FEBS Letters 582 (2008) 21022111
Minireview
Lamellipodia and lopodia in metastasis and invasion
Laura M. Machesky*
The Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, United Kingdom
Received 10 March 2008; accepted 31 March 2008
Available online 7 April 2008
Edited by Christos Stournaras
Abstract Advances in our understanding of the mechanisms of
lamellipodia and lopodia assembly have led to a better concept
of how cells move, including how the actin cytoskeleton might be
important for the motility of metastatic cancer cells. The cyto-
skeleton is a potentially interesting target for prevention of inva-
sion and metastasis. As key proteins are uncovered which
regulate the assembly of actin-based structures, these need to
be considered in light of whether they represent potential inva-
sion and metastasis proteins.
2008 Published by Elsevier B.V. on behalf of the Federation of
European Biochemical Societies.
Keywords: Actin; Invasion; Migration; Motility; Metastasis
1. Introduction
Invasion and metastasis are among the last great frontiers in
cancer research and preventing or eliminating metastasis is one
of the biggest challenges for therapeutic intervention. Very lit-
tle is known about how cancer cells progress through the var-
ious stages of metastasis and about how a few cells achieve
successful colonisation at a distant site. It is clear that metas-
tasis is a collaboration between the tumor and the host and
that the tumor cells must undergo multiple changes in gene
expression and behaviour during metastatic progression.
Migration of both tumor cells and host cells is a key part of
metastasis and relies on changes in the actin cytoskeleton. This
review aims to summarise some of the most recent advances on
our understanding of how proteins that regulate the assembly
of lamellipodia and lopodia contribute to invasion and
metastasis of cancer cells. In particular, the Arp2/3 complex
and Scar/WAVE proteins in lamellipodia and fascin and
Mena/VASP proteins in lopodia, seem to be major control-
lers of cell motility that could be of key importance in metas-
tasis.
Cancer cells must use their cytoskeletons to adapt to and
survive in a variety of sometimes hostile environments to suc-
cessfully metastasise (Fig. 1A). Firstly, they must leave the site
of the primary tumor and enter the blood or lymphatic system.
They are often aided by angiogenesis in and around the tumor
and also by stromal cells such as broblasts and macrophages
that both remodel the extracellular matrix and signal to the tu-
*
Fax: +44 141 942 6521.
E-mail address: l.machesky@beatson.gla.ac.uk
0014-5793/$34.00 2008 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
doi:10.1016/j.febslet.2008.03.039
mor cells to migrate [15]. One of the key cellular processes that
triggers the beginning of an invasive or metastatic programme
in many cancers is the dedierentiation of epithelial cells to-
ward a mesenchymal state. This process is referred to as epi-
thelial to mesenchymal transition or EMT [9]. Having
undergone EMT, cells become more broblast like. Changes
in gene expression patterns include losing expression of E-cad-
herin and breaking down the cellcell junctions. The broblas-
tic morphology allows polarised assembly of the actin
cytoskeleton into protrusive and invasive structures (Fig. 1A)
that help the cell to navigate through the extracellular matrix
(ECM) and through/into the vasculature. It also helps invasive
cells to break away from the invasive front and thus change
from collective to individual migration/invasion programmes.
The cancer cell, having entered the blood or lymph system,
then has to survive in the circulation and is transported to dis-
tant sites in the body. During this time, the vast majority of
cancer cells will be torn to shreds in the circulation or will
die entrapped in a vessel, but a few will have productive inter-
actions with blood cells and platelets. The actin cytoskeleton
likely plays a role in protecting cancer cells from destruction
and promoting junction formation with various types of blood
cells. These heterotypic cell interactions likely have important
signalling and survival roles, as well as oering physical
strength and protection from immune surveillance. The next
step for a few cancer cells will be extravasation, which is escape
from the vasculature. This process requires adherence to the
endothelium and passage through the basement membrane
into the new tissue to establish growth as a metastasis. The
cancer cell uses its actin cytoskeleton to orchestrate protrusive
migration and to burrow through the ECM to establish a new
tumor site (Fig. 1A). The new tumor cells will frequently revert
from a mesenchymal phenotype back towards something that
more closely resembles the original tumor. Colonisation de-
pends on the new tissue (the soil) being properly prepared to
accept the cancer cell (the seed) (reviewed in [19,24]). The prep-
aration of the soil tissue appears to begin in the early stages of
establishment of the primary tumor prior to metastasis but the
mechanisms by which tumor cells communicate to prepare
sites for metastasis is currently not well understood. Addition-
ally, the regrowth of the metastasis may take tens of years, but
it is not known what is happening to the pre-metastatic cancer
cells during this time or where they reside in the body during
this long incubation period. During this time, the cytoskeleton
is thought to be quiet and there is no reason to think that cells
in micrometastases migrate or invade.
