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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Trends Cell Biol. Author manuscript; available in PMC 2012 July 23.
Published in final edited form as:
PMCID: PMC3402095
NIHMSID: NIHMS252486

Metastasis: tumor cells becoming Menacing

Abstract

During breast cancer metastasis, cells emigrate from the primary tumor to the bloodstream, which carries them to distant sites where they infiltrate and sometimes form metastases within target organs. These cells must penetrate the dense extracellular matrix comprising the basement membrane of the mammary duct/acinus and migrate toward blood and lymphatic vessels, processes that mammary tumor cells execute using primarily Epidermal Growth Factor (EGF)-dependent protrusive and migratory activity. Here, we focus on how the actin regulatory protein Mena affects EGF-elicited movement, invasion and metastasis. Recent findings indicate that, in invasive migratory tumor cells, Mena isoforms that endow heightened sensitivity to EGF and increased protrusive and migratory abilities are up-regulated, while other isoforms are selectively down-regulated. This change in Mena isoform expression enables tumor cells to invade in response to otherwise benign EGF stimulus levels and may offer an opportunity to identify metastatic risk in patients.

Introduction

A traditional view of metastasis holds that metastases results from a process similar to Darwinian evolution involving the natural selection of tumor cells that are capable of migration and survival at distant sites. In this model, the selection of tumor cells exhibiting stable genetic changes occurs; these selected cells are very rare and cause metastasis late in tumor progression [1]. The recent development of new technologies, including high-density microarray-based expression profiling, intravital imaging and the collection of invasive tumor cells from live tumors, have challenged this traditional model of metastasis. These technologies have also supplied new diagnostic and therapeutic markers of metastatic disease. Studies of mammary tumors in mice [2, 3, 4, 5], expression profiling of whole human breast tumors [6, 7] and collection and profiling of the invasive subpopulation of tumor cells isolated from rat and mouse mammary tumors [8, 9, 10] indicate that metastatic ability is acquired at much earlier stages of tumor progression than predicted by the Darwinian model, is encoded throughout the bulk of the primary tumor, and involves transient changes in gene expression.

These results may be reconciled with the Darwinian model if the selection of stable genetic changes in the primary tumor during progression contributes the microenvironments necessary to induce the transient changes in gene expression that support the invasive and metastatic phenotype. The stable genetic changes required for induction of the microenvironments of invasion and cell dissemination could occur early in progression and throughout the tumor. The Tumor Microenvironment Invasion Model, which is based on this idea, holds that the tumor microenvironment initiates the expression of genes that induce cell motility, invasion and metastasis [9, 10, 11]. In this model, it is proposed that oncogenic mutations in tumor cells in the primary tumor lead to microenvironments that induce cell motility in tumor cells and stromal cells. Examples of such microenvironments are increased microvascular density [12] inflammation [13] and hypoxia [14]. These micro-environments are speculated to elicit transient and epigenetic changes in gene expression in tumor and stromal cells that resemble programs of gene expression used to drive morphogenetic cell movements in the developing embryonic organ. When the primary tumor is located in an adult organ, tumor microenvironments may trigger the embryonic program of gene expression of this organ leading to epithelial to mesenchymal transition (EMT) and the morphogenetic-like movements of cells clinically referred to as invasion and metastasis.

The Tumor Microenvironment Invasion Model predicts that microenvironments causing invasion and metastasis could appear randomly in time and location in the primary tumor leading to repeated episodes of invasion and systemic tumor cell dissemination (potentially leading to metastasis) throughout tumor progression [9]. Consistent with this model, intravital imaging of experimental mammary tumors demonstrates that only a small proportion of tumor cells are motile but are distributed throughout the tumor and are observed most frequently localized in certain areas of the tumor, particularly around peri-vascular macrophages [15, 16, 17]. Furthermore, genes correlating with metastatic outcome in a variety of solid tumors appear to be expressed early and throughout the bulk of the tumor [6, 7] and invasive mammary tumor cells can be collected throughout tumors with chemoattractant-containing needles [5, 10]. The model is also supported by the observation that micrometastases are often genetically heterogeneous, suggesting that invasive behavior is not stably specified [18]. Finally, the Tumor Microenvironment Invasion Model is generally consistent with our current understanding of how the tumor micro-environment contributes to invasion and metastasis [19].

Expression profiling of invasive tumor cells collected from primary mammary tumors reveal an invasion signature — a list of genes whose expression is uniquely altered in invasive tumor cells — involving motility pathways that account for the migratory and chemotactic activity of these cells in vivo [8-11]. One of the molecules that was highly up-regulated in the invasive mammary tumor cells collected in vivo is Mena [10], consistent with observations that high Mena levels are associated with poor clinical outcome in breast cancer patients [32, 34]. Mena, an actin regulatory protein, influences several of the motility pathways of the invasion signature, by controlling actin polymerization that is initiated in common by these pathways [10, 49]. The frequency of a tripartite structure comprised of peri-vascular tumor cells expressing high Mena levels juxtaposed to peri-vascular macrophages within breast cancer patient samples correlates well with the likelihood of metastatic disease, suggesting that Mena will be a useful prognostic biomarker for metastasis [33].

The goal of this review is to outline recent approaches developed in mammary tumors to investigate the properties of the tumor microenvironment and how tumor cells in this setting can acquire an invasive, metastatic phenotype by changes in gene expression, provide a background on the Mena protein and summarize recent findings indicating that changes in Mena expression promote metastatic progression and discuss the possible mechanisms that underlie Mena's effects on tumor cell behavior.

Identifying and collecting invasive tumor cells

To detect tumor cell behaviors in primary mammary tumors that resemble “morphogenetic” cell movements, as described above, much effort has been expended to develop intravital imaging methods that enable detailed characterization of the behavior of carcinoma and stromal cells within intact primary tumors [15, 20-24]. The resulting methods yield quantitative information about individual cell behavior in vivo, permitting analysis of parameters such as: directional migration toward histological landmarks such as blood vessels; frequency, velocity and persistence of cell motility; interactions between tumor cells, extracellular matrix and stromal cells that lead to invasion; and intravasation and extravasation. These imaging methods are valuable in defining cell behaviors necessary for invasion, intravasation and extravasation, phenotypes of cells harboring specific mutations, polarized motility and chemotaxis of cells in vivo, and the definition, size and regulation of microenvironments in vivo.

