The actin cytoskeleton in cancer cell motility
Cancer cell metastasis is a multi-stage process involving invasion into surrounding tissue, intravasation, transit in the blood or lymph, extravasation, and growth at a new site. Many of these steps require cell motility, which is driven by cycles of actin polymerization, cell adhesion and acto-myosin contraction. These processes have been studied in cancer cells in vitro for many years, often with seemingly contradictory results. The challenge now is to understand how the multitude of in vitro observations relates to the movement of cancer cells in living tumour tissue. In this review we will concentrate on actin protrusion and acto-myosin contraction. We will begin by presenting some general principles summarizing the widely-accepted mechanisms for the co-ordinated regulation of actin polymerization and contraction. We will then discuss more recent studies that investigate how experimental manipulation of actin dynamics affects cancer cell invasion in complex environments and in vivo.
Actin related proteins 2 and 3
PKC-activated protein phosphatase-1 inhibitor
Death-associated protein kinase
Myotonic dystrophy protein kinase
DAP kinase-related apoptosis-inducing protein kinase
Green fluorescent protein
Integrin linked kinase
Myosin light chain
Myosin light chain kinase
Myotonic dystrophy kinase-related Cdc42–binding kinase
Myosin phosphatase target subunit
Neural Wiskott-Aldrich syndrome protein
Phosphoinositide dependent protein kinase 1
Myosin protein phosphatase 1
Rho-associated coiled-coil containing kinase
Reactive oxygen species
Wiskott-Aldrich syndrome protein
WASP family verprolin-homologous protein
Zipper-interacting protein kinase
Actin polymerization drives cancer cell motility
The activity of the polymerization machinery is very tightly regulated. The Arp2/3 complex is regulated by its association with the WAVE and WASP family of WH2 domain containing proteins (WAVE1, 2, & 3, WASP and N-WASP) that can bind both the Arp2/3 complex and actin monomers (Fig. 2) [6, 12]. This helps to bring actin monomers very close in proximity to the Arp2/3 complex and thereby increases the rate of Arp2/3-mediated actin polymerization. WASP family proteins also bind profilin through poly-proline motifs and this further aids recruitment of actin monomers to the Arp2/3 complex . WH2 domain proteins are themselves subject to very tight regulation through a conformational switch [14, 15]. The VCA domains including the WH2 domain can be masked by intramolecular interactions (Fig. 2); this autoinhibited conformation can be relieved through a range of protein–ligand interactions. Interaction with the GTP-bound form of Cdc42, PIP2 or adaptor molecules such as WIP and Nck, have all been shown to promote the active ‘open’ conformation of WASP or N-WASP . Similarly, interactions with a multimeric complex containing Abi/Nap/PIR121 or IRSp53 can enhance the activity of WAVE proteins, in both cases GTP-bound Rac1 is a key determinant of localization of these complexes [12, 16]. The actin binding protein cortactin also binds to Arp3 and this helps to locate active Arp2/3 complexes to the sides of existing actin filaments leading to branched arrays of F-actin .
Like WAVE and WASP family proteins, FH proteins also switch between an auto-inhibited ‘closed’ conformation and an active ‘open’ conformation . Interaction with numerous GTP-binding proteins, including Cdc42, RhoA, RhoB and Rif, and adaptor proteins such as DIP/WISH can stabilise the open conformation thereby promoting actin polymerization driven by the FH2 domain [18–22]. Structural studies indicate that FH2 domains function as dimers with one FH2 domain binding a monomer in the existing actin filament and the other FH2 domain recruiting a new G-actin monomer for polymerisation .
Actin regulators implicated in cancer cell motility
Experimental evidence for role in cancer motility
Deregulation in human cancer
Nucleate actin filaments
+lu, br, co (with Wave 2)
Actin polymerisation on barbed ends
Promote actin polymerisation on barbed ends
Mena + in br
Activates LIMK and N-WASP
Activates LIMK and WAVE
Wave2 + hcc, lu, br, co (with Arp3)
Sever actin filaments/generate barbed ends
Cooperates with Arp2/3
Located on 11q amplicon
Increase Arp2/3 activity
link F-actin to PM
Ez & Moe + in many cancers
+scc, pa, −ov
Maintain reservoir of G-actin
−br, pa, hcc
Maintain reservoir of G-actin
Activates ROCK1, 2 and some DRF’s
RhoA, C + in many ca.