Cell motility and chemotaxis are essential during many of
the steps of metastasis and cell motility genes are often upreg-
L.M. Machesky / FEBS Letters 582 (2008) 21022111 2103

Fig. 1. (A) Schematic of tumor metastasis and some of the possible uses of the actin cytoskeleton during this process. Actin is shown in red to
illustrate times/places where tumor cells might need to assemble specialised actin structures that help in invasion, migration and metastasis. (B)
Simplied drawing of the role of Arp2/3 complex at the protruding edge of a cell. Scar/WAVE complex is recruited to the plasma membrane, possibly
via the small GTPase Rac1 (blue circle) where it activates Arp2/3 complex to form branches o the sides of actin laments near the plasma
membrane. Branches grow and push against the plasma membrane. (C) The Scar/WAVE complex shown in schematic, with each protein interacting
with partners that have been shown experimentally. The Rac1 GTPase is thought to provide a link to the plasma membrane. Abi and HSPC300
interact with the N-terminal SHD of Scar/WAVE.

ulated at certain stages during the progression (Fig. 1A). Che- factors, such as epidermal growth factor (EGF) are secreted
mokines secreted in host tissues are thought to be main attrac- by stromal cells and used to increase the motility and the direc-
tants between the host and the cancer cells. Also, growth ted migration of the tumor cells (for recent reviews see [24,45]).
2104
Tumor cells, in turn, secrete factors that attract certain stromal
cells and modify their behaviour to enhance the migration and
survival of the tumor cells. For example, tumor-associated
macrophages secrete EGF to enhance cancer cell survival
and motility and receive secreted CSF-1 from the cancer cells
[13]. This paracrine loop seems to increase the mobility of
the cancer cells out of the tumor and into the stroma and to
contribute to changes in the macrophages from ghting
against the cancer cells, into benecial partners that will be less
cytotoxic and more promoting of motility and survival [13,23].
Moving cells assemble and turn over their actin cytoskele-
tons rapidly and in particular, they make and retract protru-
sive actin structures that lie just beneath the plasma
membrane, such as lamellipodia and lopodia. A major con-
troller of actin assembly in lamellipodia is the seven protein
complex, the Arp2/3 complex, which contains two actin-re-
lated proteins (Arp2 and Arp3) and ve structural subunits
(ArpC1, ArpC2, ArpC3 and ArpC4). The Arp2/3 complex is
a major initiator of new actin laments in cells, and thus is
essential to the assembly of actin networks in lamellipodia. It
forms branches on the sides of preexisting actin laments
and is itself regulated by a variety of proteins that ultimately
receive signals from plasma membrane receptors. The main
controller of Arp2/3 complex in most cell types is the Scar/
WAVE complex (also known as WANP, see Fig. 1B, C and re-
viewed in [38]) which consists of ve protein subunits- Scar/
WAVE 1, 2 or 3; HSPC300; Abi-1, 2 or 3; Hem1, 2; CYFIP1,
2. The subunits have multiple isoforms in some cases and var-
ious names in the literature, making their study confusing (See
Table 1). This rather large complex interacts with the small
GTPase Rac1 via CYFIP1 and it has been proposed to be re-
cruited to the plasma membrane at sites of lamellipodial
assembly by Rac1 [40,47]. All three mammalian Scar/WAVE
proteins directly bind to and activate the Arp2/3 complex,
leading to increased actin nucleation to form branched actin
structures such as are found in lamellipodia (Fig. 1B). Mem-
bers of this complex have roles other than the recruitment of
Scar/WAVE to the plasma membrane but the Scar/WAVE
complex appears to be a stable and ubiquitous complex [29].
Filopodia are actin-containing spikes that also aid in cell
migration and are controlled by proteins such as fascin, diaph-
anous, and Mena/VASP. Fascin is an actin bundling protein
that brings together laments in lamellipodial networks to
encourage lopodial growth [76]. Its actin bundling activity
is regulated by phosphorylation and by the small GTPases
Rac and Cdc42 [1,25,33,77,87]. Murine diaphanous, mDia,
Table 1
Nomenclature for the members of the Scar/WAVE protein complex in various model organisms and in humans
Dictyostelium Human
Scar Scar/WAVE1 = WASF1 = unigene ID 75850
Scar/WAVE2 = WASF2 = unigene ID 590909
Scar/WAVE3 = WASF3 = unigene ID 635221
Nap1 NckAP1 = Nap1 = KIAA0587 = Hem2 = unigene ID 603732
Hem1 = unigene ID 182014
PIR121 PIR121 = P140Sra = CYFIP2 = KIAA1168 = POP = unigene ID 519702
CYFIP1 = unigene ID 26704
HSPC300 HSPC300 = unigene ID 649307
Abi1 Abi1 = unigene ID 508148
Abi2 = unigene ID 471156
NESH (Abi3) = unigene ID 477015
For the human protein, a human unigene ID is given to facilitate searching for information about these genes/proteins on the NCBI database online.
