role of integrins in cell invasion and migration - Semantic Scholar

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2002 Macmillan Magazines Ltd ... As cancer cells undergo metastasis — invasion and migration of a new ..... The SHC family is a group of adaptor proteins.
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ROLE OF INTEGRINS IN CELL INVASION AND MIGRATION John D. Hood and David A. Cheresh As cancer cells undergo metastasis — invasion and migration of a new tissue — they penetrate and attach to the target tissue’s basal matrix. This allows the cancer cell to pull itself forward into the tissue. The attachment is mediated by cell-surface receptors known as integrins, which bind to components of the extracellular matrix. Integrins are crucial for cell invasion and migration, not only for physically tethering cells to the matrix, but also for sending and receiving molecular signals that regulate these processes. INTEGRIN AFFINITY

The strength of the attraction of an integrin for its ligand. Signalling at the integrin’s cytoplasmic tail alters the conformation of its extracellular domain, changing the affinity of the integrin for its ligand and the adhesive capacity of the cell. INTEGRIN AVIDITY

An increase in the overall strength of cell adhesion, which is caused by the facilitation of lateral diffusion and/or clustering of integrins into multimeric complexes. STROMA

Loose, largely acellular, connective tissue. LAMELLIPODIUM

A broad membrane projection at the leading edge of the cell in the direction of movement.

The Scripps Research Institute, Departments of Immunology and Vascular Biology, 10550 North Torrey Pines Road, La Jolla, California 92037, USA. e-mail: [email protected] DOI: 10.1038/nrc727

In order for tumours to metastasize and grow, neoplastic and endothelial cells must invade and migrate into surrounding tissues. Metastatic tumours spread to different organs and are the primary cause of death in cancer patients. The ability to block the migratory and invasive capacity of tumour cells offers a new approach to treating patients with malignant disease. But metastasis is a complex process, which involves the coordination of several signal-transduction pathways that allow cancer cells to proliferate, remodel their surrounding environment, invade and migrate through new tissues, and differentiate. As cancer cells become metastatic and as endothelial cells become angiogenic, they develop altered AFFINITY and AVIDITY for their extracellular matrix (ECM). The phenotypic change is initially mediated by alterations in the expression of cell-surface molecules known as integrins, release of proteases that remodel the ECM and the deposition of new ECM molecules. These activate signalling cascades that regulate gene expression, cytoskeletal organization, cell adhesion and cell survival. As a result, cancer cells become more invasive, migratory and better able to survive in different microenvironments. Cellular invasion and migration are governed at both the extracellular and intracellular levels by several factors, and depend on the cell’s carefully balanced dynamic interaction with the ECM. For example, during invasion, cells release proteases that degrade and remodel the ECM, promoting cell

passage through to the STROMA and entrance into new tissue. This proteolytic process must be tightly controlled, such that the ECM is sufficiently degraded to facilitate cell passage, but not so degraded that cellular traction is lost. During migration, cells project LAMELLIPODIA that attach to the ECM, and simultaneously break existing ECM contacts at their trailing edge. This allows the cell to pull itself forward1 (BOX 1). Extension of lamellipodia is induced by actin polymerization and facilitated by a localized decrease in membrane tension2. Retraction of the cell edge is dependent on the adhesive environment and occurs either by fracturing the cell–ECM linkage in highly adhesive environments (during slow migration) or by simple dissociation of integrins — the cellular receptors for ECM molecules— in less adhesive environments (during fast migration)3,4. Integrins are a diverse family of glycoproteins that form heterodimeric receptors for ECM molecules. The family can form at least 25 distinct pairings of its 18 α-subunits and 8 β-subunits, with each pairing being specific for a unique set of ligands5. For example, integrin αvβ3 binds a wide range of ECM molecules, including fibronectin, fibrinogen, von Willebrand factor, vitronectin, and proteolysed forms of collagen and laminin, whereas integrin α5β1 selectively binds fibronectin 5. Each integrin generally consists of a noncovalently linked α- and β-subunit, with each subunit having a large extracellular domain, a single membrane-spanning domain, and a short, non-catalytic cytoplasmic tail.

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Summary • As cancer cells become metastatic and as endothelial cells become angiogenic, they develop altered affinity and avidity for their extracellular matrix. Some of these changes are mediated by alterations in the expression of cell-surface molecules known as integrins. • Numerous studies have documented dramatic differences in surface expression and distribution of integrins in malignant cells compared with pre-neoplastic tumours of the same type. • Integrins are also involved in regulating the activities of proteolytic enzymes that degrade the basement membrane — the initial barrier to surrounding tissue. • Integrins are essential for cell migration and invasion, not only because they directly mediate adhesion to the extracellular matrix, but also because they regulate intracellular signalling pathways that control cytoskeletal organization, force generation and survival. • Integrins not only send signals to the cell in response to the extracellular environment, but they also respond to intracellular cues and alter the way that they interact with the extracellular environment. • Integrin binding to ligands in the extracellular matrix initiates several pro-survival mechanisms to prevent apoptosis. • Over the past several years, research has led to the development of integrin and protease inhibitors that are now being tested in clinical trials.

In addition to regulating cell adhesion to the ECM, integrins relay molecular cues regarding the cellular environment that influence cell shape, survival, proliferation, gene transcription and migration6. Following ligand binding, integrins cluster into focal contacts that contain many different actin-associated proteins, such as α-actinin, vinculin, tensin and paxillin, which link the integrin to the

cytoskeleton 7. Integrins also activate kinases that phosphory-late cytoskeletal proteins, regulating stress-fibre formation, cell shape and migration. Furthermore, the cytoplasmic tail of integrins can recruit signalling proteins that regulate integrin adhesiveness to the ECM. But how do cancer cells use these integrin activities to regulate cell motility — along with other aspects of cell migration such as ECM proteolysis and growth-factor-receptor signalling — to take on a fully metastatic phenotype? Integrin expression and function

In addition to dysregulated protease activity, invasive cells also undergo dramatic alterations in levels of integrin expression and integrin affinity for ECM substrates. Numerous studies have documented marked differences in surface expression and distribution of integrins in malignant tumours compared with pre-neoplastic tumours of the same type8. For example, the integrin αvβ3 is strongly expressed at the invasive front of malignant melanoma cells and angiogenic blood vessels9, but weakly expressed on pre-neoplastic melanomas and quiescent blood vessels 9. Furthermore, inducing expression of the αv (REF. 10) or β3 (REF. 11) integrin subunit in a melanoma cell line increases metastatic potential. Similarly, the laminin-binding integrin, α6β4, is not expressed in normal thyroid cells, but induction of its expression correlates with the progression to invasive thyroid carcinoma12. Integrin α6β4 is upregulated in other carcinomas, such as papillomas13, and is mobilized from HEMIDESMOSOMES to migratory regions of the cell in others14,15.

