Abstract
The formin protein dishevelled-associated activator of morphogenesis 1 (DAAM1) polymerizes straight actin filaments and mediates migration of cancer cells. However, how DAAM1 governs cell haptotaxis in response to collagen remains unexplored in breast cancer cells. We hypothesized that DAAM1 mediates invadopodia extension and cell haptotaxis in response to type IV collagen in association with integrin receptors. Using Boyden chamber membranes coated with type IV collagen, we show here that type IV collagen activates both DAAM1 and Ras homolog family member A (RHOA) and promotes haptotaxis of MDA-MB-231 and MDA-MB-453 breast cancer cells, a process abolished by treatment with the integrin αvβ3 inhibitor cyclo(-RGDfK). shRNA-mediated knockdown of DAAM1 or a dominant-negative DAAM1 mutation (N-DAAM1) significantly decreased collagen-induced RHOA activity and the assembly of stress fibers, invadopodia extension, and cell haptotaxis. Immunoprecipitation and pulldown assays revealed that integrin αvβ3 is associated with, but only indirectly binds to, the C-terminal DAD domain of DAAM1 in mammalian cells. Blockade of RHOA activation with a specific inhibitor (CCG-1423) or via a dominant-negative RHOA mutation (RHOA-N19) suppressed collagen-induced invadopodia extension and haptotaxis of the MDA-MB-231 and MDA-MB-453 cells. Immunoblotting and immunofluorescence assays indicated high DAAM1 and RHOA expression in invadopodia, which was abolished by cyclo(-RGDfK) treatment or DAAM1 knockdown. These findings have uncovered an integrin αvβ3/DAAM1/RHOA signaling pathway for type IV collagen-induced invadopodia extension and haptotaxis in breast cancer cells. Targeting this pathway may be a means for reducing invasiveness and metastasis of breast cancer.
Keywords:
DAAM1; RHOA; actin; breast cancer; haptotaxis; integrin; integrin αvβ3; invadopodia; migration.