Signaling by Integrin Receptors

C Chandra Kumar

Department of Tumor Biology, Schering-Plough Research Institute, 2015, Galloping Hill Road, Kenilworth, New Jersey 07033, USA

Adhesive interactions are critical for the proliferation, receptor complexes (Hynes, 1992; Ruoslahti, 1991, survival and function of all cells. Integrin receptors as 1996). The a and b subunits contain a large the major family of adhesion receptors have been the extracellular domain, a short transmembrane domain focus of study for more than a decade. These studies and a cytoplasmic carboxy terminal domain of variable have tremendously enhanced our understanding of the length. The extracellular domains of both the a and b integrin-mediated adhesive interactions and have un- chains form the ligand binding domain. In mammals, raveled novel integrin functions in cell survival mechan- 17 a subunits and 8 b subunits are known. These a and isms and in the activation of divergent signaling b subunits heterodimerize to produce 22 dierent pathways. The signals from integrin receptors are receptors (Figure 1). Despite this high degree of integrated from those originating from growth factor redundancy, most integrins seem to have speci®c receptors in order to organize the cytoskeleton, biological functions raising the possibility of signaling stimulate cell proliferation and rescue cells from matrix dierences between integrins. Integrins not only bind detachment-induced programmed cell death. These ligands present in the extracellular matrix such as functions are critical in the regulation of multiple ®bronectin, collagen, vitronectin etc., certain integrins processes such as tissue development, in¯ammation, can also bind to soluble ligands such as ®brinogen or angiogenesis, tumor cell growth and metastasis and to counter-receptors such as intracellular adhesion programmed cell death. molecules (ICAMs) on adjacent cells. Many of the integrins recognize the RGD (Arg-Gly- Keywords: integrins; cell adhesion; cell survival; focal Asp) sequence in their matrix ligands (Ruoslahti, adhesion complexes 1996). Nevertheless, they are capable of distinguishing dierent RGD-containing proteins such that some bind primarily to ®bronectin and others to vitronectin. Integrins are also expressed in a cell-type speci®c

manner; thus one group of integrins such as a5 b1, av b3

Introduction and av b6 is associated with migration and proliferation in various cell types. Many other integrins are The term Integrin was coined about a decade ago to expressed in selective cell types. Examples of cell-type describe cell surface receptors that mediate cellular speci®c integrins include aIIb b3 in platelets and a6 b4 in adhesion to extracellular matrix proteins and to other epithelial cells. Integrins are capable of generating both cells (Tamkun et al., 1986; Ruoslahti and Pierschba- common signals and signals speci®c for individual cher, 1987). However, it has become clear that the integrins. The signals from these integrin receptors are biological signi®cance of integrin-mediated adhesive integrated with those originating from growth factor events goes beyond simple physical linkage of cells to and cytokine receptors in order to organize the extracellular matrix proteins or to other cells. Adhesion cytoskeleton, stimulate mitogen activated protein is shown to be important for survival and proliferation (MAP) kinase cascades and regulate immediately early of various cell types (Hynes, 1994; Ruoslahti and Reed, gene expression. It has also been recognized that 1994; Meredith et al., 1993; Meredith and Schwartz, information can also ¯ow in the reverse direction; the 1997). A remarkable number of classical signaling integrin ligand binding activity is also regulated from pathways are now known to be activated by the inside of the cell (Schwartz et al., 1995; Kolamus interactions of cells with matrix proteins via integrins and Seed, 1997). Thus integrin receptors are highly (Juliano and Haskill, 1993; Schwartz et al., 1995; versatile proteins mediating a variety of biological Clarke and Brugge, 1995). Regulation of adhesion by processes. integrins is also central to a number of specialized In this review, I will summarize the recent studies on pathways in the hematopoietic systems, allowing three signaling pathways activated by integrin recep- attachment of platelets to soluble ligands and of tors. Speci®cally the role of integrins in (1) cytoskeletal lymphocytes to antigen-presenting cells and the organization, (2) in cell proliferation signaling path- phagocytosis of complement opsonized targets by ways and (3) in cell survival pathways will be reviewed. granulocytes and macrophages. Cell-matrix interac- A brief summary of the role of integrins in cell tions result in a cascade of cellular responses which transformation and therapeutic applications is also ultimately promote not only cell binding but also lead included. to cell spreading. Both cell attachment and spreading are critical for cell survival because cells that adhere, but can not spread, may undergo programmed cell Role of integrins in cytoskeletal organization death (Bates et al., 1994; Re et al., 1994) Integrin receptors are composed of noncovalently The binding of ligands to integrin receptors leads to associated a and b chains which form heterodimeric cross-linking or clustering of integrins. This promotes Signaling by integrin receptors CC Kumar 1366