Box 1 | Mechanism of cell migration Cell migration can be viewed as a series of discrete processes that result in net cell-body movement57. a,b | Initially, cells take on a polarized phenotype with a distinct cell front and rear. At the leading edge of the cell, actin polymerization and localized decreases in cell-membrane tension lead to the projection of a lamellipodium2. This is seen in Hep3 adenocarcinoma cells, which respond to insulin stimulation by becoming polarized and extending lamellipodia towards the chemotactic gradient (see Web movie). This polarized form reflects the asymmetric distribution of the intracellular molecules and forces that are necessary for migration. Receptors for chemotactic molecules118, integrins19 and cytoskeletal proteins that interact with integrins119 localize at the leading edge of the cell, thereby loading the region of the cell that is extended in the direction of migration with the receptors and signalling molecules that are necessary to form and react to new adhesive contacts. c,d,e,f | For the cell to complete its translocation, adhesive contacts at the rear of the cell are released (denoted by *), either by cleaving of the integrins from the cell body using enzymes such as calpain120, or by reduction of integrin affinity for the extracellular matrix (ECM). After forming new cell–ECM contacts, activation of intracellular actin–myosin motor units generates contractile forces that lead to the cell body advancing forward121. Polarization

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A specialized cell junction that is found on the basal surface of epithelial cells that anchors these cells to the basal lamina.

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Box 2 | The basement membrane Basement membranes are present in nearly all multicellular organisms and they are the first extracellular matrices that are produced during embryogenesis122. Epithelial cells, endothelial cells and many mesenchymal cells are supported by this thin (20–200 nm) sheet-like extracellular matrix structure. The membrane acts as a solid-phase regulator of cell attachment, differentiation and growth, as well as a passive barrier that segregates tissue compartments. Metastatic cancer cells must penetrate the tumour’s own basement membrane, that of the vasculature and that of the target tissue to establish secondary colonies. The basement membrane has a complex molecular architecture that largely consists of laminins, type IV collagen, osteonectin, entactin and heparan sulphate proteoglycans. Laminins are flexible four-armed glycoproteins with a molecular weight of ~850 kDa. Laminin monomers polymerize into three-dimensional structures in a time- and concentration-dependent manner. As an initial step towards metastasis, many epithelial tumours alter expression or localization of laminin-binding integrins, such as α6β4. This seems to promote both invasion through the basement membrane and increased motility in the stroma, where tumour cells frequently remodel the matrix by depositing laminin. Independent of the laminin network of proteins is a type IV collagen network — the most prevalent protein in the basement membrane. Type IV collagen is a flexible thread-like molecule that self-assembles into a heterotrimer involving both hydrophobic and disulphide bonds. The covalent bonding of collagen provides a great deal of the mechanical stability of the basement membrane. Proteins such as entactin, which forms connections between networks of collagen and laminin, further stabilize the basement membrane. Many of the proteases that are upregulated in metastatic tumours show high enzymatic activity against type IV collagen, and inhibition of these enzymes inhibits tumour invasion. The basement membrane is suffused with heparan sulphate proteoglycans — a class of molecules that contains a protein core covalently linked to heparan sulphate chains. These molecules are linked to the membrane through interactions with laminin and might influence tumour biology by acting as a repository for growth factors such as fibroblast growth factor.

Whereas the expression of some integrins, such as α6β4 and αvβ3, is increased during tumorigenesis, expression of others decreases. For example, the fibronectin-binding integrin α5β1 disappears from the surface of cell lines that are transformed with Rous sarcoma virus16 and its expression in cell lines markedly reduces tumorigenesis17. Similarly, reduced expression of the α1, α6, β1 or β4 integrin subunit is associated with the formation of neoplasms in breast epithelial tissue8. Increasing the affinity of integrins for their ligand is another mechanism by which cells can alter their adhesive profile to assume a more migratory phenotype. For instance, αvβ3 affinity has been reported to be modulated in a number of cells18, and selectively blocking high-affinity αvβ3 has been reported to impair the directed migration of endothelial cells19. So cells that assume a more invasive and migratory phenotype might modulate their adhesive capacity and intracellular signalling activities by altering their integrin expression and affinity profiles. Matrix degradation and remodelling

Integrins are also involved in regulating the activities of proteolytic enzymes that degrade the basement membrane — the initial barrier to surrounding tissue. The basement membrane forms an acellular support for cells, and is made up of a complex mix of ECM proteins, including laminins, collagens and proteoglycans (BOX 2). In fact, disruption of the organization or integrity of the basement membrane is a key histological marker of a tumour’s transition to an invasive carcinoma. Cancer cells produce, activate and release several different types of protease that specifically cleave ECM molecules. Localized degradation of the basement membrane requires the expression of proteases that are specific

