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. Author manuscript; available in PMC: 2014 Feb 20.
Published in final edited form as: Biochem J. 2012 Nov 15;448(1):35–42. doi: 10.1042/BJ20112206

Scar/WAVE3 contributes to motility and plasticity of lamellipodial dynamics but not invasion in 3D

Heather J Spence 1, Paul Timpson 1, Hao Ran Tang 1, Robert H Insall 1, Laura M Machesky 1
PMCID: PMC3929901  EMSID: EMS56885  PMID: 22909346

Abstract

The Scar/WAVE complex plays a major role in the motility of cells by activating the Arp2/3 complex, which initiates actin branching and drives protrusions. Mammals have three Scar/WAVE isoforms, which show some tissue specific expression, but their functions have not been differentiated. Here we show that depletion of the Scar/WAVE3 in the mammalian breast cancer cells MDA-MB-231 results in larger and less dynamic lamellipodia. Scar/WAVE3 depleted cells move more slowly but more persistently on a 2-dimensional matrix and they typically only show one lamellipod. However, Scar/WAVE3 appears to have no role in driving invasiveness in a 3D Matrigel invasion assay or a 3D collagen invasion assay, suggesting that lamellipodial persistence as seen in 2D is not crucial in 3D environments.

Keywords: lamellipodia, migration, invasion, Scar/WAVE complex, actin dynamics, breast cancer cells

Introduction

The mammalian Scar/WAVE proteins are ubiquitous activators of the Arp2/3 complex that reside in a stable five-protein complex with ABI1/2/3, CYFIP1/2 (PIR121 or Sra-1 respectively), NAP1/2 and Hspc300 [1, 2]. Activation of the Scar/WAVE complex leads to the formation of new lamellipodia [3]. Mammals have 3 Scar/WAVE proteins: Scar/WAVE1, Scar/WAVE2 and Scar/WAVE3 [4], which can all assemble into the Scar/WAVE complex [2] and which thus far have not been differentiated between functionally. Scar/WAVE1 and Scar/WAVE3 are predominantly expressed in the brain while Scar/WAVE2 is ubiquitously expressed, but we show here that some cancer cells in culture express all three Scar/WAVE proteins and our data suggest that they have non-redundant functions.

The Scar/WAVE complex may have both positive and negative roles in tumour cell invasion, likely depending on the context [5-8]. In a 3D environment, cells show plasticity of movement and can sometimes assume very elongated and protrusive motility referred to as “mesenchymal” and other times assume more rounded bleb-based motility, often referred to as “amoeboid” [9, 10]. Scar/WAVE2 drives the formation of pseudopodia during mesenchymal cell invasion [11]. In addition, Scar/WAVE2 has been reported to have a role in motility and lamellipodia generation in mouse melanoma cell lines, human hepatocellular carcinomas and in human breast carcinomas [6, 7, 12-15]. Scar/WAVE3, although usually low in epithelial tissues is upregulated in the breast cancer cell line MDA-MB-231 and was reported by one group to be required for efficient cell invasion in Boyden chamber based Matrigel invasion assays [16, 17]. However, there is apparently no significant increase in Scar/WAVE3 expression in breast tumours [18]. Another Scar/WAVE complex member, CYFIP1 appears to be a tumour invasion suppressor in epithelial cells and can cooperate with activated Ras to drive invasion [8]. Here we investigate further the role of Scar/Wave3 in both 2D and 3D motility of cancer cells in vitro and report that it has a major role in 2D motility but not in 3D in vitro tumour cell invasion.

Materials and Methods

MDA-MB-231, A375 and MV3 cells were grown in Dulbecco’s modified Eagle medium (DMEM Invitrogen, Paisley, UK) supplemented with 10% fetal calf serum and 2mM L-Glutamine, at 37°C with 5% CO2. All cells were passaged before cells became confluent.

RNAi

For transient siRNA tranfections, cells were seeded at 3 × 105 in a 6 well plate and transfected with siRNA at a final concentration of 10 nM, using HiPerFect (Qiagen, Sussex, UK), according to manufactures instructions. Cells were subjected to two rounds of transfection, on days 1 and 3. The Qiagen siRNAs used were as follows; HS_WASF3_1 HP cat number si00090636 and HS_WASF3-3HP catalogue number si00090650.

Stable cell lines were generated using a control shRNAmir pSM retroviral plasmid (openbiosystems cat number RHS4971), and two human Scar/WAVE3 specific shRNAmir pSM2 retroviral plasmids (openbiosystems cat. number RHS1764-9702150 and RHS1764-9217288). Clones were generated using phoenix ampho packaging system [19]. Clones 1a, 1b and 1c were derived from shRNA mir pSM2 RHS1764-9217288 and clones 2a and 2b from RHS1764-9702150.