In mammary carcinomas, intravital imaging in mice and rats revealed the microenvironments in which tumor cells undergo migration and intravasation, and the importance of macrophages in these events (reviewed in [13, 15, 25, 26]). In particular, chemotaxis of tumor cells toward macrophages was found to be essential for invasion in primary mammary tumors [5, 27], while chemotaxis of tumor cells toward peri-vascular macrophages was required for intravasation [16]. Furthermore, invasion, intravasation and metastasis all involve a paracrine loop between macrophages and tumor cells which secrete EGF and CSF1, respectively [5, 27].

The point at which tumor cells migrate through the endothelium of blood vessels was identified as the site of blood vessel docking of at least one peri-vascular macrophage [16]. This detailed information about how tumor cells are attracted to blood vessels led to the development of the “In vivo Invasion Assay.” This assay mimics a blood vessel's micro-environment, being comprised of a tube filled with matrigel and EGF or CSF1 to attract invasive tumor cells and their associated macrophages in vivo as a migrating population of cells. The In vivo Invasion Assay has enabled the capture of live invasive tumor cells directly from the microenvironment for expression profiling [8, 10].

Expression profiling of invasive mammary tumor cells collected in vivo defines an Invasion Signature

Expression profiling of invasive tumor cells obtained from mammary tumors using the In vivo Invasion Assay revealed the genes correlated with survival, adjuvant-resistance and chemotaxis of invasive cancer cells inside living mammary tumors [8, 10, 28-31]. These genes, known collectively as the “Invasion Signature”, fall into well-defined pathways and are coordinately regulated in metastatic tumor cells [9-11] (Figure 1).

Figure 1
The motility and chemotaxis pathways of the Invasion Signature

The relevance of the Invasion Signature to the chemotactic migratory behavior of metastatic cancer cells during invasion and intravasation has been examined in a number of studies. A major insight to emerge from this body of work is that the motility pathways of the Invasion Signature define the mechanisms for tumor cell migration in vivo [11]. One of the genes highly up-regulated in the motility pathways of the Invasion Signature of invasive tumor cells collected from rat and mouse mammary tumors is Mena [8, 10]. Mena is also up-regulated in human breast cancer [32-34] as well as pancreatic, colon, gastric and cervical cancers [35-38].

Mena and the Tumor Microenvironment of Metastasis

The above results suggest that the level of Mena expression in tumors will be a useful biomarker for the evaluation of enhanced tumor cell motility and invasion, and metastasis in human tumors. In addition, as summarized above, invasive carcinoma cells in mouse and rat mammary tumors intravasate when associated with peri-vascular macrophages, thereby identifying a metastasis microenvironment as an anatomical structure in tumors [16, 17]. Therefore, we define the tripartite arrangement — identified by triple immunohistochemistry — of an invasive carcinoma cell (marked by Mena over-expression), a macrophage, and an endothelial cell as “TMEM,” for Tumor Microenvironment of Metastasis. TMEM has been identified in human breast tumors using this technique [33]. In a retrospective study, TMEM density in human breast carcinoma samples was found to predict the development of systemic, hematogenous metastases. In this study, a case-control analysis was performed on thirty patients who developed metastatic breast cancer and thirty patients without metastatic disease. Cases were matched to controls based on currently used prognostic criteria. Primary breast cancer samples were stained using the triple immunohistochemical method to identify and count TMEM density. Two pathologists, blinded to outcome, evaluated the number of TMEM per twenty high-power fields. TMEM density was not correlated with tumor size, lymph node metastasis, lymphovascular invasion, or hormone receptor status. However, TMEM density was greater in patients who developed systemic metastases compared to the patients with only localized breast cancer. In addition, for every increase in TMEM of 10, the risk of systemic metastasis increased by 90%. TMEM is therefore a novel prognostic marker for hematogenous metastasis of human breast tumors [33]. This work also illustrates the power of combining multi-photon imaging with mouse models of breast cancer in the development of new insights into, and markers for predicting, metastasis, and the microenvironments essential to dissemination of tumor cells in vivo. The insights into metastasis provided by multi-photon imaging also help to refine or challenge existing models for the molecular mechanisms underlying metastatic progression and to develop hypotheses to be tested using cell biological and molecular approaches in vitro.

Mena and the Ena/VASP family in actin dynamics

As noted above, Mena (also referred to as “ENAH” by the HUGO nomenclature committee database) is up-regulated in human breast cancer and is a part of the cell motility pathways identified in the mammary tumor Invasion Signature. Mena is the mammalian ortholog of Drosphila Enabled (Ena), identified originally as a genetic suppressor of phenotypes caused by mutations in the Drosophila c-Abl tyrosine kinase homolog [39]. Mena, along with the highly-related VASP and EVL proteins, comprise the vertebrate members of the Ena/VASP family, molecules that regulate cell movement, shape and adhesion [40, 41], processes required during invasion and metastasis. Caenorhabditis elegans and Drosophila melanogaster each contain a single Ena/VASP ortholog [41]; genetic analysis in both systems revealed roles for Ena/VASP in neural development [53] and epithelial morphogenesis [57, 58]. The vertebrate Ena/VASP proteins play pivotal roles in controlling the movement and morpholology [41] of a variety of cell types including fibroblasts [42], endothelial cells [48], epithelial cells [57, 59, 60] and neurons [44, 55, 48, 52, 55, 60, 61]. Ena/VASP proteins are also required for a variety of chemotactic responses [49, 56, 62], such as to the axon guidance factors Netrin (a chemoattractant) and SLIT (a repulsive cue) [63]. Subsequent work showed that Ena/VASP proteins are required in the early stages of neurite formation to generate filopodia. Unexpectedly, Ena/VASP proteins enable exocytosis, mediated specifically by the v-Snare VAMP2, which delivers membrane needed for the massive increase in surface area that accompanies neurite formation [99]. Interestingly, one way in which Mena expression drives metastasis is by increasing the amount of secreted protease activity by carcinoma cells [49].

Most cell types express one or more of the Ena/VASP proteins, which in turn localize to the leading edges of lamellipodia, the tips of filopodia, focal adhesions, cell-cell junctions and, in some cell types, in a sarcomeric pattern along stress fibers [41] (Figure 2). Ena/VASP proteins promote formation of long, sparsely branched actin filament networks [41, 42, 45-47] that modulate the morphology and dynamics of membrane protrusions and ultimately affect cell shape and motility [42-44]. The number of free barbed ends detected by incubating permeabilized cells with labeled actin correlates directly with Ena/VASP levels [48, 49]. Since Ena/VASP proteins do not create new barbed ends by nucleating new actin filaments under physiological conditions, these findings point to a role for Ena/VASP in maintaining polymerization-competent barbed ends in vivo [45, 50]. The molecular mechanism underlying the ability of Ena/VASP to regulate the geometry of actin network assembly has been recently reviewed (Box 1).