Figure 1 ab pairings of integrin receptors

the formation of structures at the cell membrane known as focal adhesions where integrins link the outside matrix to intracellular cytoskeletal complexes (Burridge et al., 1988). These protein assemblies play an important role in modulating cell adhesion and inducing cell shape changes involved in cell spreading and locomotion. Focal adhesion complexes are readily seen by immuno¯uoresence as tear shaped plaques at the ends of actin ®lament based stress ®bers. A diverse number of structural and signaling proteins, such as integrins, cytoskeletal proteins, protein kinases and signaling molecules are known to be concentrated at these sites (Craig and Johnson, 1996; Yamada and Miyamoto, 1995; Lo and Chen, 1994) (Figure 2). Actin binding proteins that colocalize with integrins at focal adhesion plaques include, a-actinin, talin, tensin, paxillin, vinculin and tensin. Protein kinases that colocalize with these structures include focal adhesion kinase (FAK), c-src, protein kinase C and integrin linked kinase (ILK) etc., (Yamada and Miyamoto, 1995; Dedhar and Hannigan, 1996). Figure 2 Model for the arrangements of dierent proteins in a focal adhesion complex. Arrangements of cytoskeletal proteins that colocalize to focal adhesion complexes in vivo and bind to integrins in vitro are shown. Ligand occupancy, integrin Focal adhesion kinase aggregation and tyrosine phosphorylation lead to the accumula- tion of F-actin and associated cytoskeletal proteins in a massive Unlike growth factor receptors, integrins lack intrinsic adhesive and signaling complex. ECM: Extracellular matrix, tyrosine kinase activity. Yet, an early event during RGD: Arg-Gly-Asp, JNK: Jun Kinase integrin signaling is the tyrosine phosphorylation of the non-receptor tyrosine kinase, FAK in response to cell adhesion (Schaller and Parsons, 1994; Richardson and

Parsons, 1995; Parsons, 1996). All b1 and av containing possibly by folding back onto the catalytic domain integrins share the ability to promote the assembly of (Richardson and Parsons, 1996). Thus the conforma- focal adhesions and activate FAK. FAK is an unusual tional transition may be a prerequisite for FAK's nonreceptor tyrosine kinase and lacks src homology catalytic activity. Integrin aggregation by ligand (SH)2 and SH3 domains. The mechanism by which binding would then result in oligomerization of FAK integrins activate FAK is incompletely understood. It is and activation of FAK by trans-autophosphorylation. clear that this process is tightly coupled to the process Autophosphorylation of FAK at residue 397 results in of assembly of focal adhesion complexes. The the binding of SH2 domain of Src and Fyn (Schaller et phosphorylation of FAK is believed to initiate a al., 1994). The Src family kinases such as Src and Fyn cascade of phosphorylation events and new protein then phosphorylate a number of FAK-associated interactions required for adhesion-dependent signaling proteins including paxilin, tensin and p130 CAS (Vuori complexes. Talin binds directly to the carboxy-terminal et al., 1996; Schlaepfer et al., 1997). Src can also domain of FAK (Chen et al., 1995) and also interacts phosphorylate FAK at tyrosine residue 925, creating a with vinculin and thereby paxilin (Brown et al., 1996). binding site for the Grb2-mSOS complex (Schlaepfer Paxilin in turn binds to a distinct site in the carboxy and Hunter, 1997). Finally autophosphorylated FAK terminus of FAK (Schaller et al., 1995). Thus it is can combine with and activate phosphatidyl-inositide-3 believed that the initial recruitment of FAK to kinase (PI-3K) (Chen et al., 1996). FAK is thus linked activated integrins is indirect and mediated by talin. to a number of intracellular signaling pathways. The It is proposed that upon recruitment to focal adhesion analysis of FAK knockout mice has provided complexes by talin, FAK undergoes conformational important information on the biological function of change and interacts through its amino terminal FAK. Embryonic ®broblasts derived from these mice domain with the b subunit tail. The amino terminal form numerous small focal contacts, but fail to form domain of FAK plays a negative autoregulatory role, the large peripheral focal adhesions and these cells Signaling by integrin receptors CC Kumar 1367 migrate less eciently than control cells (Ilic et al., has been known for some time that integrins can 1995). The observation that overexpression of FAK stimulate the production of Phosphatidyl Inositol increases cell migration while a dominant negative biphosphate (PIP2) and recent studies have provided form of the kinase inhibits it are also consistent with a evidence that this eect is mediated by Rho (Chong et role for this kinase in dynamic regulation of focal al., 1994; Hartwig et al., 1995), possibly through its adhesions during cell migration (Cary et al., 1996; interaction with a type I isoform of phosphatidyl Gilmore and Romer, 1996). inositol 4-phosphate 5-kinase (PIP4-5K) (Ren et al.,

