title: Association of MMP-2 activation potential with metastatic progression in human breast cancer cell lines independent of MMP-2 production.
abstract: BACKGROUND
  Expression of matrix metalloproteinase-2 (MMP-2), the 72-kd type IV collagenase/gelatinase, by cancer cells has been implicated in metastasis through cancer cell invasion of basement membranes mediated by degradation of collagen IV. However, the abundance of this latent proenzyme in normal tissues and fluids suggests that MMP-2 proenzyme 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 (vitrogen) gels.
  
  
  OBJECTIVE
  Our purposes in this study were 1) to determine whether MMP-2 activation is restricted to the more invasive human breast cancer cell lines and 2) to localize the activating mechanism.
  
  
  METHODS
  Zymography was used to monitor MMP-2 activation through detection of latent MMP-2 (72 kd) and mature species of smaller molecular weight (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 synthesis inhibitor, or to protease inhibitors to determine the nature of the MMP-2-activating mechanism. Triton X-114 (TX-114) detergent extracts from cells cultured on collagen I or plastic were incubated with latent 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, vimentin-positive human breast cancer cell lines (Hs578T, MDA-MB-436, BT549, MDA-MB-231, MDA-MB-435, MCF-7 ADR) and was independent of MMP-2 production. MMP-2 activation was detected in cells cultured on collagen I gels but not in those cultured on gelatin gels, Matrigel, or thin layers of collagen I or IV, gelatin, or fibronectin. Collagen-induced activation was specific for the enzyme species MMP-2, since MMP-9, the 92-kd type IV collagenase/gelatinase, was not activatable under similar conditions. MMP-2 activation was inhibited by cycloheximide and was sensitive to a metalloproteinase inhibitor but not to aspartyl, serine, or cysteinyl protease inhibitors. MMP-2 activation was detected in the hydrophobic, plasma membrane-enriched, TX-114 extracts from invasive collagen I-cultured cells.
  
  
  CONCLUSIONS
  Collagen I-induced MMP-2 activation is restricted to highly invasive estrogen receptor-negative, vimentin-positive human breast cancer cell lines, is independent of MMP-2 production, and is associated with metastatic potential. Our findings are consistent with plasma membrane localization of the activator.
  
  
  CONCLUSIONS
  The MMP-2 activation mechanism may represent a new target for diagnosis, prognosis, and treatment of human breast cancer.
authors.affiliation: Vincent T. Lombardi Cancer Research Center, Georgetown University Medical Center, Washington DC, 20007.
authors.forename: H S
authors.initials: HS
authors.lastname: Azzam
authors.forename: G
authors.initials: G
authors.lastname: Arand
authors.forename: M E
authors.initials: ME
authors.lastname: Lippman
authors.forename: E W
authors.initials: EW
authors.lastname: Thompson
mesh_terms: D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D003094:Collagen / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D005455:Fluorescent Antibody Technique; D018093:Gelatinases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D020778:Matrix Metalloproteinase 2; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D009362:Neoplasm Metastasis; D011480:Protease Inhibitors / Q000494:pharmacology; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
chemical_list: D011480:Protease Inhibitors; D003094:Collagen; D018093:Gelatinases; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2