Timelapse video microscopy

Timelapse-video microscopy was performed on a Nikon TE2000 microscope by plating 2 × 105 cells on to each well of a six well plate that was coated with 5ug/ml of fibronectin. Cells were imaged as described in the text. Cell tracking experiments were performed in Image j. Persistence represents the ratio between Euclidean distance and the total distance travelled.

2 × 105 cells were seeded on to cell-derived matrices (CDM) and timelapse video microscopy was performed. CDMs were made according to Caswell et al 2007 [20]. Briefly, gelatin-coated tissue culture dishes were crosslinked with glutaraldehyde, quenched with Tris-Cl, and equilibrated in DMEM containing 10% FCS. Human dermal fibroblasts (HDFs) or NIH 3T3 fibroblasts were seeded at near confluence (~2 × 104 cells/cm2) and grown for 10 days (HDFs) or 8 days (NIH 3T3s) in DMEM containing 10% FCS and 50 μg/ml ascorbic acid. Cells were removed by incubation with PBS containing 20 mM NH4OH and 0.5% Triton X-100, and DNA residue was removed by incubation with DNaseI. Matrices were blocked with 0.1% heat-denatured BSA prior to seeding of cells. Cell tracking experiments were performed using Image j.

Invasion assays

Inverted invasion assays were carried out as previously described by Henning et al. 1994 [21]. Matrigel was allowed to polymerise in transwell inserts (Corning, Sigma, Dorsett, UK) at 37°C with 5% CO2 for 1 hour. 4 × 104 cells were then seeded onto the inverted transwell and allowed to attach at 37°C with 5% CO2 for 2 hrs. Inserts were then placed in serum-free DMEM and DMEM containing 10% serum and 20 μg/ml of EGF was added to the top of the matrigel plug. Cells were left to invade for 4 days at 37°C with 5% CO2. Cells were then stained with Calcein-AM and images of cells in Matrigel plug were obtained using a Leica SPMP confocal microscope. Collagen gel invasion assays were carried as described in Timpson et al. [22, 23]. These assays were performed using 4 × 104 MDA MBA-MB231 cells and cells were allowed to invade in to the collagen matrix for 10 days, before fixation in 4% formaldehyde. Boyden chamber invasion assays were performed using BD Matrigel Invasion Chambers. The assay was performed using the manufacturers instructions. 2.5 × 104 cells were seeded into each invasion chamber, with 10 % serum supplemented with 20μg/ml of EGF placed below the chamber as a chemoattractant. Cells were left at 37°C with 5% CO2 for 24 hrs. The cells that had invaded were stained with Calcein-AM and the number of cells per field were counted.

Western Blots

Western bolts were performed using either Chemiluminescence or Fluorescence. Protein lysates on 4-10% gradient NuPAGE gels (Invitrogen, Paisley, UK). Proteins were transferred onto polyvinylidene difluoride membranes (Immunobilon P, Millipore, Watford, UK) or Hybond-c-extra (Amersham Biosciences) and blocked in blocking buffer (PBS containing 0.1% Tween 20 and 5% non fat milk) for 15 min. The membranes were then probed with the following primary antibodies in blocking buffer: Scar/WAVE3 1in 500 (New England Biolabs, Hitchin, UK); Scar/WAVE2 1 in 200 (Santa Cruz, Heidelberg, Germany); Scar/WAVE1 1 in 500 (Santa Cruz); Abi1 1 in 500 (MBL); Abi2 1 in 500 (Santa Cruz); Sra1 1 in 500 (Millipore, Watford, UK); Nap 1 in 1000 (Millipore) and tubulin 1 in 1000 (Sigma, Poole, UK). The appropriate secondary antibody was then added and blots were developed with SuperSignal West Pico Chemiluminescent substrate (Thermo Scientific, Northumberland, UK) or for fluorescence secondary antibodies signals were detected using the Odyssey infrared imaging system (Li-Cor Biosciences, Cambridge, UK).

Results

Scar/WAVE3 depletion does not affect levels of other Scar/WAVE components

Loss of any subunit of the Scar/WAVE complex, such as Cyfip1 or Nap1, causes depletion of other Scar/WAVE components in multiple cell types [24-27]. We thus tested the effects of depleting Scar/WAVE3 on endogenous Scar/WAVE1/2 expression and on levels of Scar/WAVE complex components. Firstly, two siRNA specific to Scar/WAVE3 were individually, transiently expressed in MDA-MB-231 cells, causing a reduction in endogenous Scar/Wave3 detectable by western blotting (Figure 1A). Secondly, we derived stable clones using 2 retroviruses expressing shRNAmir specific to Scar/WAVE3, both of which successfully resulted in endogenous Scar/WAVE3 depletion (Figure 1B). We selected two stable Scar/Wave3 knockdown clones st Scar3-2a and st Scar3-1b, which we subsequently used for our main experiments; these displayed a mean knockdown of 75 % +/− 8 and 87% +/− 6 respectively. We also did not see any significant effect of reducing Scar/WAVE3 on the expression levels of other subunits of the endogenous Scar/WAVE complex using a fluorescence-based western blot detection method that gave a linear signal in the range of protein concentrations that we tested (Figure 1C and Methods).