for the resident ECM, and the concomitant expression of protease inhibitors to prevent excess matrix degradation. For example, two members of the matrix metalloproteinase (MMP) family of pericellular proteinases — MMP-2 (gelatinase A, 72 kDa) and MMP-9 (gelatinase B, 92 kDa) — have the highest enzymatic activities against type IV collagen, which is the main constituent of the basement membrane 20. The activation of these enzymes and cellular invasion are correlated under many pathophysiological settings. For instance, stromal cells close to the invasive front of human melanomas21 and metastatic breast tumours have elevated levels of MMP activity compared with non-malignant control cells 22, as do invading angiogenic endothelial cells23. Integrins might be involved in activating specific MMPs. MMPs are secreted as inactive zymogens (proMMPs) that require proteolytic activation by extracellular proteases24. This allows precise regulation of the activation and localization of otherwise soluble MMPs. For example, MMP-2 is activated on the cell surface by a multimeric complex that is composed of MMP-2, membrane type 1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase 2 (TIMP2). Recent studies on melanomas, gliomas and angiogenic endothelial cells indicate that this multiprotein MMP-activating complex also includes integrin αvβ3 (REFS 23,25). Initially, the carboxy-terminal domain of MMP-2 binds to TIMP2 (REF. 26), which, in turn, associates with the membrane-bound MT1-MMP27. MT1-MMP then cleaves the amino-terminal propeptide of MMP-2, resulting in an intermediate form that is capable of binding integrin αvβ3 at the cell surface28. This interaction activates MMP-2, thereby localizing its proteolytic activity to the invasive front of cells23. So, during invasion, integrin αvβ3 functions

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Cytoskeletal alterations, contraction, gene transcription, and integrin modulation

Cell invasion and migration

Figure 1 | Regulation of intracellular signalling by integrins. Integrin ligation induces a complex network of signalling pathways to control cell migration. Integrin (α and β) binding to ligands in the extracellular matrix (ECM) activates focal adhesion kinase (FAK), which binds and activates multiple signalling proteins. FAK autophosphorylation at tyrosine 925 causes it to bind growth-factor-receptor-bound protein 2 (GRB2) (REF. 123) and activate another small G protein, RAS. FAK activation also promotes SRC-dependent phosphorylation of SHC124, leading to GRB2 recruitment and RAS activation. Activated RAS recruits RAF to the cytoplasmic membrane, where it can be activated by protein kinases such as SRC125, thereby leading to mitogen-activated protein kinase kinase (MEK) and extracellular-signal-regulated kinase (ERK) activation. Once activated by FAK or SHC, RAS can activate phosphatidylinositol 3-kinase (PI3K) and RAF. Activated SRC can also phosphorylate CRK-associated substrate (CAS), enabling it to bind CRK and dedicator of cytokinesis 180 (DOCK-180), leading to RAC activation. Activated RAC, in conjunction with activated CDC42, can regulate numerous biochemical pathways, including activation of p21-activated kinase (PAK). PAK affects numerous pathways, and also activates RAF’s kinase activity. MEK, once activated by RAS and RAF, can phosphorylate and activate ERK. ERK activation leads to transcriptional activity, alterations in integrin affinity for ligand, and myosin-light-chain kinase (MLCK) activity. Independent of FAK activation, signalling molecules such as SHC and PKC are also activated by integrin adhesion events. Activating these molecules can also eventually lead to RAS and RAF activation (not shown), along with the alterations in the cytoskeleton that are necessary for the migratory phenotype.

MEMBRANE RUFFLE

A characteristic phenotype of migrating cells in which the cell has regions of the leading edge that are dynamically alternating between adherent and nonadherent states, thereby giving the impression of a ‘ruffling’ edge.

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the MMP-2-dependent generation of hemopexin fragments, termed PEX, which block protease activation by competing with MMP-2 for binding to integrin αvβ3 (REF. 29). In fact, inhibitors of the interaction between MMP-2 and αvβ3 potently suppress the growth of melanomas and gliomas, along with angiogenesis, in animal models30,31.

not only as an adhesion/migration receptor, but also in activating and localizing proteases that are required for ECM degradation. Negative-feedback regulation of integrin/protease binding is required to prevent excessive degradation of the ECM, which could reduce cellular traction and inhibit migration. Proteolysis is negatively regulated by

Integrins are essential for cell migration and invasion, not only because they directly mediate adhesion to the extracellular matrix, but also because they regulate intracellular signalling pathways that control cytoskeletal organization, force generation and survival (FIG. 1). During migration, cells are constantly making and breaking integrin contacts. Depending on the composition of the ECM, integrins activate one or more intra-cellular signalling pathways. These pathways typically involve phosphorylation of focal adhesion kinase (FAK), recruitment of adaptor proteins, activation of small GTPases and subsequent activation of downstream effector molecules. These signals, in concert with signals derived from growth factors, regulate cell behaviour in a complex tissue microenvironment. FAK. FAK is a cytoplasmic protein kinase that co-localizes with integrins at structures called focal adhesions — areas of the cell surface that interact with the ECM and are heavily enriched in structural and signalling molecules. Integrin binding of ECM ligands induces integrin clustering and FAK activation32. Activated FAK binds to several signalling molecules32, mediating integrin-33 or serum-induced34 activation of the RAS–extrallular-signal-regulated kinase (ERK) pathway, which promotes cell proliferation. FAK activation has also been shown to promote cell survival35 and cell migration that is induced by integrins33 or growth factors36. FAK is expressed at higher levels in invasive tumours than in benign pre-neoplastic tumours37,38. Fak-null mice die before birth, and cells isolated from these embryos have migration defects and an impaired ability to remodel cell–ECM contacts39. How FAK regulates cell migration is not completely understood. After integrin clustering, FAK is autophosphorylated at tyrosine 397 and recruits SRC family kinases to focal adhesions. Recruitment of Src to focal adhesions has been shown to be required for integrin-mediated cell motility in fibroblasts and Chinese hamster ovary (CHO) cells33,40,41. In agreement with these findings, cell motility induced by transgenic FAK expression in FAK-null fibroblasts is impaired by SRC inhibition33, and Src expression rescues cell motility in Fak-deficient cells42. Similarly, phosphorylated FAK can activate ERK by recruiting adaptor proteins such as growth-factor-receptorbound protein 2 (GRB2) and CRK-associated substrate (CAS), as well as by activating protein kinases such as the SRC-family kinases32. FAK-mediated activation of these pathways can, in turn, regulate cell proliferation and migration.