L.M. Machesky / FEBS Letters 582 (2008) 21022111
has three isoforms and is is involved in multiple dierent cyto-
skeletal activities, including interaction with microtubules [26].
However, its action in lopodia is thought to be primarily
mediated by its ability to act as an actin lament nucleator
and elongation promotion factor. Mena/VASP proteins also
have the ability to promote the elongation of long parallel bun-
dles of actin in lopodia [2,8]. All of these proteins have been
implicated in promotion of cancer metastasis and invasion, so
it seems that both lamellipodial and lopodial actin dynamics
may be important during this process.
2. Analysis of the role of the cytoskeleton in metastasis
The study of the role of the actin cytoskeleton in cancer
metastasis is complicated by the need for improved methods
to recreate the complex environments encountered by tumor
cells in the body and by the need for improved technology
for intravital imaging and live time-lapse imaging in three
dimensions. Furthermore, since metastasis is such a lengthy
and complex process, cells undergo multiple changes in gene
expression during the process, making it dicult to sample
the relevant populations of cells when trying to determine
which genes are up or downregulated. For example, comparing
just the primary tumor and the distant metastasis ignores all of
the genes transiently up- or downregulated during the initial
invasion and the trac through the blood or lymph systems,
as tumor cells tend to revert toward the expression programme
of the original tumor when they settle down in a new site as a
metastasis.
Perhaps the simplest in vitro system to study the role of actin
structures in invasion involves plating cancer cells onto a
reconstituted thin ECM on glass and imaging the holes made
by the cancer cells as well as the protrusions into the thin ma-
trix (Fig. 2). This has led to the identication of structures now
referred to as invadopodia which are perhaps analogous to a
focal adhesion complex in cells spread on a thin matrix on
glass in two dimensions. Invadopodia assays have led to the
identication of several actin-associated proteins as well as
proteases and other cellular components as important for the
formation of matrix-degrading protrusions.
The study of invadopodia has implicated N-WASP, Arp2/3
complex, dynamin and cortactin and associated proteins as key
players in the actin assembly that occurs when cells break
through the matrix and protrude into the holes [4,11,30].
Invadopodia are sites of tyrosine phosphorylation and require
Arabidopsis C. elegans
Scar Scar
Nap125 Gex3
PIROGI = KLUNKER Gex2
BRICK1 unknown
unknown Abi1
L.M. Machesky / FEBS Letters 582 (2008) 21022111
Fig. 2. An SCC9 head and neck squamous cell carcinoma cell making invadopodia on a gelatin matrix. The merge shows gelatin in red and cortactin
in green. The gelatin image shows black holes where the cell has degraded the matrix and the cortactin image shows concentration of this cytoskeletal
and endocytosis protein at sites of matrix degradation (Photo credit John Dawson, Beatson Institute for Cancer Research).
the activity of Src kinase [5]. Like focal adhesion complexes,
invadopodia contain a large number of dierent kinds of pro-
teins, however, there does appear to be specicity in their orga-
nisation and invadopodia are distinct from focal adhesions in
many ways. Invadopodia do not seem to be sites for the strong
concentration of adhesion proteins such as vinculin and talin,
but they do perhaps have specialised integrin components indi-
cating a specialised role in adhesion or interface with the ma-
trix [20,5557].
Invadopodia also appear to contain specic actin assembly
proteins. Scar/WAVE proteins have not been found in invado-
podia, but N-WASP is concentrated within them and is essen-
tial for their assembly [52,85,86]. Dynamin, an actin and
endocytosis protein is also found in invadopodia. The role of
dynamin in invadopodia may be as a scaold for recruitment
of N-WASP and cortactin to assemble actin comet tails [6],
which could provide a driving force for protrusion of the cell
into holes in the matrix. There also must be a mechanism for
the tracking of proteases to the cell surface at these sites
and for the tracking of degraded and engulfed matrix into
the cell, as invadopodia have been observed to be sites of
phagocytosis [17]. The correlation between invadopodia and
structures found when cancer cells invade in a 3D matrix is
not yet well understood, but it would appear that cancer cells
have the facility to degrade holes in a rigid matrix and push
through them using a machinery based on Arp2/3 nucleated
actin assembly and N-WASP-cortactin-dynamin.