Figure 1. Reduction of endogenous Scar/WAVE3 by siRNA and shRNAmir in MDA-MB-231 cells.

Figure 1

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A) Two independent Scar/WAVE3 specific siRNA oligos (t Scar3 3-1 and t Scar3 3-3, where t= transient) were transfected into MDA-MB-231 cells and cell lysates were used to perform immunoblotting with Scar/WAVE3 and tubulin antisera. B) Cell lysates derived from MDA-MB-231 stable (st) Scar3 kd cells (1a, 1b, 1c, 2a and 2b) and cells expressing shRNAmir control construct were used for immunoblotting with Scar/Wave3 and tubulin antisera. C) Western blot analysis of Scar/Wave complex members of cell lysates from clone stScar3-2a and nt cells. The mean % intensities of stScar3-a compared to nt cells were as follows, for Scar3 25% +/− 8, Scar1 107% +/− 13, Scar2 100% +/− 5, Sra 88% +/− 10, Nap 105% +/− 4, Abi1 108% +/− 9, Abi2 95% +/− 13 and tubulin as control 110% +/− 5. The results represent 3 western blot experiments.

Scar/WAVE3 reduction results in large stable lamellipodia, increased persistence and decreased speed of motility

Both stable and transient Scar/WAVE3 knockdown cells showed a striking morphology change from having complex shapes with several lamellipodial protrusions (Figure 2A) to rather uniform shapes with one leading lamellipod and a rounded rear. Knockdown cells frequently assumed shapes reminiscent of a goldfish keratocyte (Figure 2A and see for example, [28]). Time-lapse video microscopy and tracking of the cells plated on fibronectin revealed that both the stable and transient knockdown cells moved in very persistent paths and only rarely changed direction or split their large lamellipods (Figure 2A,B and C). Scar/WAVE3 knockdown cells also moved at a slower speed, 1.2 and 0.87 μm/min for transient kd and 1.3 and 1.2 μm/min for stable kd μm/min compared to 1.7μm/min for control nt cells (Figure 2C).

Figure 2. 2D time-lapse video microscopy of Scar/WAVE3 knockdown cells.

Figure 2

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Time-lapse video microscopy was performed on MDA-MB-231 cells plated on fibronectin and images were taken every 15 min for 19 hrs. Shown in A are a selection of stills at 0, 15, 30 and 45 min of MDA-MB-231 nt cells, Scar/Wave3 stable (st) and transient (t) MDA-MB-231 kd cells (clone 2a). See movies 1-3. B) Spider plots of tracking individual cells for 2.5 hrs. C) Quantification of spider plots depicting the speed and persistence of cells. Persistence represents the ratio between Euclidean distance and total distance travelled. Shown in each instance is the mean and SEM. Results represent 3 independent experiments. D and E) Analysis of time-lapse video microscopy performed on MDA-MB-231 nt and MDA-MB-231 Scar/WAVE3 stable kd cells (clone 2a) where images were taken every minute for 200 minutes. D) Average number of lamellipodia present at each time point. E) Average number of new protrusions formed at each time point. Results represent tracking of 10 cells. * represents a p value of < 0.02, ** p value of < 0.005 and *** p value of <0.0006 in the Student’s T-test.

To analyse in more detail the lamellipodial dynamics of Scar/WAVE3 depleted cells, we collected video time-lapse images every minute for 200 minutes and counted the total number of lamellipodia and new protrusions formed at each time point. The control nt cells had on average more lamellipodia present at each time point compared to the stable Scar/WAVE3 knockdown cells (Figure 2D). In addition, the control nt cells had 8 times as many new protrusions over the course of the experiment compared to the Scar/Wave3 kd cells (Figure 2E). These data suggest that Scar3 knockdown cells tend to have a single broad stable lamellipod protrusion and move very persistently, but more slowly than cells expressing Scar/WAVE3, which have a more plastic shape and more dynamic protrusions.