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REVIEWS CAS–CRK. Activated FAK associates with the adaptor protein CAS43. FAK and SRC both phosphorylate CAS at multiple tyrosine residues, causing CAS to associate with another adaptor protein, CRK44,45, and localize to MEM46 BRANE RUFFLES . The CAS–CRK complex is a component of the molecular migration machinery that is located at the leading edge of motile cells46. Inhibiting the formation of the CAS–CRK complex prevents both HAPTOTAXIS and CHEMOTAXIS. This complex seems to act as a molecular switch, inducing cell motility by activating dedicator of cytokinesis 180 (DOCK-180), leading to the activation of the small GTP-binding protein RAC47. In support of this notion, pancreatic carcinoma cells that undergo adhesion-dependent CAS phosphorylation are more motile and metastatic than cells that do not undergo adhesiondependent CAS phosphorylation46. Furthermore, the migration-enhancing effects of the CAS–CRK complex are attenuated by co-expression of dominant-negative forms of RAC46. The dominant-negative form of RAC is likely to interfere with the ability of wild-type RAC to interact with and become activated by DOCK-180, CAS and CRK48. Mutation of the SRC homology 3 (SH3) domain of CRK prevents DOCK-180 from binding to CRK, as well as preventing RAC activation, thereby inhibiting cell migration48. Because both CAS and CRK have a large number of potential binding partners, the CAS–CRK complex potentially regulates, and is regulated by, numerous proteins. For example, the tyrosine kinase ABL phosphorylates CRK at tyrosine 221, inhibiting formation of the CAS–CRK complex and cell migration49. Paradoxically, adhesion and cytokines can activate both ABL and CAS–CRK complex formation. So the migratory phenotype of a cell might be regulated by the balance of integrin-induced signals. For example, a cell might activate ABL, which blocks formation of the CAS–CRK complex, yet also activate FAK-mediated signals that promote CAS–CRK coupling.

HAPTOTAXIS

Migration on an extracellular matrix gradient in the absence of chemotactic stimuli. CHEMOTAXIS

Migration by a cell in the direction of a chemical gradient. FILOPODIUM

A small membrane projection that emanates from the leading edge of the cell in the direction of movement.

SHC. The SHC family is a group of adaptor proteins that are recruited to activated tyrosine kinases in response to ligation of integrins α1β1, α6β4, α5β1 or αvβ3, and to some extracellular receptors in response to growth-factor binding50,51. Formation of this complex leads to SHC-dependent cell-cycle progression, migration, anti-apoptotic signals and ERK activation50,51. These proteins are composed of three interaction domains: a carboxy-terminal SRC homology 2 (SH2) domain, a central collagen homology (CH) domain, and an amino-terminal phosphotyrosine binding (PTB) domain52,53. Shc-deficient mice die during embryogenesis with defects in blood-vessel formation and cardiovascular development54. Furthermore, fibroblasts derived from these mice show impaired ability to respond to ECM cues by activating the ERK pathway or reorganizing their cytoskeleton54, as would be required for migration. It has recently been reported that SHC overexpression in a glioblastoma line increases the ‘random motility’ component of migration, in which cells move rapidly in random directions. FAK and CAS overexpression, by contrast, resulted in

cells that migrated more slowly, but in a persistent direction55. This indicates that the differential activation of SHC and other signalling molecules impacts both whether a cell migrates and how it migrates. As they become malignant, pre-neoplastic tumours undergo a dramatic transformation from a highly proliferative cellular phenotype to a spontaneous highly haptotactic phenotype. Little is known about the molecular events that govern the regulation of cell migration versus proliferation, but recent studies indicate that SHC might be involved in this transition 56. Whereas tyrosine phosphorylation of SHC is necessary for both DNA synthesis and cell migration, mutations in the SH2 domain of SHC selectively inhibit DNA synthesis, and mutations in the PTB domain of SHC selectively inhibit migration56. Integrin ligation is normally required to induce SHC phosphorylation, but constitutive phosphorylation of SHC is found in many highly metastatic carcinoma cells, thereby promoting haptotaxis56. Constitutive phosphorylation of the SHC PTB domain might therefore be a crucial signalling element in transformation from a pre-neoplastic to a metastatic phenotype. Small G proteins. Highly motile cells, such as malignant carcinomas, have dramatic alterations in their cytoskeletal organization that facilitates their invasive and migratory behaviour. For example, initiation of migration is characterized by the rapid reorganization of actin to the cell edge, the protrusion of a leading lamellipodium and membrane ruffling at the advancing front of the lamellipodium57. The RHO family of small GTPases — including RHO, CDC42 and RAC — can regulate these changes58 (see also the review by Sahai and Marshall on page 133 in this issue). RAC and CDC42 are activated by ligation of integrins59, whereas RHO activation is dependent on integrins, syndecan-4 and additional cell-surface receptors60–62. Activation of RHO leads to the formation of actin stress fibres and the assembly of focal adhesions63, whereas activation of CDC42 is involved in the formation of FILOPODIA64, and activation of RAC promotes membrane ruffling and migration65. Expression of constitutively active RAC and CDC42 is sufficient to depolarize differentiated mammary epithelial cells and induce integrin-mediated invasion through a threedimensional collagen matrix66. RAC activation also potentiates ERK signalling and increases cellular sensitivity to growth factors67, whereas activation of RAC and CDC42 activates p21-activated kinase (PAK)68, which phosphorylates RAF kinase69. This provides another mechanism of ERK activation. Consistent with a role for PAK-mediated ERK activation in RACand CDC42-induced migration, inhibition of PAK with a dominant-negative mutant blocks endothelial migration by inhibiting cell contractile forces and detachment70. So, adhesion events that lead to RAC activation promote actin reorganization, as well as facilitating mitogen-activated ERK activity, which can lead to actin/myosin force generation and the release

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Figure 2 | Regulation of integrin function by intracellular signals. Integrin (α and β) adhesiveness to extracellular matrix (ECM) components is regulated by altering the integrin’s affinity for its ECM ligands and/or by altering the integrin’s avidity. Small GTP-binding proteins seem to have a prevalent role in these processes. The activation of the small GTP-binding protein RRAS can lead to increases in integrin affinity for the ECM, whereas HRAS activation can lead to decreases in integrin affinity (no ECM binding). Similarly, activation of the small GTP-binding proteins RAC and CDC42, and protein kinase C (PKC), can lead to clustering of integrins, thereby increasing integrin avidity. The careful regulation of each of these events is necessary for efficient cell migration.

of integrin contacts. RHO-family GTPases are therefore not only necessary for invasion and migration — they might also be sufficient to induce the signalling and cellular responses that are required to reorganize the actin cytoskeleton and the actin/myosin motor unit into an invasive and migratory phenotype.