In contrast to FAK which is activated in response to 1996). The increase in PIP2 synthesis by Rho A is cell adhesion, the activity of the serine/threonine-kinase potentially relevant to focal adhesion assembly because termed integrin-linked kinase (ILK) appears to be the actin binding activity of several cytoskeletal inhibited in response to integrin ligation. ILK was proteins such as pro®lin and gelsolin is modulated by originally isolated in the yeast two hybrid system by PIP2 in vitro (Hartwig et al., 1995) and PIP2 is enriched virtue of its ability to interact with the integrin b1 in focal adhesion plaques. In light of this, Gilmore and subunit (Hannigan et al., 1996; Dedhar and Hannigan, Burridge (1996) reported that the association of PIP2 1996). ILK also colocalizes to the focal adhesions like with vinculin induces a conformational change in FAK. In contrast to FAK, ILK can be readily vinculin, allowing it to interact with talin, which binds coimmunoprecipitated with various b integrins (Han- actin. Furthermore they showed that injection of anti- nigan et al., 1996; Radeva et al., 1997). Overexpression PIP2 antibodies into ®broblasts inhibited LPA/Rho- of ILK in epithelial cells has been shown to induce induced stress ®bers and focal adhesion formation, anchorage-independent growth and malignant trans- suggesting a role for PIP2 in focal adhesion and stress formation (Radeva et al., 1997). A third protein termed ®ber assembly. Second, dominant negative Rho has b3 endonexin has also been identi®ed from yeast two been shown to partially suppress the activation of hybrid screens as a b3 interacting protein (Shattil et al., ERK in response to ligation of integrins, suggesting

1995). The physiological role of b3 endonexin is that a Rho regulated pathway is necessary for the full currently unknown. activation of ERK following integrin mediated cell adhesion (Renshaw et al., 1996). How does activation of Rho A lead to recruitment Role of Rho family of proteins in focal adhesion of Focal adhesion proteins and assembly of focal assembly adhesion? This question still remains unanswered Alan Hall's group was the ®rst to describe the completely. However, exciting progress has been made assembly of focal adhesions and stress ®bers in serum in recent years. The involvement of phosphoinositide starved Swiss 3T3 cells. These cells retain few stress kinases in Rho signaling has been reported. The ®bers and are devoid of focal adhesions and its eector proteins for Rho A include PIP4-5K, the components. Readdition of serum or LPA stimulates recently identi®ed serine/threonine kinase Rho-asso- the formation of stress ®bers and focal adhesion ciated coil-coil containing protein kinase (ROCK) and complexes within 2 ± 5 min (Ridley and Hall, 1992; protein kinase N which all associate with and are Ridley et al., 1992; Nobes and Hall, 1995). Other activated by RhoA (Amano et al., 1997; Van Aelst and growth factors such as platelet derived growth factor D'Souza-Schorey, 1997). In addition, the p190 Rho (PDGF), epidermal growth factor (EGF) insulin, GTPase-activating protein (GAP) may also regulate bombesin also stimulate focal adhesion assembly, but integrin-clustering (Settleman et al., 1992). Constitutive with a longer time course. The induction of a variety of activation of p190 Rho-GAP disrupts integrin cluster- cortical actin assemblies can be ascribed to the action ing and focal plaque formation. PI-3 kinase which of individual Rho proteins. Rho family of proteins are phosphorylates PI(4) phosphate (PIP) or PI(4,5)

RAS-related GTP binding proteins consisting of Rho biphosphate (PIP2) to generate PI (3,4)P2 or PI

