Inhibition of migration and invasion of carcinoma cells by urokinase-derived antagonists of alphavbeta5 integrin activation.
We previously showed that, while binding to urokinase receptor (uPAR) through its growth factor domain (GFD, residues 1-49), urokinase (uPA) can engage alphavbeta5 integrin through an internal domain (CP, residues 132-158). This novel uPA/alphavbeta5 interaction promotes cytoskeletal rearrangements and directional cell migration (Franco et al., J Cell Sci 2006;119:3424-34). We now show that treatment of cells with phosphomimic uPA (uPA138E/303E, serine 138 and 303 substituted with glutamic acid) strongly inhibits matrix-induced cell migration. Unlike uPA, binding of uPA138E/303E to cell surface did not induce F-actin enriched protruding structures and caused a 5-fold reduction in cell translocation speed, as determined by video tracking of living cells. Inhibition of migration was found to be independent of uPAR, since uPA variants lacking the GFD domain, but carrying the relevant Ser to Glu substitutions were as effective inhibitor as uPA138E/303E. Through several independent approaches, we established that the phosphomimics specifically bind to alphavbeta5 integrin through the CP region carrying the S138E mutation. This interaction blocks integrin activation, as determined by a decreased affinity of alphavbeta5 to vitronectin and a reduced association of the beta5 cytoplasmic tail with talin. Finally, stable expression of uPA138E/303E in human squamous carcinoma cells prevented tumor cell invasion in vivo. Thus, when expressed in cancer cells, the inhibitory phosphomimic effect was dominant over the effect of endogenously produced uPA. These results shed light on the regulation of cell migration by uPA phosphorylation and provide a realistic opportunity for a novel antiinvasive/metastatic therapeutic intervention.
Institute of Genetics and Biophysics Adriano Buzzati-Traverso, National Research Council, Via Castellino, 111, Naples, Italy.
Immacolata
I
Vocca
Paola
P
Franco
Daniela
D
Alfano
Giuseppina
G
Votta
Maria Vincenza
MV
Carriero
Yeriel
Y
Estrada
Mario
M
Caputi
Paolo A
PA
Netti
Liliana
L
Ossowski
Maria Patrizia
MP
Stoppelli
D002277:Carcinoma / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D002465:Cell Movement; D002633:Chemotaxis; D003599:Cytoskeleton / Q000378:metabolism; D020868:Gene Silencing; D006801:Humans; D016023:Integrins / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D009361:Neoplasm Invasiveness; D017434:Protein Structure, Tertiary; D019030:Receptors, Vitronectin / Q000037:antagonists & inhibitors* / Q000737:chemistry; D016608:Talin / Q000737:chemistry; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry*
D016023:Integrins; D019030:Receptors, Vitronectin; D016608:Talin; C064660:integrin alphaVbeta5; D014568:Urokinase-Type Plasminogen Activator