Targeting the WASF3 complex to suppress metastasis

Abstract

Wiskott-Aldrich syndrome protein family members (WASF) regulate the dynamics of the actin cytoskeleton, which plays an instrumental role in cancer metastasis and invasion. WASF1/2/3 forms a hetero-pentameric complex with CYFIP1/2, NCKAP1/1 L, Abi1/2/3 and BRK1 called the WASF Regulatory Complex (WRC), which cooperatively regulates actin nucleation by WASF1/2/3. Activation of the WRC enables actin networking and provides the mechanical force required for the formation of lamellipodia and invadopodia. Although the WRC drives cell motility essential for several routine physiological functions, its aberrant deployment is observed in cancer metastasis and invasion. WASF3 expression is correlated with metastatic potential in several cancers and inversely correlates with overall progression-free survival. Therefore, disruption of the WRC may serve as a novel strategy for targeting metastasis. Given the complexity involved in the formation of the WRC which is largely comprised of large protein-protein interfaces, there are currently no inhibitors for WASF3. However, several constrained peptide mimics of the various protein-protein interaction interfaces within the WRC were found to successfully disrupt WASF3-mediated migration and invasion. This review explores the role of the WASF3 WRC in driving metastasis and how it may be selectively targeted for suppression of metastasis.

Keywords: Metastasis; Stapled peptide; WASF Regulatory Complex; Wiskott-Aldrich syndrome protein family 3.

Fig. 1:

Structural and schematic view of the WASF Regulatory Complex (WRC) A. Schematic representation of the domain structure of the three WASF family members. Conserved domains are highlighted. B. Crystal structure of WASF1 (PDB ID: 3P8C) highlighting the domain topology and conserved structural features of the WASF proteins. C. Crystal structure (left, PDB ID: 3P8C) of the WASF regulatory complex in its autoinhibited state showing CYFIP1 (orange) and NCKAP1 (cyan) in a surface view. WASF1 (green), Abi2 (purple) and BRK1 (red) are shown as ribbon structures. D. An illustration of the WASF regulatory complex based on the crystal structure is shown on the right.

Fig. 2:

The WASF3 WRC regulates cell motility and invasion In its inactive state, the WRC is located at the cell membrane with the VCA domain of WASF3 sequestered within the complex. Stimuli lead to phosphorylation of key tyrosine residues on WASF3, leading to the binding of Rac1 GTPases to the WRC. This triggers a conformational change where the VCA domain of WASF3 is released from its inhibited state. The free VCA domain then binds to Arp2/3, thereby allowing F-actin nucleation that catalyzes the formation of branching actin filaments. These actin filaments generate and propagate an outward mechanical force to the cell membrane through the WRC that drives the formation of cell protrusions and filopodia. Signaling events downstream of the WRC also lead to the expression of MMP9 that is subsequently secreted through these cell protrusions and aids in the degradation of the basement membrane to facilitate invasion.

Fig. 3.

Design and development of first-generation WASF3 mimic stapled peptides A. Crystal structure of the WRC (PDB ID: 3P8C) highlighting the N-terminal region of WASF1 (WASF3 paralog) (inlet) that served as a template for the development of WASF Helical Mimic (WAHM) peptides (side chains are represented as sticks with carbon atoms in white, nitrogen atoms in blue, oxygens atoms in red and sulfur atoms in yellow). B. Helical wheel representation of residues 26 through 41 of WASF3 along with the native sequence and two stapled versions derived from the native sequence, WAHM1 and WAHM2. Polar basic residues are shown in cyan, polar acidic residues in blue, polar uncharged residues in lilac and nonpolar residues in yellow. WAHM1 and 2 are shown where (s)-2-(4-pentenyl)alanine is positioned at i, i+4 positions and is represented by red asterisks while the hydrocarbon staple is represented by a red line linking the asterisks.

Fig. 4.

Design and development of in silico optimized WASF3 mimic stapled peptides A. A zoom-in view of the WRC (PDB ID: 3P8C) highlighting the region of WASF3 (paralog of WASF1) identified using Rosetta PeptiDerive. The WASF fragment, termed WAHMIS (WAHM In Silico), is shown with side chains in stick representation (carbon atoms are shown in white, nitrogen atoms in blue, oxygens atoms in red and sulfur atoms in yellow). B. Helical view of the native WAHMIS sequence (polar basic residues are shown in cyan, polar acidic residues in blue, polar uncharged residues in lilac and nonpolar residues in yellow) along with lead stapled peptide WAHMIS-2. The stapled WAHMIS-2 peptide is shown where (s)-2-(4-pentenyl)alanine is introduced at i, i+4 positions and is represented by red asterisks. The hydrocarbon staple is represented by a red line linking the asterisks.

Fig. 5.

Design and development of NCKAP1 mimic stapled peptides A. Front and rear view of the WRC with NCAKP1 (cyan) shown in cartoon view. Inlet shows a zoomed-in view of the region of NCKAP1 used to develop a stapled peptide mimic. Side chains are stick representations with carbon atoms shown in white, nitrogen atoms in blue, oxygens atoms in red and sulfur atoms in yellow. B. Helical wheel representation of residues 1110 through 1121 of NCKAP1 where polar basic residues are shown in cyan, polar acidic residues in blue, polar uncharged residues in lilac and nonpolar residues in yellow. The stapled WANT3 peptide is shown where (s)-2-(4-pentenyl)alanine is introduced at i, i+4 positions and is represented by red asterisks. The hydrocarbon staple is represented by a red line linking the asterisks.