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

Abstract

Inactivation of the WASF3 gene suppresses invasion and metastasis of breast cancer cells. WASF3 function is regulated through a protein complex that includes the NCKAP1 and CYFIP1 proteins. Here, we report that silencing NCKAP1 destabilizes the WASF3 complex, resulting in a suppression of the invasive capacity of breast, prostate, and colon cancer cells. In an in vivo model of spontaneous metastasis in immunocompromized mice, loss of NCKAP1 also suppresses metastasis. Activation of the WASF protein complex occurs through interaction with RAC1, and inactivation of NCKAP1 prevents the association of RAC1 with the WASF3 complex. Thus, WASF3 depends on NCKAP1 to promote invasion and metastasis. Here, we show that stapled peptides targeting the interface between NCKAP1 and CYFIP1 destabilize the WASF3 complex and suppress RAC1 binding, thereby suppressing invasion. Using a complex-disrupting compound identified in this study termed WANT3, our results offer a mechanistic proof of concept to target this interaction as a novel approach to inhibit breast cancer metastasis. Cancer Res; 76(17); 5133-42. ©2016 AACR.

Figure 1. NCKAP1 interacts with WASF3

(a) Following immunoprecipitation (IP) of WASF3 from MDA-MB-231 and Hs578T breast cancer cells, western blot analysis identified NCKAP1 in the IP. The interaction between NCKAP1 and WASF3 was further demonstrated in a GST fusion-protein pulldown assays (b). Lysates from MDA-MB-231 cells were incubated with the GST-tagged WASF3 prepared in BL21 bacterial cells, where the correct size fusion protein was confirmed using anti-GST antibodies (below). The presence of NCKAP1 was then demonstrated in the WASF3-GST (GST-W3) complex using anti-NCKAP1 antibodies. Interaction between NCKAP1 and WASF3 was also demonstrated in vivo following transfection of the NCKAP1-venus1 (NCKAP1-v1) and WASF3-venus2 (WASF3-v2) constructs into MDA-MB-231 cells (c). After 12 hours, GFP was detected by fluorescence microscopy in cells where both constructs were expressed but not in cells where either of the constructs was expressed alone. In the co-transfected cells, a membrane localization of the GFP signal could be seen (arrows).When the WASF3 complex was recovered using immunoprecipitation from MDA-MB-231 cells grown in the presence or absence of FBS (d), NCKAP1 was detected in the complex whether FBS was present or not. The presence of NCK1, however, was only seen in cells treated with FBS, where WASF3 (P-WASF3) was activated.

Figure 2. Molecular and cell invasion analysis following NCKAP1 knockdown

Breast cancer MDA-MB-231 and Hs578T cells in which NCKAP1 had been stably knocked down (shNCKAP1-1 and shNCKAP1-2) show significantly reduced levels of WASF3 (a) compared with cells carrying a control shRNA (shGFP). Similarly, reduced levels of the WASF1 and WASF2 proteins were also seen in the NCKAP1 knockdown cells. When NCKAP1 knockdown cells were analyzed using Transwell invasion assays (b), their invasion potential was suppressed. Immunoprecipitation of HA-tagged WASF3 from MDA-MB-231 cells in which NCKAP1 had been knocked down shows the absence of RAC1 in the WASF3 immunocomplex (c), compared with parental cells expressing the control shRNA (shGFP). When WASF3 was overexpressed in NCKAP1 knockdown MDA-MB-231 and Hs578T cells, there was no recovery of invasion potential (d). *p<0.05, **p<0.01 and ns indicates no statistical significance.

Figure 3. Metastasis in vivo is suppressed following NCKAP1 knockdown

Kaplan-Meier plot analyses with the log-rank test, shows that higher NCKAP1 expression was associated with lower relapse-free survival rates compared with low NCKAP1 expression (a). When MDA-MB-231 cells were implanted subcutaneously into six-week-old female NSG mice (b) primary tumor growth was not affected by knockdown of NCKAP1 (shNCKAP1-1 and shNCKAP1-2), compared to control knockdown (shGFP) cells. When the lungs were removed from these mice, however, the number of nodules on the surface of the lungs was significantly reduced in the NCKAP1 knockdown cells (c). Histological analysis of these lungs demonstrated that, while animals receiving the control cells showed extensive tumor infiltration throughout the lung (d) the NCKAP1 knockdown cells showed relatively few, small tumor foci. Images on the right derived from the boxed areas on the left. **p<0.01.