p > MLC, p-MYPT1, p > CPI-17, p > LIMK
ROCK + in pr
p > MLC, p-MYPT1, p > LIMK
p > MLC
+nsclc, co, br, gl
p > MLC
−scc, co, le, lu
p > MLC, p-MYPT1, p > CPI-17
+nsclc, pa, co
p > MLC, p > LIMK
PAK1 + in many ca., PAK4+
Myosin II binding
+bl, br, co, pa, mel, rcc, scc, nsclc, ga
Stabilize actin filaments
TPM1––br, nb. TPM2 + pa, scc
Co-operation and plasticity in actin polymerization mechanisms
The molecular machinery that regulates the different facets of actin polymerization functions coordinately in most cell types. For example, cofilin and Arp2/3 co-operate to drive maximal actin polymerization in breast cancer cells. A localized increase in cofilin activity leads to increased numbers of barbed ends for actin polymerization while Arp2/3 promote the nucleation of new filaments [34, 35]. Cofilin activity is localized to a region close the plasma membrane because this is the site where PIP2 is hydrolysed and because of its intrinsic preference for recently polymerised ATP-actin filaments , while activation of membrane tethered small G proteins leads to increased Arp2/3 activity at the plasma membrane [37, 38]. It should also be noted that in other contexts (such as if ADP-actin filaments are severed or other co-operating mechanism are not active) cofilin-mediated filament severing can reduce F-actin levels. Numerous separate studies have shown that key actin regulators become deregulated during cancer progression (Table 1), i.e. they appear to be coordinately up-regulated in a sub-set of motile cancer cells . This coordination makes sense if one considers the multi-step and cyclical nature of cell motility; up-regulation of any one of the key regulatory steps in isolation would simply result in other regulators becoming rate-limiting thereby producing little or no overall increase in cell motility. Conversely, disruption of any one regulatory pathway would likely have an effect on motility; there is wealth of literature documenting the effects of disrupting Arp2/3, cofilin, FH proteins and Ena/VASP on cell migration [4–7]. However, if one examines the data in more detail it becomes clear that disruption of any particular actin regulatory mechanism fails to completely abrogate motility, most likely due to compensatory mechanisms maintaining actin polymerization and turn-over, thereby supporting motility, albeit at reduced rates. A particularly striking example, if the generation of branched actin filaments by the Arp2/3 complex is blocked, then extensive filopodia formation is observed which sustains cell motility . Conversely, if the function of Ena/VASP proteins that normally promote filopodia formation is blocked, then cells extend a more persistent and uniform lamellipod leading to increased cell speed . These studies reveal some important principles: that the different mechanisms of actin polymerization co-operate to generate the F-actin structures used for cell motility, and that there is plasticity in the regulation of these mechanisms that enables cells to adapt to interference with any one mechanism. Different relative activities of various regulators of actin polymerisation most likely explain the diverse range of morphologies that can be observed in motile cancer cells (Fig. 3).
The ability of cancer cells to move requires force generation to overcome factors that oppose movement (e.g. cell–cell and cell–matrix adhesions, drag, etc.). F-actin assembles with myosin II filaments composed of heavy and regulatory light chains to form a protein complex that uses energy from ATP hydrolysis to power actin–myosin contraction [32, 42]. The resultant generation of contractile force drives the morphological reorganization and extracellular matrix remodelling that facilitate cell movement. Given the profound effects that actin–myosin contractility can have, it is not surprising that there is a sophisticated network of regulatory components that hold a tight rein over this process.
Phosphorylation of the myosin II light chains (MLC) is a key mechanism for regulation of actin–myosin contractility . MLC phosphorylation promotes the release of the myosin heavy chain tail allowing for assembly into filaments, and facilitates the association of the myosin head with F-actin. The myosin head uses ATP to ‘walk’ towards the barbed end. When multimeric myosin is associated with more than one actin filament this causes the filaments to move relative to each another, thereby generating contractile force. MLC phosphorylation has been reported to be mediated by numerous kinases including: the Rho-regulated ROCK1 and ROCK2 , the ROCK-regulated ZIPK , MRCKα and MRCKβ [46, 47], ILK , DAPK 1  and 2 , DRAK 1 and 2 , PAK [52, 53] and MLCK  (Table 1). The ability of these various kinases to phosphorylate MLC allows for multiple signalling pathways to converge on the regulation of actin–myosin contractility. Although it would be difficult to define every condition and cell type in which a specific kinase phosphorylates MLC, studies with small molecule inhibitors indicate that ROCK1 and ROCK2 are the major calcium-independent kinases while MLCK is the major calcium-dependent kinase.
