Role of urokinase plasminogen activator receptor in thrombospondin 1-mediated tumor cell invasion

We previously showed that thrombospondin 1 (TSP-I) upregulates the plasmino gen/plasmin system and promotes breast tumor cell invasion. Preliminary dat a from our laboratory using neutralizing antibodies suggested that the upre gulation in breast tumor cell invasion seen in response to TSP-1 involved t he urokinase plasminogen activator receptor (uPAR). To confirm these findin gs in MDA-MB-231 breast cancer cells, we developed three other strategies t o study the role of uPAR in tumor cell adhesion and TSP-1-mediated tumor ce ll invasion: (a) enzymatic cleavage of uPAR with glycosylphosphatidylinosit ol-specific phospholipase C; (b) inhibition at the mRNA level with a uPAR a ntisense construct (cells named LKAS-MDA); (c) inhibition of plasminogen bi nding with the lysine analogue epsilon-aminocaproic acid. Adhesion to lamin in and type I and type TV collagen with and without the addition of epsilon -aminocaproic acid was studied. Tumor cell invasion was studied in a modifi ed Boyden chamber collagen invasion assay. Antisense uPAR inhibition decrea sed uPAR expression by 48-66% and cell-associated urokinase plasminogen act ivator (uPA) by 30-68%. Additionally, antisense uPAR inhibition induced a 6 8-70% reduction in uPA and plasmin activities. Antisense uPAR transfection increased tumor cell adhesion by 46-53%. A similar effect was observed in e psilon-aminocaproic acid-treated MDA-MB-231 cells. TSP-1-mediated tumor cel l invasion was almost completely inhibited by either antisense uPAR inhibit ion or treatment with phospholipase C or epsilon-aminocaproic acid. We conc lude that uPAR plays a crucial role in the regulation of tumor cell adhesio n and TSP-l-mediated tumor cell invasion. (C) 1999 Academic Press.