The Role of the Plasminogen Activation System in Angiogenesis and Metastasis

CANCER METASTASIS: BIOLOGICAL AND CLINICAL ASPECTS

THE ROLE OF THE PLASMINOGEN ACTIVATION SYSTEM IN ANGIOGENESIS AND METASTASIS Shafaat A. Rabbani, MD, and Andrew P. Mazar, PhD

Tumors grow and disseminate by recruiting and forming new blood supplies through the process of tumor-mediated angiogenesi~.~~ Although recent evidence suggests that tumor cells also form blood-carrying structures that resemble vessels and may be able to compensate for a lack of angiogene~is;~there is still a significantbody of data that supports the requirement for true neovessel formation for tumor progression. Numerous preclinical animal models have demonstrated the use of antiangiogenic approaches in suppressing the growth and metastasis of tumors30 and some antiangiogenic compounds with antiangiogenic activity in preclinical models have begun to demonstrate similar activity in human clinical trials.137 Angiogenesis is a prerequisite for the hematogenous dissemination of tumor cells. Although primary metastasis can occur through the lymphatic system, the most predominant mode of tumor cell egress is intra~asation.~~ Thus, agents that inhibit angiogenesis may also inhibit metastasis. Further, metastasis and angiogenesis share many common phenotypic features that lead to the motility of tumor cells (in the case of metastasis) and endothelial cells (in the case of angiogenesis). These include the upregulation of protease and integrin expression, loss of cell-cell and cell-matrix contacts, an increase in responsiveness to growth and differentiation factors and the remodeling of extracellular matrix (ECM) and basement membrane This work was supported by Grant MT-16209 to SAR from the Medical Research Council of Canada.

From the Departments of Oncology and Physiology, McGill University Health Centre, Montreal, Quebec, Canada (SAR); and Attenuon, LLC, San Diego, California (APM)

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( ~ ~ ) . 784,114 2 . jyumerous molecules have been implicated in these processes and many of these molecules have provided the basis for targeted therapeutic and diagnostic approaches for the treatment and detection of ~ a n c e r . ' ~ , ~ ~ The authors' research over the past 11 years has focused on the role of the urokinase plasminogen activator (uPA) system in metastasis and angiogenesis, a system that the authors believe to be central to many of the cascades that have been demonstrated to be important in tumor progression (Fig. l).The uPA system, apart from its ability to promote extracellular fibrinolysis, plays an important role in initiating cascades that result in the activation of plasminogen,l12several matrix metalloproteases (MMPs),4O,73 the release and processing of latent growth factors,69, 92, lo6and the remodeling

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Angiogenesis Metastasis Mitogenesis Figure 1. Model of the role of urokinase (uPA) system in tumor progression. uPA is localized within the tumor cell environment by binding to uPA receptor (uPAR) to promote activation of plasmin, matrix metalloproteases (MMPs), and growth factors resulting in increased angiogenesis, tumor metastases, and mitogenesis.

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of BM and ECM.62,91 Further, the uPA system can also initiate localized fibrin turnover and this may contribute to the role of the uPA system in angiogenesis. Although the proteolytic activity of uPA has long been accepted as being important in the degradation of ECM, it is only recently that the nonproteolytic activities of uPA and its receptor, uPAR, have become recognized for their role in cell motility, including the migration of endothelial cells and the invasion of tumor cells. In this article, the authors summarize recent data on the integration of the various activities of the uPA system and how these contribute to tumor metastasis and angiogenesis. uPA-DEPENDENT PROTEOLYSIS