Dephosphorylation of MLC is catalyzed by the PP1M phosphatase complex, which is comprised of a PP1Cδ catalytic subunit, a myosin light chain binding subunit (MBS) and a smaller M20 subunit of unknown function . The MBS is a critical component of the complex as it brings together the phosphatase catalytic subunit with its cognate substrate and because of the role it plays in regulating phosphatase activity. An interesting recent development is the discovery that there are five proteins that may act as the MBS (MYPT1, MYPT2, MYPT3, MBS85 and TIMAP) . The best characterized MBS is the ubiquitously-expressed MYPT1 protein, it appears that the more tissue-restricted MYPT2 likely functions and is regulated similarly . The other MBS proteins have not been studied extensively and their roles in regulating MLC phosphorylation remains to be determined. The major site of MYPT1 phosphorylation is Threonine 696 (numbering relates to the human form), which inhibits phosphatase function , possibly by blocking the active site or by disrupting interaction of the catalytic subunit with phosphorylated substrate . Kinases that have been reported to phosphorylate Thr696 include: ROCK1 and ROCK2 , MRCKα and MRCKβ [47, 59], ILK [60, 61], ZIPK  and the DMPK . Phosphorylation of Threonine 853 by ROCK has also been reported to inhibit MLC dephosphorylation by decreasing MLC binding [57, 64].
MLC phosphorylation is also regulated by the CPI-17 protein  (Table 1), which when phosphorylated on Threonine 38 potently inhibits PP1M activity by masking the active site in the catalytic PP1Cδ subunit . A number of the same kinases that phosphorylate MYPT1 have also been shown to phosphorylate CPI-17, including ROCK1 and ROCK2 , ZIPK  and ILK , raising the possibility that kinases which inhibit PP1M activity do so by targeting multiple regulatory proteins. The closely related proteins KEPI and PHI-1 [70, 71] also appear to inhibit PP1C activity in a phosphorylation-dependent manner, but their possible roles in regulating MLC phosphorylation have not been characterized in detail. Elevated expression of CPI-17 in several tumour cell lines has been reported, where inhibition of PP1M led to inactivation of the Merlin tumour suppressor protein and consequent oncogenic transformation . An additional possibility is that elevated CPI-17 expression and/or phosphorylation would contribute to the metastatic ability of tumour cells.
A number of kinases, including ROCK, apparently have two modes for elevating MLC phosphorylation, by acting as direct MLC kinases and by inhibiting PP1M activity. There has not been a great deal of effort spent in trying to dissect the relative contribution of these two pathways to MLC phosphorylation induced by a given kinase. However, one possibility is that the major pathway for some kinases is the phosphorylation of MYPT1 and consequent inhibition of PP1M. As a result, a net gain in MLC phosphorylation would actually require less kinase activity directed towards MLC than under conditions in which PP1M was not inhibited. A manifestation of this effect is the increased calcium sensitivity of MLC phosphorylation and the consequent actin–myosin contractile response that can be induced by ROCK . In this example, it would imply that Ca2+ and/or calcium-regulated kinases such as MLCK or DAPK would cooperate with ROCK to promote contractile force generation, and contribute to metastatic behaviour.
As well as a role in facilitating MLC phosphorylation, calcium may contribute to cancer cell metastasis by binding to proteins such as S100A4 . There is very strong evidence from clinical and experimental studies which indicates a significant role for S100A4 overexpression in increased metastasis and poor prognosis for a wide variety of cancers including; breast, colorectal, pancreatic and renal (Table 1). Intriguingly, S100A4 has an extracellular role in promoting metastasis, possibly by inducing remodelling of the extracellular matrix and/or through interactions with a cell surface receptor, as well as an intracellular role. It has been proposed that S100A4 acts by binding to the myosin II heavy chain  and promotes increased directional motility by shifting the balance towards forward protrusions and away from side protrusions . In addition, S100A4 may also affect actin–myosin contractility by direct binding to F-actin  and to the actin-binding protein tropomyosin .
Tropomyosins are derived from four distinct genes (α, β, γ, δ) that are transcribed and spliced into over 40 isoforms [33, 79]. Although they play key roles in the calcium-responsive contraction of striated muscle, their roles in non-muscle cells are less well defined. Different isoforms appear to have distinct biological functions, as a result the patterns of expression affect how tropomyosins might affect the actin cytoskeleton. The expression of tropomyosin isoforms is frequently altered in tumours (Table 1). Some isoforms appear to recruit myosin to actin filaments , and influence the activity of the myosin head ATPase and contractility . Tropomyosin has also been reported to increase actin filament stiffness  and protect F-actin from the actions of cofilin  and gelsolin . However, some isoforms actually reduce active myosin levels and promote the association of cofilin with actin filaments, resulting in the formation of lamellipodia . To add further complexity, isoforms are sorted to different cellular compartments, and these distributions may change during development or in tumour cells. As a result, actin–myosin regulation may be affected by factors in addition to tropomyosin expression levels. Further research is necessary to determine how both isoform expression and subcellular distribution patterns contribute to tumour cell metastasis.