While invadopodia are a useful 2-dimensional model for
cancer invasion, there also exist 3-dimensional culture systems
which may more closely resemble the tumor environment. For
example, tumor/normal cells grow as balls called spheroplasts
in collagen gels or Matrigel or as stratied layers of cells on top
of collagen gels suspended over culture medium [58,67]. The
movement and invasion of those ball structures or stratied
layers mimics in vivo invasion and matrix remodeling. Sahai
and colleagues have recently demonstrated the importance of
stromal cells in cancer invasion using a squamous cell model
where the cancer invades using collective nger-like protru-
sions into a layer of articial ECM containing broblasts
[28]. Fibroblasts associated with the tumor were discovered
to actively carve paths into the matrix and lead strings of can-
cer cells into the matrix and thus allow the cancer cells to in-
vade. The broblasts utilised Rho-mediated contractility and
specialised integrins to remodel the matrix to make pathways
for invasion by the cancer cells. The cancer cells did not require
Rho-mediated contractility to enter the matrix behind bro-
blasts, but rather followed behind the broblasts using
dierent signaling mechanisms. In this type of system, multi-
2105
photon confocal microscopy allows the high resolution imag-
ing of the cancer cells and the broblasts (or other cell types)
cooperating to form an invasion front as well as visualisation
of the extracellular matrix bres [66,69]. Culture systems where
multiple cell types can be mixed together in a reconstituted
extracellular matrix oers considerable exibility and allows
the study of molecular details of invasion and metastasis
in vitro in a relatively physiological setting.
Approaches to the study metastasis intravitally have also
considerably advanced in recent years [66]. The Condeelis
and Segall labs have developed a system for the isolation
and imaging of tumor cells migrating into capillary tubes
placed into tumors of live animals [1416]. This is the rst
model that allows study of the molecular mechanisms by which
tumor cells make their initial egress from a solid tumor into the
surrounding stroma and toward the vasculature. Migratory tu-
mor cells were isolated and microarray analysis was performed
to determine the sets of genes that are upregulated specically
in a more highly motile chemotactic population of cancer cells
[81]. This is a huge step forward for the eld, since it is the rst
isolation of the relevant population of cells for migration and
chemotaxis away from a primary tumor. Many proteins in the
Scar/WAVE and Arp2/3 complex pathway to actin assembly
as well as colin and actin capping proteins are upregulated
in the more highly migratory tumor cells. This data has been
supported by extensive in vitro studies implicating the N-
WASP-Arp2/3 complex-cortactin machinery as well as colin
and capping protein in invasion and migration (reviewed in
[45]). Migration of tumor cells can now be imaged in vivo
and the interactions of tumor cells with macrophages is strik-
ing [13,46,69,83,84]. This type of study is starting to reveal
gene expression signatures and other properties of the earliest
metastatic cells and raises hopes for developing methods to
both predict and prevent early metastasis. However, we are
still left with the dilemma that metastasis has likely occurred
before a primary tumor is detected, especially for breast can-
cer.
3. Regulation of the Arp2/3 complex during metastatic
progression
While signicant progress is being made in developing model
systems that will allow the study of metastasis and invasion in
the lab, clinical samples from patient tumors provide direct
evidence that the actin cytoskeleton is modulated during can-
cer progression. There is some evidence that expression of sub-
units of the Arp2/3 complex is modulated during metastatic
2106
progression in human patients. Two studies from the same
group demonstrate the downregulation of the seven subunits
of the Arp2/3 complex in gastric cancer. ARPC1, also named
p41-Arc, was found to be downregulated in a screen for dier-
entially methylated DNA fragments in gastric cancer
[43,44,53]. This group went on to show that the methylation
status was not likely a key regulatory point for Arp2/3 complex
gene expression, but that indeed all seven subunits of the Arp2/
3 complex were signicantly downregulated in primary gastric
cancers compared with normal surrounding gastric epithelium.
However, the expression status of Arp2/3 subunits may de-
pend on the invasive state of the cancer, as other studies have
shown upregulation of subunits during progression towards
more aggressive and invasive status [42]. This also agrees with
the murine microarray studies from Condeelis and colleagues
who nd Arp2/3 subunits upregulated in more migratory and
chemotactically active tumor cells [15]. Interestingly, the sur-
rounding stromal cells in some invasive tumors also showed in-
creased Arp2/3 complex expression, indicating that stromal
cells might be facilitiating the mobility of the cancer cells by
becoming more motile themselves. It might not always be pos-
sible to distinguish stromal cells from mesenchymal tumor cells
however and in some cases tumor cells are thought to contrib-
ute to the stroma of a tumor [9]. Perhaps surprisingly, Arp2/3
complex staining was reported as absent from the normal co-
lonic stroma and mucosa, but was observed in vascular endo-
thelial cells and lymphocytes. However, based on tissue
distribution data, [54] it seems more likely that normal cells
have a small amount of Arp2/3 complex, but they do not re-
quire the amounts needed in more motile free-living cells such
as macrophages or broblasts. Upregulation of Arp2/3 com-
plex expression also appears to be potentially predictive of
poor outcome in breast cancers [41].