TETRASPANIN

Transmembrane-4 superfamily proteins, including CD9, CD53, CD81, CD82 and CD151. These proteins regulate the association of protein kinase C with specific integrins.

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ERK pathway. During cell migration in vivo, the formation of new integrin contacts provides the cell with positional and molecular signals that activate ERK signalling. Integrins regulate ERK activity both directly and by modulating growth-factor-stimulated ERK activity71. As discussed above, integrins directly activate ERK through several FAK-dependent and -independent pathways. Integrins also co-localize with growth-factor receptors on the surface of cells72 and impact many convergent signalling pathways 6, including the ERK pathway. For example, blocking integrin ligation 73,74 or FAK 34 in vitro prevents full growth-factor-induced activation of RAF or mitogenactivated-protein-kinase kinase (MEK), thereby abrogating growth-factor-induced ERK activation. Similarly, exposing angiogenic tissues to monoclonal antibodies against αvβ3 integrin blocks the secondary sustained phase of fibroblast growth factor (FGF)-mediated ERK activation 75. This sustained phase of ERK activity corresponds to a period of intense migratory activity by the endothelial cells9 that is necessary for angiogenesis75. ERK activation is likely to impact invasion and migration through many pathways by influencing gene transcription and survival, as well as by directly regulating the enzymes that are necessary for cell locomotion. For example, during migration the ERK signal decreases integrin-mediated adhesion and phosphorylates myosin-light-chain kinase (MLCK)76, a key regulator of contractile force within the cell. MLCK phosphorylates and activates myosin II, an ATPase that is located in leading lamellae and posterior regions of motile cells, which generates force by promoting translational movement along actin cables77. Activated myosin generates contractile forces that pull the cell forward towards newly formed integrin contacts and breaks adhesive

contacts at the trailing edge of the cell76. ERK also regulates calpain, a calcium-activated protease that is responsible for breaking rear contacts during migration78. Therefore, ERK can biochemically regulate both release of the trailing cell body and the subsequent forward movement of the cell. PKC. The protein kinase C (PKC) family of serine/threonine kinases are important for regulating integrin function and signalling. For example, ligation of fibronectin by integrins and syndecan-4 recruits PKC to membrane adhesions79—81, where it is required for focal-adhesion formation82, phosphorylation of FAK81, cell spreading81, SHC-dependent ERK activation80 and migration83. Similarly, PKC is important for αvβ5-integrin-mediated focal-adhesion formation and cell migration on vitronectin-containing substrates84, along with vascular endothelial growth factor (VEGF)-induced angiogenesis85. Recent reports indicate that PKC interacts with β1 integrins using TETRASPANIN-family proteins as bridging molecules86, and that PKC regulates β1 transport within the cell87. Furthermore, PKC activation is required to mobilize integrin α6β4 from hemidesmosomes to the lamellar protrusions, where it is required for migration by many epithelial-cell tumours88. PI3K. Phosphatidylinositol 3-kinase (PI3K) regulates integrin-dependent cell motility by modulating integrin responses in both normal and neoplastic tissue. In kidney epithelial cells and breast carcinomas, PI3K is activated following integrin ligation89,90. In breast carcinoma cells, for example, α6β4 integrin ligation activates PI3K to promote formation of lamellae and invasion90. Furthermore, PI3K activity is required for CDC42 and RAC-induced cell motility and invasiveness in mammary epithelial cells66. Similarly, enhanced β1-integrinmediated adhesion and migration by breast carcinomas in response to epidermal growth factor (EGF) and its family member, heregulin (which are expressed by metastatic breast cancer cells), is dependent on PI3K activity91. Paradoxically, normal epithelial cells might use PI3K to limit migration. For example, α6β4-integrininduced PI3K activation in keratinocytes attenuates haptotaxis that is mediated by α3β1 — the integrin that mediates adhesion to the basement membrane in normal epithelial cells92. PI3K activation might therefore assist in maintaining the non-motile phenotype of normal cells. But in cancer cells that have mobilized α6β4 from the hemidesmosome, the same signalling molecule can stimulate invasion and migration. Inside-out signalling

Adhesion to extracellular matrix molecules through integrin heterodimers is a fundamental requirement for cells to acquire the traction necessary for movement. Interestingly, the maximum rate of cell migration occurs at intermediate levels of adhesiveness93,94. At low levels of adhesiveness, weakly attached cells cannot generate sufficient traction to move efficiently. At high levels of adhesiveness, cells cannot break contact and are therefore immobile. Intermediate

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Figure 3 | Integrin binding to extracellular matrix ligands prevents apoptosis. One hurdle that neoplastic cells must overcome to establish metastases is the ability of the extracellular matrix (ECM) of host tissue to induce ‘integrin-mediated death’ in invading cells. Each integrin heterodimer binds distinct sets of ECM molecules, and ECM composition is tissue-type specific. ECM composition therefore governs cell survival as a function of the integrins that are expressed on the surface of a cell that is occupying new territory. a | If the cell is migrating through a microenvironment in which the ECM does not contain a suitable ligand (yellow circles) for its integrins, the β1- or β3-chain (blue) of the integrin cytoplasmic tail recruits caspase-8 to the membrane, inducing apoptosis115. b | If the ECM contains the appropriate ligands, the integrin is properly ligated, thereby inducing pro-survival signals. This provides a mechanism by which aberrant cell migration might be controlled in pre-neoplastic cells and is the reason why certain types of cancer cell establish metastases in specific organs.

