Targeting the WASF3–CYFIP1 Complex Using Stapled Peptides Suppresses Cancer Cell Invasion

Abstract

Activation of the WASF3 protein by extracellular stimuli promotes actin cytoskeleton reorganization and facilitates cancer cell invasion, whereas WASF3 depletion suppresses invasion and metastasis. In quiescent cells, the interaction between WASF3 and a complex of proteins including CYFIP1 acts as a conformational restraint to prevent WASF3 activation. Therefore, we took advantage of this endogenous regulatory mechanism to investigate potential sites that disrupt WASF3 function. 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. 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. Treatment of highly invasive breast and prostate cancer cells with WAHM inhibitor peptides significantly reduced motility and invasion in vitro. Mechanistic investigations revealed that these inhibitors suppressed the interaction between Rac and the WASF3 complex, which has been shown to promote cell migration. Furthermore, peptide-mediated inhibition of WASF3 also resulted in the dysregulation of known downstream targets such as MMP-9 and KISS1. 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. Collectively, our findings suggest that targeting WASF3 function with WAHM peptides could represent a promising therapeutic strategy for preventing tumor invasion and metastasis.

The WASF3–NCKAP1–CYFIP1 Complex Is Essential for Breast Cancer Metastasis

“…Thus, NCKAP1 is also required for the stability of these proteins. Invasion potential, however, is not affected by knockdown of WASF1 and WASF2 in MDA-MB-231 cells (30). …”

“…Knockdown of NCKAP1 in highly invasive cancer cells leads to suppression of invasion (Figure 2b), which is accompanied by decreased WASF3 levels. Reduced WASF3 levels are also seen after knockdown of CYFIP1 (30), which associates with WASF3 as a dimer with NCKAP1 (42–43). Loss of either member of this trimeric complex leads to loss of WASF3 protein levels (Supplementary Figure 5) and suppresses invasion.…”

“…Currently, no inhibitors target WASF3 function directly, but targeting the WRC may provide a means to suppress invasion. We have previously reported, for example, that targeting a direct protein-protein interaction between CYFIP1 and WASF3 using stapled peptides (SPs) can effectively suppress invasion in WASF3 overexpressing cancer cells (30). It is possible, therefore, that disrupting the WRC by targeting NCKAP1 might have a similar consequence.…”

“…Since targeting the WASF3-CYFIP1 interface also led to disruption of the WRC and suppression of invasion (30), we investigated the relative ability of targeting the WASF3-CYFIP1 (using the WHAM1 peptide) or CYFIP1-NCKAP1 (using the WANT3 peptide) interaction to disrupt the WRC. Following treatment of MDA-MB-231cells with WHAM1 or WANT3, it was clear that targeting the CYFIP1-NCKAP1 interface was more efficient in decreasing WASF3 levels compared to targeting the WASF3-CYFIP1 interface (Figure 6f).…”

“…MDA-MB-231 and T47D and have been verified using SNP-CGH (7,27) for characteristic cytogenetic changes. The ATCC Cell Authentication Testing service confirmed the identity of Hs578T, PC3 and SW620 (August 2015) using STR DNA fingerprinting analysis (30). Standard cell culture, transient transfections, RT-PCR, western blotting, immunoprecipitation (IP), flow cytometry, Biotin-Avidin pulldown, lentiviral transduction, cell proliferation and Transwell invasion assays were carried out as described previously (7–9,30).…”