uPA is a serine protease that is highly specific for the activation of plasminogen. It is secreted in a proenzyme or single chain uPA (scuPA). ScuPA can be activated proteolytically by plasmin (to form catalyticallyactive uPA) or by several other protease such as cathepsins B and L, plasma kallikrein, and mast cell t r y p t a ~ e . ~ ~Physiologically, ,~~, plasmin is the most efficient activator of scuPA although other enzymes could potentially activate scuPA physiologically in the absence of plasmin. ScuPA also forms a catalytically active moiety in the absence of proteolytic activation by plasmin when it is bound to uPAR either on the monocyte cell surface70 or in solution.44This process may require the presence of certain c o f a ~ t o r and s~~ the physiologic relevance of this nonproteolytic activation is not presently understood. ScuPA bound to cell-surface uPAR is activated by plasmin much more efficientlythan scuPA in solution and thus, one role for suPAR in mediating cell-surface proteolysis is to potentiate the activation of SCUPA.~~ Plasminogen and plasmin can also bind to the cell surfaces of numerous cell types including tumor cells and thus scuPA/uPA and plasminogen/plasmin can exert their proteolytic effects while anchored to the cell surface. Because uPAR is often polarized and localized to the migrating or invading edge of a cell, uPA-dependent cascades are focally directed in the direction of cell movement rather than being uniformly activated all over the cell surface. Once plasminogen is activated to plasmin, several divergent pathways are initiated. Plasmin itself can cleave matrix componentssuch as fibrin and fibronectin.'j7, In addition, plasmin can activate several proMMPs (collagenases) such as proMMP2 and proMMP9 either alone or in combination with the activity of membrane-type (MT) MMPs such as MT-MMP1.40, 73, 82 This extends the activity of the pathway activated by uPA to the remodeling of type IV collagen and other matrix components through activated collagenases (MMPs).Type IV collagen is the major constituent of the basement membrane and is remodeled during angiogenesis.'05Plasmin can also process angiogenic growth factors such as vascular endothelial growth factor (VEGF)56or release latent forms of other growth factors (such as fibroblast growth factor 2 (FGF-2) and transforming growth factor B (TGFB))from

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their binding sites in the matrix.92,'06 These growth factors can also function in a paracrine manner to regulate the expression of the uPA system, an aspect that is discussed in the section on the activation and release of growth factors. THE uPA SYSTEM IN MIGRATION, INVASION, AND ANGIOGENESIS

The exvression of uPA and uPAR has been demonstrated in numerous tumor types including prostate, breast, colon, glioblastoma, hepatocellular, The ~ ,levels ~ ~ , ~of~uPA and uPAR are typically inand renal cell c a r c i n ~ m a . ~ creased in more aggressive forms of disease and are often associated with ~ , ~ ~ ~recent studies have demonthe invasive front of the t ~ m o r . ' Several strated strong immunohistochemical staining for uPAR in blood vessels associated with the invasive front of breast (Fig. 2), colon, and renal cell c a r ~ i n o m a . ~Because , ~ ~ , ~angiogenesis ~~ most likely occurs at the tumorhost interface, these results implicate uPAR in the angiogenic process. High levels of uPA in breast cancer patients correlates with poor prognosis and this has also been observed in other cancer types such as head and neck and colon cancer.18, The expression of uPA and uPAR has also been observed on tumor-associated macrophages in several tumor types.23,55 uPA is chemotactic for monocytes and mediates adhesion and migration of these cells.31Adhesion and migration require only uPAR occupancy in the absence of uPA catalytic activity. uPAR occupancy, however, stimulates differential effects depending on the differentiation state of the monocytic Thus, less differentiated, more monocyte-like cells are stimulated to migrate in response to uPA binding (as might occur if a histiocyte was exposed to a uPA gradient in the region of a tumor) whereas a more differentiated macrophageslike cell might be induced to adhere in the presence of uPA (as might occur when the cell has reached the tumor). The role of macrophages in tumor progression has been controversial although recent evidence from patient biopsies demonstrates that macrophages are associated with areas of neovascularization in the tumor.46Thus, the uPA system may stimulate tumor angiogenesis by recruiting the primary inflammatory response to the tumor. Hildenbrand et a1 have demonstrated that only tumor-associated macrophages cultured from invasive breast carcinomas or ductal carcinoma in situ (DCIS) expressed high levels of uPAR whereas resident macrophages from normal breast tissue did not.47 Macrophages are known to secrete angiogenic factors such as VEGF, FGF-2 and interleukin-8 (IL-8) and regions of inflammation have been demonstrated to preferentially support tumor growth in model systems.47It is not clear why the inflammatory response does not lead to an immune response against the tumor but one hypothesis may be that tumors have evolved in such a way as to uncouple inflammation, which can promote tumor progression, from the immune response. Thus, the uPA system may initiate tumor angiogenesis by regulating the inflammatory response in tumors.

