WASPs, WAVEs, and cell motility

When members of the WASP/WAVE family are active, the VCA region is exposed, allowing the Arp2/3 complex and an actin monomer to bind to it, leading to nucleation of a new actin filament.

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Activation of WASP/WAVE family members has been implicated in many biological processes where reorganization of the actin cytoskeleton is required, such as vesicle trafficking, microbial infection, and cell-substrate adhesion (see reviews by Takenawa and Suetsugu9 and Takenawa and Miki23).

The main focus of this review is their role in the control of cell motility, with a particular focus on cancer cell motility and metastasis.

WASPs, WAVEs, and cell protrusions

The motility of eukaryotic cells is accomplished by dynamic remodeling of the actin cytoskeleton. Cell motility in two-dimensional environments, such as on culture dishes or over the ECM, is driven by formation of lamellipodia and filopodia at the leading edge.

It is traditionally believed that lamellipo­dia are the key structures that provide the main driving force in two-dimensional cell motility. This was first demonstrated by a simple experiment where a section excised from a fish keratinocyte lamellipodium was seen migrating at a speed similar to that of an intact cell.40

Filopodia, although they are not implicated as being the major force behind cell motility, seem to have a role in sensing the environment outside the cell and guide cell migration and forming of adhesions with the ECM.41,42 The organization of actin filaments is different in the various protrusions that drive cell motility.

Actin filaments assemble in a branching network in lamellipodia, whereas in filopodia they form long parallel bundles.43

WASP/WAVE family proteins interact with the Arp2/3 complex to induce formation of branched actin networks, where actin filaments are assembled at a 70° angle from the sides of pre-existing filaments, which are the main structures responsible for formation of lamellipodia.44

Although the main mode of cancer cell migration is through three-dimensional matrices, which are discussed later in this review, many advances in our understanding of the mechanics of cell motility and the role of WASP/WAVE family proteins have been as a result of studying features of cells that move in two dimensions. Some of these findings are briefly discussed here.

Initial work investigating the role of WASP/WAVE family proteins in cell motility found that Cdc42 signaling requires nWASP activity in order to form the actin structures seen in filopodia, and WAVE1 activity downstream of Rac is required for formation of lamellipodia.17,31

However, it is now clear that these studies give a limited understanding of the role of the WASP/WAVE family, as nWASP activity is not essential for the formation of filopodia in response to Cdc42 signaling in fibroblasts.45

Moreover, many reports have implicated nWASP in the control of the actin dynamics in lamellipodia through demonstrating nWASP activity localized at the leading edge of lamellipodia in a variety of cell types.46–48

However, several studies have also shown that depletion of WASP or nWASP activity does not affect the formation of lamellipodia in several cell types, including mice fibroblasts, HeLa cells, and Drosophila S2 cells.49–51

Instead, many reports support the idea that members of the WAVE subfamily are key in the control of lamellipodia formation in various cell types.50–53

In particular, induced WAVE2 deficiency in mouse endothe­lial, embryonic fibroblast, melanoma, and macrophage cells resulted in impaired ability of the cells to form lamellipodia and a decrease in cell motility.53–56

These findings collectively suggest that the WAVE subfamily, particularly WAVE2, has a more important role in the formation of lamellipodia protru­sions and mesenchymal migration, and hence some aspects of cell motility, than the WASP subfamily, but that there is evidence that the contribution of the WASP/WAVE family members to the control of actin dynamics varies in different cell types.

Most of these studies analyzing the generation of filopodia and lamellipodia have used cells cultured on rigid two-dimensional substrates which can help us understand the mechanisms involved in cell motility.

While formation of these types of cell structures does take place in motile cells in vitro, cells in vivo, and especially cancer cells, generally use different methods of migration as they have to invade through a three-dimensional matrix.57

Nevertheless, cancer cells do still utilize actin-based protrusions to allow them to invade through the ECM and migrate during the process of metastasis. The WASP/WAVE family proteins have been shown to be important in the formation of these protrusions, which are more specific to motility in three dimensions and in migration of cancer cells, as well as in two-dimensional motility. Interestingly, a recent report has suggested that the WASP/WAVE family members may have different roles depending on the mode of cellular migration.58

This group targeted the WAVE complex using small interfering RNA (siRNA) silencing techniques and found that although it promotes motility in two dimensions, disruption of the WAVE complex promotes cell invasion and enhances focal adhesion kinase activity and nWASP localization at invasive protru­sion sites.

They also proposed that the WAVE complex and nWASP have opposing roles in three-dimensional epithelial cell invasion and that there may be an interplay between WASP/WAVE family members in the control of various modes of cell motility.

Reports that examine the role of the WASP/WAVE family in three-dimensional motility, and in particular the motile behavior of cancer cells, are the main focus of the rest of this review.

WASPs, WAVEs, and cancer cell protrusions

Podosomes and invadopodia are specialized membrane-surface structures that have a role in the migration of invasive cells and degradation of the ECM.

Podosomes are formed in various cell types, including monocytic cell lineages, smooth muscle cells, and endothelial cells, whereas invadopodia usually refer to protrusions similar in structure and func­tion to podosomes but are found in invasive cancer cells. Invadopodia contain a core of actin filaments, as well as adhesion proteins, proteinases for ECM degradation, and many signaling molecules.5,59

nWASP and invadopodia

nWASP and the Arp2/3 complex have been implicated in the formation of invadopodia. Using a biosensor, nWASP has been shown to be active at invadopodia in MTLn3 rat mammary adenocarcinoma cells.46

nWASP staining has also shown clear localization with invadopodia in these highly metastatic cells, and also other cancer cell types.

Furthermore, RNA interference techniques have been used to show that nWASP and some of its interaction partners, in particular the Arp2/3 complex, are crucial for formation of invadopodia. WAVE1 and WAVE2 showed no invadopodium-specific staining and were not shown to be essential for their formation.60

Such findings agree with other studies in their conclusion that nWASP is involved in formation of inva­dopodia in various cell types, including rat fibroblasts and adenocarcinoma cells.61,62

More recently, a study has proposed that nWASP-mediated formation of invadopodia is essential in breast cancer invasion, intravasation, and metastasis to the lungs.

This research group induced MTLn3 cells to overexpress the dominant negative form of nWASP, which lacks the ability to activate the Arp2/3 complex and so competitively inhibits endogenous nWASP, and reduced nWASP protein expression in these cells by 75% using short-hairpin RNA (shRNA) silencing techniques.

Interference with endogenous nWASP activity in these cells significantly reduced formation of invadopodia and their proteolytic activity when cultured on a gelatin matrix.63

Since formation of invadopodia correlates with the invasive capacity of cancer cells, it is clear that the nWASP pathway could be considered a therapeutic target for inhibi­tion of invasion in various cancers, in particular breast cancer, as demonstrated by use of mammary adenocarcinoma cells in the mentioned studies.64