CHELATE AND RE-BINDING EFFECTS

The factors that underlie the increased cell adhesive strength caused by avidity changes. Chelate denotes that a large extracellular matrix protein, which presents many potential integrin-binding sites, can bind several integrins if they are in sufficiently close proximity. Rebinding effects are those in which a ligand that is displaced from one integrin will rapidly re-bind a neighbouring integrin if it is in sufficiently close proximity. ANOIKIS

Apoptosis that is induced when anchorage-dependent cells detach from their extracellular matrix.

levels of adhesion allow traction at the cell front while releasing contacts at the rear, resulting in net forward movement. Integrins not only send signals to the cell in response to the extracellular environment, but they also respond to intracellular cues and alter the way in which they interact with the extracellular environment95. This process — termed ‘inside-out’ signalling — regulates integrin adhesiveness by modulating the affinity and avidity of integrins for their ECM ligands, regulating cell invasion and migration. Binding of proteins at the cytoplasmic tail is believed to induce a shift in an integrin’s quartenary structure, resulting in the head groups becoming ‘open’ and available for ligand binding96,97. Integrin clustering, which increases avidity, is observed in focal adhesions, where it is thought to promote ECM ligand binding through CHELATE 98 AND RE-BINDING EFFECTS . Relocalization of integrins to adhesive migratory structures is another mechanism by which cells can alter their binding to the ECM. For example, for a non-motile epithelial cell to become a migrating epithelial cell, it loses polarity: α6β4 integrin is released from hemidesmosomes and is frequently recruited to lamellipodia99. Similarly, activated integrin αvβ3 localizes to the leading edge of lamellipodia in endothelial cells in two-dimensional cultures on fibrinogen19, and to the

leading edge of phosphorylated FAK-containing cell extensions in cell-derived three-dimensional matrices100. The signalling mechanisms that regulate integrin affinity and avidity are likely to vary for different integrin heterodimers, but small GTP-binding proteins are known to be involved (FIG. 2). Pharmacological inhibition of RHO kinase has been shown to prevent integrin-dependent lymphoblastoid aggregation 101 and lymphocyte adhesion102, whereas overexpression of mutant inactive forms of RHO prevent fibroblast spreading103, presumably through alterations in cell avidity104. Furthermore, expression of active RAC and CDC42 in mammary epithelial cells leads to disrupted cell polarization and induces a PI3K-dependent invasive, motile phenotype that is indicative of a large shift in integrin localization66. RRAS and HRAS, by contrast, modulate the adhesiveness of integrins for their substrate. For example, overexpression of constitutively active RRAS increases myeloid-cell adhesiveness by increasing the affinity of multiple integrins, including α4β1, α5β1 and αvβ3, for the ECM105. Similarly, overexpression of RRAS also increases the adhesivenes of CHO cells by increasing the affinity of integrin αIIbβ3, indicating that this might be a mechanism of increasing integrin affinity that is common to many different cells and integrins105. By contrast, overexpression of constitutively active HRAS in CHO cells signals integrins α5β1, α6Aβ1, α6Aβ3 or α6Bβ3 — through their cytoplasmic tails — to decrease affinity106. Survival during invasion

For invasive cells to successfully invade and migrate to distal tissues, they must be able to activate survival mechanisms to prevent induction of apoptosis 107. Accordingly, inducing apoptosis in displaced cells prevents aberrant cell migration107. Integrin binding to ECM ligands initiates several pro-survival mechanisms to prevent apoptosis (FIG. 3). Notably, many anti-apoptotic pathways are the same as those that are involved in regulating migration. For example, ligation of integrins α5β1 or αvβ3 leads to SHC-, PI3K- and FAK-mediated survival of CHO cells under serum-free conditions108, whereas expression of constitutively activated FAK is sufficient for anchorage-independent survival35. This indicates that FAK has a role as a survival factor that is independent of matrix signalling. Several targets of FAK signalling have also been implicated in survival, including RAS 89, RAC 109, PI3K 89 and ERK 109, along with CAS–CRK coupling 109. Accordingly, disruption of FAK signalling by overexpressing inactive forms of FAK in breast carcinoma cells induces caspase-8dependent apoptosis in cells that are otherwise anchorage independent110. Similarly, FAK cleavage by caspases is an early event in apoptosis that might eliminate survival cues from the ECM111. Correspondingly, anchorage-dependent cells that are deprived of the ability to bind immobilized ECM undergo apoptosis, a process referred to as ANOIKIS112. However, the antagonism of individual integrins,

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Table 1 | Proteinase and integrin antagonists in clinical trials Drug

Sponsor

Stage

Marimastat

British Biotech

Phase III

COL-3

Collagenex

Phase I

Neovastat

Aeterna

Phase III

AG3340

Agouron

Phase III

BMS-275291

Bristol-Myers Squibb

Phase I

CGS 27023A

Novartis

Phase I/II

Vitaxin

MedImmune

Phase II

MED1522

Merck KgaA

Phase I/II

MMP inhibitors in clinical trials

Integrin inhibitors in clinical trials

MMP, matrix metalloproteinase.

such as αvβ3, is sufficient to induce apoptosis and block invasive events such as liver metastasis and angiogenesis113,114. In fact, when adherent cells express specific integrin complexes in matrices that lack the cognate ligand, they undergo a form of apoptosis termed ‘integrin-mediated death’ (IMD) 115. For example, expression of integrin αvβ3 on cells in a collagen gel that lacks an αvβ3 ligand leads to the recruitment of caspase-8 to the membrane, leading to caspase-8 activation and cell death115. IMD might provide mechanistic insights into why antagonists of integrin αvβ3 prevent endothelial-cell

1.

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4.

5. 6.

7.

8.

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11.

12.

13.