4. Scar/WAVE3 in invasion and metastasis
Although it is at present unclear if functional dierences ex-
ist between Scar/WAVE isoforms, Scar/WAVE3 has been
strongly implicated in promotion of cancer metastasis by one
group. It was originally found to be truncated and inactivated
in a patient with ganglioneuroblastoma [74]. Inactivation of
Scar/WAVE3 was hypothesised to lead to an accumulation
of neural crest cells that are defective in migration yet retain
some potential for proliferation. The resulting enlarged ganglia
would contain accumulated undierentiated proliferating cells
that could potentially be dierentiated if given the right signals
at a later time. This is a typical manifestation of ganglioneuro-
blastoma [74]. It was noted by Cowell and colleagues that Scar/
WAVE2 is located on chromosome 1, at 1p35-36, a region that
is frequently lost or deleted in advanced stages of neuroblas-
toma. However, they could not detect loss of Scar/WAVE2
in the tumor specimens that were available to them [74]. Look-
ing at Scar/WAVE2 status may be interesting for future studies
on a larger scale.
It may not be surprising that Scar/WAVE3 is implicated in a
cancer of the brain, as Scar/WAVE3 and Scar/WAVE1 are
both important in brain development and function
[18,70,74]. Scar/WAVE3 appears to have a function in the
dynamics of dendritic processes and protrusions in hippocam-
pal neurons, similar to the role of Scar/WAVE1 in brain
[18,70]. The Scar/WAVE complex localises to dendritic spines
L.M. Machesky / FEBS Letters 582 (2008) 21022111
and protrusions in primary hippocampal neurons in culture
[61]. In live cell studies, Scar/WAVE3 was shown to accumu-
late at puncta, which appeared to be precursors to new lopo-
dia and protrusions that became dendritic spines [61]. These
puncta were shown to be places of accumulation of F-actin
and overexpression of Scar/WAVE3 in hippocampal neurons
induced accumulation of F-actin [61].
Following on from their neuroblastoma work, Cowell and
colleagues found that in general, Scar/WAVE3 has a positive
rather than a negative role in promoting the metastasis and
invasion of cancer cells. This was also conrmed by the work
of Condeelis and colleagues who found an upregulation of
Scar/WAVE3 in their microarray analysis of motile tumor cells
[15]. Scar/WAVE3 expression appears to be increased in vari-
ous cancer cell lines and in human breast cancer samples
[71,73,74]. In one study, Scar/WAVE3 was linked to expres-
sion of metalloproteases MMP-1, -3 and -9 in cancer cell inva-
sion [72]. Silencing of Scar/WAVE3 using siRNA showed a
reduced motility and invasiveness of cells in Matrigel assays
[73] similar to what has been previously shown for Scar/
WAVE-1 and -2 [75]. The loss of Scar/WAVE3 led to an in-
crease in actin stress bres and focal adhesion complexes, indi-
cating the possibility that the Scar/WAVE3 or the balance of
Scar/WAVE proteins in the cell could regulate the mobility
or contractility of the actin cytoskeketon. Reduction of Scar/
WAVE3 levels also led to a decrease in the number of lung
metastases of breast cancer in SCID mice and inhibited pri-
mary tumor establishment of MDA-MB-231 cells in SCID
mice [73].
5. Implication of Scar/WAVE2 in invasion and metastasis
Although Scar/WAVE3 has had the most attention as a reg-
ulator of migration in metastasis, there is growing evidence
that Scar/WAVE2 may also have a role. Coexpression of
Scar/WAVE2 and Arp2 in adenocarcinoma of the lung and
in breast and colorectal carcinoma has been linked to metasta-
sis and aggressive cancer phenotypes [41,42,68]. When high
levels of Arp2 and Scar/WAVE2 were found in lung adenocar-
cinomas this could be correlated with poor survival. Curiously,
the Scar/WAVE2 and Arp2/3 positive cells appeared randomly
scattered throughout the tumors and were very rich in Scar/
WAVE2 and Arp2/3 in relation to the surrounding tumor cells.