Coordinating polymerization and contraction
A common theme in these examples is that precise localized regulation of polymerization and contraction is critical. Excessive or global activation can be as detrimental to motility as lack of activity and this should be considered when attempting to reconcile apparently contradictory findings. For example, both excess and reduced levels of LIMK-mediated phosphorylation of cofilin have been reported to reduce cell motility , this could be explained if low levels are required at sites of actin polymerization to allow cofilin to generate new barbed ends for polymerization but high levels are in contractile zones to prevent the severing of filaments required for myosin-mediated contractility . Global LIMK activation would reduce polymerization, while global inactivation would reduce the number of filaments available for the contractile machinery.
Actin organisation in complex environments
Most studies analyzing the generation of filopodia and lamellipodia have used cells cultured on rigid 2D substrates; however, these conditions are clearly different from the environment through which cells move in vivo. Recently, significant effort has been focused on trying to understand how F-actin is organized in cancer cells moving in more complex environments . On thicker substrates composed of matrix proteins, many cancer cells form ventral actin-rich structures called invadopodia that are associated with ECM proteolytic activity [90, 91]. These structures have many similarities with podosomes that are found in cells of monocytic origin. The formation of invadopodia requires the activity of the actin nucleating Arp2/3 complex, regulated by N-WASP and cortactin, and the actin severing action of cofilin . The ability of cancer cells to make invadopodia often correlates with their ability to enter the vasculature . However, the holes generated typically in the ECM by invadopodia (1–2 microns) are small compared to the size of the cell, and cancer cells have not been observed to move through the areas of matrix degradation produced by invadopodia. This may merely reflect a limitation of the experimental systems used, but until this issue is resolved the relationship between invadopodia and cancer cell invasion through matrix barriers will remain a topic of lively debate.
Invading A431 squamous cell carcinoma cells do not have a single distinct F-actin protrusion but instead have numerous filopodia  (shown in 2D in Fig. 3). It is tempting to speculate that these structures ‘sense’ the surrounding matrix and those that extend in a favourable direction then guide cell movement . However, this will remain a hypothesis until confirmed by experimental studies.
It is also clear that the organization of the acto-myosin contractile machinery can be quite different in more complex environments. Most studies have focused on the regulation of stress fibres, which are prominent in cells cultured on rigid substrates (Fig. 3); however, these structures are much less prominent when cells are in 3D environments . In many cases the contractile machinery is associated with the sub-membranous cortical actin cytoskeleton. Much less is understood about the regulation of this F-actin network; nonetheless it is clear that RhoA and the ROCK kinases are critical for its maintenance. In addition, it has recently been shown that modulation of Dia2  and PDK1 [97, 98] activity can affect cortical actin. High levels of RhoA, RhoC or ROCK activity promote contraction of the cortical actin that is associated with membrane blebbing .
Observation of cancer cells moving in 3D environments has suggested that cancer cells can move using series of membrane blebs. Although this type of motility had been observed in vivo in developing fish embryos during the 1970s by Trinkaus and colleagues , it received little attention until recently . Strong actin–myosin contraction in one part of a cell may also produce a compressive force that leads to increased hydrostatic pressure and a localized detachment of the plasma membrane from the cortical cytoskeleton which results in bleb protrusion . The cortical actin–myosin network generates a basal level of tension across a cell surface . However, unlike a soap bubble in which surface tension is more or less uniform, local differences in cortical actin–myosin contraction produce variations in tension that affect cell shape. Surface area will increase in regions of localized relaxation whereas contraction will decrease surface area. Consistent with this, ROCK and MLC are localised at the rear of cells moving in this manner . The generation of hydrostatic pressure would require that the contractile machinery be attached to the plasma membrane; in fact, interference with ERM proteins which link acto-myosin cytoskeletal structures with the plasma membrane reduces blebbing-mediated invasion . Although increased intracellular pressure has been observed in blebbing mitotic cells in vitro , direct demonstration of the role of hydrostatic pressure in cells moving in 3D environments is problematic, at least in part because techniques that measure force and elasticity such as atomic force microscopy can not easily be used in these environments.