Motility of Scar/WAVE3 depleted cells on cell-derived matrix

In a 3D environment, such as in a collagen-rich stroma, cells move along fibres and tend to assume a much more elongated shape than on a flat rigid 2D surface. We thus performed time-lapse video microscopy experiments on Scar/WAVE3 knockdown and control cells plated on cell-derived matrices (CDM), which are matrices that are rich in fibronectin and fibrillar collagen[20, 29]. CDMs were used as they allow direct imaging of cells, compared to cells in the 3D invasion assays which are buried in the matrix and are difficult to image. Images of the Scar/WAVE3 knockdown and control nt cells were taken every 15 min for 19 hrs and manual tracking experiments of cells over 2.5 hrs revealed that the Scar/WAVE3 knockdown and control cells had a more similar morphology compared to the strikingly different morphology they have in a 2D environment (Figure 3A-C and supplementary movies 4 and 5). The Scar/WAVE3 knockdown cells travelled at a reduced speed, 0.5, 0.3, 0.4 and 0.5 μm/min compared to 0.8 μm/min for the control nt cells (Figure 3B). In contrast to cell motility on a 2D environment, the control nt cells moved in a more persistent manner (Figure 3B). The knockdown cells continued to move in a persistent manner on CDM as they did in a 2D environment.

Figure 3. Scar/WAVE3 knockdown cells on 3 D cell-derived matrices.

Figure 3

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Scar/WAVE3 MDA-MB-231 knockdown and control nt cells were plated onto 3D cell-derived matrices and video time-lapse microscopy was performed with images taken every 15 min for 19 hrs. See Movies 4 and 5. A) Still photos of MDA-MB-231 nt and stable Scar/WAVE3 knockdown cells (clone 2a) on CDMs. B) Quantification of the persistence and velocity of cells. Shown is the mean and SEM. C) Quantification of the shape of cells on CDM matrices. Value of 1 represents circular and 0 represents elongated. Shown is the mean and SEM. Results in A, B and C represent 3 independent experiments. * represents a p value of < 0.02 and *** represents value of <0.0006 in the Student’s T-test.

Depletion of Scar/WAVE3 does not affect invasion into Matrigel or collagen 3D matricies

To determine if knockdown of Scar/WAVE3 in MDA-MB-231 cells resulted in a reduction in the invasion of the cells in 3D environment, we performed Boyden chamber invasion assays using thin Matrigel coated filters. We could not see any reduction in invasion of both stable and transient knockdown of Scar/Wave3 (figure 4A). These results contradict previous findings where Scar3/Wave3 depletion reduced invasion [17]. In addition to the Boyden chamber invasion assay we also performed inverted invasion assays into thick matrigel plugs [21]. Control nt cells and Scar/WAVE3 knockdown cells invaded to a similar extent, with around half of the cells invading beyond 10 uM into the gel (Figure 4B). We also performed identical assays with MV3 and A375 metastatic melanoma cells invading into Matrigel following Scar/WAVE3 depletion (Supplementary Figure 1) and saw no effect of depleted Scar/WAVE3 (Figure S1 A,B,C and D). Thus, the effect is not cell type specific and invasion into 3D Matrigel is not inhibited by depletion of Scar/WAVE3.

Figure 4. Scar/Wave3 MDA-MB-231 knockdown and control nt cells in Boyden chamber invasion assays, inverted invasion assays and collagen invasion assays.

Figure 4

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A) Boyden chamber invasion assays were performed on control, transient and stable Scar/WAVE kd MDA-MB-231 cells. Images show cells that have invaded and quantification of the mean invasion and SEM. B) Inverted invasion assays were performed on control, transient and stable Scar/WAVE kd MDA-MB-231 cells. Results show confocal microscopy images of invading cells and quantification of the mean % invasion and SEM. C) Haematoxylin and eosin stained paraffin sections and quantification of control and Scar/WAVE3 knockdown MDA-MB-231 cells in collagen plug invasion assays. Shown is the mean invasion with S.EM. The number of cells that invaded as colonies of more than 5 cells is also quantified. Data in A, B and C represents 3 individual experiments per value on the graphs. In C * represents a p value of < 0.02 and *** represents value of <0.0006 in the Student’s T-test.

To further confirm whether invasive motility was affected by knockdown of Scar/WAVE3, we performed a third type of invasion assay. This assay Involves placing the cancer cells on to the top of the matrix derived of a mixture of collagen and primary human fibroblasts, and the percentage of cells that invade into the matrix is measured [22, 23]. Both Scar/WAVE3 knockdown cells and control cells were equally invasive in this invasion assay (Figure 4C). One striking feature of the Scar/WAVE3 knockdown cells was that they showed more tendency toward collective invasion than the control cells (Figure 4C). Quantification of the change to collective invasion is shown in figure 4C, where we determined the number of cells that invaded in groups of more than 5 cells [30].