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Figure 2. lmmunolocalization of uPAR in breast cancer. Normal (A) and malignant human tissue sections were stained with rabbit antihuman uPAR IgG to localize breast cancer (8) uPAR (original magnification x 400).

In addition to monocyte and macrophage chemotaxis, the uPA system plays a role in the migration and invasion of endothelial cells during angiogenesis. One of the major results of localized inflammatory response is the stimulation of vessel leaking by VEGF, which is secreted by macrophages and platelets that adhere in regions of inflammati~n.~~? This allows fibrinogen (and other plasma proteins) to diffuse into the extravascular space where it is converted to fibrin through the action of tissue factor (TF)-mediated clotting pathways.75Fibrin forms the transitional matrix on which endothelial cells migrate during angiogene~is.~~ uPA and uPAR have been localized to the invading, tube-forming front of microvessel endothelial cells in a fibrin matrix in ~ i t r o . ~ ~

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The role of uPA in the angiogenic process was observed initially in models of corneal vasc~larization~~ and recent studies have extended this early work to implicate uPAR and uPA in the activities associated with neovascularization. Endothelial cells engineered to over-express uPA are more invasive than parental cells in ~ i t r oEndothelial .~~ cells can be induced to migrate in vitro by various proangiogenic factors such as basic FGF (bFGF) and VEGF. These factors upregulate uPA and uPAR in these cells.21,71 Hypoxia, which may stimulate tumor neovascularizationin vivo, also regulates uPAR expression in human umbilical vein endothelial cells (HUVEC) in vitro and this process appears to be mediated by a heme protein-dependent pathway.36An antagonist of uPA binding to uPAR has been demonstrated to inhibit endothelial cell branching morphogenesis in vitron and inhibitors of uPA expression in endothelial cells (such as testosterone and dexamethasone) also inhibit tube f ~ r m a t i o nThis . ~ ~ inhibition by steroid hormones could be reversed by the addition of exogenous uPA to the model system. In vivo evidence also supports a role for the hormonal regulation of angiogenesis. This has important implications for the growth of hormone-dependent tumors, many of which eventually become hormone independent after hormone ablation therapy. uPA and uPAR expression and tumor aggressiveness also increase as a tumor becomes hormone independent.loO Shionogicarcinoma cells (grownin a dorsal skin-fold chamber in SCID mice) depend on androgen for survival. Castration of the SCID hosts leads to tumor cell apoptosis, which is preceded by rarefaction of tumor-associated blood vessels.53After several weeks, however, a second wave of angiogenesis and tumor growth is observed in the castrated animals. An increase in uPA and uPAR levels in hormone-indevendent tumors may be one of the mechanisms responsible for relapse af
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Figure 3. Effect of uPA expression on skeletal metastases. Rat prostate cancer cells Mat Ly Lu overexpressing uPA (uPA-S) were inoculated into the left ventricle of syngeneic male Copenhagen rats. Animals were monitored for the development of hind limb paralysis. Comparison was made with control group of animals inoculated with wild type vector transfected Mat Ly Lu cells (top). Histologic analysis of these skeletal metastases from uPA-S inoculated cells showed an osteoblastic response at these affected lumbar vertebra (bottom).

r e p ~ r t e d . l ~The l , ' ~mitogenic ~ effects of the ATF have been reported in ovarian tumor cells and in bone cell^.^^^'^' ATF was originally identified from prostatic cancer cell conditioned media as a growth factor for bone cells10' and this observation was extended in vivo through gain of function experiments, which demonstrated that the over-expression of uPA in prostatic cancer cells led to the early onset of skeletal metastases to cause hind limb paralysis of experimental animals. Furthermore, because of the mitogenic effects of ATF (of uPA), these skeletal metastases found in the lumbar vertebra were of the osteoblastic variety2 (Fig. 3). These effects required the expression of uPAR on the target cells and the stimulation of bone cells with ATF resulted in the induction of early response genes such as c-fos, c-jun, and ~ - m y c . The ' ~ ~ maximum induction was seen in c-fos mRNA expression, effects which could be abrogated by coincubation of ATF with soluble uPAR to indicate that this response was mediated via the uPAR on osteoblasts (Fig.4). Similarly, uPA-mediated invasion through Matrigel was also dependent on the ability of tumor cells to bind to uPAR and the simple displacement of uPA from its receptor inhibited invasion in several