Figure 2
Distribution of Mena in a primary hippocampal growth cone (top) and an MTLn3 carcinoma cell stimulated with EGF (bottom)

Box 1

Ena/VASP and the regulation of actin dynamics

How Ena/VASP proteins regulate actin dynamics has been debated in the literature, and, as this topic has been reviewed recently [45, 109], we will present only a brief overview of the subject. Multiple distinct models for Ena/VASP function have been proposed over the past five years. One model proposes that Ena/VASP interacts with the barbed ends of F-actin filaments and enhances the rate of F-actin polymerization, and delays capping by barbed end capping proteins (“anti-capping”) [94, 50, 42]. An extension of this model suggests that the Ena/VASP interaction with profilin:actin complexes facilitates direct monomer transfer to the barbed ends, increases the rate of filament elongation and enhances the anti-capping activity [94, 85, 50]. Another study proposed that Ena/VASP has no effect on filament elongation, suggesting instead that Ena/VASP acts solely to bundle filaments that are nucleated by formins, which nucleate linear actin filaments and act as processive (remaining attached to the filament) barbed end elongating factors [97]. Yet a third model proposed that Ena/VASP enhances filament elongation but does not have anti-capping activity in solution or utilize profilin for monomer transfer to filaments [95]. In addition, this study suggested that Ena/VASP could block capping of filaments, but only upon dense clustering on beads that also induced a shift to processive filament elongation [95].

All of the studies listed above used either bulk polymerization assays or visual assays in which the actin, but not the Ena/VASP, was labeled. Direct insight into Ena/VASP function by visual, single-molecule assays has been missing from the field. A recent study employing a visual assay with labeled VASP found that VASP binding to the barbed ends of filaments is strongly enhanced by the presence of actin monomer, suggesting that the F-actin binding activity in VASP combined with monomer binding impaired all F-actin binding except to the barbed end, which could accommodate the monomer. Labeled VASP was observed at the tips of elongating filaments and enhanced the rate of filament growth [111]. Therefore, VASP is in fact a processive actin polymerase. VASP also enhances the rate of filament elongation in the presence of profilin, supporting the direct monomer transfer model. Importantly, VASP delayed the rate of filament capping by capping protein six-fold, proving definitively that it has anti-capping activity [111]. The development of a visual assay for Ena/VASP activity will allow the field to move beyond this debate over whether VASP has anti-capping activity, whether it can utilize profilin-actin for polymerization, and whether it is a processive elongation factor. It will be interesting to see how the other Ena/VASP proteins, and in particular the various Mena isoforms, behave in similar assays.

A further interesting twist to the study of Ena/VASP function comes from several studies that suggest various Ena/VASP family members interact genetically with members of the formin family of actin nucleation/elongation factors. In some cases, Ena/VASP proteins can be co-immunoprecipitated with formin family proteins including mDia1 [96], DdDia2 [97] and Drosophila Diaphonous [98]. It is not clear how much of the total pools of Ena/VASP and the various formins are in complex together, but it is likely to represent a relatively small fraction of each. While it seems unlikely that Ena/VASP act simply to bundle filaments behind formins, it will be interesting to determine the functional role of these Ena/VASP:formin complexes.

Given the effects of Ena/VASP on actin networks, it is not surprising that one major function of Ena/VASP is to regulate the dynamics of the unbranched, parallel and bundled actin filaments that comprise filopodia, and that loss of Ena/VASP function greatly impairs or eliminates filopodia formation in neurons and a variety of other cell types [51-56]. In some cell types such as fibroblasts, elevated Ena/VASP activity leads to frequent failure in effective lamellipodial protrusion due to the relatively long sparsely branched actin networks that buckle in response to the countervailing forces of membrane tension [42]. Other cell types, including carcinoma cells, are equipped to translate the effects of elevated Ena/VASP activity into productive protrusions that lead to cell translocation [49].

Mena isoforms in motility and invasion

Mena, like the other Ena/VASP proteins, contains two conserved domains called “EVH1” and “EVH2” and a central unstructured proline-rich region (Figure 3, and Box 2). The EVH1 domain mediates protein-protein interactions important for Ena/VASP localization and regulation (Box 3). The polyproline-rich region and EVH2 interact with the actin monomer binding protein profilin and directly with G- and F-actin, respectively [45].

Figure 3
Mena Domain Structure

Box 2

Organization of Ena/VASP proteins

The N-terminal EVH1 domain (for Ena/VASP homology) binds to proteins containing a specific proline-rich motif that helps localize Ena/VASP proteins and recruit them into complexes with signaling proteins.

The middle portion of Ena/VASP proteins consists of a proline-rich region that binds a number of SH3- and WW-domain containing proteins including IRSp53, an I-Bar protein and Cdc42 effector that promotes filopodial formation [81, 82]. The proline-rich region also binds to the actin monomer binding profilin proteins, which play diverse roles in regulating actin dynamics (see recent reviews [83, 84]), including the ability to transfer bound monomer onto free F-actin barbed ends. Profilin can bind actin monomer and interact simultaneously with Ena/VASP through a high-affinity profilin-binding site (termed “loading site” [85]) with the consenus PPP[AP]PPLP [68, 83, 85, 86]. Importantly, profilin:actin complexes have a higher affinity for the loading site than does profilin alone, suggesting that once actin monomer is transferred from profilin to a barbed end, exchange of the profilin bound to the loading site for a new profilin:actin complex would be favored [86]. Interestingly, while VASP and Evl each have a single loading site, Mena contains four, suggesting it may be capable of more profilin:actin complexes than its paralogs. Importantly, the poly-Pro loading sites in Ena/VASP proteins are located adjacent to actin binding motifs contained in the EVH2 domain.

The C-terminal EVH2 domain of Ena/VASP contains binding sites for G- and F-actin [87, 88], called “GAB” and “FAB”, respectively. The proximity of a poly-Pro loading site permits Ena/VASP to bind profilin+G-actin complexes through two interfaces simultaneously: profilin-PPP[AP]PPLP and the adjacent G-actin+GAB. The G-actin in this complex is oriented towards the FAB motif of Ena/VASP, presumably positioned to be added on to growing filaments [85]. The organization of binding sites for profilin, actin monomer and F-actin lead to a model in which profilin:actin binding to the loading site+GAB is followed by direct transfer of the monomer onto the adjacent F-actin barbed end and subsequent exchange of profilin for profilin:actin [83][89].