Discussion

We have investigated the effect of loss of Scar/WAVE3 on the motility and invasiveness of cancer cells and we find surprisingly that Scar/WAVE3 is important for cells to maintain plasticity of shape and speed of movement rather than for invasion. Scar/WAVE3 is not usually expressed at detectable levels in epithelial cells, but can be upregulated in cancer cells ([31]. We found that MDA-MB-231 breast cancer cells express all three Scar/WAVE isoforms and that knockdown of Scar/WAVE3 gives a specific phenotype resulting in slower migration of cells in 2D and the protrusion of large and very persistent lamellipodia. While all three Scar/WAVE isoforms are thought to participate in the Scar/WAVE complex in mammalian cells [2], it is unknown whether they have redundant or specific functions. We find that Scar/WAVE3 has a nonredundant function in lamellipodial persistence in MDA-MB-231 breast cancer cells.

Dictyostelium cells and neutrophils (and likely other cells types moving in 2-dimensions) move by splitting their pseudopods and choosing a more successful pseudopod to bias the direction of motility for chemotaxis [32]. The causes of splitting are unknown but could result from local inhibition of Scar/WAVE complex within leading edge lamellipods. The lack of lamellipodial plasticity in Scar/WAVE3 knockdown cells suggests that Scar/WAVE3 functions to promote this local inhibition. Scar/WAVE3 could be directly competing with Scar/WAVE1 and 2 for activation factors, but be less efficient at generating actin networks (and therefore inhibitory) or it could be indirectly binding to and sequestering activation factors to tip the balance away from continuous actin assembly and toward discontinuous plastic behaviour. It would have been interesting to overexpress Scar/WAVE3 and examine the effects on migration, but in our experience, overexpression of Scar/WAVE proteins disrupts the actin cytoskeleton, so is not really a physiologically relevant method [3]. Another interesting test would be to localize fluorescently labelled Scar/WAVE proteins simultaneously in living cells and see whether Scar/WAVE3 preferentially localized to regions of lamellipodia where splits were occurring. This is not yet technically possible, as addition of a GFP or similar tag to either the N- or C- terminus of Scar/WAVE proteins disrupts their activity (RHI and LMM, unpublished observation). Dictyostelium only have one conventional Scar/WAVE isoform and loss of this leads to a similar shift toward slow and persistent motility [33].

Despite previous reports that Scar/WAVE3 was important for invasion through Matrigel matrix [16, 34, 35], we found no effect of transient or stable knockdown of Scar/WAVE3 on invasion into 3D Matrigel or collagen gels. We also performed Matrigel transwell assays to look for invasion defects but did not find any effect of Scar/WAVE3 depletion on the ability of cells to cross Matrigel coated filters. The various assays used to measure invasion measure both the ability of the cells to penetrate through matricies of different composition and to migrate in 3D toward various attractants. In our assays, we generally included EGF as an attractant, as this is highly implicated in breast cancer invasion and metastasis by many basic and clinical studies (e.g. see [36]). Previously, Sossey-Alaoui and colleagues have used BD Biosciences Matrigel coated membrane chambers where cells migrated through a filter and a thin layer of Matrigel toward a 10% serum gradient [16, 35] or toward 50ng/ml PDGF [34]. We performed this assay, using 10% serum supplemented with 20μg/ml EGF (Figure 4) but we did not find any effect of Scar3 knockdown. We also performed a number of other assays, such as an inverted invasion assay (Figure 4) where cells cross a filter and invade fully into Matrigel toward a gradient of EGF. MDA-MB-231 cells invade readily in this assay as collective strands [37]. We still saw no effect of Scar3 depletion, using two independent transient oligos and two stable knockdowns. We also confirmed these results with melanoma cell lines MV3 and A375, so we do not think that this is just a cell-type specific lack of effect of Scar/WAVE3 depletion on invasion. We further employed a collagen plug invasion assay that is sometimes referred to as organotypic, because it uses fibroblasts embedded into a collagen gel and the gel is thus contracted and crosslinked by the fibroblasts [38] and thus is considered a mimic for stroma. This assay is highly dependent on the cancer cells being able to use proteases and to cooperate with fibroblasts to degrade the matrix and does not use a gradient of serum or EGF [37]. The effect of Scar3 depletion here is not to impede invasion, but rather to shift cells toward a more collective invasion style, as evidenced by clusters of more than 5 cells residing deep in the gels. We previously observed a similar effect with overexpression of the Mtss1 protein [30]. Thus, although it could be argued that subtle differences in experimental procedures could account for the differences between our results and previous reports, our experimental evidence argues against a robust requirement for Scar/WAVE3 in invasion of cells into matrix.

