Supplementary Figure S1. Real-time RT-PCR analysis shows that knockdown of CYFIP1 does not affect WASF1/2/3 transcript levels. Supplementary Figure S2. As a result of the knockdown for any of the three genes (shW1-1 and shW1-2 for WASF1, shW2-1 and shW2-2 for WASF2, shW3 and shW3-2 for WASF3), there was no change in proliferation compared to control knockdown cells (shGFP). Supplementary Figure S3. Expression levels of WASF3 (W3) are unchanged over a twenty-four hour period following treatment of MDA-MB-231 cells with either scrambled (SCR) or WAHM peptides. Supplementary Figure S4. Proliferation of MDA-MB-231 and PC3 cells are not significantly affected by treatment with either of the scrambled (SCR) peptides or WAHM peptides at two different concentrations, compared with vehicle alone treated (DMSO) cells. Supplementary Figure S5. In scratch wound assays (A) cell motility over 24 hours was recorded for four different cell lines; MDA-MB-231 and HS578T (breast cancer cells) and PC3 and DU145 (prostate cancer cells). Supplementary Figure S6. WAHM peptides suppress cell invasion in a dose-dependent manner. Supplementary Figure S7. Loss of phosphoactivated WASF3 in the absence of serum. MDA-MB-231 cells express high levels of WASF3 and PC3 cells express lower levels (input). Supplementary Figure S8. Rac2 is identified as a novel WASF3-interacting protein.

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. Cancer Res; 76(4); 965–73. ©2015 AACR.