Mobility and invasiveness of metastatic esophageal cancer are potentiated by shear stress in a ROCK- and Ras-dependent manner

1 Department of Clinical Pharmacology and 2 Department of Biochemistry, Royal College of Surgeons, Dublin; and 3 Cork Cancer Research Centre, Mercy University Hospital, University College Cork, Cork, Ireland Submitted 14 December 2005 ; accepted in final form 22 February 2006 To metastasize, tumor cells must adopt different morphological responses to resist shear forces encountered in circulating blood and invade through basement membranes. The Rho and Ras GTPases play a critical role in regulating this dynamic behavior. Recently, we demonstrated shear-induced activation of adherent esophageal metastatic cells, characterized by formation of dynamic membrane blebs. Although membrane blebbing has only recently been characterized as a rounded mode of cellular invasion promoted through Rho kinase (ROCK), the role of shear forces in modulating membrane blebbing activity is unknown. To further characterize membrane blebbing in esophageal metastatic cells (OC-1 cell line), we investigated the role of shear in cytoskeletal remodeling and signaling through ROCK and Ras. Our results show that actin and tubulin colocalize to the cortical ring of the OC-1 cell under static conditions. However, under shear, actin acquires a punctuate distribution and tubulin localizes to the leading edge of the OC-1 cell. We show for the first time that dynamic bleb formation is induced by shear alone independent of integrin-mediated adhesion ( P < 0.001, compared with OC-1 cells). Y-27632, a specific inhibitor of ROCK, causes a significant reduction in shear-induced bleb formation and inhibits integrin v 3 -Ras colocalization at the leading edge of the cell. Direct measurement of Ras activation shows that the level of GTP-bound Ras is elevated in sheared OC-1 cells and that the shear-induced increase in Ras activity is inhibited by Y-27632. Finally, we show that shear stress significantly increases OC-1 cell invasion ( P < 0.007), an effect negated by the presence of Y-27632. Together our findings suggest a novel physiological role for ROCK and Ras in metastatic cell behavior. cytoskeletal remodeling; dynamic blebs Address for reprint requests and other correspondence: D. Kenny, Dept. of Clinical Pharmacology, Royal College of Surgeons, 123 St. Stephens Green, Dublin 2, Ireland (e-mail: dkenny{at}rcsi.ie )