FilGAP, a GAP for Rac1, down-regulates invadopodia formation in breast cancer cells

Abstract

Invadopodia are protrusive structures that mediate the extracellular matrix (ECM) degradation required for tumor invasion and metastasis. Rho small GTPases regulate invadopodia formation, but the molecular mechanisms of how Rho small GTPase activities are regulated at the invadopodia remain unclear. Here we have identified FilGAP, a GTPase-activating protein (GAP) for Rac1, as a negative regulator of invadopodia formation in tumor cells. Depletion of FilGAP in breast cancer cells increased ECM degradation and conversely, overexpression of FilGAP decreased it. FilGAP depletion promoted the formation of invadopodia with ECM degradation. In addition, FilGAP depletion and Rac1 overexpression increased the emergence of invadopodia induced by epidermal growth factor, whereas FilGAP overexpression suppressed it. Overexpression of GAP-deficient FilGAP mutant enhanced invadopodia emergence as well as FilGAP depletion. The pleckstrin-homology (PH) domain of FilGAP binds phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2], which is distributed on membranes of the invadopodia. FilGAP localized to invadopodia in breast cancer cells on the ECM, but FilGAP mutant lacking PI(3,4)P2-binding showed low localization. Similarly, the decrease of PI(3,4)P2 production reduced the FilGAP localization. Our results suggest that FilGAP localizes to invadopodia through its PH domain binding to PI(3,4)P2 and down-regulates invadopodia formation by inactivating Rac1, inhibiting ECM degradation in invasive tumor cells.