EVH2-mediated interactions with growing ends of actin filaments are required for stable targeting of Ena/VASP to the leading edge of lamellipodia [42, 51, 91]. The GAB motif stabilizes Ena/VASP at the tips of filopodia suggesting that it plays a role in recognizing barbed ends analogous to the barbed end capture activity in the highly-related WH2 domain within N-WASP [51]. Both G- and F-actin interactions are disrupted by phosphorylation at sites within the EVH2 domain [50, 90], including a protein kinase G site found in both Mena and VASP [40, 91].

At the very C-terminus of EVH2, a right-handed coiled-coil mediates both homo-tetramerization and the formation of mixed tetramers containing different family members [92, 93]. The combination of tetramerization and F-actin binding allows Ena/VASP to bundle actin filaments [87]; this bundling activity acts to cluster the tips of elongating filaments during filopodial formation and extension [51], however, a physiological role for Ena/VASP bundling along the length of filaments in cells has not been demonstrated.

Box 3

EVH1-mediated interactions

EVH1 domains bind proteins that contain the consensus: [FL]PX[var phi]P, where [var phi] is any hydrophobic residue [65, 66]. There are a growing number of proteins with EVH1-binding sites and a full discussion of all such molecules is beyond the scope of this review, therefore only a few examples will be presented. The first characterized EVH1-ligand was ActA, a protein found on the surface of the intracellular bacterial pathogen Listeria monocytogenes that contains four EVH1-binding motifs that recruit host cell Ena/VASP proteins to the bacterial surface [66]. Listeria employ host cell proteins to trigger actin polymerization on the bacterial surface to produce a propulsive force that drives their movement [67] and Ena/VASP recruitment by ActA greatly enhances actin polymerization and bacterial movement [68, 69]. Zyxin, which helps recruit Ena/VASP to focal adhesions and stress fibers, contains four EVH1-binding sites [110]. Lamellipodin (Lpd), an adaptor protein containing RA and PH domains that bind Ras and PI(3,4)P2, respectively, harbors six EVH1-binding sites and plays an important role in recruiting Ena/VASP to lamellipodia [70, 71]. Silencing Lpd in B16 cells produces a dramatic reduction in F-actin content, thereby eliminating normal lamellipodial protrusion. Lpd is a target for Abl/Arg tyrosine kinases and is required along with Ena/VASP for PDGF-induced dorsal ruffling in fibroblasts [72] and the Drosophila Lpd is required for normal epithelial morphogenesis [73]. Mig-10, the C.elegans Lpd ortholog, is required for cell polarization in response to Netrin and for axon guidance responses to Netrin and Slit [74-76]. The Slit receptor, Robo, binds to Ena/VASP through EVH1-binding sites in its cytoplasmic tail [63]. Palladin, an actin binding protein and EVH1 ligand [77] has been implicated in metastatic progression; it is upregulated 3.2-fold in the invasion signature [11], contributes to breast cancer cell invasion [78] and is a target for the anti-metastatic kinase Akt-1, which blocks Palladin-driven invasion [79]. Finally, the putative tumor suppressor TES is an unconventional Mena-specific EVH1 ligand that binds via a LIM domain to a region that overlaps with the [FL]PX[var phi]P binding pocket [80].

Mena has several unique features not found in the other Ena/VASP proteins that endow it with the ability to potentiate carcinoma metastasis dramatically. Importantly, alternate splicing of Mena produces distinct protein isoforms, including an invasion-specific isoform, “MenaINV” (discussed further below), that has no counterpart in VASP or EVL, and which is found exclusively in invasive tumor cells.

Analysis of the invasion signature of mammary carcinoma cells revealed that Mena expression was up-regulated in invasive cells compared to average primary tumor cells [8, 10]. Increased Mena levels were also observed in invasive human breast cancers compared to normal mammary tissue [32]. As noted above, perivascular tumor cells expressing high Mena levels are a component of TMEM, a structure whose density in clinical samples correlates with increased risk of metastatic outcome in breast cancer patients [33]. In addition to breast cancer, Mena up-regulation has been observed in advanced pancreatic, colon and cervical carcinomas [35-37, 100].

Mena has a number of features that its paralogs VASP and EVL do not share. The first is an extended repeat region spanning 70 residues with most of the repeats containing the consensus, [LM]-E-[QR]-[EQ]-[QR] (abbreviated as “LERER” repeat), which is predicted to form a coiled-coil structure [101]. The repeat is located between the EVH1 domain and the proline-rich region. In addition to this unique feature, the Mena message undergoes extensive alternate splicing to give rise to multiple protein isoforms that are expressed in specific tissues and cell-types [40] (Figure 3). In contrast, EVL has 1 alternately included exon and VASP has none. There are 14 constitutively included exons in Mena and 5 alternately included exons that can all encode protein sequence in frame. There has not been a comprehensive analysis of which of the possible combinations of alternately included exons are actually produced as mRNA, nor do we know all of the cell types which produce the various Mena isoforms.

Cloning Mena cDNA from a breast cancer cell line identified the Mena11a isoform [102]. Analysis of RNA from primary mammary tumor cells collected by FACs, compared to that expressed in invasive mammary tumor cells collected using the in vivo invasion assay, revealed that the 11a exon is expressed in tumor cells making up the bulk of the primary tumor, but this exon is essentially undetectable in the Mena message from invasive tumor cells [103]. Consistent with this finding, the 11a exon is specific to Mena isoforms expressed in epithelial cell lines and is not found in mesenchymal cells [100, 102]. In fact, 11a becomes excluded in human mammary epithelial cells that are driven to undergo epithelial to mesenchymal transition (EMT) by expression of the EMT inducing transcription factor Twist [104]. The presence of 11a in epithelial cells is driven in part by the activity of the recently identified epithelial-specific splicing factors ESRP1 and ESRP2 [105]. Mena11a is also expressed in normal ovarian tissue where its inclusion is promoted by the Fox2 splicing factor [106]. Interestingly, analysis of 21 aggressive ovarian tumors revealed a reduction in Fox2 levels compared to normal tissue and a concomitant loss of 11a inclusion in Mena [106]. Therefore, Mena11a appears to be included in epithelial cells and primary carcinomas but excluded from mesenchymal cells as well as invasive/aggressive tumor cells.