A similar phenomenon was seen in liver metastases from colon
cancer and in the primary tumor sections as well. Bulk mRNA
levels for Arp2, Arp3 and Scar/WAVE2 were all found to be
higher in the samples that showed Arp2/3 and Scar/WAVE2
colocalisation. The colocalisation in primary tumors was
found to be a signicant predictor of liver metastasis. The evi-
dence provided by these studies suggests that, if a few cells
within a primary tumor gain increased mobility via a co-upreg-
ulation of Scar/WAVE2 and Arp2/3 complex, these cells may
have increased metastatic potential.
Scar/WAVE2 expression has been studied in the Fidler mod-
el of melanoma, using non-metastatic parental melanoma cells,
slightly metastatic B16F1 cells and highly metastatic B16F10
cells [27,48]. In this model, expression of both Scar/WAVE1
and Scar/WAVE2 increased as the cells became more aggres-
sive. Scar/WAVE3 was reported not to be expressed in these
cell lines. Levels of active Rac1 GTPase were also increased
in the more highly aggressive cells, indicating that the Rac1-
L.M. Machesky / FEBS Letters 582 (2008) 21022111
Scar/WAVE pathway might be upregulated overall with
increasing metastatic potential. Scar/WAVE2 was important
for invasion into Matrigel and motility in 3D gel matricies
and lung metastasis of B16F10 cells [48]. Thus Scar/WAVE2
appears to promote metastatic behaviour of melanoma cells,
at least in the Fidler model. In an unrelated study, the expres-
sion of a tumor metastatic suppressor gene MRP-1/CD9,
which encodes a tetraspanin protein, was found to downregu-
late the expression of Scar/WAVE2 in HT1080 human bro-
sarcoma cells [37]. Cells expressing CD9 appeared rounded
and showed less lamellipodial activity, but no mechanism
was given for this downregulation.
6. Implication of Scar/WAVE complex members in cancer
Scar/WAVE proteins are associated in a stable and ubiqui-
tous complex containing four other subunits (Fig. 1C). In gen-
eral, these subunits are required for the stability of the whole
complex, so it has been dicult to determine whether there
are any specic functions attributable to any particular com-
plex member [10,47]. A number of these proteins have been
implicated in cancer metastasis and migration/invasion, but
in cases where a particular protein is lost, it is likely that the
whole Scar/WAVE complex and therefore Scar/WAVE and
Arp2/3 complex are aected. Below is a summary of the recent
literature surrounding the role of Scar/WAVE complex mem-
bers in various cancer models or patient samples.
The smallest member of the Scar/WAVE complex is
HSPC300, a 110 amino acid polypeptide that interacts with
the N-terminus of Scar/WAVE (Fig. 1C) [29,40]. Loss of
HSPC300 in patients with Von Hippel-Lindau Syndrome
(VHL) has been found to be protective against clear renal cell
carcinoma, the most common malignancy of the kidney [12]. It
is not known why loss of HSPC300 in these patients can be
protective, but it is speculated that aberrant cytoskeletal orga-
nisation in VHL tumors may prevent migration of the tumor
cells and thus slow metastasis. Additionally, a cytokinesis de-
fect, resulting in slower cell division, may lead to slower tumor
cell proliferation. Patients with the double deletion of
HSPC300 and VHL develop mainly hemangioblastomas and
not renal cell carcinoma.
Abelson interactor protein-1 (Abi-1) binds directly to the N-
terminus of Scar/WAVE proteins and appears to be essential
for lamellipodia assembly triggered by the Scar/WAVE com-
plex [40]. It also interacts with Eps8 in a separate trimolecular
complex together with SOS and regulates the activation of Rac
downstream of tyrosine kinase receptor signaling [39]. Wang
and colleagues found that of several cancer cell lines tested,
those that were the least invasive also expressed on average
the least Abi1 [79]. They hypothesised that Abi contributes
to invasiveness. For the most part, low levels of Abi protein
correlated with low expression of the whole Scar/WAVE com-
plex, so it is likely that this is not a direct eect of low Abi-1 in
each case, but perhaps destabilisation of the whole complex
[10,47]. Downregulation of Abi-1 by RNA interference caused
the cells to appear rounder and inhibited lamellipodia, adhe-
sion, proliferation, anchorage-dependent colony formation in
soft agar and passage through the cell cycle. The migration
and invasion in a transwell lter assay was also reduced in cells
that had lower levels of Abi-1. Perhaps more surprisingly, lev-
els of AKT protein were decreased in Abi-1 downregulated
2107
cells, as were the levels of phospho-PDK-1 and phospho-
Raf. It is not known whether total levels of PDK-1 and Raf
were aected. This study implicates Abi-1, as a member of
both the SOS-Eps8-Abi complex and the Scar/WAVE com-
plex, in cancer metastasis. Clearly loss of Abi-1 has cata-
strophic eects on the cells, as many proteins depend on
Abi-1 for stability and activity. A recent study by Politt and
Insall also implicated Abi-1 in regulation of cytokinesis but
found that loss of Abi-1 did not completely abolish Scar/
WAVE activity [62] suggesting that the context and the cell
type might be important determinants of the stability of each
complex member.