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I B 45 min Figure 4. Effect of ATF on oncogene expression. A, Treatment of human osteoblast-derived osteosarcoma cells SaOS, with ATF (2.5 pg/mL) for 45 minutes resulted in a significant induction of c-myc, c-jun, and c-fos mRNA expression. B, Coincubationof SaOS, cells with ATF and soluble uPAR (S-uPAR) (10 pg/mL) resulted in neutralizing the ability of ATF to induce c-fos mRNA expression.

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in vitro system^.^^,^^^ Several studies in vivo have also demonstrated that inhibiting the catalytic activity of uPA directly135or displacing uPA from its receptor19lead to a significant decrease in tumor growth and metastasis. Delivery of uPAR antisense using an adenovirus construct into established sc U87MG glioma tumors not only inhibited tumor progression but also induced regre~sion.~' These results were consistentwith data from Aguirre Ghiso et a1 that demonstrated the down-regulation of uPAR in Hep4 cells resulted in the dormancy of these cells when grown in a CAM model.3A polyclonal antibody raised against the N-terminus of rat uPAR inhibited tumor growth and resulted in tumor regression in a syngeneic rat breast cancer model.lo4 Although the therapeutic potential of inhibiting the uPA system in treating cancer has been evident for several years, the role of the uPA-uPAR interaction in tumor progression has been difficult to address in vivo. A species barrier prevents rodent uPA from binding to human uPAR and vice versa and this has proved problematic for testing antagonists developed to inhibit human uPAR in mouse tumor models. Human uPAR-specific antagonists would not be expected to bind to mouse uPAR expressed on macrophages and endothelial cells and could therefore not probe the contribution of the host stromal cells to tumor progression. This issue was addressed recently in an elegant proof-of-principle study that demonstrated the full therapeutic potential of inhibiting the binding of uPA to uPAR in a syngeneic system. The delivery of an adenovirus-encoded murine ATF directly into tumors resulted in the suppression of neovascularization and tumor growth arrest.64In addition, tumor growth arrest was observed in syngeneic and xenograft models, indicating that the inhibition was mediated through the suppression of the host angiogenic response (a murine ATF would not be expected to bind to human tumor cell uPAR but only to host endothelial cells and leukocytes). Although most studies have focused on either the uPAR-binding region of uPA or its catalytic activity, the authors have recently identified a novel epitope from within the connecting peptide region of uPA (amino acids 136-158 in the mature uPA sequence) which mediates cell motility and ~ o n t r a c t i l i t y .The ~~,~ authors ~ have also demonstrated that a peptide derived from this region of uPA (A6, comprised of amino acids 136-143) inhibits tumor cell invasion38and smooth muscle contraction in ~ i t r oEx.~~ perimental animals inoculated with rat (Mat B-111) or human (MDA-231) breast cancer cells receiving A6 showed a marked reduction in their tumor volume throughout the course of this study because of the antiangiogenic effects of A6 in vivo (Fig. 5). uPA that is phosphorylated in this region (Ser 138) is no longer chemotactic for m o n ~ c y t e s Proteolytic .~~ cleavage within this region has been demonstrated also to abrogate the stimulatory activity of uPA on vascular smooth muscle cell (SMC) proliferation3' and the ability of uPA to promote the binding of soluble uPAR to hematopoietic cells.79Mukhina et a1 have also recently demonstrated that the recombinant kringle of uPA containing the connecting peptide was still able to bind to SMC (as observed in competition studies) and stimulate their

