TY - JOUR
T1 - Inhibition of migration and invasion of carcinoma cells by urokinase-derived antagonists of αvβ5 integrin activation
AU - Vocca, Immacolata
AU - Franco, Paola
AU - Alfano, Daniela
AU - Votta, Giuseppina
AU - Carriero, Maria Vincenza
AU - Estrada, Yeriel
AU - Caputi, Mario
AU - Netti, Paolo A.
AU - Ossowski, Liliana
AU - Stoppelli, Maria Patrizia
PY - 2009/1/15
Y1 - 2009/1/15
N2 - We previously showed that, while binding to urokinase receptor (uPAR) through its growth factor domain (GFD, residues 1-49), urokinase (uPA) can engage αvβ5 integrin through an internal domain (CP, residues 132-158). This novel uPA/αvβ5 interaction promotes cytoskeletal rearrangements and directional cell migration (Franco et al., J Cell Sci 2006;119:3424-34). We nowshow that treatment of cells with phosphomimic uPA (uPA 138E/303E, serine 138 and 303 substituted with glutamic acid) strongly inhibits matrix-induced cell migration. Unlike uPA, binding of uPA 138E/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 uPA 138E/303E. Through several independent approaches, we established that the phosphomimics specifically bind to αvβ5 integrin through the CP region carrying the S138E mutation. This interaction blocks integrin activation, as determined by a decreased affinity of αvβ5 to vitronectin and a reduced association of the β5 cytoplasmic tail with talin. Finally, stable expression of uPA 138E/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.
AB - We previously showed that, while binding to urokinase receptor (uPAR) through its growth factor domain (GFD, residues 1-49), urokinase (uPA) can engage αvβ5 integrin through an internal domain (CP, residues 132-158). This novel uPA/αvβ5 interaction promotes cytoskeletal rearrangements and directional cell migration (Franco et al., J Cell Sci 2006;119:3424-34). We nowshow that treatment of cells with phosphomimic uPA (uPA 138E/303E, serine 138 and 303 substituted with glutamic acid) strongly inhibits matrix-induced cell migration. Unlike uPA, binding of uPA 138E/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 uPA 138E/303E. Through several independent approaches, we established that the phosphomimics specifically bind to αvβ5 integrin through the CP region carrying the S138E mutation. This interaction blocks integrin activation, as determined by a decreased affinity of αvβ5 to vitronectin and a reduced association of the β5 cytoplasmic tail with talin. Finally, stable expression of uPA 138E/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.
KW - αVβ5 antagonist
KW - Cell cytoskeleton
KW - Cell migration inhibitor
KW - In vivo carcinoma invasion
KW - Upa phosphorylation
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U2 - 10.1002/ijc.23933
DO - 10.1002/ijc.23933
M3 - Article
C2 - 18844213
AN - SCOPUS:58249092372
VL - 124
SP - 316
EP - 325
JO - International Journal of Cancer
JF - International Journal of Cancer
SN - 0020-7136
IS - 2
ER -