98

Sheetz, M. P., Felsenfeld, D., Galbraith, C. G. & Choquet, D. Cell migration as a five-step cycle. Biochem. Soc. Symp. 65, 233–243 (1999). Raucher, D. & Sheetz, M. P. Cell spreading and lamellipodial extension rate is regulated by membrane tension. J. Cell Biol. 148, 127–136 (2000). Palecek, S. P., Horwitz, A. F. & Lauffenburger, D. A. Kinetic model for integrin-mediated adhesion release during cell migration. Ann. Biomed. Eng. 27, 219–235 (1999). Palecek, S. P., Huttenlocher, A., Horwitz, A. F. & Lauffenburger, D. A. Physical and biochemical regulation of integrin release during rear detachment of migrating cells. J. Cell Sci. 111, 929–940 (1998). van der Flier, A. & Sonnenberg, A. Function and interactions of integrins. Cell Tissue Res. 305, 285–298 (2001). Aplin, A. E., Howe, A. K. & Juliano, R. L. Cell adhesion molecules, signal transduction and cell growth. Curr. Opin. Cell Biol. 11, 737–744 (1999). Sastry, S. K. & Burridge, K. Focal adhesions: a nexus for intracellular signaling and cytoskeletal dynamics. Exp. Cell Res. 261, 25–36 (2000). Mizejewski, G. J. Role of integrins in cancer: survey of expression patterns. Proc. Soc. Exp. Biol. Med. 222, 124–138 (1999). Brooks, P. C., Clark, R. A. & Cheresh, D. A. Requirement of vascular integrin αvβ3 for angiogenesis. Science 264, 569–571 (1994). Felding-Habermann, B., Mueller, B. M., Romerdahl, C. A. & Cheresh, D. A. Involvement of integrin αv gene expression in human melanoma tumorigenicity. J. Clin. Invest. 89, 2018–2022 (1992). Filardo, E. J., Brooks, P. C., Deming, S. L., Damsky, C. & Cheresh, D. A. Requirement of the NPXY motif in the integrin-β3 subunit cytoplasmic tail for melanoma cell migration in vitro and in vivo. J. Cell Biol. 130, 441–450 (1995). Serini, G. et al. Changes in integrin and E-cadherin expression in neoplastic versus normal thyroid tissue. J. Natl Cancer Inst. 88, 442–449 (1996). Tennenbaum, T. et al. The suprabasal expression of α6β4 integrin is associated with a high risk for malignant progression in mouse skin carcinogenesis. Cancer Res. 53, 4803–4810 (1993).

invasion during angiogenesis, whereas humans or mice that are deficient in this integrin undergo apparently normal angiogenesis 116. An antagonist that blocks ligation of integrins such as αvβ3 would induce IMD, whereas preventing integrin expression would remove a possible trigger for apoptosis. Deletion of an apoptotic signal would also have physiological consequences. For example, mice that lack αvβ3 and/or αvβ5 display increased tumour growth and angiogenesis117. Although it seems to contradict results from experiments with integrin inhibitors, this finding is consistent with the idea that unligated integrins promote IMD115 — the removal of such an integrin (in knockout mice) would increase the number of invasive endothelial cells in a tissue site by decreasing their level of apoptosis. Therefore, several mechanisms regulate cell migration — including the ability to survive in a new microenvironment. Therapeutics that are designed to modify cellular invasive and migratory activities might be useful in treating pathologies that are associated with these cell phenotypes, such as cancer-cell metastasis, angiogenesis and inflammatory disease. Over the past several years, research has led to the development of integrin and protease inhibitors that are now being tested in clinical trials (TABLE 1). Continuing research into the pathways that are intrinsic to the invasive and migratory phenotype hold the promise for the development of new, more effective cancer therapeutics.

14. Rabinovitz, I. & Mercurio, A. M. The integrin α6β4 functions in carcinoma cell migration on laminin-1 by mediating the formation and stabilization of actin-containing motility structures. J. Cell Biol. 139, 1873–1884 (1997). 15. O’Connor, K. L., Shaw, L. M. & Mercurio, A. M. Release of cAMP gating by the α6β4 integrin stimulates lamellae formation and the chemotactic migration of invasive carcinoma cells. J. Cell Biol. 143, 1749–1760 (1998). References 14 and 15 describe a dramatic functional shift in which immotile cells that were using integrin α6β4 to remain immotile alter location and function of this integrin to promote migration. 16. Plantefaber, L. C. & Hynes, R. O. Changes in integrin receptors on oncogenically transformed cells. Cell 56, 281–290 (1989). 17. Varner, J. A., Emerson, D. A. & Juliano, R. L. Integrin α5β1 expression negatively regulates cell growth: reversal by attachment to fibronectin. Mol. Biol. Cell 6, 725–740 (1995). 18. Pampori, N. et al. Mechanisms and consequences of affinity modulation of integrin αvβ3 detected with a novel patchengineered monovalent ligand. J. Biol. Chem. 274, 21609–21616 (1999). 19. Kiosses, W. B., Shattil, S. J., Pampori, N. & Schwartz, M. A. Rac recruits high-affinity integrin αvβ3 to lamellipodia in endothelial cell migration. Nature Cell Biol. 3, 316–320 (2001). Describes the RAC-mediated selective relocalization of activated integrins to migratory structures. 20. Collier, I. E. et al. H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen. J. Biol. Chem. 263, 6579–6587 (1988). 21. Pyke, C. et al. Localization of messenger RNA for Mr 72,000 and 92,000 type IV collagenases in human skin cancers by in situ hybridization. Cancer Res. 52, 1336–1341 (1992). 22. Monteagudo, C., Merino, M. J., San-Juan, J., Liotta, L. A. & Stetler-Stevenson, W. G. Immunohistochemical distribution of type IV collagenase in normal, benign, and malignant breast tissue. Am. J. Pathol. 136, 585–592 (1990). 23. Brooks, P. C. et al. Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin αvβ3. Cell 85, 683–693 (1996).