A, B, C, D and E; Racs 1 and 2 and Rac E; and (3,4,5)P3 respectively has been shown to associate with Cdc42, Rho G and TC10 (Van Aelst and D'Souza- integrin-associated focal adhesion complexes (Chen et Schorey, 1997; Hall, 1998). Rho is involved in the al., 1996). The SH2 domain of the p85 subunit of PI-3 organization of focal adhesions and stress ®bers in K may be important for its association with tyrosine response to LPA; Rac is directly responsible for the phosphorylated FAK. The role of PI3K in integrin membrane ruing and extension of lamellipodia and induced changes in cell behaviour is not understood; Cdc42 uniquely controls the formation of ®lipodia however the evidence that inhibition of PI-3 K blocks (Hall, 1994, 1998; Van Aelst and D'Souza-Schorey, growth factor induced actin arrangements suggests a 1997). There is evidence for the interconnection of possible involvement in integrin regulated cytoskeletal these family members in a hierarchial manner: Cdc42 rearrangements. activates Rac and Rac activates Rho. Rho can also activate a serine/threonine protein In addition to the growth factor receptors, Rho kinase known as Rho Kinase, which plays an family members can also be activated by integrin important role in the assembly of focal adhesions ligation and clustering. Hotchin and Hall (1995) have (Amano et al., 1997). This kinase phosphorylates the demonstrated that the interaction of integrins with myosin binding subunit (MBS) of myosin light chain extracellular matrix is not sucient to induce integrin phosphatase, thereby suppressing the activity of the clustering and focal complex formation, but requires enzyme (Kimura et al., 1996). The resulting increase in the activity of Rho family members. The molecular myosin light chain phosphorylation is believed to basis for this regulation is not clear but may involve induce a conformational change in myosin, thereby eector proteins which associate with and become increasing its binding to actin ®laments and promote activated by GTP-bound RhoA. Two lines of evidence actomyosin contractility and formation of focal support the notion that integrins can activate Rho. It complexes and stress ®bers (Chirzanowska-Wondnicka Signaling by integrin receptors CC Kumar 1368

the lamin/collagen receptor a1b1, the ®bronectin

receptor a5b1 and the vitronectin receptor avb3 are linked to the RAS-ERK signaling pathway by the adaptor protein Shc (Wary et al., 1996; Giancotti, 1997). Shc is an SH2 and phosphotyrosine binding (PTB) domain adaptor protein which links tyrosine- phosphorylated signal transducers to Ras (Pawson, 1995). Upon recruitment to activated receptors, Shc is phosphorylated on tyrosine and binds to the Grb2- mSOS complex. This process results in the juxtaposi- tion of the GTP exchange factor domain of mSOS to RAS, leading to its activation. The role of FAK in the activation of RAS-ERK pathway is not clearly