Our results agree with a previous report that depletion of Scar/WAVE complex members (p140Sra and PIR121) did not reduce and even enhanced the ability of cancer cells to invade into matrix [8]. In contrast, Wang et al. reported that depletion of Abi-1 inhibited invasion of breast cancer cells and reduced proliferation and anchorage independent growth [39] and Dubielecka et al. reported that loss of Abi-1 severely impaired migration and WAVE complex integrity [25]. It is unclear whether the reduced cell viability observed by Wang et al. accounted for the impairment of the cells in transwell invasion assays [39] or whether other activities of Abi-1 outside of the Scar/WAVE complex, such as the interaction between N-WASP and Abi1 might also contribute to this phenotype [40].

Overall, our data on suggest that Scar/WAVE3 is not likely to be important as a mediator of tumour cell invasion. It was interesting that Scar/WAVE3 depletion resulted in a more collective appearance of invasion into collagen gels (Figure 4C), which might indicate that cells are also less plastic and more likely to move in groups due to reduced ability to change direction and break away from a cluster than cells containing Scar/WAVE3. It will be interesting to explore the expression of Scar/WAVE3 in more cancer types and see if there is a connection with collective invasion or any other feature of invasion or metastasis.

Supplementary Material

Supplementary figure 1
Supplementary material
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Supplementary movie 2
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Supplementary movie 3
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Supplementary movie 4
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Supplementary movie 5
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References