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Figure 5. Effect of a6 on tumor volume. A, Syngeneic female Fischer rats were inoculated with rat breast cancer cells (mat B-Ill) and received either vehicle control (CTL) or 75 mglkgld of A6 by intraperitoneal injection (B). MDA-231 tumor bearing BALBIc (nulnu) female mice were injected with vehicle alone (CTL) or A6 (75 mg/kg/d) (B) and tumor volume was measured at timed intervals. Results represent the mean & SEM of 6 starting animals in each group in 4 different experiments. Significant difference in tumor-volume from control tumorbearing animals after ~6 treatment is shown by asterisks (P < .05).

migration with an EC50 6 nM, suggesting either a novel receptor for this domain or a secondary interaction of uPA with ul'AR.'l Although it is not known whether the connecting peptide directly modulates tumor progression when it is part of uPA or whether the uPA/uPAR interac@onsin a tumor are the same as those observed on SMC, the effect of the A6 peptide, isolated from this region, on tumor growth and vascular contractility suggest that it is its important in tumor progression as well.

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PLASMINOGEN ACTIVATOR INHIBITORS

Modulation of uPA proteolytic activity in tumors can also be exerted through specific plasminogen activator inhibitors (PAIs).Of this family of serpin inhibitors PAI-1 is probably the most relevant to tumor progression. Counter-intuitively, however, high PAI-1 levels are also associated with a poor prognosis and advanced disease in many human tumors including: breast ~ancer.'~ The activation of vroteolvtic cascades must be temvoraEy controlled in order for tumor ptogressi~nand angiogenesis to ockr. Uncontrolled and unfocused uPA-dependent proteolysis is not productive from the standpoint of tumor progression. Excessive proteolytic activity in angiogenic endothelial cells leads to hemangioma rather than tube formation, indicating a requirement for a balance between protease and inhibitor." Fibrin is the major component of transitional matrix and promotes angiogenesis when it is deposited in the extravascular space iA inflammation and in tumors.48PAI-1 may serve to protect the fibrin matrix in a tumor such that angiogenesis can occur. The preservation of fibrin in the angiogenic process may be especially critical to angiogenesis because fibrin is the major ligand for the integrin a,/?3, which is present on angiogenic but not on quiescent endothelial cells.33In addition, fibrin induces the expression of IL-8 and intercellular adhesioin molecule 1 (ICAM-1) in endothelial cells.99,104 IL-8 is a potent angiogenic factor and chemokine and mediates the recruitment of neutrophils (PMN)to tumor^.^ Neutrophils also secrete VEGF, thereby amplifying tumor angi~genesis.~~ In addition, many leukocytes (e.g., monocytes, macrophages, neutrophils) secrete chemokines, many of which have recently been demonstrated to be associated with tumors and to induce angiogenesis as ell.^,''^ The recent demonstration of CXCR4 (a major chemokine receptor) expression on endothelial cells further strengthens the link between inflammation and tumor angi~genesis.~~ Finally, fibrin itself is a major mediator of inflammatory cell recruitment through ligation of /?, integrins such as CD11/18 (Mac-1) on the surfaces of neutrophils and macrophages.lo9The presence of numerous inflammatory cells in many human tumors has been well documented and these cells may be fueling the angiogenic response rather than seeking out and destroying tumor cells. Thus, the role of the uPA system (and specifically PAI-1) in angiogenesis involves at least partially the initiation and preservation of the inflammatory response in tumors. PAI-1 is expressed by endothelial cells in the Matrigel system and is probably required for proper tube formation.l13 Pepper et a1 recently demonstrated that a new member of the VEGF family, VEGF-C, up-regulates PAI-1 expression in several endothelial cell lines.94PAI-1 expression has been demonstrated in fibroblasts contacting endothelial cells, which can occur when the basement membrane has been degraded as part of the angiogenic process and the endothelial cells are in the process of migrating5 Tumor progression and angiogenesis is also inhibited in PAI-1-deficient mice in ~ i v oPAI-1-deficient .~ mice, however, develop without any overt vascular abnormalities, and endothelial cell migration after denudation is not inhibited in these animals.95TNP-470, an antiangiogenic drug, inhibits