The alternately included 11a exon encodes 21 amino acids that are inserted in the EVH2 domain, between the FAB sequence and the coiled-coil tetramerization domain. The Mena paralog EVL also has an alternately included 21 amino acid insertion (“EVL-I”) in an identical relative location as the 11a insertion site, but the sequences share no similarity [107]. The site of 11a insertion is adjacent to the F- and G- actin binding sites, and the 11a insertion can be phosphorylated [102], potentially disrupting actin binding. Therefore, it is possible that the 11a inclusion affects the way in which Mena interacts with barbed ends and adds an extra site for phospho-regulation of Mena function.

Three alternately included Mena exons were identified by screening a mouse brain cDNA library [40]. The largest exon, denoted as “+,” falls adjacent to the proline-rich region and is itself quite rich in proline. Mena+ is a 798 residue protein (the most widely expressed form of Mena, denoted “Menaclassic,” is 541 amino acids), however, due to their high proline content both Mena+ and Menaclassic migrate aberrantly on SDS-PAGE gels at approximately 140kDa and 80kDa, respectively. Western blot analysis of adult tissues has shown that the 140kDa isoform is only readily detected in the brain compared to other organs and tissues [61]. Two other short exons, denoted “++” and “+++” and encoding 4 and 19 residues, respectively, were identified in brain cDNAs containing the “+” exon. Both ++ and +++ are inserted at the same site just C-terminal to the EVH1 domain and between the LERER repeat. No tissue-specific expression has been identified for Mena++ and Mena+++. Interestingly, the +++ exon is highly conserved in mammals but is not found in other vertebrates.

The majority of Mena mRNA up-regulated in the invasive subpopulation of tumor cells isolated from rat, mouse and human mammary tumors using the In vivo Invasion Assay contains either the ++ or +++ exon, while strong downregulation of Mena 11a occurs in the same invasive tumor cells. The upregulation of the ++ or +++ exons persists in circulating tumor cells isolated from blood [103]. These results suggest that Mena+++ and Mena++ are the isoforms that may function in metastatic progression.

This prediction has recently been tested [49] (Roussos et al., unpublished) and findings suggest that expression of Menaclassic, and Mena+++ (referred to as the “invasion isoform” or MenaINV) in particular, promotes carcinoma cell invasion in three-dimensional collagen gels and increases carcinoma cell motility in vivo [49]. Menaclassic and MenaINV localize to and stabilize invadopodia, actin-rich protrusions required for degradation and movement through extracellular matrix and possibly invasion across basement membranes, thereby increasing the invasive and metastatic potential of tumor cells.

Finally, MenaINV plays a sensitizing role in the chemotactic and motility responses of tumor cells to EGF as expression of MenaINV sensitizes mammary tumor cells to EGF signals by at least 25- to 50-fold, causing tumor cells to respond to otherwise undetectable EGF levels [49] (Roussos et al., unpublished). MenaINV regulates the lifetime of actin filament barbed ends produced by EGF-elicited protrusion; within as little as 20 seconds of stimulation, cells expressing MenaINV have 80% more free barbed ends than control cells or cells expressing Menaclassic [49]. The stimulatory effect of MenaINV requires cofilin severing but precedes the accumulation of Arp2/3 in lamellipodia, indicating that MenaINV acts directly on barbed ends generated by cofilin severing. Therefore, we propose that MenaINV exerts this stimulatory effect by delaying barbed end capping (Figure 4). This is an important finding because cofilin-generated barbed ends of actin filaments are needed to initiate invasive protrusions during chemotaxis and maintain the motility of crawling tumor cells [10, 49, 108]. The mechanisms underlying the effect of the additional 19 amino acids in the MenaINV isoform, and the ability of this isoform to potentiate EGF-dependent motility responses, are under investigation. The present findings, however, indicate that we have identified a master gene that makes breast cancer cells aMENAble to metastasis.

Figure 4
Proposed model for Mena anti-capping/elongation activity in carcinoma cell invasion

Concluding remarks and future directions

The identification of MenaINV and direct observation of its effects on tumor cell invasion and metastasis were made possible through the use of multiphoton imaging and the in vivo invasion assay. The next challenge is to turn these new insights into tools that can be used to diagnose, and potentially treat, metastatic disease. As a component of TMEM, Mena expression is already being used to develop prognostic tests. The development of new probes to the INV and 11a sequences may prove to be even more powerful and straightforward predictors of metastatic spread. Furthermore, since Mena deficiency in mice is compatible with viability, inhibitors of Mena function may be useful tools to prevent metastatic disease. Finally, given the powerful effects of alternate splicing on Mena, it is likely that regulation by splicing will alter the properties of many molecules relevant to morphogenetic cell movements, and cancer onset and progression. Regulation by splicing may be as, or even more, functionally significant than regulation at the level of gene expression. Through the use of new sequencing technologies, it should be possible to use the in vivo invasion assay to identify the entire repertoire of invasion isoforms.

Acknowledgments

This work is supported by grants from the NCI CA100324, CA150344, CA113395, CA126511 to JC and NIH GM58801, Integrated Cancer Biology Program grant 1-U54- CA11296, and funds from the Ludwig Center for Metastasis at MIT research to FBG. We thank Shannon Alford and Stephanie Gupton for contributing the images in Figure 2, and Scott Hansen and Dyche Mullins for sharing results prior to publication.