established. Ligation of a1b1, a5b1 and avb3 integrins which are all linked to Shc results in Erk activation, but ligation of other integrins does not produce this eect, even though FAK is stimulated (Wary et al., 1996). A dominant negative mutant Shc was found to suppress ERK activation in response to integrin ligation (Wary et al., 1996), but two distinct Figure 3 Signaling pathways involving integrin receptors and dominant-negative mutants of FAK do not (Lin et Rho family GTPases leading to focal adhesion complex and actin stress ®ber formation. The integrins activate Rac via Ras and PI- al., 1997; Giancotti, 1997). These results indicate that 3K and Rho through unknown mechanisms. Rac can also be Shc is necessary and sucient to link speci®c integrins activated by growth factors such as EGF, PDGF, and Insulin and to the RAS-ERK signaling pathway. However, they do Rho can be activated by LPA. Rac can also weakly activate Rho not exclude the possibility that FAK may cooperate by an unknown mechanism. Rac and Rho interact with a number with Shc to fully activate ERK in response to integrin of target eectors. Some of these such as Por1, PIP-5Kinase and Rho kinase are responsible for organizing cytoskeletal structures ligation. As discussed above, integrin-mediated adhe- such as lamellipodia and focal adhesions sion signals the autophosphorylation of FAK on Tyrosine 397, generating a binding site for SH2 domains of Src and related kinases (Calab et al., 1995). The further phosphorylation of FAK at Tyr et al., 1996). More recently, evidence has been 925, presumably by Src generates a binding site for the presented that Rho kinase can also phosphorylate SH2 domain of the adaptor protein GRB2 (Schlaepfer MLC at the same site phosphorylated by MLC kinase et al., 1994). GRB2 signals through the RAS GDP/ (Amano et al., 1996). A model proposed for integrin- GTP exchange protein SOS, which has also been mediated and Rho-induced stress ®ber formation is detected in the adhesion-dependent focal adhesion depicted in Figure 3. complexes containing FAK, GRB2 and c-Src (Yama- In addition to the Rho family members, R-Ras, a da and Miyamoto, 1995) (Figure 4). The activation of GTP-binding protein homologous to Ha-Ras but with RAS-ERK pathway by integrin ligation has been activities distinct from those of the transforming RAS shown to require an intact cytoskeleton suggesting proteins, has been shown to activate integrins. that the integrin-dependent cytoskeletal complexes are Expression of constitutively active R-Ras induced instrumental in the activation of the MAP kinase suspension cells to adhere to the extracellular matrix pathway. proteins by converting integrins from a low anity to a MAP kinase in turn activates a number of high anity state (Zhang et al., 1996). Dominant transcription factors such as SRF, c-Myc that are negative R-Ras reduced the adhesiveness of cells involved in regulating growth and dierentiation. suggesting that endogenous R-Ras can control integ- Studies have shown that in primary cells, engagement rin-ligand anity states. The molecular basis for this of integrins linked to Shc activates transcription from regulation is not understood at the present time. the serum response element (SRE) and promotes progression through the G1 phase of the cell cycle in response to growth factors. Since growth factors also Role of integrins in cell-proliferation signaling pathways stimulate the RAS-MAP kinase pathway, it is likely that the integrins and growth factor receptors may In addition to growth factors and nutrients, many synergize to enhance RAS-MAP kinase activation. normal cells require adhesion to the extracellular In addition to the participation of integrins in the matrix to proliferate. The adhesion-dependent activa- activation of the RAS-ERK pathway via Shc, recent tion of MAP kinase appears to be important in the studies have shown that speci®c integrins may also pathway by which integrins regulate cell proliferation. cooperate with growth factor receptors directly. The

A number of signaling proteins have been found to be avb3 integrin receptor has been found to uniquely associated with the focal adhesion complexes suggest- associate with cytoplasmic signal transduction media- ing the involvement of integrin engagement in the tors of the insulin (and insulin like growth factor, activation of signaling pathways. It is now generally (IGF) receptors (Vuori and Ruoslahti, 1994). One such accepted that the activation of ERK in response to signal transduction mediator is insulin receptor integrin ligation requires Ras signaling (Schlaepfer and substrate (IRS)-1. IRS-1 is tyrosine phosphorylated Hunter, 1997; Wary et al., 1996). Evidence is presented by the activated insulin (and IGF) receptor and as a

that certain integrins such as the laminin receptor a6b4, result binds a number of other signaling molecules. In Signaling by integrin receptors CC Kumar 1369 that ERK can phosphorylate MLCK and ERK- phosphorylated MLCK displays an enhanced capacity to phosphorylate MLC in a calmodulin dependent manner. In summary, it is clear that growth factor receptors as well as integrin receptors in¯uence the activity of one or more members of the MAP kinase family of signaling molecules. While MAP kinase signaling has been associated with the regulation of gene transcription events, David Cheresh's work clearly demonstrates that MAP kinase can promote cell migration on the ECM in a transcription-independent manner.

Role of integrins in cell survival signaling mechanisms

One important function of the integrin signaling pathways is to regulate anchorage dependence. Many mammalian cells are dependent on adhesion to the extracellular matrix for their continued survival. Early studies used ®broblasts which become growth inhibited in suspension, but remain viable. More recent studies Figure 4 Integrin-mediated signaling pathways leading to cell have shown that certain specialized cell types such as proliferation and cell survival. Activation of Ras-ERK signaling endothelial cells and epithelial cells, when detached pathway by integrins involves the recruitment of Shc via a from substrate undergo apoptosis (Frisch and Francis, transmembrane adaptor protein. Although all b1 and av integrins can regulate focal adhesions via FAK, only some of them can 1994; Meredith et al., 1993; Frisch and Ruoslahti, recruit Shc and activate Ras-ERK signaling and promote cell 1997; Meredith and Schwartz, 1997). This phenomenon cycle progression. Cell survival is promoted by integrins via Ras- is referred to as `anoikis' from the greek for home- ERK and Akt. Jnk may inhibit cell survival if ERK is not lessness. Anoikis is a mechanism to insure that cells activated. There is also evidence for the involvement of PI-3K in integrin-mediated activation of Raf/ERK pathway. See the text which are displaced from their natural environment are for details eliminated. Tumor cells are characterized by their ability to grow in the absence of contacts with the extracellular matrix. Cell-detachment induced anoikis cells that have adhered to a substrate, such as does not occur in cells expressing activated Src or Ras vitronectin through the avb3 integrin, a subset of IRS- oncogenes. Hence, these oncogenes must therefore be 1 binds to the integrin. This interaction substantially able to provide a constitutive signal normally enhances the growth stimulating eects of insulin and originating from integrin receptors. IGF. This interaction appears to be speci®c to integrin The ability of the extracellular matrix to promote avb3 receptor. A 190 Kda protein that is phosphory- cell survival appears to be mediated by at least two lated in tyrosines as a result of PDGF receptor activity distinct signaling pathways. Studies on the role of Shc also binds to avb3 receptor, suggesting that there may in integrin signaling have indicated that in cells be cooperation between this integrin and the PDGF adhesion mediated by integrins not linked to Shc, pathway (Schneller et al., 1997). results not only in cell cycle arrest, but also in apoptotic cell death (Wary et al., 1996; Giancotti, 1997). In agreement with a critical role for Shc Regulation of cell cycle progression signaling in protection from apoptosis, a dominant Recent studies have indicated that cell adhesion is negative form of Shc has been shown to induce speci®cally required for the induction of cyclin D1 and apoptotic cell death in primary endothelial cells plated for the activation of cyclin E-cdk2 complex in early- on ®bronectin in the presence of mitogens (Wary et al., mid G1 phase (Fang et al., 1996). Although cyclin D1 1996; Giancotti, 1997). is regulated by cell adhesion at both the transcriptional and translational levels, the eect of cell adhesion on Role of phosphoinositide 3-Kinase and PKB/Akt in cyclin E-cdk2 activity appears to be indirect and anoikis mediated by downregulation of the cdk inhibitors p21 and p27 (Zhu et al., 1996). These ®ndings suggest Julian Downward's group studied the mechanism by several mechanisms by which integrin engagement which activated RAS oncogene protects MDCK could regulate the cell cycle. epithelial cells from anoikis upon removal from extracellular matrix (Khwaja et al., 1997). Their results suggest a following model of integrin signaling Regulation of cell migration to the apoptotic machinery in untransformed epithelial Recently David Cheresh and coworkers have provided cells. Cellular adhesion through integrins results in the evidence that integrin mediated activation of ERKs activation of PI-3 kinase independently of signals from in¯uence the cells' motility machinery by phosphor- serum factors. The PI-3 kinase lipid products provide a ylating and thereby enhancing myosin light chain protective signal acting through PKB/Akt which blocks kinase (MLCK) activity leading to phosphorylation entry into apoptosis. When epithelial cells are detached of MLC (Klemke et al., 1997). In vitro assays indicate from the matrix, PI-3 kinase and PKB/Akt become Signaling by integrin receptors CC Kumar 1370 inactive even in the presence of serum factors and an constant while forcing cells either to remain round or apoptotic pathway is engaged. RAS activation leads to spread out (Chen et al., 1997). This was accomplished the constitutive activation of PI-3 kinase, PKB/Akt by the use of microfabricated surfaces composed of pathway accounting for the survival of RAS-trans- islands of adhesive surface interspersed at varying formed cells in the absence of adhesion to extracellular distances from one another. When endothelial cells matrix. PI-3 kinase acting through PKB/Akt is were cultured on these surfaces, round cells were found therefore implicated as a key mediator of the aberrant to be susceptible to apoptosis, whereas cells forced to survival of RAS-transformed epithelial cells in the stretch over widely dispersed small islands survived and absence of attachment. Interestingly, introduction of proliferated (Chen et al., 1997). These results again activated forms of the PI3 K p110 subunit, or of AKT provide evidence that cell spreading following cell rescued the MDCK cells from anoikis. attachment is crucial to rescue cells from anoikis. As with other signaling mechanisms, the PI-3 kinase Dierent cells may dier in their ability to utilize and PKB/Akt pathway is also activated upon growth speci®c integrins to activate the antiapoptotic mechan- factor stimulation and mediates the cell survival ism. Recent studies using CHO cells engineered to

mechanism in the absence of growth factor stimula- express either a5b1 or avb1 integrin as their ®bronectin tion (Kennedy et al., 1997; Marte and Downward, receptor have shown that only those cells expressing

1997). The mechanism by which integrin engagement a5b1 survived in serum free cultures (Zhang et al., leads to PI-3 kinase activation in normal cells is not 1995). This response was associated with an elevated known. One mechanism may be through FAK as this expression of the antiapoptotic protein Bcl-2. Other

kinase has been found to associate with the SH2 integrins such as avb1 or avb3 and various b1 integrins domains of p85 of PI-3K, so it may contribute to the fail to rescue cells from apoptosis under these constitutive activation of PI-3 kinase. Alternatively, as conditions. It is possible that under other conditions PI3-K is a major target eector of RAS (Rodriguez- cells depend on other integrins for survival (Brooks et Viciana et al., 1994), the activation of PI3-K by al., 1994; Boudreau et al., 1995). Dierent cell types integrins could be mediated by the ability of Shc to may dier in their integrin dependence under the same activate RAS. Recent studies by King et al. (1997) have conditions. Thus vascular endothelial cells seem to

shown that integrin-mediated Erk-2, Mek-1 and Raf-1 depend on avb3 receptor for their survival (Brooks et

activation but not Ras-GTP loading, was inhibited by al., 1994). Inhibition of avb3 function during angiogen-

PI-3K inhibitors demonstrating that PI-3K may esis with anti-avb3 antibodies both inhibited prolifera- function upstream of Raf-1 in integrin-mediated tion and induced apoptosis, whereas treatment with

activation of Erk-2. Growth factor stimulation and antibodies that block b1 integrin function did not integrin engagement leads to the stimulation of PI-3 (Brooks et al., 1994). Similarly, prevention of kinase activity which results in the production of interactions of mammary epithelium with the matrix

phosphatidyl inositol (3,4,5) triphosphate and Ptd by anti-b1 integrin antibodies both inhibited prolifera-

Inositol (3,4)P2 (Franke et al., 1995). The binding of tion and induced apoptosis (Boudreau et al., 1995). pleckstrin homology (PH) domain of PKB/Akt to these These results suggest that the same integrin might phosphoinositides both recruites PKB/Akt to the connect to dierent pathways in dierent cell types and plasma membrane and in the case of PI(3,4)P2 directly that a given integrin could provide a survival signal in stimulates its kinase activity (Franke et al., 1997a, b). one cell type and an anoikis signal in another. A Full activation of PKB/Akt requires phosphorylation possible physiological signi®cance of the integrin- of Thr 308 by PDK1 and Ser 473by PDK2 (Alessi et selective apoptosis phenomenon is that it may prevent al., 1996, 1997). Activated PKB/Akt phosphorylates cells from attaching to inappropriate places in the substrates resulting in a variety of biological eects body, because attachment through the wrong integrin including suppression of apoptosis. Figure 4 sum- would induce apoptosis. marizes the integrin-mediated signaling pathways leading to cell proliferation and cell survival. Role of integrins in cell transformation Role of cell shape in anoikis It is clear from the above discussion that cells require Cell adhesion to matrix causes dramatic changes in cell both adhesion to immobilized matrix proteins and shape. Therefore, the question arises, does the cytoske- stimulation by serum or growth factors in order to letal organization and associated cell shape ± as opposed proliferate. An important conclusion of these studies to integrin signaling per se ± suppress anoikis? Two series reviewed above is that signaling pathways activated by of studies speci®cally address this issue of cell shape and growth factor receptors and integrin receptors, cell spreading in anoikis. Endothelial cells that were ultimately converge to determine cell cycle progres- placed in suspension underwent anoikis. Addition of sion. By contrast, tumor cell proliferation is generally microbeads coated with RGD peptides that were too independent of both adhesion and serum though small to permit cell spreading did not rescue these cells. anchorage independence is the feature that correlates However, plastic surfaces coated with RGD peptides best with tumorigenicity in vivo (Freedman and Shin, rescued cells from anoikis eciently (Re et al., 1994). 1974). Proteins encoded by the oncogenes are generally These results suggest that mere attachment of cells components of the normal growth regulatory pathways through integrins is insucient for rescue and that cell that when overexpressed or mutated lead to irreversible shape changes are required. activation of these pathways. Many oncogene encoded Recently in an interesting study the role of cell proteins also activate the integrin mediated pathways spreading was studied using a system in which the total mainly through the Rho family proteins. Speci®cally, surface area available for cell attachment was kept activated RAS has been shown to activate the cell Signaling by integrin receptors CC Kumar 1371 morphology pathway through RAC and recent studies learned about integrin signaling, important questions have shown that activation of both the cell morphol- remain concerning the circuitry responsible for the ogy pathway and ERK pathway are essential for crosstalk among signals triggered by dierent integrins transformation (Joneson et al., 1996). Constitutive and integration of signals from integrins and growth activation of both integrin-mediated and growth factor receptors. Finally, an analysis of mechanisms by factor pathways would be necessary to elicit serum which tumor cells survive and proliferate in the absence of and anchorage-independent status to the transformed signals from the integrins will be an important area of cells. Activation of a step on the growth factor portion investigation. only should lead to enhanced proliferation but not Research on integrin receptors has also led to a anchorage-independent growth. Thus ®broblasts trans- number of therapeutic applications and targets for formed by Raf and constitutively active MEK grow tumor therapy. One integrin-directed drug, an anti-b3 very poorly in an anchorage-independent manner integrin antibody for the prevention of arterial (Mansour et al., 1994, and CC Kumar unpublished restenosis, has reached the market place. Several other data). Activated Rac and Rho proteins have been integrin based drugs such as the peptides containing shown to synergize with Raf to eciently transform the integrin-binding RGD sequence (Ruoslahti, 1996), ®broblast cells, suggesting that Rac and Rho proteins and mimics of such peptides that speci®cally block are components of a separate pathway. Activation of a individual integrins are also under development. These step on an integrin pathway prior to convergence drugs target thrombosis (aIIbb3 in platelets (Pierschba- would be expected to lead to anchorage-independent cher et al., 1994), osteoporosis (avb3 in osteoclsts but serum-dependent growth. Thus oncogenes such as (Flores et al., 1992)) and tumor-induced angiogenesis dbl and lbc which function as constitutively active (avb3 in neovascular endothelial cells (Brooks et al., guanine nucleotide exchange factors for the Rho family 1994). In a recent exciting study, Ruoslahti's group of proteins, induce anchorage-independent but serum- used the in vivo selection of phage display libraries to dependent growth (Schwartz et al., 1996). lbc and dbl isolate peptides that home speci®cally to tumor blood were identi®ed by their ability to induce focus vessels. Two of these peptides ± one containing av formation and tumorigenicity in vivo when expressed integrin-binding Arg-Gly-Asp motif and the other an in 3T3 cells (Toksoz and Williams, 1994; Eva and Asn-Gly-Arg motif ± were conjugated to the anticancer Aaronson, 1985). Thus both lbc and dbl transformed drug doxorubicin to demonstrate enhanced ecacy of cells show the same serum-dependence as control cells. the drug against human breast cancer xenografts in These studies show that anchorage-independence and nude mice (Pasqualimi et al., 1997). These peptide- serum-independence can be separated. Understanding drug conjugates exhibited signi®cantly less toxicity, the mechanisms by which tumor cells gain the suggesting that it may be possible to develop targeted anchorage-independence status is an important area chemotherapeutic strategies based on the selective of investigation and may lead to the identi®cation of expression of integrin receptors in tumor vasculature. novel targets for drug discovery. In summary, recent studies on integrins have greatly increased our understanding of the central biological phenomena, such as anchorage-dependence, tumor- Summary and therapeutic applications induced angiogenesis and tumor metastsis, as well as generated a number of possible approaches to new An important conclusion from the studies reviewed above therapies for cancer. is that the signaling pathways activated by growth factor receptors and integrin receptors are extensively inter- twined, most importantly at the level of Ras, PI-3K and FAK. In addition, certain integrins physically form complexes with growth factor receptors. Many signaling Acknowledgements intermediates interact with and receive signals from I would like to thank Drs Martin McMahon and Lydia various upstream elements and bind to a large number Armstrong for useful discussions and for their comments of downstream target eectors. Although much has been on the manuscript.

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