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the expression of uPA and PAI-1 in human lung cancer cell lines51although it is not known if this down-regulation contributes to the antiangiogenic mechanism of this drug. Mice deficient in the various components of the uPA system (plasminogen, uPA, uPAR, PAI-1) have also been used to assess the role of these of components in angiogenesis and tumor growth and dissemination. Wound healing is impaired in uPA-deficient mice, at least in part because of reduced migration by leukocytes, fibroblasts, and smooth muscle cells. Re-endothelialization of denuded vessels, however, is not affected in mice deficient in uPA, uPAR PAI-1 or ~ l a s m i n o g e nIn . ~these ~ systems, the lack of fibrinolytic components results in an increased deposition of fibrin, which provides a strong stimulus for angi~genesis.~~ In tumors, it may be necessary to have profibrinolytic and procoagulant activities operating simultaneously but in different regions of the tumor or cell. Because uPA-dependentproteolysis is focalized, one can imagine an endothelial cell that is actively degrading matrix at one end while interacting with fibrin at the other end. Alternatively,tumor-associated fibrinolysis and coagulation may cycle. Increased levels of PAI-1 would promote fibrin deposition by inhibiting plasminogen activation. This would potentiate the procoagulant activity of TF, which is expressed by many tumor cells and endothelial cells and may initiate fibrin deposition in the tumor milieu.75TF also directly regulates VEGF expressionby tumor cells.' PAI-1 expression in tumor cells is stimulated by TGFB, which is itself released from binding sites within the matrix through the activity of the uPA cascade. TGFB directly inhibits endothelial cell proliferation in .~ vitro by inducing apoptosis but stimulates angiogenesis in v i v ~ TGFB also stimulates VEGF expression in fibroblasts, especially in conjunction with hypoxia, which is often present in a tumor.12The combination of VEGF and fibrin (formedbecause of the inhibition of uPA-dependent fibrinolysis) may stimulate endothelial cell migration, proliferation, and angiogenesis. The binding of factor VIIa to TF has also recently been demonstrated to upregulate uPAR expression in pancreatic cancer cells1" suggesting an emerging paradigm in which cross-talk between procoagulant and profibrinolytic pathways fuels tumor invasion, metastasis, and angiogenesis. One hypothesis of how both systems might function simultaneously is that an invasive or migrating cell may be considered polarized because the leading edge and the trailing edge are in contact with different milieus. Migrating cells usually display calcium gradients that range from high (Ca+') coincident with regions of actin depolymerization (at the trailing edge) to low (Ca+2)coincident with regions of actin polymerization (leading edge), indicating different environments at each end of the cell. Similarly, haptotactic contacts, which promote migration, may also be different within a procoagulant environment (fibrin, high PAI-1) at one end of the cell and a profibrinolytic (high uPA, uPAR) at the other end of the cell. Plasminogen (Plg) also has been implicated in the negative control of angiogenesis, primarily through the activity of its degradation product, angiostatin.1° Angiostatin is produced through the proteolytic cleavage

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of Plg and numerous enzymes have been implicated in this process.93, 118 O'Mahony et a1 recently demonstrated that PAI-1 inhibited the production of angiostatin in the presence of the pancreatic cell line, ASPC-1,88 which may provide an explanation hypothesis for the pro-angiogenic role of PAI-1. The induction of apoptosis and the inhibition of migration in endothelial cells by angiostatin have been d e ~ c r i b e d . ~Recently, ',~~ a second fragment of plasminogen, kringle 5, has also been demonstrated to inhibit endothelial cell and rnigrati~n.~' This fragment of plasminogen is generated through the activity of macrophage elastase, which could lead to its production in the tumor milieu as well. Thus, various components of the uPA system may alternately elicit positive and negative regulatory effects on angiogenesis although the control of these effectsat the molecular level is not understood. THE uPA SYSTEM AND GROWTH FACTOR ACTIVITY

