., St. Louis, MO. Recombinant human CBD consist- ing of all three fibronectin Type II modules (Gly417-Cys631 of human MMP-2) was expressed and purified as published previously (11). Cells. The breast cancer cell lines MDA-MB-231 and T47D were grown in RPMI containing 10% (volume for volume) FCS, 0.25 units/ml insulin, 20 mM HEPES (pH 7.0), and 4 mM L-glutamine as described previously (18). Normal Received 6/18/01; accepted 10/31/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the Millennium Foundation and the University of Sydney Cancer Fund. Christopher M. Overall was supported by a Canada Research Chair in Metalloproteinase Biology and a grant from the Canadian Institutes of Health Research. 2 To whom requests for reprints should be addressed, at Westmead Institute for Cancer Research, Westmead Millennium Institute, Westmead Hospital, Westmead. NSW. 2145, Australia. Phone: 61 2 9845 9069; Fax: 61 2 9845 9102; E-mail: linda_bendall@wmi. usyd.edu.au. 3 The abbreviations used are: MMP, matrix metalloproteinase; CBD, collagen-binding domain; MT-MMP, membrane-type matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; BMF, bone marrow fibroblast; SAMF, sheep antimouse fluorescein. 283