Another Scar/WAVE complex member, Nap1 (Fig. 1C), is
essential during mouse development and its loss causes severe
defects in cell migration in the early embryo, suggesting that
the Scar/WAVE complex is important for the regulation of cell
migration in vivo [63]. Genes that are important for migration
during embryogenesis are good candidates for promotion of
motility during metastasis. Nap1 knockout mice do not survive
past E9.0 and the embryos show various abnormalities indicat-
ing migration defects. At E8.5 the embryos accumulate disor-
ganised mesenchymal cells at the primitive streak, indicating a
defect of migration of the mesoderm away from the primitive
streak. Many tissues failed to fuse or close normally, including
the heart primorida. The endoderm failed to close to form the
foregut pocket and the neural ectoderm failed to close. About
a quarter of the embryos had dramatic duplications of struc-
tures along the body axis. These defects all could be attributed
to dierent types of abnormal cell migration. It is interesting
that in these Nap1 mutants, no Scar/WAVE1 or Scar/WAVE2
could be detected, so the authors propose that this mutant rep-
resents a total loss of function of Scar/WAVE signaling to ac-
tin. However, cells in the mutant still can move, they are just
less ecient and have aberrant shapes (more rounded than
polarised) so this implies that there are multiple pathways
important for cell motility in addition to Scar/WAVE
in vivo. It seems possible that the type of motility seen in the
Nap1 mutants could be similar to the Rho-ROCK-mediated
amoeboid motility observed in cancer cells migrating in
three-dimensional spaces when protease-dependent motility
has been blocked [65]. This would agree with the more
rounded shapes observed for cells of the Nap1 knockout em-
bryos. This also agrees with the nding that Dictyostelium
lacking Scar/WAVE proteins can still move but more slowly
and with altered shape [7].
7. Regulation of the lopodial proteins fascin and Mena+11a in
cancer
Ena/VASP proteins are important regulators of actin assem-
bly in lopodia as well as lamellipodia. They are found near
the tips of lopods and promote lopod actin lament assem-
bly (for an excellent review see [31]). Mena is overexpressed in
a subset of human benign breast lesions and its expression cor-
relates with increased risk of transformation [22]. The upregu-
lation of Mena correlates with other prognostic indicators of
human breast cancer such as proliferation index, Her-2 overex-
pression and hormonal receptor status [22]. The Ena/VASP
family protein variant Mena+11a is both upregulated in
expression level and phosphorylated in breast cancer cell lines,
suggesting that it may be involved in the progression or en-
2108
hanced motility of cancer cells [21]. Mena+11a is nearly unde-
tectable in a number of normal tissues, including normal hu-
man mammary gland, cecum and cervix, but is expressed in
many cancer cell lines. The expression is responsive to EGF
treatment and EGF treatment also induces phorphorylation,
which may alter the activities of Mena+11a. Other studies have
shown that murine Mena is upregulated during Her-2-induced
breast cancer progression [3] and that the upregulation is
EGF-responsive [80]. Transfection of exogenous Mena+11a
into MCF7 cells induces an increase in proliferation rate and
in p42/44 MAPK activity. The mechanism for this increase is
unclear and could hint at an interesting role for Mena+11a,
which has not yet been discovered [21,22].
Fascin is another key protein in lopod assembly, regulating
the formation of actin bundles and recruiting protein kinase C
during migration and lopod assembly [33]. Fascin expression
is transiently upregulated during the early stages of colon can-
cer invasion in response to b-catenin-TCF signalling in the
Wnt pathway when cells start to make a transition from an
epithelial to a mesenchymal phenotype [77,78]. Fascin upregu-
lation confers a 10-fold increase in the ability of HT-29 colon
cancer cells injected into the tail vein to colonise in the lung
[77]. Fascin is highly expressed in the invasive fronts of high
grade and later stage human colon carcinoma and largely ab-
sent from distant metastases and its expression correlates with
poor survival [35]. This indicates that the upregulation is tran-
sient, during the stages of active invasion and metastasis and
then fascin is downregulated when the metastasis becomes
established. Fascin upregulation has also been correlated with
poor survival in other cancers, such as gastric cancer [34],
esophageal cancer [32], extrahepatic bile duct cancer [59], oral
squamous cell carcinoma [49], ovarian tumors [36], lung cancer
[60] and breast cancer [64]. Thus fascin upregulation appears
to be generally predictive of increased metastatic or invasive
potential in cancer and this may be mediated by increased
lopodial activity and mobility of the cells.