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57. Horwitz, A. R. & Parsons, J. T. Cell migration — movin’ on. Science 286, 1102–1103 (1999). 58. Nobes, C. D. & Hall, A. Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81, 53–62 (1995). References 58 and 63 describe the regulation of cell shape changes by low-molecular-weight GTPases. 59. Price, L. S., Leng, J., Schwartz, M. A. & Bokoch, G. M. Activation of Rac and Cdc42 by integrins mediates cell spreading. Mol. Biol. Cell 9, 1863–1871 (1998). 60. Ren, X. D., Kiosses, W. B. & Schwartz, M. A. Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J. 18, 578–585 (1999). 61. Saoncella, S. et al. Syndecan-4 signals cooperatively with integrins in a Rho-dependent manner in the assembly of focal adhesions and actin stress fibers. Proc. Natl Acad. Sci. USA 96, 2805–2810 (1999). 62. Ishiguro, K. et al. Syndecan-4 deficiency impairs focal adhesion formation only under restricted conditions. J. Biol. Chem. 275, 5249–5252 (2000). 63. Ridley, A. J. & Hall, A. The small GTP-binding protein Rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70, 389–399 (1992). 64. Kozma, R., Ahmed, S., Best, A. & Lim, L. The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts. Mol. Cell Biol. 15, 1942–1952 (1995). 65. Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D. & Hall, A. The small GTP-binding protein Rac regulates growth factor-induced membrane ruffling. Cell 70, 401–410 (1992). 66. Keely, P. J., Westwick, J. K., Whitehead, I. P., Der, C. J. & Parise, L. V. Cdc42 and Rac1 induce integrin-mediated cell motility and invasiveness through PI(3)K. Nature 390, 632–636 (1997). Directly links small GTPase function to invasive and migratory activity. 67. Leng, J., Klemke, R. L., Reddy, A. C. & Cheresh, D. A. Potentiation of cell migration by adhesion-dependent cooperative signals from the GTPase Rac and Raf kinase. J. Biol. Chem. 274, 37855–37861 (1999). 68. Manser, E., Leung, T., Salihuddin, H., Zhao, Z. S. & Lim, L. A brain serine/threonine protein kinase activated by Cdc42 and Rac1. Nature 367, 40–46 (1994). 69. King, A. J. et al. The protein kinase Pak3 positively regulates Raf-1 activity through phosphorylation of serine 338. Nature 396, 180–183 (1998). 70. Kiosses, W. B., Daniels, R. H., Otey, C., Bokoch, G. M. & Schwartz, M. A. A role for p21-activated kinase in endothelial cell migration. J. Cell Biol. 147, 831–844 (1999). 71. Keely, P., Parise, L. & Juliano, R. Integrins and GTPases in tumour cell growth, motility and invasion. Trends Cell Biol. 8, 101–106 (1998). 72. Miyamoto, S., Teramoto, H., Gutkind, J. S. & Yamada, K. M. Integrins can collaborate with growth factors for phosphorylation of receptor tyrosine kinases and MAP kinase activation: roles of integrin aggregation and occupancy of receptors. J. Cell Biol. 135, 1633–1642 (1996). 73. Renshaw, M. W., Ren, X. D. & Schwartz, M. A. Growth factor activation of MAP kinase requires cell adhesion. EMBO J. 16, 5592–5599 (1997). 74. Chen, Q., Lin, T. H., Der, C. J. & Juliano, R. L. Integrinmediated activation of MEK and mitogen-activated protein kinase is independent of Ras. J. Biol. Chem. 271, 18122–18127 (1996). 75. Eliceiri, B. P., Klemke, R., Stromblad, S. & Cheresh, D. A. Integrin αvβ3 requirement for sustained mitogen-activated protein kinase activity during angiogenesis. J. Cell Biol. 140, 1255–1263 (1998). Shows a time-dependent requirement for integrin signalling during an in vivo tissue remodelling process. 76. Klemke, R. L. et al. Regulation of cell motility by mitogenactivated protein kinase. J. Cell Biol. 137, 481–492 (1997). 77. Conrad, P. A. et al. Relative distribution of actin, myosin I, and myosin II during the wound healing response of fibroblasts. J. Cell Biol. 120, 1381–1391 (1993). 78. Glading, A., Uberall, F., Keyse, S. M., Lauffenburger, D. A. & Wells, A. Membrane proximal ERK signaling is required for M-calpain activation downstream of epidermal growth factor receptor signaling. J. Biol. Chem. 276, 23341–23348 (2001). 79. Couchman, J. R. & Woods, A. Syndecan-4 and integrins: combinatorial signaling in cell adhesion. J. Cell Sci. 112, 3415–3420 (1999). 80. Miranti, C. K., Ohno, S. & Brugge, J. S. Protein kinase C regulates integrin-induced activation of the extracellular regulated kinase pathway upstream of Shc. J. Biol. Chem. 274, 10571–10581 (1999).

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Acknowledgements The authors would like to thank K. Spencer, J. Condelis and M. Bailey for assistance with figures.

Online links DATABASES The following terms in this article are linked online to: CancerNet: http://cancernet.nci.nih.gov/ breast tumours | gliomas | melanoma | pancreatic carcinoma | thyroid carcinoma LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ α-actinin | ABL | actin | calpain | CAS | caspase-8 | CDC42 | collagen | CRK | DOCK-180 | EGF | entactin | ERK | FAK | FGF | fibrinogen | fibronectin | GRB2 | heregulin | HRAS | α1β1 integrin | α4β1 integrin | α5β1 integrin | α6β4 integrin | α6Aβ1 integrin | α6Aβ3 integrin | α6Bβ3 integrin | αIIbβ3 integrin | αvβ3 integrin | laminin | MEK | MLCK | MMP-2 | MMP-9 | MT1-MMP | myosin | osteonectin | PAK | paxillin | PI3K | PKC | RAC | RAF | RRAS | SHC | SRC | syndecan-4 | tensin | TIMP2 | VEGF | vinculin | vitronectin | von Willebrand factor FURTHER INFORMATION The integrin page: http://www.geocities.com/CapeCanaveral/9629/ Photos of the basal lamina: http://www.heuserlab.wustl.edu/BasalLaminaLink.html The WWW virtual library of cell biology — the extracellular matrix: http://vlib.org/Science/Cell_Biology/cell_adhesion_ecm.shtml Access to this interactive links box is free online.

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