As discussed above, some cancer cells invade with a rounded morphology associated with high levels of Rho–ROCK activity driving cortical acto-myosin contraction; in contrast other cancer cells move with an elongated morphology that does not require Rho–ROCK function [104, 106]. Instead the ROCK related kinases, MRCKα and β function redundantly with ROCK1 and 2 to regulate acto-myosin . ROCK and MLCK have been shown to play distinct but complementary roles in the regulation of MLC phosphorylation, actin structures and motility of cells in 2D tissue culture conditions [107–110]. Elevated MLCK expression has been detected in numerous tumour types [111–114] and cancer cell lines [115–117] suggesting that increased MLCK activity resulting from overexpression or increased calcium transients might act to drive cancer cell motility in vivo, possibly in co-operation with ROCK. ZIPK has recently been shown to be phosphorylated and activated by ROCK , and can phosphorylate common substrates including MLC , MYPT1  and CPI-17 . These findings suggest that ZIPK could amplify a Rho–ROCK signal or that elevated ZIPK activity might substitute for Rho–ROCK activity, to promote metastasis. However, the exact roles played by MLCK and ZIPK in regulating acto-myosin function in 3D environments remains to be determined.
In addition to the diverse patterns of F-actin organization observed in motile cancer cells  (Fig. 3), it is now clear that many cancer cells exhibit significant plasticity in the mechanisms they use to move . This presents a particular challenge when designing inhibitor strategies to block cell movement; for example inhibition of extracellular proteases causes many cancer cells to move with a rounded, blebbing morphology [104, 121]. Constriction of the cortical acto-myosin enables these cells to squeeze through gaps in the surrounding matrix or deform the matrix and thereby invade without the need for protease function . To date, this plasticity has been observed in the experimental context; however, it may also enable cancer cells to overcome the diverse challenges of the metastatic process in human patients. Moving through dense connective tissue, crossing a thin endothelial layer and surviving the shear stresses in the circulation are likely to require different cytoskeletal organizations. Therefore, it may be that a high degree of plasticity in actin organization is particularly favourable during metastasis.
Actin dynamics in living tumours
Live tumour cell imaging will also allow the organization of the cytoskeleton to be analysed and should help to address questions such as; the prevalence of filopodia and invadopodia in motile cells in vivo, and if there are structures analogous to lamellipodia in vivo. By imaging GFP-tagged myosin light chain, the acto-myosin contractile machinery was found to be located around the cortex of motile cancer cells in vivo . Furthermore, the organization of MLC and the motility of these cells was ROCK-dependent . It will be fascinating to extend this type of analysis to regulators of the actin polymerization machinery. The behaviour of cells with increased Rho–ROCK function following stable knockdown of the Smurf1 E3 ubiquitin ligase, which targets RhoA for degradation , also has been imaged in vivo. Smurf1 knockdown led to locally increased Rho activity around the cell cortex resulting in a more rounded morphology of motile cells within the tumours and an increased number of cells observed within the vasculature . Taken together, these observations support the notion that high levels of cortical acto-myosin contraction are associated with amoeboid or rounded cancer cell motility and the metastatic process. This could potentially explain the elevated expression levels of many of the molecules involved in the regulation of acto-myosin contraction in metastatic human cancers.
In this review we have tried to summarize current thinking about regulation of the actin cytoskeleton in invading cancer cells and highlight some areas of current debate. It is clear that there is still much we do not know, but can we speculate what we might hope to learn in next two or three years? Recently the number of known molecules that can promote actin polymerization has increased but many of these have not yet been studied in the context of cancer biology. In fact only the regulators of the Arp2/3 complex and cofilin have been extensively studied in cancer models. It will be fascinating to learn about the role of the various FH proteins and other actin nucleators, such as spire and cordon bleu, in the migration of cancer cells and to determine if they become aberrantly regulated in tumours. Another area of growing interest is diverse range of morphologies or ‘modes of motility’ exhibited by cancer cells; these range from amoeboid, to elongated and collective patterns of invasion. Many human tumours show strand like patterns of invasion with cells often retaining cell-cell adhesions . This adds considerable complexity to the problem of cell invasion; we need to understand how the behaviour of many cells is coordinated so that they invade in one direction, and explore the possibility that distinct cells in the strands have different roles . Greater knowledge of the molecular pathways that determine the mode of motility used by cancer cells and how switching between different actin architectures is regulated will be very beneficial in understanding why and how cancer cells exit primary tumours.
This work was supported by Cancer Research UK (Erik Sahai and Michael F. Olson) and by the National Institutes of Health (Michael F. Olson; R01 CA030721).