ASSOCIATION OF MMP-2 ACTIVATION POTENTIAL WITH METASTATIC PROGRESSIONIN HUMAN BREAST-CANCER CELL-LINES INDEPENDENT OF MMP-2 PRODUCTION

Background: Expression of matrix metalloproteinase-2 (MMP-2), the 72-k d type IV collagenase/gelatinase, by cancer cells has been implicated in metastasis through cancer cell invasion of basement membranes media ted by degradation of collagen IV. However, the abundance of this late nt proenzyme in normal tissues and fluids suggests that MMP-2 pro-enzy me utilization is limited by its physiological activation rather than expression alone. We previously reported activation of this proenzyme by normal and malignant fibroblastoid cells cultured on collagen I (vi trogen) gels. Purpose: Our purposes in this study were 1) to determine whether MMP-2 activation is restricted to the more invasive human bre ast cancer cell lines and 2) to localize the activating mechanism. Met hods: Zymography was used to monitor MMP-2 activation through detectio n of latent MMP-2 (72 kd) and mature species of smaller molecular weig ht (59 or 62 kd). Human breast cancer cell lines cultured on plastic, vitrogen, and other matrices were thus screened for MMP-2 activation. Collagen I-cultured cells were exposed to cycloheximide, a protein syn thesis inhibitor, or to protease inhibitors to determine the nature of the MMP-2-activating mechanism. Triton X-114 (TX-114) detergent extra cts from cells cultured on collagen I or plastic were incubated with l atent MMP-2 and analyzed by zymography to localize the MMP-2 activator . Results: MMP-2 activation was only induced by collagen I culture in the more aggressive, highly invasive estrogen receptor-negative, vimen tin-positive human breast cancer cell lines (Hs578T, MDA-MB-436, BT549 , MDA-MB-231, MDA-MB-435, MCF-7ADR) and was independent of MMP-2 produ ction. MMP-2 activation was detected in cells cultured on collagen I g els but not in those cultured on gelatin gels, Matrigel, or thin layer s of collagen I or IV, gelatin, or fibronectin. Collagen-induced activ ation was specific for the enzyme species MMP-2, since MMP-9, the 92-k d type IV collagenase/gelatinase, was not activatable under similar co nditions. MMP-2 activation was inhibited by cycloheximide and was sens itive to a metalloproteinase inhibitor but not to aspartyl, serine, or cysteinyl protease inhibitors. MMP-2 activation was detected in the h ydrophobic, plasma membrane-enriched, TX-114 extracts from invasive co llagen I-cultured cells. Conclusion: Collagen I-induced MMP-2 activati on is restricted to highly invasive estrogen receptor-negative, viment in-positive human breast cancer cell lines, is independent of MMP-2 pr oduction, and is associated with metastatic potential. Our findings ar e consistent with plasma membrane localization of the activator. Impli cations: The MMP-2 activation mechanism may represent a new target for diagnosis, prognosis, and treatment of human breast cancer.