Footnotes

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References

1. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100:57–70. [PubMed]
2. Mantovani A, et al. Characterization of tumor lines derived from spontaneous metastases of a transplanted murine sarcoma. Eur J Cancer. 1981;17:71–76. [PubMed]
3. Giavazzi R, et al. Metastasizing capacity of tumour cells from spontaneous metastases of transplanted murine tumours. Br J Cancer. 1980;42:462–472. [PMC free article] [PubMed]
4. Milas L, et al. Spontaneous metastasis: random or selective? Clin Exp Metastasis. 1983;1:309–315. [PubMed]
5. Wyckoff J, et al. A paracrine loop between tumor cells and macrophages is required for tumor cell migration in mammary tumors. Cancer research. 2004;64:7022–7029. [PubMed]
6. van't Veer LJ, et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature. 2002;415:530–536. [PubMed]
7. Ramaswamy S, et al. A molecular signature of metastasis in primary solid tumors. Nat Genet. 2003;33:49–54. [PubMed]
8. Wang W, et al. Identification and testing of a gene expression signature of invasive carcinoma cells within primary mammary tumors. Cancer research. 2004;64:8585–8594. [PubMed]
9. Wang W, et al. Tumor cells caught in the act of invading: their strategy for enhanced cell motility. Trends Cell Biol. 2005;15:138–145. [PubMed]
10. Wang W, et al. Coordinated regulation of pathways for enhanced cell motility and chemotaxis is conserved in rat and mouse mammary tumors. Cancer research. 2007;67:3505–3511. [PubMed]
11. Condeelis J, et al. The great escape: when cancer cells hijack the genes for chemotaxis and motility. Annu Rev Cell Dev Biol. 2005;21:695–718. [PubMed]
12. Leek RD, Harris AL. Tumor-associated macrophages in breast cancer. J Mammary Gland Biol Neoplasia. 2002;7:177–189. [PubMed]
13. Condeelis J, Pollard JW. Macrophages: obligate partners for tumor cell migration, invasion, and metastasis. Cell. 2006;124:263–266. [PubMed]
14. Giraudo E, et al. An amino-bisphosphonate targets MMP-9-expressing macrophages and angiogenesis to impair cervical carcinogenesis. J Clin Invest. 2004;114:623–633. [PMC free article] [PubMed]
15. Condeelis J, Segall JE. Intravital imaging of cell movement in tumours. Nat Rev Cancer. 2003;3:921–930. [PubMed]
16. Wyckoff JB, et al. Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors. Cancer research. 2007;67:2649–2656. [PubMed]
17. Kedrin D, et al. Intravital imaging of metastatic behavior through a mammary imaging window. Nat Methods. 2008;5:1019–1021. [PMC free article] [PubMed]
18. Klein CA, et al. Genetic heterogeneity of single disseminated tumour cells in minimal residual cancer. Lancet. 2002;360:683–689. [PubMed]
19. Radisky D, et al. Tumors are unique organs defined by abnormal signaling and context. Semin Cancer Biol. 2001;11:87–95. [PubMed]
20. Sidani M, et al. Probing the microenvironment of mammary tumors using multiphoton microscopy. J Mammary Gland Biol Neoplasia. 2006;11:151–163. [PubMed]
21. Sahai E. Illuminating the metastatic process. Nat Rev Cancer. 2007;7:737–749. [PubMed]
22. Timpson P, et al. Quantitative real-time imaging of molecular dynamics during cancer cell invasion and metastasis in vivo. Cell Adh Migr. 2009;3:351–354. [PMC free article] [PubMed]
23. Perentes JY, et al. In vivo imaging of extracellular matrix remodeling by tumor-associated fibroblasts. Nat Methods. 2009;6:143–145. [PMC free article] [PubMed]
24. Egeblad M, et al. Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy. Dis Model Mech. 2008;1:155–167. discussion 165. [PMC free article] [PubMed]
25. Yamaguchi H, et al. Invadopodia and podosomes in tumor invasion. Eur J Cell Biol. 2006;85:213–218. [PubMed]
26. Kedrin D, et al. Imaging tumor cell movement in vivo. Curr Protoc Cell Biol. 2007;Chapter 19 Unit 19 17. [PubMed]
27. Goswami S, et al. Macrophages promote the invasion of breast carcinoma cells via a colony-stimulating factor-1/epidermal growth factor paracrine loop. Cancer research. 2005;65:5278–5283. [PubMed]
28. Wang W, et al. Gene expression analysis on small numbers of invasive cells collected by chemotaxis from primary mammary tumors of the mouse. BMC Biotechnol. 2003;3:13. [PMC free article] [PubMed]
29. Goswami S, et al. Breast cancer cells isolated by chemotaxis from primary tumors show increased survival and resistance to chemotherapy. Cancer research. 2004;64:7664–7667. [PubMed]
30. Wang W, et al. The activity status of cofilin is directly related to invasion, intravasation, and metastasis of mammary tumors. J Cell Biol. 2006;173:395–404. [PMC free article] [PubMed]
31. Xue C, et al. Epidermal growth factor receptor overexpression results in increased tumor cell motility in vivo coordinately with enhanced intravasation and metastasis. Cancer research. 2006;66:192–197. [PubMed]
32. Di Modugno F, et al. Human Mena protein, a serex-defined antigen overexpressed in breast cancer eliciting both humoral and CD8+ T-cell immune response. Int J Cancer. 2004;109:909–918. [PubMed]
33. Robinson BD, et al. Tumor microenvironment of metastasis in human breast carcinoma: a potential prognostic marker linked to hematogenous dissemination. Clin Cancer Res. 2009;15:2433–2441. [PMC free article] [PubMed]
34. Di Modugno F, et al. The cytoskeleton regulatory protein hMena (ENAH) is overexpressed in human benign breast lesions with high risk of transformation and human epidermal growth factor receptor-2-positive/hormonal receptor-negative tumors. Clin Cancer Res. 2006;12:1470–1478. [PubMed]
35. Gurzu S, et al. The immunohistochemical aspects of protein Mena in cervical lesions. Rom J Morphol Embryol. 2009;50:213–216. [PubMed]
36. Gurzu S, et al. The expression of cytoskeleton regulatory protein Mena in colorectal lesions. Rom J Morphol Embryol. 2008;49:345–349. [PubMed]
37. Toyoda A, et al. Aberrant expression of human ortholog of mammalian enabled (hMena) in human colorectal carcinomas: implications for its role in tumor progression. Int J Oncol. 2009;34:53–60. [PubMed]
38. Junnila S, et al. Genome-wide gene copy number and expression analysis of primary gastric tumors and gastric cancer cell lines. BMC Cancer. 10:73. [PMC free article] [PubMed]
