Adhesion of tumor cells to host cell layers and subsequent transcellular migration are pivotal steps in cancer invasion and metastasis. The small GTPase Rho controls cell adhesion and motility through reorganization of the actin cytoskeleton and regulation of actomyosin contractility. Cultured rat MM1 hepatoma cells migrate through a mesothelial cell monolayer in vitro in a serum-dependent, Rho-mediated manners. Among several proteins isolated as putative target molecules of Rho, the ROCK (ROK) family of Rho-associated serine-threonine protein kinases are thought to participate in the induction of focal adhesions and stress fibers in cultured cells, and to mediate calcium sensitization of smooth muscle contraction by enhancing phosphorylation of the regulatory light chain of myosin. Transfection of MM1 cells with cDNA encoding a dominant active mutant of ROCK conferred invasive activity independently of serum and Rho. In contrast, expression of a dominant negative, kinase-defective ROCK mutant substantially attenuated the invasive phenotype. A specific ROCK inhibitor (Y-27632) blocked both Rho-mediated activation of actomyosin and invasive activity of these cells. Furthermore, continuous delivery of this inhibitor using osmotic pumps considerably reduced the dissemination of MM1 cells implanted into the peritoneal cavity of syngeneic rats. These results indicate that ROCK plays an essential part in tumor cell invasion, and demonstrate its potential as a therapeutic target for the prevention of cancer invasion and metastasis.

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Protein tyrosine phosphorylation accompanies and is essential for integrin signaling. We have shown that tyrosine phosphorylation of paxillin ␣ and Crk-associated substrate (p130 Cas ) is a prominent event on integrin activation in normal murine mammary gland epithelial cells. Tyrosine phosphorylation of p130 Cas has been demonstrated to facilitate cell migration. We show here that tyrosine phosphorylation of paxillin ␣ acts to reduce haptotactic cell migrations as well as transcellular invasive activities in several different experimental cell systems, whereas tyrosine phosphorylation of p130 Cas exerts opposing effects to those of paxillin ␣. Each of the phosphorylation-null mutants acts as a dominant negative for each phenotype. Moreover, we found that overexpression of paxillin ␣ reduced the cell saturation density of normal murine mammary gland cells, whereas overexpression of p130 Cas increased it. These effects also seemed to depend on tyrosine phosphorylation events. Cell growth rates and morphologies at growing phases were not significantly altered, nor were cells transformed. Addition of epidermal growth factor increased saturation density of the paxillin ␣-overexpressing cells, whereas no further increment was observed in p130 Cas -overexpressing cells. We propose that tyrosine phosphorylation of paxillin ␣ and p130 Cas exerts opposing effects on several integrin-mediated cellular events, possibly through different signaling pathways.