CRMP4a suppresses cell motility by sequestering RhoA activity in prostate cancer cells

Abstract

ABSTRACTObjectives: Distant metastasis is a critical factor for cancer-associated death. Our previous studies identified collapsin response mediator protein 4a (CRMP4a) as a metastasis suppressor in prostate cancers. Enhancing CRMP4 expression by promoter-targeted small activating RNAs reduced cell migration in vitro and abolished distal metastasis in mouse xenograft models. In this study, we investigated the mechanism for CRMP4a-mediated suppression of cell migration.Methods: PC-3 cells were stably infected with lentiviruses expressing CRMP4a cDNA or a shRNA sequence. Cytoskeletal organization was analyzed by measuring cellular focal adhesion area and number, percentage of cell area and lamellipodia numbers after phalloidin staining or anti-vinculin immunocytofluorescent staining. Cell migration was evaluated with TranswellTM chambers coated with MatriGel. RhoA activation was determined with a Rhotekin RBD agarose bead-based assay kit. Lentiviruses harboring RhoA-Q63L or RhoA-T19N mutant constructs were used to overexpress mutant RhoA proteins.Results: CRMP4a overexpression largely reduced while CRMP4a knockdown remarkably increased cytoskeletal organization in PC-3 cells. CRMP4a immunoprecipitation pulled down RhoA but not cdc42 or Rac1 proteins. Manipulating CRMP4a expression levels reversely altered active RhoA levels. Overexpression of RhoA active (Q63L) but not inactive (T19N) mutants reversed CRMP4a-mediated reduction of cancer cell migration while RhoA inhibitor Rhosin diminished CRMP4a shRNA-induced increase of cancer cell migration. CRMP4a overexpression also largely reduced cell spreading that was abolished by overexpressing RhoA active mutant.Conclusion: Our data demonstrated that CRMP4a interacts with RhoA and sequesters its activity, resulting in suppression of cytoskeletal organization, cell migration and spreading.