39. Gertler FB, et al. Genetic suppression of mutations in the Drosophila abl proto-oncogene homolog. Science. 1990;248:857–860. [PubMed]
40. Gertler FB, et al. Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics. Cell. 1996;87:227–239. [PubMed]
41. Krause M, et al. Ena/VASP proteins: regulators of the actin cytoskeleton and cell migration. Annu Rev Cell Dev Biol. 2003;19:541–564. [PubMed]
42. Bear JE, et al. Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility. Cell. 2002;109:509–521. [PubMed]
43. Lacayo CI, et al. Emergence of large-scale cell morphology and movement from local actin filament growth dynamics. PLoS Biol. 2007;5:e233. [PubMed]
44. Lebrand C, et al. Critical role of Ena/VASP proteins for filopodia formation in neurons and in function downstream of netrin-1. Neuron. 2004;42:37–49. [PubMed]
45. Bear JE, Gertler FB. Ena/VASP: towards resolving a pointed controversy at the barbed end. J Cell Sci. 2009;122:1947–1953. [PubMed]
46. Skoble J, et al. Pivotal role of VASP in Arp2/3 complex-mediated actin nucleation, actin branch-formation, and Listeria monocytogenes motility. J Cell Biol. 2001;155:89–100. [PMC free article] [PubMed]
47. Plastino J, et al. Actin filaments align into hollow comets for rapid VASP-mediated propulsion. Curr Biol. 2004;14:1766–1771. [PubMed]
48. Furman C, et al. Ena/VASP is required for endothelial barrier function in vivo. J Cell Biol. 2007;179:761–775. [PMC free article] [PubMed]
49. Philippar U, et al. A Mena invasion isoform potentiates EGF-induced carcinoma cell invasion and metastasis. Dev Cell. 2008;15:813–828. [PMC free article] [PubMed]
50. Barzik M, et al. Ena/VASP proteins enhance actin polymerization in the presence of barbed end capping proteins. J Biol Chem. 2005;280:28653–28662. [PMC free article] [PubMed]
51. Applewhite DA, et al. Ena/VASP proteins have an anti-capping independent function in filopodia formation. Mol Biol Cell. 2007;18:2579–2591. [PMC free article] [PubMed]
52. Dent EW, et al. Filopodia are required for cortical neurite initiation. Nat Cell Biol. 2007;9:1347–1359. [PubMed]
53. Drees F, Gertler FB. Ena/VASP: proteins at the tip of the nervous system. Curr Opin Neurobiol. 2008;18:53–59. [PMC free article] [PubMed]
54. Gupton SL, Gertler FB. Filopodia: the fingers that do the walking. Sci STKE. 2007;2007:re5. [PubMed]
55. Kwiatkowski AV, et al. Ena/VASP Is Required for neuritogenesis in the developing cortex. Neuron. 2007;56:441–455. [PubMed]
56. Han YH, et al. Requirement of a vasodilator-stimulated phosphoprotein family member for cell adhesion, the formation of filopodia, and chemotaxis in dictyostelium. J Biol Chem. 2002;277:49877–49887. [PubMed]
57. Gates J, et al. Enabled plays key roles in embryonic epithelial morphogenesis in Drosophila. Development. 2007;134:2027–2039. [PubMed]
58. Sheffield M, et al. C. elegans Enabled exhibits novel interactions with N-WASP, Abl, and cell-cell junctions. Curr Biol. 2007;17:1791–1796. [PMC free article] [PubMed]
59. Scott JA, et al. Ena/VASP proteins can regulate distinct modes of actin organization at cadherin-adhesive contacts. Mol Biol Cell. 2006;17:1085–1095. [PMC free article] [PubMed]
60. Menzies AS, et al. Mena and vasodilator-stimulated phosphoprotein are required for multiple actin-dependent processes that shape the vertebrate nervous system. J Neurosci. 2004;24:8029–8038. [PubMed]
61. Lanier LM, et al. Mena is required for neurulation and commissure formation. Neuron. 1999;22:313–325. [PubMed]
62. Neel NF, et al. VASP is a CXCR2-interacting protein that regulates CXCR2-mediated polarization and chemotaxis. J Cell Sci. 2009;122:1882–1894. [PubMed]
63. Chédotal A. Slits and their receptors. Adv Exp Med Biol. 2007;621:65–80. [PubMed]
64. Strickland P, et al. Slit2 and netrin 1 act synergistically as adhesive cues to generate tubular bi-layers during ductal morphogenesis. Development. 2006;133:823–832. [PubMed]
65. Ball LJ, et al. EVH1 domains: structure, function and interactions. FEBS Lett. 2002;513:45–52. [PubMed]
66. Niebuhr K, et al. A novel proline-rich motif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVH1 domain, a protein module present in the Ena/VASP family. EMBO J. 1997;16:5433–5444. [PubMed]
67. Smith GA, Portnoy DA. How the Listeria monocytogenes ActA protein converts actin polymerization into a motile force. Trends Microbiol. 1997;5:272–276. [PubMed]
68. Geese M, et al. Contribution of Ena/VASP proteins to intracellular motility of listeria requires phosphorylation and proline-rich core but not F-actin binding or multimerization. Mol Biol Cell. 2002;13:2383–2396. [PMC free article] [PubMed]
69. Auerbuch V, et al. Ena/VASP proteins contribute to Listeria monocytogenes pathogenesis by controlling temporal and spatial persistence of bacterial actin-based motility. Mol Microbiol. 2003;49:1361–1375. [PubMed]
70. Krause M, et al. Lamellipodin, an Ena/VASP ligand, is implicated in the regulation of lamellipodial dynamics. Dev Cell. 2004;7:571–583. [PubMed]
71. Rodriguez-Viciana P, et al. Signaling specificity by Ras family GTPases is determined by the full spectrum of effectors they regulate. Mol Cell Biol. 2004;24:4943–4954. [PMC free article] [PubMed]
72. Michael M, et al. c-Abl, Lamellipodin, and Ena/VASP proteins cooperate in dorsal ruffling of fibroblasts and axonal morphogenesis. Curr Biol. 2010;20:783–791. [PMC free article] [PubMed]
73. Lyulcheva E, et al. Drosophila pico and its mammalian ortholog lamellipodin activate serum response factor and promote cell proliferation. Dev Cell. 2008;15:680–690. [PMC free article] [PubMed]
74. Adler CE, et al. UNC-6/Netrin induces neuronal asymmetry and defines the site of axon formation. Nat Neurosci. 2006;9:511–518. [PMC free article] [PubMed]
75. Chang C, et al. MIG-10/lamellipodin and AGE-1/PI3K promote axon guidance and outgrowth in response to slit and netrin. Curr Biol. 2006;16:854–862. [PubMed]
76. Quinn CC, et al. UNC-6/netrin and SLT-1/slit guidance cues orient axon outgrowth mediated by MIG-10/RIAM/lamellipodin. Curr Biol. 2006;16:845–853. [PubMed]
77. Boukhelifa M, et al. Palladin is a novel binding partner for Ena/VASP family members. Cell Motil Cytoskeleton. 2004;58:17–29. [PubMed]