uPA (directly, or through Plm formation) leads to the release or activation of many angiogenic growth factors such as bFGF, VEGF, HGF, IGF, EGF, and TGFB.56,83,92,106,142~he release of FGF-2 through the activity of the uPA system has also recently been demonstrated in vivo in the CAM model.lo7uPA itself is able to directly cleave VEGF to one of its active forms, VEGF189, and directly activates HGFS3in a stoichiometric complex which then reacts with the HGF receptor, c-met.14' Tumor promoting/ proangiogenic agents such as HGF, EGF, and IGF upregulate expression of the uPA-uPAR system in other tumor-associated cell population^.'^^^'^^^^^^ Agents that down-regulate the expression of the HGF receptor, c-met (e.g., the geldanamycins) also inhibited the expression of uPA and uPAR in SKLMS-1cells.129The HGF-stimulated motility of these cells was also suppressed using these compounds,presumably through the down-regulation of the uPA pathway. In addition to recruiting, focusing, and enhancing the activation of plasminogen by uPA locally on cell surfaces, uPAR also participates in the regulation of this activity through the internalization of uPA-PA1 complexes. While scuPA and uPA remain stably bound to cell surface uPAR, ternary complexes of uPAR-uPA-PA1 are rapidly endocytosed through recognition and uptake by the lipoprotein receptor-related protein (LRP, a,-macroglobulin receptor). The uPA and PA1 components are degraded while uPAR is recycled to the cell surface.87Cells deficient in LRP migrated to a much greater extent than cells that express LRP, indicating that the presence of a constitutive uPA-uPAR complex on the cell surface promotes migration and invasion.130In that study, however, no PAI-1 was added to quench the system and in the presence of PAI-1, LRP-mediated internalization of the uPA-uPAR complex would probably allow the system to be refreshed through uPAR re~yc1ing.l~~ Also implicit in this cycling is an oscillating interaction of uPAR with its adhesion partners although it is not known whether uPAR that is bound to both uPA and an integrin or matrix component will internalize in the presence of PAI-1.

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SIGNALING THROUGH uPAR

uPA bound to uPAR also mediates intracellular signal transduction in endothelial cells. ATF alone is able to mediate certain aspects of capillary tube formation, as described above. Tang et a1 have demonstrated that uPAR occupancy on endothelial cells results in the phosphorylation of focal adhesion proteins and the activation of MAP kinase.lZ3Antisense oligonucleotides which target uPAR expression inhibited endothelial cell proliferation, migration, and invasion and diacylglycerol was identified as a second messenger, leading to activation of the protein kinase C (PKC).26 The down-regulation of uPAR expression in this study would be expected to inhibit signaling pathways that were dependent on uPA catalytic activity and uPAR occupancy. It is possible that uPA catalytic activity can lead to the activation of PKC (through the remodeling of matrix and the disruption of integrin ligation) whereas uPAR occupancy can signal through the MAP kinase pathway. These two uPA-dependent pathways could operate simultaneously or be regulated in a temporal fashion in a migrating or invading cell. Studies with cells other than endothelial cells have also implicated the MAP kinase (MAPK) pathway in uPAR-mediated cell migration in vitro. In MCF-7 breast cancer cells, uPAR occupancy resulted in cell migration, which occurred through the activation of ERKl/ERK2. An inhibitor targeting MAPK redundant k-kinase, a member of the JAK family of kinases, suppressed ligand-induced uPAR-dependent activation of ERKl / E m 2 in these cells.85This pathway has also been implicated in the migration and invasion of HT1080 fibrosarcoma cells.60The JAK/STAT pathway may also be involved in the uPAR-dependent migration of vascular smooth muscle cells.24A second, uPAR-dependent signaling pathway involving Src-like protein tyrosine kinases (TKs) has also been described in these cells, although the functional relevance of this second pathway is not yet understood in smooth muscle cells. In U937 myelomonocytic cells, however, activation of Src-family TKs in undifferentiated U937 cells led to enhanced migration whereas down-regulation of the expression of these same TKs in differentiating cells led to increased adhesion.17The JAK/STAT pathway was also activated by clustering the uPA-uPAR complex using a monoclonal antibody in the human kidney epithelial cell line TCL-598, resulting in the migration of this cell line.61The MAPK/JAK/STAT pathway is activated in cytokine-mediated signaling and has been implicated as a major signal-transducing pathway (stimulated by VEGF and bFGF) in angiogene~is.~~, lZ2 INTEGRINS AND uPAR SIGNALING

