Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell diapedesis
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Background The Rho GTPases comprise one of the eight subfamilies of the Ras superfamily of monomeric GTP-binding proteins and are involved in cytoskeletal organization. Previously, using a dominant negative construct, we demonstrated a role for RhoC GTPase in conferring invasive capabilities to PC-3 human prostate cancer cells. Further, we demonstrated that inactivation of RhoC led to morphological changes commensurate with epithelial to mesenchymal transition (EMT) and was accompanied by increased random, linear motility and decreased directed migration and invasion. EMT was related positively to sustained expression and activity of Rac GTPase. In the current study we analyze the individual roles of RhoA, RhoC and Rac1 GTPases in PC-3 cell directed migration, invasion and tumor cell diapedesis across a human bone marrow endothelial cell layer in vitro. Results Use of specific shRNA directed against RhoA, RhoC or Rac1 GTPases demonstrated a role for each protein in maintaining cell morphology. Furthermore, we demonstrate that RhoC expression and activation is required for directed migration and invasion, while Rac1 expression and activation is required for tumor cell diapedesis. Inhibition of RhoA expression produced a slight increase in invasion and tumor cell diapedesis. Conclusions Individual Rho GTPases are required for critical aspects of migration, invasion and tumor cell diapedesis. These data suggest that coordinated activation of individual Rho proteins is required for cells to successfully complete the extravasation process; a key step in distant metastasis.
KeywordsProstate cancer Invasion Diapedesis RhoA GTPase RhoC GTPase Rac GTPase
Fetal bovine serum
Bone marrow endothelial cell
Phosphate buffered saline
Normal goat serum
Small hairpin RNA
Epithelial to mesenchymal transition
We would like to thank Drs. Kirk Czymmek and Harry Yao for technical assistance, Dr. Robert Gorman for help with statistical analysis and the University of Delaware Prostate Cancer Working Group for thoughtful input. This study was funded in part by the Department of Defense Prostate Cancer Research Program (W81XWH-04-1-0225 and W81XWH-05-1-0005) and the University of Delaware Research Fund (to K.L.v.G.).
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