Activation of WASF proteins is facilitated by binding Rac proteins and phosphorylation of tyrosine residues, which leads to relaxation of the conformational constraints and, in the cases of WASF3, results in increased migration, invasion, and metastasis ( 5). In resting cells, WASF proteins are maintained in a conformation-restricted, inactive form as a result of binding the WASF regulatory complex (WRC) comprised of the CYFIP1/SRA1 (or the PIR121/CYFIP2 ortholog), NCKAP1/NAP1/HEM2 (or the HEM1 ortholog), ABI2 (or the ABI1 and ABI3 orthologs), and HSPC300/BRICK1 proteins ( 5–7). The protein C-termini carry motifs (VCA) that bind to monomeric actin and the ARP2/3 complex, to facilitate actin polymerization and cytoskeleton reorganization ( 1). WASF3 ( 1) is one of three genes in the Wiskott–Aldrich syndrome family that has been implicated in the regulation of cell movement related to wound healing, neuronal migration, chemotaxis, and immune cell activation, through control of membrane protrusions resulting from reorganization of the actin cytoskeleton ( 2–4). Collectively, our findings suggest that targeting WASF3 function with WAHM peptides could represent a promising therapeutic strategy for preventing tumor invasion and metastasis. Finally, we demonstrate that this invasive phenotype is specific to WASF3 as depletion of WASF1 and WASF2, which can also bind to CYFIP1, did not affect invasion. Furthermore, peptide-mediated inhibition of WASF3 also resulted in the dysregulation of known downstream targets such as MMP-9 and KISS1. Mechanistic investigations revealed that these inhibitors suppressed the interaction between Rac and the WASF3 complex, which has been shown to promote cell migration. Treatment of highly invasive breast and prostate cancer cells with WAHM inhibitor peptides significantly reduced motility and invasion in vitro. Based on existing crystallographic data, we developed stapled peptides, referred to as WASF Helix Mimics (WAHM), that target an α-helical interface between WASF3 and CYFIP1. Here, we show that genetic knockdown of CYFIP1 in cancer cells led to the destabilization of the WASF3 complex, loss of WASF3 function, and suppressed invasion. Therefore, we took advantage of this endogenous regulatory mechanism to investigate potential sites that disrupt WASF3 function. In quiescent cells, the interaction between WASF3 and a complex of proteins, including CYFIP1, acts as a conformational restraint to prevent WASF3 activation. Activation of the WASF3 protein by extracellular stimuli promotes actin cytoskeleton reorganization and facilitates cancer cell invasion, whereas WASF3 depletion suppresses invasion and metastasis.
0 kommentar(er)