Recent evidence suggests that uPAR-dependent signal transduction and migration are mediated through integrins (Fig. 6). In addition to PIand &-integrin involvement in the uPAR-dependent adhesion and migra, ~interaction ' ~ , ~ ~ ~ of uPAR with integrins has also tion of l e u k o ~ y t e s , l ~ ~the

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Figure 6. uPA-uPAR interaction with integrins. Single chain uPA (scuPA), by binding to its cell surface receptor, can interactwith integrins through domain 2 and 3 of uPAR. Similarly through domain 1 and 2 scuPA-uPAR can complex with vitronectin to collectively induce intracellular signals to promote adhesion, migration, invasion, and differentiation.

been demonstrated on tumor cells. a,& binds to fibrin and vitronectin Xue et a1 (VN), and may regulate the expression of uPAR and PAI-1.57,86 have demonstrated the interaction of uPAR on HT1080 cells with various a and @ integrins including B1 and B3 and a,a3, a5, and a6.13$Migration, but not adhesion, of FG cells, which express a,B,, on VN was uPA-uPAR dependent.141Migration of several melanoma cell lines, however, that express only a,B3 occurred independently of uPAR.'~~ Transfection of HT1080 cells with VN resulted in localization of uPA to a,B5-containing focal adhesions, resulting in increased levels of cell adhesion concomitant with decreased levels of plasminogen a~tivati0n.l~~ Adhesion of myogenic cells to PAI-1 coated plates depended on both a,B3 and uPAR.~~ Yebra et a1have

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demonstrated that ligation of uPAR (with ATF or uPA) enhances the migration of uPAR-transfected LnCAP cells on fibronectin (FN) (which was a5B1dependent) and leads to the phosphorylation of two proteins (FAK that are involved in integrin signaling.140 uPAR and coand p13OCaS) irnmunoprecipitate only after uPAR ligation. The mechanism of forming this signaling complex is unclear, however. uPAR ligation may lead to conformational changes in uPAR that allow it to interact with the integrin or it could allow for the reorganization of adhesive interactions that allow the uPAR-integin complex to form. In Hep3 cells, the uPAR-a5B1interaction was required for the adhesion of these cells to FN. The interaction with FN led to a persistent stimulation of ERK and this activation could be enhanced by the ligation of uPAR with scuPA. Inhibition of the uPAR-a5B1 interaction led to tumor cell dormancy in the CAM ~ y s t e mThe . ~ ligation of uPAR leading to enhanced adhesion and migration does not require the presence of the catalytic domain and has now been demonstrated to mediate migration in several systems including tumor T lymphocytes13and most recently in neutrophil migration in response to a neutrophil chemotactic agent in vivo.lZ8Similarly, in breast cancer cells that migrate on vitronectin, uPAR associated with the vitronectin-bindingintegrin, a,B5 .16Thus, the nature of the uPAR-integrin association may depend on the nature of the cellmatrix component involved in the interaction. Physiologically, the same cell may interact with different matrix components temporally during cell migration, invasion, and growth and the nature of the uPAR-integrin interaction may change to support these various cellular activities. Very little is known about how the uPAR-integrin complex initiates signal transduction. A recent study by Wei et a1 using smooth muscle cells suggests that uPAR helps stabilize a caveolin dependent complex between B1 integrins and Src family kinases.131 Disruption of this complex either through down-regulation of caveolin expression or through the disruption of the B1-uPAR interaction reduces the ability of these cells to migrate and blocks the B1-Src kinase association. Unfortunately, there are few data available for tumor and endothelial cells and for non-& integrindependent associations of uPAR on cell surfaces. Thus, it is difficult to speculate on whether uPAR-dependent signal transduction is generally dependent on caveolin. SUMMARY

A substantial body of data now supports the central role of the uPA system in tumor angiogenesis and metastasis. The components of this system have both negative and positive regulatory roles in these processes, which may depend on time, place, and location of the cell and the expression of the components of this system. A major challenge in the uPA field is how to translate the wealth of biological information now available to obtain therapeutically useful agents. As with other important biological cascades (e.g., MMPs) that have been implicated in tumor progression and angiogenesis, the relevance of the uPA system to human disease will

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