8. WASH, A new potential player in actin dynamics and cancer
It has only come to light fairly recently that proteins of the
actin cytoskeleton may play an important role in metastatic
progression of cancers. Normal cells in an adult epithelium
do not usually need to migrate extensively and so are likely
to have low levels of many migration-associated proteins and
relatively quiet cytoskeletons. When a metastatic programme
is initiated, cells which have upregulation of certain motility
genes may have an advantage in invasion into matrix, through
endothelial cell layers and into new sites for tumor formation.
Both lamellipodial and lopodial proteins are increasingly
implicated in tumor metastasis and invasion and it seems likely
that over the coming years new important players will continue
to be uncovered. The cytoskeleton is a complex nework of
multiple proteins that is also specialised in dierent cell types
to carry out precise and varied functions. Just recently, a
new member of the WASP-family has been discovered,
WASH, which in humans (but not primates or other mam-
mals) resides in subtelomeric regions and so may be actively in-
volved in evolutionary processes [51]. Humans have several
copies of WASH genes, only one of which is thought to repre-
sent an actual coding region. WASH is widely conserved, and
is found in amoebas, Drosophila, andC. elegans, and it is essen-
L.M. Machesky / FEBS Letters 582 (2008) 21022111
tial in Drosophila. WASH contains a VCA sequence (refer to
Fig. 1C) similar to WASP and Scar/WAVE proteins and a un-
ique N-terminus [51]. It was reported to be overexpressed in a
breast cancer cell line SKBR3, in a study looking for potential
prognostic biomarkers [50]. This is just one example of a rela-
tively new addition to the growing set of potential targets for
much needed improvements in both prognostic and therapeu-
tic strategies for cancer metastasis.
9. Discussion
While the sections of this review have been roughly divided
between proteins that are implicated in the assembly of lamel-
lipodia (Scar/WAVEs and Arp2/3 complex) or lopodia (Me-
na+11a and fascin), it seems likely that in vivo cells will use
dierent structures which may combine features of lopodia
and lamellipodia and invadopodia- terms like invasive pseu-
dopodia might be useful when trying to describe what
researchers can see in three dimensional networks [82]. As we
try to come to grips with improved technology for viewing cells
in three dimensional space and in vivo, we will likely more pre-
cisely dene dierent kinds of invasive pseudopods and more
clearly understand the functions of cytoskeletal proteins that
have largely been characterised in a test tube or in cells moving
on a at surface.
It is clear that there is much crosstalk between the stroma
and the tumor and that the contributions of stromal cells to tu-
mor cell activities is extremely important, as is the potential for
the tumor to contribute both signals and cells to the stroma.
How the body responds to cancer and how cancer changes
the organisms responses are extremely complex and fascinat-
ing areas of research. The role of the cytoskeleton and migra-
tion is only one small part of a larger quest to understand both
normal and diseased cell behaviours.
The study of the actin cytoskeleton in cancer metastasis
seems to have come of age and the time is ripe for increasing
our understanding of this fascinating aspect of biology and hu-
man disease. It is time for the elds of cell migration/motility
and cancer metastasis to form alliances and to uncover those
genes important in aspects of cancer spread. While consider-
able work has been done, it is hard to evaluate which aspects
will be of clinical relevance and whether the actin assembly
machineries of lopodia and lamellipodia represents a valid
prognostic or potential therapeutic target. On the negative
side, metastasis of many cancers has probably already oc-
curred by the time a primary tumor has been discovered, so
unless the micrometastases can be identied and destroyed,
therapy against future metastatic spread will not be useful.
Furthermore, once micrometastases are established, they typi-
cally revert toward the phenotype of the original tumor or
adapt to their new environment and things such as motility
and invasion genes are often downregulated again. However,
analysis of the regulation of the actin machinery of cells of
the primary tumor might give clues about whether or how
likely micrometastases are and whether to use more aggressive
therapy (also discussed in [45]). There might also be benet in
prevention of metastasis and invasion for tumors that are not
completely able to be removed by surgery. Furthermore, as ge-
netic tools to predict predispositions to certain types of cancers
become more accurate and more widely used, preventative
therapies for metastasis might also become possible.
L.M. Machesky / FEBS Letters 582 (2008) 21022111
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