Figure 4. RAC1 binding to the WASF3 complex is required for NCKAP1-mediated invasion of breast cancer cells

NCKAP1 overexpression in MDA-MB-231 cells does not affect WASF3 levels and, in T47D cells which do not express WASF3, overexpression of NCKAP1 does not increase WASF3 levels (a). Transwell assays demonstrate that overexpressing NCKAP1 in MDA-MB-231 cells significantly increases invasion potential, although T47D cells are unaffected (b). IP of WASF3 (W3) from MDA-MB-231 cells shows increased RAC1 levels in the WASF3 complex and increased WASF3 phosphorylation when NCKAP1 is overexpressed (c). Treatment of MDA-MB-231 and Hs578T breast cancer cells with the NSC23766 RAC1 inhibitor, leads to a dose-dependent reduction in invasion potential (d) but does not affect protein levels of either WASF3, NCKAP1 or RAC1 (e). IP of WASF3 (W3) from MDA-MB-231 cells treated with NSC23766 shows that, at high (50 uM) concentration, activation of WASF3 is suppressed and RAC1 engagement in the complex is virtually eliminated (f). When a dominant-negative RAC1 (RAC1DN) is introduced into MDA-MB-231 cells overexpressing NCKAP1, levels of phosphoactivated WASF3 are significantly reduced in concert with reduced RAC1 levels (g). In Transwell assays, NSC23766 leads to a significant reduction in invasion in both MDA-MB-231 parental cells containing the empty vector (EV) and cells overexpressing NCKAP1 (h). Similarly, the RAC1 dominant-negative construct (RAC1DN) significantly suppresses invasion in MDA-MB-231 cells overexpressing NCKAP1 (i). *p<0.05 and **p<0.01.

Figure 5. Invasion and metastasis analysis after NCKAP1 overexpression in WASF3 knockdown cells

When NCKAP1 was overexpressed in WASF3 knockdown MDA-MB-231 and Hs578T cells (a), cell invasion was not significantly affected (b). Following subcutaneous implantation of MDA-MB-231 cells overexpressing NCKAP1 into NSG mice, the number of nodules on the surface of the lungs after 8 weeks in these animals was not significantly different compared with the WASF3 (shW3) knockdown cells (c). Histological analyses showed the same distribution of tumors in the lungs of these mice carrying the NCKAP1 overexpressing cells as seen for the WASF3 knockdown cells (d). **p<0.01 and ns indicates no statistical significance.

Figure 6. Targeting the NCKAP1-WASF3 complex using stapled peptides leads to loss of invasion in breast cancer cells

Sequence of amino acid regions 631–642, 933–944 and 1110–1121 in NCKAP1 (a) used to design stapled peptides (above). The three stapled peptides WANT1, 2 and 3 were designed to target interaction surfaces between CYFIP1 and NCKAP1 where (*) represent the position of the non-natural amino acids (below). The scrambled peptide was used as a negative control. Transwell invasion assays show that only WANT3 significantly suppresses MDA-MB-231 cell invasion (b) and suppresses both WASF3 and NCKAP1 protein levels (c). A time course of WANT3-FITC uptake using flow cytometry over the first 30 minute of exposure (d) shows progressive fluorescein labeling in breast cancer MDA-MB-231 cells. WANT3 suppresses WASF3 protein levels in a dose-dependent manner (e). WANT3 suppresses phosphoactivation of WASF3 more significantly than the WASF3-CYFIP1 peptide mimic WAHM1 (f). Using a high dose of WANT3 (20 µM) leads to a more remarkable reduction in MDA-MD-231 cell invasion compared with low dose treatment (g). WANT3 peptides were preincubated in serum-containing medium at 37°C for 1–7 days. When this medium was then used in invasion assays, significant suppression of invasion in MDA-MB-231 cells was still observed for up to three days (h).