78. Goicoechea SM, et al. Palladin contributes to invasive motility in human breast cancer cells. Oncogene. 2009;28:587–598. [PMC free article] [PubMed]
79. Chin YR, Toker A. The Actin-Bundling Protein Palladin Is an Akt1-Specific Substrate that Regulates Breast Cancer Cell Migration. Molecular Cell. 2010;38:333–344. [PMC free article] [PubMed]
80. Boeda B, et al. Tes, a specific Mena interacting partner, breaks the rules for EVH1 binding. Mol Cell. 2007;28:1071–1082. [PubMed]
81. Lim KB, et al. The Cdc42 effector IRSp53 generates filopodia by coupling membrane protrusion with actin dynamics. J Biol Chem. 2008;283:20454–20472. [PubMed]
82. Robens JM, et al. Regulation of IRSp53-dependent filopodial dynamics by antagonism between 14-3-3 binding and SH3-mediated localization. Mol Cell Biol. 2010;30:829–844. [PMC free article] [PubMed]
83. Dominguez R. Actin filament nucleation and elongation factors--structure-function relationships. Crit Rev Biochem Mol Biol. 2009;44:351–366. [PMC free article] [PubMed]
84. Birbach A. Profilin, a multi-modal regulator of neuronal plasticity. Bioessays. 2008;30:994–1002. [PubMed]
85. Ferron F, et al. Structural basis for the recruitment of profilin-actin complexes during filament elongation by Ena/VASP. EMBO J 2007 [PubMed]
86. Chereau D, Dominguez R. Understanding the role of the G-actin-binding domain of Ena/VASP in actin assembly. J Struct Biol. 2006;155:195–201. [PubMed]
87. Bachmann C, et al. The EVH2 domain of the vasodilator-stimulated phosphoprotein mediates tetramerization, F-actin binding, and actin bundle formation. J Biol Chem. 1999;274:23549–23557. [PubMed]
88. Huttelmaier S, et al. Characterization of the actin binding properties of the vasodilator-stimulated phosphoprotein VASP. FEBS Lett. 1999;451:68–74. [PubMed]
89. Kang F, et al. Profilin interacts with the Gly-Pro-Pro-Pro-Pro-Pro sequences of vasodilator-stimulated phosphoprotein (VASP): implications for actin-based Listeria motility. Biochemistry. 1997;36:8384–8392. [PubMed]
90. Harbeck B, et al. Phosphorylation of the vasodilator-stimulated phosphoprotein regulates its interaction with actin. J Biol Chem. 2000;275:30817–30825. [PubMed]
91. Loureiro JJ, et al. Critical roles of phosphorylation and actin binding motifs, but not the central proline-rich region, for Ena/vasodilator-stimulated phosphoprotein (VASP) function during cell migration. Mol Biol Cell. 2002;13:2533–2546. [PMC free article] [PubMed]
92. Ahern-Djamali SM, et al. Mutations in Drosophila enabled and rescue by human vasodilator-stimulated phosphoprotein (VASP) indicate important functional roles for Ena/VASP homology domain 1 (EVH1) and EVH2 domains. Mol Biol Cell. 1998;9:2157–2171. [PMC free article] [PubMed]
93. Kuhnel K, et al. The VASP tetramerization domain is a right-handed coiled coil based on a 15-residue repeat. Proc Natl Acad Sci U S A. 2004;101:17027–17032. [PubMed]
94. Pasic L, et al. Ena/VASP proteins capture actin filament barbed ends. J Biol Chem 2008 [PubMed]
95. Breitsprecher D, et al. Clustering of VASP actively drives processive, WH2 domain-mediated actin filament elongation. EMBO J. 2008;27:2943–2954. [PubMed]
96. Grosse R, et al. A role for VASP in RhoA-Diaphanous signalling to actin dynamics and SRF activity. EMBO J. 2003;22:3050–3061. [PubMed]
97. Schirenbeck A, et al. The bundling activity of vasodilator-stimulated phosphoprotein is required for filopodium formation. Proc Natl Acad Sci U S A. 2006;103:7694–7699. [PubMed]
98. Homem CC, Peifer M. Exploring the roles of diaphanous and enabled activity in shaping the balance between filopodia and lamellipodia. Mol Biol Cell. 2009;20:5138–5155. [PMC free article] [PubMed]
99. Gupton S, Gertler FB. Integrin Signaling Switches the Cytoskeletal and Exocytic Machinery That Drives Neuritogenesis. Dev Cell. 2010 in press. [PMC free article] [PubMed]
100. Pino MS, et al. Human Mena+11a isoform serves as a marker of epithelial phenotype and sensitivity to epidermal growth factor receptor inhibition in human pancreatic cancer cell lines. Clin Cancer Res. 2008;14:4943–4950. [PMC free article] [PubMed]
101. McDonnell AV, et al. Paircoil2: improved prediction of coiled coils from sequence. Bioinformatics. 2006;22:356–358. [PubMed]
102. Di Modugno F, et al. Molecular cloning of hMena (ENAH) and its splice variant hMena+11a: epidermal growth factor increases their expression and stimulates hMena+11a phosphorylation in breast cancer cell lines. Cancer research. 2007;67:2657–2665. [PMC free article] [PubMed]
103. Goswami S, et al. Identification of invasion specific splice variants of the cytoskeletal protein Mena present in mammary tumor cells during invasion in vivo. Clin Exp Metastasis. 2009;26:153–159. [PMC free article] [PubMed]
104. Warzecha CC, et al. ESRP1 and ESRP2 are epithelial cell-type-specific regulators of FGFR2 splicing. Mol Cell. 2009;33:591–601. [PMC free article] [PubMed]
105. Shen S, et al. MADS+: discovery of differential splicing events from Affymetrix exon junction array data. Bioinformatics. 26:268–269. [PMC free article] [PubMed]
106. Venables JP, et al. Cancer-associated regulation of alternative splicing. Nat Struct Mol Biol. 2009;16:670–676. [PubMed]
107. Lambrechts A, et al. cAMP-dependent protein kinase phosphorylation of EVL, a Mena/VASP relative, regulates its interaction with actin and SH3 domains. J Biol Chem. 2000;275:36143–36151. [PubMed]
108. Wang W, et al. The cofilin pathway in breast cancer invasion and metastasis. Nat Rev Cancer. 2007;7:429–440. [PMC free article] [PubMed]
109. Trichet L, et al. Relaxing the actin cytoskeleton for adhesion and movement with Ena/VASP. J Cell Biol. 2008;181:19–25. [PMC free article] [PubMed]
110. Hoffman LM, et al. Genetic ablation of zyxin causes Mena/VASP mislocalization, increased motility, and deficits in actin remodeling. The Journal of Cell Biology. 2006;172:771–782. [PMC free article] [PubMed]
111. Hansen S, Mullins D. VASP is a processive actin polymerase that requires monomeric actin for barbed end association. The Journal of Cell Biology. 2010 In Press. [PMC free article] [PubMed]