title: Urokinase-type plasminogen activation in three human breast cancer cell lines correlates with their in vitro invasiveness.
abstract: In order to invade and spread cancer cells must degrade extracellular matrix proteins. This degradation is catalysed by the concerted action of several enzymes, including the serine protease plasmin. Several experimental studies have shown that inhibition of plasmin formation reduces cancer cell invasion and metastasis, indicating a critical role of this proteolytic pathway in these processes. In order to further study the role of plasmin in cancer progression, we have characterized urokinase-type plasminogen activator (uPA) mediated plasmin formation in three human breast cancer cell lines. Using monoclonal antibodies against uPA and its receptor uPAR, we have investigated the contribution of uPA and uPAR to invasive capacity in an in vitro invasion assay. MDA-MB-231 BAG cells were found to express high protein levels of uPA, uPAR and PAI-1. MDA-MB 435 BAG cells produced low amounts of uPA, PAI-1 and moderate amounts of uPAR, whereas MCF-7 BAG cells showed low levels of uPA, uPAR and PAI-1 protein. In a plasmin generation assay MDA-MB-231 BAG cells were highly active in mediating plasmin formation, which could be abolished by adding either an anticatalytic monoclonal antibody to uPA (clone 5) or an anti-uPAR monoclonal antibody (clone R3), which blocks binding of uPA to uPAR. The two other cell lines lacked the capacity to mediate plasmin formation. In the Matrigel invasion assay the cells showed activity in this order: MCF-7 BAG < MDA-MB-435 BAG < MDA-MB-231 BAG. Testing MDA-MB-231 BAG cells in the Matrigel invasion assay revealed that invasion could be inhibited in a dose-dependent manner either by the clone 5 uPA antibody or by the clone R3 uPAR antibody, suggesting that the cell surface uPA system is actively involved in this invasive process. It is concluded that these three cell lines constitute a valuable model system for in vitro studies of the role of cell surface uPA in cancer cell invasion and has application in the search for novel compounds which inhibit mechanisms involved in uPA-mediated plasmin generation on cancer cells.
authors.affiliation: The Finsen Laboratory, Copenhagen, Denmark.
authors.forename: C
authors.initials: C
authors.lastname: Holst-Hansen
authors.forename: B
authors.initials: B
authors.lastname: Johannessen
authors.forename: G
authors.initials: G
authors.lastname: Høyer-Hansen
authors.forename: J
authors.initials: J
authors.lastname: Rømer
authors.forename: V
authors.initials: V
authors.lastname: Ellis
authors.forename: N
authors.initials: N
authors.lastname: Brünner
mesh_terms: D000911:Antibodies, Monoclonal; D001665:Binding Sites; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D003432:Cross-Linking Reagents; D017077:Culture Media, Conditioned; D005260:Female; D005341:Fibrinolysin / Q000096:biosynthesis; D006801:Humans; D009361:Neoplasm Invasiveness; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000032:analysis; D011956:Receptors, Cell Surface / Q000032:analysis / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis / Q000276:immunology / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D017077:Culture Media, Conditioned; C524898:PLAUR protein, human; D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; C086951:urokinase-type plasminogen activator (6-135); D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator