Oncogene (2008) 27, 6285–6298 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc REVIEW Regulation of : apoptotic cues from the ECM

DA Cheresh and DG Stupack

Department of Pathology, Moores UCSD Cancer Center, UCSD School of Medicine, La Jolla, CA, USA

The extracellular matrix (ECM) acts both as a physical tion (Andreoli and Miller, 2007), or even endometriosis scaffold for cells and as a repository for growth factors. (Sha et al., 2007). In these cases, it is now well Moreover, ECM structure and physical–chemical proper- established that an aggressive neovascular response ties convey precise information to cells that profoundly can serve as a diagnostic and/or prognostic indicator influences their biology by interactions with cell surface of disease progression (Craft and Harris, 1994; Gaspar- receptors termed . During angiogenesis, the ini et al., 1996). perivascular ECM plays a critical role in determining In contrast, the loss of vascular function that follows the proliferative, invasive and survival responses of the or infarction leads to ischemia and a local vascular cells to the angiogenic growth factors. variety of harmful sequelae (Marti and Risau, 1999).In Dynamic changes in both the ECM and the local vascular these cases, revascularization of the local tissues is cells act in concert to regulate newblood vessel growth. critical to restoring tissue function.Thus, it may The digestion of ECM components by proteolysis is sometimes be desirable to promote neovascularization critical for the invasive capacity of endothelial cells, to treat disease.Whether one considers the promotion but also creates ECM fragments, which antagonize or inhibition of angiogenesis as a therapeutic strategy, the mechanosensory function of integrins, and can be an increased understanding of the highly complex apoptogenic. Here, we discuss the roles of integrins in mechanisms that regulate the survival and invasion of modulating cellular responses to a changing ECM, in angiogenic vascular cells is critical. particular the regulation of survival and invasion among invasive endothelial cells. Architecture of the resting microvasculature Oncogene (2008) 27, 6285–6298; doi:10.1038/onc.2008.304 The vascular cells resident within the , Keywords: ; ; survival; endothelial; and of the body are typically quiescent.The pericyte acts as vascular lining cells, which facilitate smooth blood flow. endothelial cells are anchored in place through interactions with the under- The initiation of angiogenesis lying vascular lamina over a very large surface area, which encompasses nearly half the cell membrane.In Angiogenesis as a determinant in disease addition, endothelial cells interact with each other in New growth, or angiogenesis, occurs during cell–cell junctions that often partially overlap each the development and maturation as well as during other.These junctions limit vascular leak, permitting critical physiological processes, including wound healing only molecules below 75 kDa to escape the circulation and reproduction.However, angiogenic processes are (Firth, 2002; Weis et al., 2007). The junctions also served also usurped in many pathologies, and thereby con- to provide structural continuity, connecting the cytos- tribute to an array of disorders including cancer, keletal structure of one cell to the next.Specialized inflammation and autoimmune diseases (Folkman, junctions, such as those in the , can limit the escape 2006).In these cases, angiogenesis typically supports of blood-borne elements further (Ueno, 2007).Inter- expanding aberrant tissues, such as a tumor or the spersed along the exterior of capillaries is a second type rheumatoid pannus.However, the hypervascularization of vascular cells; the pericyte, which extends cellular of these tissues often promotes significant aspects of processes to contact several different endothelial cells in disease pathology.For example, it may exacerbate its immediate microenvironment.These processes inter- inflammation in autoimmune disease or facilitate tumor act directly with the endothelial cell surface and both progression or dissemination in cancer.In other cases, support and stabilize the capillary structure.The angiogenesis itself is the principal factor responsible for interaction between pericyte and endothelial cells is disease pathology (Andreoli and Miller, 2007), such as is important; loss of contact provides one criteria for the the case in and macular degenera- transition from mature, nonproliferative vessels to immature, unstable angiogenic vessel. The basal proliferation of vascular cells is low, among Correspondence: Dr DA Cheresh, Department of Pathology, Moores UCSD Cancer Center, UCSD School of Medicine, 3855 Health the lowest of any cell in the body (Folkman, 2006). Sciences Drive, La Jolla, CA 92039-0803, USA. However, the combination of low proliferation, strong E-mail: [email protected] anchorage, tight cell junctions and supporting pericyte Apoptotic cues from the ECM DA Cheresh and DG Stupack 6286 contact defines the infrastructure that renders mature Integrin mobilization and subsequent interactions endothelium highly resistant to proapoptotic insults. with ECM potentiate the signaling events However, this privileged ‘resistant’ status changes critical to immediate early events in angiogenesis.In following the induction of angiogenesis. fact, the signaling activity of receptor tyrosine is dependent upon integrin engagement of ECM (Eliceiri et al., 1998). This is one important reason that the Early signaling events during the initiation of angiogenesis repertoire of integrin receptors on angiogenic vascular Angiogenesis is a local process that can be induced by cells changes during angiogenesis; to maintain appro- paracrine and autocrine growth factors.The list of priate cellular interaction with the remodeling ECM. proangiogenic agents that can stimulate quiescent The earliest interactions occur through pre-existing endothelial cells to become angiogenic is still growing integrins, such as avb5, which are activated downstream and includes growth factors, bioactive lipids and of growth factors (Klemke et al., 1994) and proceed even complex polysaccharides.Angiogenesis can be through Src family kinases and Rho , which considered to occur in a series of stages, with immediate facilitate endothelial cell retraction in concert with early events triggered by the binding of growth factors release of junctional complex.This permits circulating and cell signaling, followed by early events that change platelets direct access to the lamina underlying the genetic programming of vascular cells, followed by endothelial cell margin (Weis et al., 2004). Platelet invasive angiogenesis and finally a resolution stage, binding to these substrates activates them, leading to the where cells revert to their quiescent stage.At each step, discharge the contents of their a-granules, which include cell interaction with the local microenvironment plays a a variety of molecular effectors ranging from protease/ critical role in determining the angiogenic response. lipases to ECM components to numerous angio- Vascular endothelial cell growth factors (VEGFs) genesis-regulating factors such as VEGF and PDGF represent a family of cytokines that stimulate vascular (platelet-derived growth factor) or S1P (sphingosine-1 and lymphatic endothelium to form new vascular or phosphate). lymphatic structures, respectively.VEGFs are well- It is important to consider that these immediate-early studied growth factors that bind to cell surface heparin, events in angiogenesis proceed as a concerted intracel- and to the receptor tyrosine kinases Flk-1, lular and extracellular cascade.Events within the cells Flt-1 and Flt-3, depending upon the particular VEGF are coordinated to permit changes in the cells micro- isoform (A, B, C and D) or splice variant (Ferrara et al., environment, and to permit the cell to adapt to those 2003).VEGF-A binding to Flk-1 on endothelial cells changes.Subsequent signaling within the vascular cells triggers a cascade of immediate-early signaling events, in proceeds, releasing the ‘quiescent’ transcriptional pro- which the activation of nonreceptor tyrosine kinases gram and promotes cell cycle entry, the expression of play key roles.In particular, the Src-family kinases and new cell surface receptors and proteases, and the the members of the kinase (FAK) family upregulation of several apoptosis-regulating . are important. These early Src-family kinase-mediated events include Src-dependent of VE-, which Changes to endothelial cell programming during promotes the disruption of endothelial cell junctions angiogenesis (Potter et al., 2005), a critical factor in inducing vascular The switch from quiescent to angiogenic cell that occurs permeability (Figure 1).The induction of permeability downstream of growth factor signaling is coordinated slows blood flow, decreasing the clearance of locally by changes in homeobox expression.In resting produced angiogenic factors, while facilitating the endothelial cells, the homeobox genes Gax, HoxA5 and leakage of plasma-borne components into the local HoxD10 coordinately suppress the expression of proan- interstitial tissues.Moreover, Src-family kinases coordi- giogenic genes such as Flk-1, A1, Hif1a and nately phosphorylate FAK, which promotes the turn- COX2, whereas promoting expression of LRP1 (a over of the stable focal contacts used by resting vascular negative regulator of proteolytic activity) and throm- cells for anchorage (Eliceiri et al., 2002). The coordi- bospondin 2, a matricellular protein that inhibits nated cytoskeletal remodeling goes beyond simple angiogenesis (Myers et al., 2002; Patel et al., 2005). mobilization of integrins and disruption of cell–cell HoxA13 and its targets Ephrin A6 and A7 have also junctions.The activation of phosphoinositide 3 0 kinases been implicated in maintaining the quiescent status of at (PI3K) is critical for signaling small of the Rho least some endothelial cells (Shaut et al., 2007). This family (Cheresh et al., 1999; Hoang et al., 2004; Nagy programming favors tight interaction with neighboring and Senger, 2006), whereas the initiation of mitogen- cells, no degradation of the surrounding ECM and low activated (MAPK) signaling pushes cells proliferation. towards proliferation, and may mobilize contractile Deactivation of the quiescent program is initiated by elements important for cell invasion (Klemke et al., the induction of proangiogenic Hox genes as well as by 1997; Zhai et al., 2003). Together, the pathways permit suppression/retargeting of pre-existing Hox genes.This dynamic interaction between and newly includes transcriptional repression but also involves the forming focal contacts initiated by ligation of integrins, expression of microRNA such as miR130a, which the principle adhesion receptors for the extracellular suppress the expression of both GAX and HoxA5 matrix (ECM). (Chen and Gorski, 2008).HoxD3 was the first

Oncogene Apoptotic cues from the ECM DA Cheresh and DG Stupack 6287 Early Events in Angiogenesis

blood vessel lumen EC EC

EC

Src (off)

Src (on) Integrins VEGFR2 Ang1 VEGF Pericyte Laminin Type IV Collagen

VEGF

Vascular Leakage blood vessel lumen EC EC Src Phosphorylation of VE Cadherin Rho & Src dependent pY-Catenin retraction Src dependent FAK activation

Ang1 VEGFR2 Ang2 Depostion of Integrins Switch VEGF provisional ECM Modes PDGF VEGF Anchorage VEGF to Migration PDGFR Pericyte Retration

Figure 1 Early signaling events in angiogenesis.The resting endothelium (upper panel) consists of a monolayer of endothelial cells, which make tight connections with each other (for example, through vascular epithelium (VE) cadherin) and with pericytes (for example, through presented by the pericyte).However, this situation is disrupted by the binding of vascular endothelial cell growth factor (VEGF) to VEGFR2 (KDR/flk-1), which promotes activation of src family kinases (Src).Here, we focus on the role of Src in initiating vascular permeability and facilitating rapid changes in the local extracellular matrix (ECM) (lower panel).Src phosphorylation of VE cadherin promotes disruption of cell–cell juntions, whereas Src- and Rho-dependent contractility elicit cellular contraction, further facilitating leakage.Src also translocates to sites of integrin-mediated anchorage, associates with FAK and mobilizes the integrins for use as receptors for cell invasion.Coordinated with these early events, the production of angiopoietin 2 by the endothelial cell disrupts association with pericytes.

proangiogenic Hox gene described.HoxD3 induces the tively (Myers et al., 2000; Mace et al., 2005). HoxA9 expression of several invasion-promoting genes includ- has also been identified as critical for upregulation ing ECM receptors such as integrin avb3 and proteases of EphB4 during angiogenesis; interestingly, suppression such as urokinase plasminogen activator (Boudreau of either HoxA9 or EphB4 block neovascularization et al., 1997), whereas its paralogs HoxB3 and HoxA3 (Bruhl et al., 2004). The common recurring theme regulate through its regulation of EphA1 among these changes in Hox genes is the upregulation of or matrix metalloproteinases and UPA receptor, respec- elements that guide cellular interactions with the local

Oncogene Apoptotic cues from the ECM DA Cheresh and DG Stupack 6288 microenvironment, including proteases, integrins and These results, and the fact that avb3 was increased in the eph receptors. vasculature of angiogenic endothelium in all chordates examined, suggested that avb3 played a critical role in regulating angiogenesis. This idea was confirmed in early preclinical studies, Changes to the vascular cell surface during angiogenesis which demonstrated that blockade of avb3 with mono- clonal antibodies (Brooks et al., 1994a) or small Among the changes occurring in the programming on molecules (Brooks et al., 1994b; Storgard et al., 1999) the endothelium, the changes in the expression of cell led to endothelial cell apoptosis and vascular regression. surface adhesion receptors play prominent and guiding By contrast, no vascular abnormalities were described in roles in angiogenesis for several reasons.First, the human patients with a defective integrin b3 gene (Coller adhesion receptors are in direct contact with the et al., 1991), nor were gross vascular abnormalities extracellular milieu and are ligated to varying degrees observed when the gene was later knocked out in mice based on counter-receptors on neighboring cells or on (Hodivala-Dilke et al., 1999). It became clear that these ECM content.These receptors are therefore constantly endothelial cells were not completely normal (Reynolds sampling the extracellular environment and conveying et al., 2002; Weis et al., 2007), and in fact, increased signals to the cell in a dynamic manner.Secondly, neovascularization was observed during pathological interactions with the ECM by adhesion receptors, and forms of angiogenesis.These results were consistent with integrins in particular, modulates signaling events prior suggestions that antagonized or unligated integrins elicited by other cell surface receptors, such as pro- could signal negatively into the cell, promoting apopto- tease-activated receptors and growth factor receptors sis (Stupack et al., 2001). The conclusions that we (Giancotti and Ruoslahti, 1999).The combined inter- arrived at these studies is that avb3 integrin acts as a actions regulate downstream targets, which include biosensor, providing prosurvival signals to a cell when transcription factors and epigenetic elements; these the integrin is ligated, but eliciting proapoptotic signals signals further optimize cellular programming for the when unligated (or antagonized).This model has current microenvironment.Among the alterations oc- subsequently been extended to other integrins, and curring on the endothelial cell surface during angiogen- appears to hold true in a variety of models, depending esis, the induction of integrin avb3 expression provides a upon the specific integrin and the cellular context well-studied example of an integrin that dramatically studied (Todorovicc et al., 2005; Stupack et al., 2006; modifies cell behavior. Davis and Senger, 2008). This integrin model matches that proposed for Integrin avb3 as a prototype integrin ‘biosensor’ dependence receptors (Bredesen et al., 2005); those A principle change following stimulation with angio- receptors that signal positively in the presence of ligand, genic growth factors is the de novo expression of integrin but which promote apoptosis when an appropriate avb3, an oncofetal receptor not commonly expressed in ligand is absent.Accordingly, increased expression of a adult tissues, but expressed abundantly on vascular cells dependence receptor in the absence of an appropriate selectively during angiogenesis.Integrins are composed ligand can promote apoptosis.This situation contrasts of 1 of 18 different a and 8 different b-subunits from at with a ‘classical’ least 24 distinct a/b heterodimers; a single ECM protein such as the EGF receptor or ErbB2, where over- can serve as a ligand for several different integrins (for expression per se can be sufficient to mediate cell- example, laminin is bound by a3b1, a6b1, a6b4, a7b1as signaling events and promote survival.By contrast, well as by a1b1, and a2b1 to a lesser degree).Most absence of a dependence receptor frequently promotes integrin heterodimers on the endothelial cell surface aberrant cell survival.In this respect, it is interesting that recognize a relatively narrow range of ligands, deter- mice lacking integrin avb3 exhibit increased vascularity mined in part by the particular a–b pair being used.For during pathological forms of angiogenesis such as tumor this reason, endothelial cells express a number of growth (Reynolds et al., 2002). different integrins, such as a5b1 (a fibronectin receptor), Integrins vary from the dependence model in one a2b1 (a collagen receptor) and a6b4 (a laminin particularly important detail; integrins are mechanosen- receptor).In each case, from tens of thousands to sitive receptors (Ingber, 2002; Mammoto et al., 2008). hundreds of thousands of copies of each integrin The application of tension increases integrin affinity for heterodimer are present on the cell surface. ligand, and also serves as a critical factor in modulating By contrast, integrin avb3 is a relatively promiscuous integrin-mediated signaling within the cell.In fact, the receptor that recognizes a wide range of ligands. same ‘ligand’ can function either as an integrin However, each of the ligands recognized by avb3is antagonist or agonist, depending upon whether the either a provisional ECM component, such as vitronec- ligand is soluble or affixed to an immobilized substrate. tin, fibrinogen or fibronectin, or is a fragment of an Translating these in vitro observations in the context of interstitial/anatomical ECM protein that is produced by in vivo findings with small molecule inhibitors, it proteases during ECM remodeling (including proteoly- becomes clear why drugs that bind to the ligand-binding tic fragments of collagen or laminin).Thus, the increase site of integrins do not promote productive signaling; in avb3 expression is timed to coincide with alterations they are compromised in their capacity to present an in the local ECM that provide a number of avb3 ligands. opposing tensile force.

Oncogene Apoptotic cues from the ECM DA Cheresh and DG Stupack 6289 and ECM degradation as a central with these results, invasion-promoting proteases, such as mechanism in regulating survival MMPs, can also promote apoptogenic signals among The requirement for mechanical force to facilitate invasive endothelial cells (Boger et al., 2001; Ben-Yosef integrin signaling has additional important implications et al., 2005). to the cell.On one hand, productive signaling can be The expression of cell surface proteases, such as MT- propagated by ligated integrins, either through cyclic MMP1, or integrin-associated protease receptors, such application of mechanical tension (such as would be as uPAR, is dramatically increased on the surface of transmitted through normal blood flow) or through the angiogenic endothelial cells (van Hinsbergh and Kool- activation of internal contractile forces, such as those wijk, 2008).Integrin avb3 serves as a scaffold for MMP- effected downstream of the Rho family of small 2 (Silletti et al., 2001), reinforcing the notion that the GTPases (Wu et al., 2007a; Mammoto et al., 2008). processes of adhesion, migration and proteolysis are These signals play an important role in regulating cell tightly linked during vascular cell invasion.Angiogen- survival, particularly when growth factor signaling is esis should therefore be viewed as a highly orchestrated limited.On the other hand, growth factor initiated process in which proinvasive functions are checked and (Giancotti and Ruoslahti, 1999) or mechanically pro- balanced by cellular apoptotic pathways. pogated (Li et al., 1997) signaling events depend upon cell anchorage and co-signaling by integrins.This requirement provides a mechanism for attenuation or Mechanisms of integrin-mediated regulation of vascular ablation of signaling events based on the current status cell apoptosis of the local ECM microenvironment.Such a mechanism might be expected to be central to a rapidly remodeling The expression of integrins such as avb3 compromise ECM, wherein integrin ligation of immobilized ECM cell survival when the cells were placed in an environ- components is suppressed (or prevented). ment that provided no ligands for this integrin; native In this respect, integrin mediated adhesion to the collagen gels (Stupack et al., 2001). This occurs despite ECM can be strongly influenced by the actions of the fact that these cells expressed functional receptors MMPs and serine or cysteine proteases, including UPA, for collagen, permitting cell adhesion to, and migration MT1-MMP, soluble MMPs and cathepsins (van Hins- through, the gels.However, these cells did not ligate bergh et al., 2006). These influence cell integrin b3 and they underwent apoptosis.Thus, adhesion by two mechanisms; first and most obvious, antagonism of integrins may not be a prerequisite for they digest the underlying ECM, and thereby compro- apotposis; however, the addition of soluble ligands of mise stable integrin interactions with it.Secondly, the avb3 to cells within the collagen gels enhances cell death; by-products of ECM digestion are frequently small, and these data therefore suggest ‘antagonized’ integrins soluble ECM fragments that subsequently compete with as more efficient transducers of apoptotic signals than substrate-immobilized ligand for integrin occupancy. simple, unligated integrins.In fact, we also found this to Studies have identified a growing list of these, including be true in two-dimensional culture on collagen gels as angiogstatin, endostatin, kininostatin, restin, tumstatin well; in this case we could rescue the survival of and vastatin (O’Reilly et al., 1997; Jimenez et al., 2000; endothelial cells by first cross-linking integrin ligands Xu et al., 2001; John et al., 2005; Chen et al., 2006; Wu to the collagen gels. et al., 2007b), and it is likely that many as yet Integrin avb3 antagonists may induce apoptosis uncharacterized fragments remain to be discovered. among cells cultured on tissue culture plastic; in some When present in modest concentrations, these integrin cases, apoptosis is initiated selectively by a caspase ‘antagonists’ can actually promote cell signaling and 8-dependent mechanism, which then promotes cell migration by facilitating integrin ‘turnover.’ The effect detachment (Brassard et al., 1999; Stupack et al., 2001; can be compared to ‘greasing’ the wheels on a car; Meerovitch et al., 2003; Erdreich-Epstein et al., 2005; integrin binding to ECM is compromised in a very Zhao et al., 2005). In other cases, (particularly when limited manner, permitting somewhat fewer bound cells are attached to a substrate integrin avb3 ligand) integrins on the cell surface at any given time, but detachment can be rapid and precedes the initiation of increasing integrin cycling through focal adhesion apoptosis (Ruegg et al., 2002; Kim et al., 2007). Here, contacts (that is, lowering their residency within these cell death proceeds through anoikis, and can involve any sites).This, increased rate of re-engagement of the number of different apoptotic pathways (Frisch and ECM, in turn, enhances integrin signaling and promot- Screaton, 2001).Accordingly, the anoikis pathway ing cell survival.However, as proteolytic activity works best when other cell adhesion mechanisms (that increases and the local concentration of soluble protein is, other integrins and ligands) are limiting to the cell. fragments rises, integrin-ligand binding events are Lastly, there is the phenomenon where cells manage increasingly likely to involve antagonistic fragments to survive despite bearing unligated or antagonized rather than immobilized substrates.In this case, positive integrins.This case is cell-type and cell-context depen- signaling is compromised, and proapoptotic signaling is dent, and therefore may be the most interesting, as elicited.In fact, mice that lack endogenous protease dissection of these survival pathways may give clues as inhibitors, such as the MMP inhibitor PAI-1, often to how integrins regulate apoptosis.For example, exhibit deficiencies in angiogenesis because of unregu- endothelial cells attached and spread on the surface of lated protease activity (Bajou et al., 1998). Consistent three-dimensional collagen gels are more sensitive to

Oncogene Apoptotic cues from the ECM DA Cheresh and DG Stupack 6290 integrin avb3 antagonists than cells on two-dimensional activation observed in endothelial cells do play some collagen surfaces (Stupack et al., 2001). Ongoing studies role in maintaining cell survival, particularly under using similar systems have provided clues as to the conditions of nutrient deprivation.In these cases, major specific molecular mechanisms regulating vascular cell targets are bcl-2 proteins, p53 (Mayo and Donner, 2002) survival.These involve both early signaling events and and mTOR (Maeshima et al., 2002). downstream transcriptional targets. Integrin ligation also protects against apoptosis mediated through caspase 8 and the extrinsic cell death pathway.VEGF-mediated signaling is integrin avb5- Modification of apoptotic effectors: signaling events at the dependent, and both integrin ligation and VEGF-signaling focal adhesion complex can prevent endothelial caspase 8 activation mediated by The type of substrate present for an integrin, as well as death receptors (Ruegg et al., 1998; Alavi et al., 2003; the given quantity and type of integrin heterodimers on Bieler et al., 2007), whereas the loss of integrin adhesion a cell will influence survival directly.For example, can sensitize cells to apoptosis mediated by death receptors integrin avb3anda5b1 appear to influence each other’s (Aoudjit and Vuori, 2001).The mechanisms for this are ligand-binding activity (Blystone et al., 1999), as do unclear; lack of adhesion may upregulate recpetors for cell several other integrin pairs, although the precise surface death receptors.The protection also appears molecular mechanisms responsible for this may vary dependent on Src kinase activity (Alavi et al., 2003), from case to case.As suggested above, integrin signaling which may function by altering death ligand expression or occurs in a manner dependent on the mechanical rigidity more likely may have a direct role in regulating cell death of the substrate (Giannone and Sheetz, 2006).There- downstream of the integrins (Figure 2). fore, the fact that three-dimensional ECM gels and In this respect, we previously showed that caspase-8 tissue culture plastic differ in their rigidity by several was recruited to a complex containing (unligated) orders of magnitude is significant, and it is perhaps not integrins, where it was activated, leading to apoptosis surprising that focal adhesion complexes that accumu- (Stupack et al., 2001). One might speculate that late in tissue-cultured cells are much larger than those unligated integrins could therefore induce apoptosis found among cells migrating through a three-dimen- through caspase 8 on their own or might otherwise sional ECM (Larsen et al., 2006). The signaling lower the threshold required for caspase 8 activation by potentiated by these ‘exaggerated’ focal adhesions in death receptors.Conversely, caspase-8 mediated killing two-dimensional cultured cells impacts both the caspase by death receptors, or unligated integrins may be 9- and caspase 8-mediated signaling pathways in two- prevented by ligated integrins and focal adhesion dimensional cultures, and generally makes cells highly complex signaling.In fact, caspase 8 is phosphorylated resistant to apoptotic stimuli in vitro.Although apop- on tyrosine 380 following growth factor stimulation tosis can be triggered by different initiator caspase (Cursi et al., 2006; Senft et al., 2007) or integrin- pathways, most are influenced by integrin signaling mediated adhesion (Barbero et al., 2008). Phosphoryla- (Stupack and Cheresh, 2002).The end result of an tion of caspase 8 is mediated by Src family kinases (and apoptotic stimuli, therefore, will often depend upon possibly other kinases) within an SH2-recognition motif both the local ECM milieu and the specific integrin that can be bound by Grb2 and Src SH2 proteins heterodimers that are expressed on the cell surface. (Barbero et al., 2008), as well as the SH2 domains of the During cell spreading, integrin ligation activates the p85 subunit of PI3K (Senft et al., 2007). Phosphoryla- MAPK pathway downstream of Src, FAK and Ras tion of this site inhibits proteolytic activation of the (Schlaepfer et al., 1994). This pathway is critical for caspase 8 zymogen (Cursi et al., 2006). It is not clear angiogenesis (Eliceiri et al., 1998), and this is in part due whether this is due to phosphorylation per se, due to to a role in promoting endothelial cell survival.The SH2-mediated steric blockade of a critical enzymatic site MAPK pathway provides resistance to starvation- or simply due to sequestration of procaspase 8. mediated cell death and does so in part through the It is worth noting that the link between caspase 8 and translocation of Raf to the mitochondria, where it integrins appears to function in both directions; integrins prevents loss of mitochondrial membrane permeability can influence caspase 8 activity, and caspase 8 can (Alavi et al., 2003). Activation of MAPK opposes also influence integrin activity.Indeed, among adherent cells, the proapoptotic ‘side effects’ of Rho-kinase activation the expression of caspase 8 can promote motility (Mavria et al., 2006), likely through its capacity to signal independent of its protease activity (Senft et al., 2007), to the acto-myosin system (Klemke et al., 1997). and phospho-caspase 8 is found to be enriched at the Integrin ligation also activates PI3K by Ras and leading edge of migrating cells (Barbero et al., 2008). subsequent downstream targets such as p21-activated Therefore, caspase 8 phosphorylation or sequestration by kinases 1 and 2 (Kiosses et al., 2002; Hood et al., 2003) focal adhesion complexes (downstream of ligated integ- and Akt1 and Akt2 (Maeshima et al., 2002; Ruegg et al., rins) likely represent a mechanism to oppose caspase 8 2002).The activation of Akt may be a highly potent activation, such as that promoted by death receptors or survival signal, as dominant active forms of Akt protect unligated integrins (Stupack, 2005; Cursi et al., 2006). against a wide range of cellular insults.Although In addition to these immediate early interactions that expression of a dominant active kinase is probably not prevent activation of the caspase 8 and caspase 9 physiologically relevant to endothelial cell biology, such signaling pathways, the ligation of specific integrins studies nonetheless suggest that lower levels of Akt triggers selective signaling elements that promotes the

Oncogene Apoptotic cues from the ECM DA Cheresh and DG Stupack 6291 Protein 2. Proteolytic Activity Fragments 3. Proteolytic Fragments Compromises the ECM Antagonize Integrins

Protein Protease Fragments 1. Ligated Integrin Eph Activity Antagonized + Growth Factor Receptors (or unligated) Signaling Integrins

Death Receptors

ECM Rap

4. Modulation of Caspase 8 Integrin Signaling Growth FAKSrc Factor Ask 1 - Mt Caspase 3 Receptor Ras Caspase 9 Raf Mek Ras Erk PI3K Akt Endothelial Cell Endothelial Cell mTOR Survival vs Apoptosis

Figure 2 Roles for integrins and the extracellular matrix (ECM) in regulating cell survival.(1) Integrins that ligate an immobilized ECM promote cell signaling through growth factors, such as vascular endothelial cell growth factor (VEGF), through both the MAPK pathway and the PI3K pathway.These signals are strongly prosurvival.(2) The localization of proteases to sites of contact, through integrins or other receptors, promote degradation of the ECM and the formation of antagonistic ECM fragments.At low concentrations, the fragments promote integrin rebinding to the immobilized ECM, and can potentiate signaling.(3) Accumulation of antagonistic fragments disrupts focal adhesions, and further initiates apoptogenic signals through integrins through caspase 8.Whether a cell lives or dies is determined by its capacity for the survival pathway to oppose apoptogenic signals arising from integrins or other local insults (hypoxia, inflammation and so on.). For example, Src signaling opposes caspase 8 activation, whereas Raf binding to Ask- 1 protects cells from oxidative stress.(4) Signaling through integrins is likely to be regulated by their activation state.Integrins can be activated transiently by ligand binding.Moreover, integrin are controlled from the inside-out by the activation of the small GTPase Rap, which promotes binding of the cytoskeletal protein talin that is critical for focal contact formation and prosurvival signaling.The integration of all of these factors, compounded with the fact that each integrin heterodimer will influence each pathway to varying degrees, determines the ratio of survival to death during angiogenesis. transcriptional regulation of prosurvival proteins.For CAMs belong to a number of families, with prominent example, avb3ora5b1, but not avb1, promotes molecules on the cell surface belonging to the immu- upregulation of protective bcl-2 (Matter and Ruoslahti, noglobulin and cadherin families.As described above, 2001), whereas ligation of avb3 promotes Bcl-2 expres- VE cadherin is phosphorylated and internalized during sion, but suppresses p53 activity and thereby attenuates angiogenesis, and cell surface expression is concomi- Bax and p21WAF1/CIP1 expression (Stromblad et al., tantly dramatically decreased.This occurs as part of 1996).These prosurvival signals appear to depend in the process that facilitates vascular permeability.By part upon the MAPK cascade (Hood et al., 2003) as well contrast, immunoglobulin superfamily cell adhesion as on integrin signaling through the nuclear factor-kB molecules, such as ICAMs, VCAM-1, -CAM and pathway (Scatena and Giachelli, 2002; Courter et al., neuropilin are increased on the cell surface.VCAM is a 2005).The nuclear factor- kB pathway is activated by ligand for integrins a4b1 and a4b7, where it plays a death receptors, integrins and toll-like receptors—and role in the recruitment of circulating endothelial cell each of these receptor groups also initiates apoptosis precursors and in regulating cell–cell interactions with through caspase 8.The fact that caspase 8-deficient cells stromal cells such as pericytes (Garmy-Susini et al., can be deficient in nuclear factor-kB signaling seems to 2005; Jin et al., 2006a, b). ICAM-1 binds to fibrinogen bind these groups together at the functional level (Zheng fragment D (Lominadze et al., 2005) and similar to et al., 2006). In fact, endothelial cells appear highly other ICAMs serves as an endothelial cell ligand for the dependent upon caspase 8 during development, as b2 family of integrins.The b2 integrins are present on selective deletion of caspase 8 with ECs is lethal hematopoietic cell populations, and play a critical role (Kang et al., 2004). in the recruitment of circulating cell populations to the angiogenic milieu.ICAMs are upregulated as part of the inflammation associated with angiogenesis, either by Interactions with non-integrin receptors that influence chemokines, or VEGF itself (Croll et al., vascular cell survival 2004).L1-CAM is a homotypic adhesion molecule The endothelial cell surface contains a variety of cell that also serves as a ligand for a5b1 and avb3 adhesion molecules, including ECM proteins and (Felding-Habermann et al., 1997; Hall and Hubbell, transmembrane cell adhesion molecules (CAMs).These 2004; Hall et al., 2004). Neuropilin plays a central role

Oncogene Apoptotic cues from the ECM DA Cheresh and DG Stupack 6292 as coreceptors for VEGFs and semaphorins pathway through H-Ras and Raf (Eliceiri et al., 1998; (Vieira et al., 2007), thereby regulating permeability Alavi et al., 2003; Hood et al., 2003). Thus, in tandem and angiogenesis (Acevedo et al., 2008), but can also with positive survival signaling, a feedback mechanism serve as a cellular ligand for integrins (Fukasawa et al., that could promote integrin dissociation from ligand is 2007).Most of these immunoglobulins have short provided.This may be critical for mobilization of cytosolic domains, which interact with adaptor proteins integrin-mediated anchorage in anticipation of cellular and the cytoskeleton; thus, they serve as agonistic migration, and/or it may prime the cells so that survival integrin ligands due to their capacity to provide an depends now upon ligation of a smaller subset of opposing mechanical force.However, each can be integrins.As mentioned above, antagonized integrins dissociated from the cell surface by proteases (Silletti (which are, by definition, in the active conformation) are et al., 2000; Garton et al., 2003; Tsakadze et al., 2004; better at promoting apoptosis than unligated integrins. El-Sheikh et al., 2005; Essick et al., 2008); thus, these cell Thus, the regulation of integrin conformation may surface adhesion receptors also have soluble forms that influence proapoptotic, or ‘negative’ signaling as well. are antagonistic towards integrins.Thus, with ECM Aside from growth factor receptors, other cell surface ligands, shed forms of these CAMs can influence cell receptor tyrosine kinases also influence integrin-ligand survival during angiogenesis. binding.In particular, are known to regulate integrin activity.Ephrins work in tandem with Eph receptors on neighboring cells.Ephrin-B1 transduces Integrin conformation and activity signals to activate integrin-mediated migration, attach- Integrins adapt different conformations that influence ment and angiogenesis (Huynh-Do et al., 2002). The their ligand binding and signal-transduction activity. downstream signaling pathways appear to use reactive The conformations were originally characterized on the oxygen species as an integration point.EphrinA1 surface of lymphoid cell lines before the ability of inhibits Rac1 GTPase activity, which promotes LMW- integrins to signal was appreciated (Dustin and PTP dephosphorylation of p190RhoGAP and contrac- Springer, 1989; Dransfield et al., 1992; Stupack et al., tility through RhoA activation (Parri et al., 2007). This 1992).However, it was somewhat intuitive that promotes loss of adhesion, as EphA1 also promotes peripheral blood leukocytes required some mechanism SPAR-mediated deactivation of Rap-1 (Pasquale, 2008). by which to regulate their dichotomous functions; on These changes reduce integrin interactions with the the one hand, present in the circulation, and on the ECM, but may also ameliorate, to some degree, their other, arresting and extravasation into the surrounding capacity to bind soluble ligands. tissues.This regulation was central to the concept of immune surveillance and lymphocyte trafficking, and has direct relevance to angiogenesis both with respect to Critical roles for hematopoietic, mural and tumor cells in the recruitment of endothelial cell precursors and angiogenesis cellular mediators of inflammation (Rose et al., 2007). These studies described a number of conformation- Endothelial cells interact with the ECM and with other specific antibodies (van de Wiel-van Kemenade et al., endothelial cells during angiogenesis; however, endothe- 1992; Stupack et al., 1994; Pampori et al., 1999), which lial cell interactions with other cell types, including revealed that integrins on nonhematopoietic cells were pericytes, endothelial cell precursors and inflammatory also regulated at the level of conformation and avidity, cells may be at least as important in regulating although not to the same extreme degree as the angiogenesis.In quiescent endothelial cells, hematopoietic cell populations originally studied.This mediate intercellular interactions among ECs, whereas regulation of conformation is influenced by the external angiopoietin 1 (Ang1) mediates interactions between milieu, and in particular, divalent cations can indepen- pericytes and the endothelial cells.The overall levels of dently promote integrin maintenance of the most active cell surface immunoglobulin superfamily CAMs are low, conformation, as can the presence of bound ligand and arrest and extravasation of circulating cells is rare. (Luscinskas and Lawler, 1994). Endothelial cells precursors are a circulating popula- However, integrin conformation is also influenced by tion of myeloid cells, which are recruited to sites of cytosolic effectors.For example, although integrin angiogenesis in part by the upregulation of cell surface ligation can promote H-Ras activity and signaling CAMs such as ICAM and VCAM.Recent studies through the MAPK pathway, the activation of H-Ras suggest that (Salven et al., 2003; Garmy-Susini and in turn promotes the acquisition of the less active Varner, 2005; Hristov et al., 2007) a measurable fraction conformation of integrins by deactivation of the small of ECs, both in physiological and pathological forms of GTPase Rap (Kinbara et al., 2003; Banno and Gins- angiogenesis, can be derived from circulating endothe- berg, 2008).Rap regulates the interaction of the lial precursor cells.The paradigm of leukocyte recruit- cytoskeleton protein talin with the cytosolic domain of ment to an inflammatory site is mimicked in this integrins; this may be a common mechanism to regulate process, and EC precursors interact with cell surface integrin function, as paxillin binds to integrin a4 CAMS through and integrins; in particular, (Rose et al., 2003). This is significant in the context of a4b1 (Jin et al., 2006b). Although it is not yet clear how angiogenesis, because most angiogenic growth factors, the survival of circulating endothelial precursor cells is including bFGF and VEGF, activate the MAPK regulated during angiogenesis, it is assumed that these

Oncogene Apoptotic cues from the ECM DA Cheresh and DG Stupack 6293 cells do not require adhesion during circulation for least in myocytes, (Dallabrida et al., 2005) but this does survival.Integrin-mediated events may be critical for not appear to be the case for all cells (Brindle et al., targeting and activating these cells, at which point they 2006).It remains unclear whether integrin ligation is become adherent and dependent upon the underlying required for Tie2-mediated tyrosine kinase activity, as it ECM as a survival factor (Salven et al., 2003). This is for other receptor tyrosine kinases.Nonetheless, requirement is gained as part of the ‘precursor to EC’ integrin binding to Ang2 could play a role in sequester- differentiation process, as a requirement for ECM ing it, thereby favoring Ang1 interactions with Tie2. interaction during this process has been proposed to These interactions are critical for the resolution of occur during this process in vitro and in vivo (Moldovan, physiological angiogenic processes, as re-entry into a 2003); its likely that this process is controlled by master quiescent state and vessel maturity ultimately requires differentiation genes of the ID1/ID3 family, which are pericyte interaction.Thus, its perhaps not surprising essential for ECP recruitment (Lyden et al., 1999). This that PDGF has been identified as an angiogenic growth dependence on adhesion may occur very early in the factor.PDGF does not act directly upon endothelial recruitment process, as small molecule antagonists of cells, which lack PDGF receptors, but rather activates integrins limit the accumulation of ECP in angiogenic pericytes and other mural cells to produce VEGF sites (Loges et al., 2007). secretion; this mechanism is analogous to the induction Similar to ECPs, other populations of myeloid cells of VEGF by inflammatory mediators.VEGF is then are recruited to angiogenic sites, and can be found capable of maintaining cell survival to some degree, within the interstitium.In particular, tumor-associated although VEGF alone is insufficient for vascular macrophages play a significant role in maintaining an maturation.What is somewhat surprising is that inflammatory angiogenic milieu (Porta et al., 2007; although PDGF promotes pericyte proliferation, en- Allavena et al., 2008). These cells secrete angiogenic dothelial cells numbers far exceed pericytes numbers in growth factors directly or induce their expression in other an angiogenic environment.Given that pericytes are cells in response to -1 or tumor necrosis factor-a. ultimately critical to blood vessel stabilization and The situation is similar in tumor-like tissue, such as the maturation, it is tempting to speculate that some rheumatoid pannus in rheumatoid arthritis (Szekanecz and mechanism exists that limits pericyte accumulation, thus Koch, 2007), where macrophage-derived factors provide a preventing ‘overvascularization’ of tissues.Supporting significant portion of the angiogenic stimulus.These this notion, the addition of VEGF or angiogenic growth factors can act as survival factors for the angiogenic factors can transiently induce angiogenesis in vivo, but is endothelial cells.These cells bind to ICAMs and VCAM1 typically insufficient for a lasting (that is, mature) on the surface of endothelial cells through integrin- angiogenic response in vivo (Gounis et al., 2005). mediated interactions, subsequently invading the loal ECM.Within this milieu, tumor-associated macrophages further contribute by expressing ECM components, such Endothelial cell apoptosis as a natural conclusion to as fibronectins.However, the prosurvival signals are angiogenesis; integrating signals complemented by the potentially apoptotic signals arising Why should there be a balance that ultimately favors from ECM degradation by uPA and MMPs produced by apoptosis? During angiogenesis events, increased vascu- these cells.In some cases, the leukocytes will even trigger a lature accumulates relative to the surrounding tissues, caspase cascade directly, through presentation of death although composed of immature vessels.However, in a ligands such as Fas (Ishida et al., 2003). physiological angiogenesis event, this is a temporary The critical role of the pericyte in angiogenesis has situation, and the natural process termed ‘vascular become appreciated in recent years.Pericytes play a pruning’ subsequently occurs, wherein ‘extraneous’ critical role in stabilizing the vasculature through blood vessels undergo apoptosis (Figure 3).In fact, interactions at sites of pericyte: EC contact.These apoptosis can be detected among endothelial cells contacts involve interactions through the receptor during angiogenesis before pruning, and the ultimate tyrosine kinase Tie2 and Ang1 present within the dieback, which occurs is programmed into the cells. quiescent vasculature (Fukuhara et al., 2008; Saharinen Angiogenic blood vessels require growth factor stimula- et al., 2008). After stimulation with an angiogenic tion, which is dependent upon ongoing interactions with growth factor, endothelial cell secretion and accumula- their microenvironment through integrins; active death tion of angiopoietin 2 (Ang2) leads to direct competition is induced in those cells, which are in a highly proteolytic with Ang1 for binding to the Tie2 receptor, disrupting microenvironment wherein integrins are antagonized, or the EC-pericyte junction.This leads to pericyte to among cells in a microenvironment inappropriate to retraction and facilitates endothelial cell invasion of the their integrin complement.In the pathological angio- local interstitial matrix.Ang2 seems to function as more genesis scenario, present within a tumor, the rheumatoid than a simple decoy ligand, as Ang2 binding to the Tie2 pannus or a diabetic retina, this is essentially an ongoing receptor can function to promote signaling in certain cell state, wherein cells are constantly proliferating and types, although not among ECs (Shim et al., 2007). dying, remodeling their microenvironment and in turn Integrins act as a secondary receptor for Ang2, and being dependent on their addiction to growth factors Ang2 can promote cell invasion through integrin- and integrin-mediated signaling. mediated signals (Imanishi et al., 2007). Integrins also In these cases, the maturation associated with mediates some Ang1-mediated survival signaling, at physiological angiogenesis does not occur.Maturation

Oncogene Apoptotic cues from the ECM DA Cheresh and DG Stupack 6294 Immature Vessels Mature Vessels

Pericyte Coverage Sustains Endothelial Cell Viablity After Growth Factor Loss

Patent Vessels

Provisional ECM Replaced with Anatomical ECM

Loss of Unsupported Vasculature: “Vascular Pruning”

Figure 3 Vascular maturation involves endothelial cell apoptosis.The loss of growth factor signaling during the resolution of angiogenesis combined with ongoing dissolution of the provisional matrix creates a proapoptotic environment for the angiogenic endothelial cells.In this case, those that form extensive contacts with pericytes are protected, whereas supernumerary cells are deleted during this ‘pruning.’ The resulting mature vessels do not exhibit vascular leak, and offer improved blood flow. This process can be induced in tumors, which exhibit an immature vasculature, by VEGF-sequestering agents.Although this decreases vasculature, it increases flow, and promotes delivery of other chemotherapeutic agents.

involves a depletion of growth factors, replacement of VEGF-production, whereas ablating VEGF activity in the provisional insterstitial ECM with an anatomically the retina can have a more significant impact on appropriate ECM and the reestablishment of pericyte endothelial proliferation.But the answer may be far contacts.In fact, depletion of growth factors such as simpler; tumors may make more blood vessels than they VEGF using therapeutic antibodies or decoy receptors need.Thus, ablation of VEGF signaling leads many does induce endothelial apoptosis, yet ultimately results vessels to die, but in doing so promotes maturation of in establishment of a stable vasculature.Could the effect the others (Willett et al., 2004; Winkler et al., 2004). of these drugs simply result from facilitating a natural In the back of the eye, this will restore the normal conclusion to the angiogenic event? (pre-existing) physiology.However, it remains unclear Ultimately, the fact that so many interacting factors how this plays out in the . balance the decision between life and death in angio- Immature and leaky tumor vasculature is highly genic ECs leads one to the conclusion that angiogenic inefficient at drug deliver, but mature vessels yield endothelial cells are heavily ‘loaded’ with respect to sufficient increases in drug delivery that the lower molecules determining cell fate.A given endothelial cell vascular volume is inconsequential (Dickson et al., derives numerous signals that promote apoptosis, and 2007).By reducing VEGF in the tumor blood vessels thus becomes dependent upon survival factors to show increased maturation and blood flow associated counteract them.This lends new credence to the concept with enhanced pericyte coverage.This may facilitate originally proposed by Dr Folkman; interventions that drug delivery to tumors and explains in part the need to impact a single factor strongly enough may be sufficient give anti-VEGF therapy in combination with cytotoxic to ‘tip the balance’ irrevocably towards EC apoptosis drugs (Willett et al., 2004) (Folkman, 2006). Can integrins, as the critical receptors for the ECM and central regulators of survival and apoptosis, be exploited as targets as well? It would appear to be Conclusions and therapeutic outlook possible.Small molecule antagonists of integrins, as well as humanized monoclonal antibodies, have shown Despite spectacular results in a variety of preclinical success in preclinical models, and appear safe in clinical models, antiangiogenic agents have so far met with trials (Beekman et al., 2006) (with some reports of limited success in the clinic.Dramatic advances have dramatic effects as single agents) (Raguse et al., 2004). been achieved in ocular indications using anti-VEGF- But this story is very similar to that of the VEGF- based approaches as single therapies (Algvere et al., inhibitors, and trials with additional drugs are likely 2008), whereas in the cancer clinic no single-agent warranted to recaptitulate synergies already seen in activity has been achieved (Grothey and Ellis, 2008). preclinical models.One advantage of targeting integrins In part, one might attribute this to the fact that may be that many growth factor signaling pathways are human tumors display high levels of ongoing dependent upon them, and disrupting integrins can

Oncogene Apoptotic cues from the ECM DA Cheresh and DG Stupack 6295 thereby disrupt growth factor signaling (Eliceiri There have been many surprises arising from recent et al., 1999). Conversely, one possible limitation of studies of angiogenesis.In spite of our recent revela- the VEGF-inhibitors is that a tumor may eventually tions, angiogenesis remains a highly complex biological develop a significant capacity to induce angiogenesis process, and ultimately poorly understood process.In through other growth factors, such as bFGF, fact, we are still only beginning to understand the effects rendering VEGF-targeted therapies less effective.More- of how the regulation of vascular cell survival can over, as EC recruitment or survival requires integrins ultimately influence disease outcome.Nonetheless, (Bussolino et al., 2006; Kohl et al., 2007), it is possible initial results continue to be encouraging, and the recent that small molecule antagonists could also block direct evidence of impact in the clinic provides excite- EC accumulation, providing another antiangiogenic ment for the future prospects of controlling neovascu- mechanism. larization.

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