  • 1.Eden S, Rohatgi R, Podtelejnikov AV, Mann M, Kirschner MW. Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck. Nature. 2002;418:790–793. doi: 10.1038/nature00859. [DOI] [PubMed] [Google Scholar]
  • 2.Stovold CF, Millard TH, Machesky LM. Inclusion of Scar/WAVE3 in a similar complex to Scar/WAVE1 and 2. BMC cell biology. 2005;6:11. doi: 10.1186/1471-2121-6-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Machesky LM, Insall RH. Scar1 and the related Wiskott-Aldrich syndrome protein, WASP, regulate the actin cytoskeleton through the Arp2/3 complex. Current biology: CB. 1998;8:1347–1356. doi: 10.1016/s0960-9822(98)00015-3. [DOI] [PubMed] [Google Scholar]
  • 4.Suetsugu S, Miki H, Takenawa T. Identification of two human WAVE/SCAR homologues as general actin regulatory molecules which associate with the Arp2/3 complex. Biochemical and biophysical research communications. 1999;260:296–302. doi: 10.1006/bbrc.1999.0894. [DOI] [PubMed] [Google Scholar]
  • 5.Cai X, Xiao T, James SY, Da J, Lin D, Liu Y, Zheng Y, Zou S, Di X, Guo S, et al. Metastatic potential of lung squamous cell carcinoma associated with HSPC300 through its interaction with WAVE2. Lung Cancer. 2009;65:299–305. doi: 10.1016/j.lungcan.2009.06.001. [DOI] [PubMed] [Google Scholar]
  • 6.Iwaya K, Norio K, Mukai K. Coexpression of Arp2 and WAVE2 predicts poor outcome in invasive breast carcinoma. Modern pathology. 2007;20:339–343. doi: 10.1038/modpathol.3800741. [DOI] [PubMed] [Google Scholar]
  • 7.Iwaya K, Oikawa K, Semba S, Tsuchiya B, Mukai Y, Otsubo T, Nagao T, Izumi M, Kuroda M, Domoto H, et al. Correlation between liver metastasis of the colocalization of actin-related protein 2 and 3 complex and WAVE2 in colorectal carcinoma. Cancer science. 2007;98:992–999. doi: 10.1111/j.1349-7006.2007.00488.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Silva JM, Ezhkova E, Silva J, Heart S, Castillo M, Campos Y, Castro V, Bonilla F, Cordon-Cardo C, Muthuswamy SK, et al. Cyfip1 is a putative invasion suppressor in epithelial cancers. Cell. 2009;137:1047–1061. doi: 10.1016/j.cell.2009.04.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Wolf K, Mazo I, Leung H, Engelke K, von Andrian UH, Deryugina EI, Strongin AY, Brocker EB, Friedl P. Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis. The Journal of cell biology. 2003;160:267–277. doi: 10.1083/jcb.200209006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Friedl P, Wolf K. Tumour-cell invasion and migration: diversity and escape mechanisms. Nature reviews. Cancer. 2003;3:362–374. doi: 10.1038/nrc1075. [DOI] [PubMed] [Google Scholar]
  • 11.Sanz-Moreno V, Gadea G, Ahn J, Paterson H, Marra P, Pinner S, Sahai E, Marshall CJ. Rac activation and inactivation control plasticity of tumor cell movement. Cell. 2008;135:510–523. doi: 10.1016/j.cell.2008.09.043. [DOI] [PubMed] [Google Scholar]
  • 12.Kurisu S, Suetsugu S, Yamazaki D, Yamaguchi H, Takenawa T. Rac-WAVE2 signaling is involved in the invasive and metastatic phenotypes of murine melanoma cells. Oncogene. 2005;24:1309–1319. doi: 10.1038/sj.onc.1208177. [DOI] [PubMed] [Google Scholar]
  • 13.Yang LY, Tao YM, Ou DP, Wang W, Chang ZG, Wu F. Increased expression of Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2 correlated with poor prognosis of hepatocellular carcinoma. Clinical cancer research. 2006;12:5673–5679. doi: 10.1158/1078-0432.CCR-06-0022. [DOI] [PubMed] [Google Scholar]
  • 14.Semba S, Iwaya K, Matsubayashi J, Serizawa H, Kataba H, Hirano T, Kato H, Matsuoka T, Mukai K. Coexpression of actin-related protein 2 and Wiskott-Aldrich syndrome family verproline-homologous protein 2 in adenocarcinoma of the lung. Clinical cancer research. 2006;12:2449–2454. doi: 10.1158/1078-0432.CCR-05-2566. [DOI] [PubMed] [Google Scholar]
  • 15.Yokotsuka M, Iwaya K, Saito T, Pandiella A, Tsuboi R, Kohno N, Matsubara O, Mukai K. Overexpression of HER2 signaling to WAVE2-Arp2/3 complex activates MMP-independent migration in breast cancer. Breast cancer research and treatment. 2011;126:311–318. doi: 10.1007/s10549-010-0896-x. [DOI] [PubMed] [Google Scholar]
  • 16.Sossey-Alaoui K, Ranalli TA, Li X, Bakin AV, Cowell JK. WAVE3 promotes cell motility and invasion through the regulation of MMP-1, MMP-3, and MMP-9 expression. Experimental cell research. 2005;308:135–145. doi: 10.1016/j.yexcr.2005.04.011. [DOI] [PubMed] [Google Scholar]
  • 17.Sossey-Alaoui K, Safina A, Li X, Vaughan MM, Hicks DG, Bakin AV, Cowell JK. Down-regulation of WAVE3, a metastasis promoter gene, inhibits invasion and metastasis of breast cancer cells. The American journal of pathology. 2007;170:2112–2121. doi: 10.2353/ajpath.2007.060975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Fernando HS, Davies SR, Chhabra A, Watkins G, Douglas-Jones A, Kynaston H, Mansel RE, Jiang WG. Expression of the WASP verprolin-homologues (WAVE members) in human breast cancer. Oncology. 2007;73:376–383. doi: 10.1159/000136157. [DOI] [PubMed] [Google Scholar]
  • 19.Scott LA, Vass JK, Parkinson EK, Gillespie DA, Winnie JN, Ozanne BW. Invasion of normal human fibroblasts induced by v-Fos is independent of proliferation, immortalization, and the tumor suppressors p16INK4a and p53. Molecular and cellular biology. 2004;24:1540–1559. doi: 10.1128/MCB.24.4.1540-1559.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Caswell PT, Spence HJ, Parsons M, White DP, Clark K, Cheng KW, Mills GB, Humphries MJ, Messent AJ, Anderson KI, et al. Rab25 associates with alpha5beta1 integrin to promote invasive migration in 3D microenvironments. Developmental cell. 2007;13:496–510. doi: 10.1016/j.devcel.2007.08.012. [DOI] [PubMed] [Google Scholar]
  • 21.Hennigan RF, Hawker KL, Ozanne BW. Fos-transformation activates genes associated with invasion. Oncogene. 1994;9:3591–3600. [PubMed] [Google Scholar]
  • 22.Timpson P, McGhee EJ, Morton JP, von Kriegsheim A, Schwarz JP, Karim SA, Doyle B, Quinn JA, Carragher NO, Edward M, et al. Spatial regulation of RhoA activity during pancreatic cancer cell invasion driven by mutant p53. Cancer research. 2011;71:747–757. doi: 10.1158/0008-5472.CAN-10-2267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Edward M, Gillan C, Micha D, Tammi RH. Tumour regulation of fibroblast hyaluronan expression: a mechanism to facilitate tumour growth and invasion. Carcinogenesis. 2005;26:1215–1223. doi: 10.1093/carcin/bgi064. [DOI] [PubMed] [Google Scholar]
  • 24.Blagg SL, Stewart M, Sambles C, Insall RH. PIR121 regulates pseudopod dynamics and SCAR activity in Dictyostelium. Curr Biol. 2003;13:1480–1487. doi: 10.1016/s0960-9822(03)00580-3. [DOI] [PubMed] [Google Scholar]
  • 25.Dubielecka PM, Ladwein KI, Xiong X, Migeotte I, Chorzalska A, Anderson KV, Sawicki JA, Rottner K, Stradal TE, Kotula L. Essential role for Abi1 in embryonic survival and WAVE2 complex integrity. Proceedings of the National Academy of Sciences of the United States of America. 2011;108:7022–7027. doi: 10.1073/pnas.1016811108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Ibarra N, Blagg SL, Vazquez F, Insall RH. Nap1 regulates Dictyostelium cell motility and adhesion through SCAR-dependent and -independent pathways. Curr Biol. 2006;16:717–722. doi: 10.1016/j.cub.2006.02.068. [DOI] [PubMed] [Google Scholar]
  • 27.Kunda P, Craig G, Dominguez V, Baum B. Abi, Sra1, and Kette control the stability and localization of SCAR/WAVE to regulate the formation of actin-based protrusions. Curr Biol. 2003;13:1867–1875. doi: 10.1016/j.cub.2003.10.005. [DOI] [PubMed] [Google Scholar]
  • 28.Barnhart EL, Lee KC, Keren K, Mogilner A, Theriot JA. An adhesion-dependent switch between mechanisms that determine motile cell shape. PLoS Biol. 2011;9:e1001059. doi: 10.1371/journal.pbio.1001059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Cukierman E, Pankov R, Stevens DR, Yamada KM. Taking cell-matrix adhesions to the third dimension. Science. 2001;294:1708–1712. doi: 10.1126/science.1064829. [DOI] [PubMed] [Google Scholar]
  • 30.Dawson JC, Timpson P, Kalna G, Machesky LM. Mtss1 regulates epidermal growth factor signaling in head and neck squamous carcinoma cells. Oncogene. 2012;31:1781–1793. doi: 10.1038/onc.2011.376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Fernando HS, Sanders AJ, Kynaston HG, Jiang WG. WAVE3 is associated with invasiveness in prostate cancer cells. Urologic oncology. 2010;28:320–327. doi: 10.1016/j.urolonc.2008.12.022. [DOI] [PubMed] [Google Scholar]
  • 32.Andrew N, Insall RH. Chemotaxis in shallow gradients is mediated independently of PtdIns 3-kinase by biased choices between random protrusions. Nat Cell Biol. 2007;9:193–200. doi: 10.1038/ncb1536. [DOI] [PubMed] [Google Scholar]
  • 33.Bear JE, Rawls JF, Saxe CL., 3rd SCAR, a WASP-related protein, isolated as a suppressor of receptor defects in late Dictyostelium development. The Journal of cell biology. 1998;142:1325–1335. doi: 10.1083/jcb.142.5.1325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Sossey-Alaoui K, Li X, Ranalli TA, Cowell JK. WAVE3-mediated cell migration and lamellipodia formation are regulated downstream of phosphatidylinositol 3-kinase. The Journal of biological chemistry. 2005;280:21748–21755. doi: 10.1074/jbc.M500503200. [DOI] [PubMed] [Google Scholar]
  • 35.Sossey-Alaoui K, Safina A, Li X, Vaughan MM, Hicks DG, Bakin AV, Cowell JK. Down-regulation of WAVE3, a metastasis promoter gene, inhibits invasion and metastasis of breast cancer cells. The American journal of pathology. 2007;170:2112–2121. doi: 10.2353/ajpath.2007.060975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Roussos ET, Condeelis JS, Patsialou A. Chemotaxis in cancer. Nature reviews. Cancer. 2011;11:573–587. doi: 10.1038/nrc3078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Scott RW, Hooper S, Crighton D, Li A, Konig I, Munro J, Trivier E, Wickman G, Morin P, Croft DR, et al. LIM kinases are required for invasive path generation by tumor and tumor-associated stromal cells. The Journal of cell biology. 2010;191:169–185. doi: 10.1083/jcb.201002041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Timpson P, McGhee EJ, Erami Z, Nobis M, Quinn JA, Edward M, Anderson KI. Organotypic collagen I assay: a malleable platform to assess cell behaviour in a 3-dimensional context. Journal of visualized experiments: JoVE. 2011:e3089. doi: 10.3791/3089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Wang C, Navab R, Iakovlev V, Leng Y, Zhang J, Tsao MS, Siminovitch K, McCready DR, Done SJ. Abelson interactor protein-1 positively regulates breast cancer cell proliferation, migration, and invasion. Molecular cancer research: MCR. 2007;5:1031–1039. doi: 10.1158/1541-7786.MCR-06-0391. [DOI] [PubMed] [Google Scholar]
  • 40.Innocenti M, Gerboth S, Rottner K, Lai FP, Hertzog M, Stradal TE, Frittoli E, Didry D, Polo S, Disanza A, et al. Abi1 regulates the activity of N-WASP and WAVE in distinct actin-based processes. Nature cell biology. 2005;7:969–976. doi: 10.1038/ncb1304. [DOI] [PubMed] [Google Scholar]

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