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The Plasminogen Activation System in Cell Invasion

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The plasminogen activator/plasmin system is an enzymatic cascade involved in the control of fibrin degradation, matrix turnover and cell invasion.

Extracellular conversion of the ubiquitous inactive plasminogen to the broad spectrum serine protease plasmin results in the recruitment of an enormous reservoir of potential proteolytic activity. Plasmin is, in turn, able to degrade fibronectin, laminin, vitronectin, proteoglycans, as well as fibrin and activate latent collagenases. Plasminogen activation is catalyzed by urinary (uPA) or tissue-type (tPA) plasminogen activators (PAs), which are subjected to time- and space-dependent regulation. In particular, uPA is regarded as the critical trigger for plasmin generation during cell migration and invasion, under physiological and pathological conditions (such as cancer metastasis), whereas tPA is likely to play an important role in the control of intravascular fibrin degradation. The system includes specific plasminogen activator inhibitors (PAIs) which counteract the activity of PAs, thereby restricting the generation of plasmin for extracellular matrix (ECM) as well as for intravascular fibrin degradation.

Like the proteases of the blood coagulation system, plasminogen and PAs are complex molecules bearing large noncatalytic regions, which mediate their regulatory interactions with matrix or cellassociated proteins. Membrane receptors for all components of the PA system ensure plasminogen activation at the cell surface, thus focusing proteolysis to the immediate pericellular environment.

In addition to its role in regulating the localization of proteolytic activity, the well characterized receptor for uPA (uPAR) has the ability to bind matrix vitronectin and to transduce signals upon ligation with catalytically inactive uPA. By these means, receptorbound uPA elicits biological outcomes, such as cell adhesion, migration and growth.

Relevant information provided by the analysis of mice with specific gene deficiencies has revealed that this system has a causal role in tissue regeneration, wound healing as well as in tissue involution, immune response, angiogenesis and cancer invasion. The causal link between the multiple effects observed in vivo and the complex molecular interactions of the PA/plasmin system with metalloproteases, integrin receptors, endocytosis receptors and growth factors is the subject of current investigation. Moreover, the clearcut involvement of PAs in cancer metastasis render the whole system an attractive subject for prognostic, diagnostic and therapeutic studies.

Introduction

Cell invasion requires that motile cells cross tissue barriers through the degradation of basement membranes and extracellular matrices (ECM). This is achieved through the cooperation of tightly regulated extracellular proteolytic cascades.1

The controlled generation of plasmin from plasminogen provides an efficient proteolytic activity directed to the degradation of fibronectin, laminin, vitronectin and proteoglycans.2 In addition, plasmin may represent a physiological activator of latent metalloproteases (MMPs), thereby switching on collagen degradation. Thus, the uPA-plasmin system may play a pivotal role in the general control of matrix degradation.3,4

Further activities of plasmin include the increased availability of active basic fibroblast growth factor (bFGF), as a consequence of ECM degradation, as well as the direct activation of latent transforming growth factor-β (TGF-β).5Therefore, the role of plasmin extends beyond ECM degradation to the control of cell growth and differentiation through growth factor activation.

In vivo, the multiple effects of plasmin activity emerge from the analysis of plasminogen-deficient mice which survive embryonic development but are impaired in growth, fertility and survival.6 In the same mice, skin wound healing is severely impaired, suggesting that plasmin activity is also required for tissue regeneration.7 Functional cooperation between the plasminogen/plasmin and the metalloprotease cascades is suggested by the finding that complete inhibition of the healing process requires both plasminogen deficiency and metalloprotease inhibition.8

The two known plasminogen activators, namely urinary-type (uPA) and tissue-type (tPA), have a restricted substrate specificity, as they recognize and cleave the R560V561 peptide bond in plasminogen. However, uPA can also activate the scatter factor pro-HGF, which exhibits extensive homologies with plasminogen, and the macrophage-stimulating protein (MSP), thereby controlling cell proliferation, invasion of extracellular matrix and prevention of apoptosis.9

Unlike the simple digestive proteases, in which only a signaling peptide and a short activation domain are attached to the catalytic region, the fibrinolytic proteases bear large noncatalytic regions.10 These regions contain functionally autonomous modules, such as “kringle”, “growth factorlike” or “EGFlike” and “finger” domains, which also occur outside the serine protease family. Phylogenetic trees constructed for the “kringle” and protease domains strongly suggest an independent domain evolution through exon shuffling.11

A graphic representation of the domain arrangement in plasminogen, uPA and tPA is shown in Figure 1. Some of these noncatalytic regions, which are essential for the biological activity of these proteases, may occur in vivo as degradation products with new functional properties. A typical case is that of angiostatin, which is an internal fragment of plasminogen comprising kringle domains 14 and part of kringle 5. Interestingly, this fragment can be generated by elastase activity or by plasmin autocatalysis.12 When given systemically, angiostatin strongly inhibits tumor growth and maintains tumor cells in a dormant state defined by a balance of proliferation and apoptosis.13

Figure 1. Graphic representation of the protein domains of plasminogen (PL), tissuetype plasminogen activator (tPA) and urokinase (uPA) proenzymes.

Figure 1

Graphic representation of the protein domains of plasminogen (PL), tissuetype plasminogen activator (tPA) and urokinase (uPA) proenzymes. The kringle (K), Finger (F), EGFlike (EGF) and catalytic (CD) domains are marked. The activation sites are indicated (more...)

Plasminogen and Plasminogen Activators

Plasminogen

Human plasminogen is a 791 aminoacid zymogen which can be activated to the broadspectrum twochain plasmin by a single proteolytic cleavage of Arg560Val562 peptide bond. The large aminoterminal region of plasminogen includes a “finger” module and five “kringle” domains, involved in regulatory interaction with fibrin and matrix proteins; the carboxyterminal domain is responsible for the serine protease activity (Fig. 1). Plasminogen occurs in blood plasma at the concentration of 12 μM and is also largely present in tissues. This enormous reservoir of proteolytic activity is controlled by urokinasetype and tissuetype plasminogen activators, which were originally identified in urine and tissue extracts, respectively.2

On the other hand, no significant additional pathways for physiological plasminogen activation can be envisaged in mice, as the phenotype of plasminogen-deficient mice is similar to that of mice carrying both uPA and tPA deficiencies.6

The Urokinasetype Plasminogen Activator (uPA)

The single chain prourokinase (prouPA) is secreted as a 411 aminoacids zymogen form and becomes activated by plasmin cleavage of K158I159 peptide bridge, thus generating uPA, a two-chain molecule held together by a single disulfide bond. Pro-uPA can be also cleaved by cathepsin B or kallikrein, which may indirectly control plasmin generation. In vivo, pro-uPA may be activated by glandular kallikrein mGK6, as shown in the urine from plasminogen-deficient mice.14

A further cleavage at K135K136 releases prouPA aminoterminal domain (ATF, aminoacids 1135), which includes an “EGFlike” and a “kringle” domain. The remaining carboxyterminal region (LMW urokinase, amino acids 135-411) retains full catalytic activity (Fig. 1). Pro-uPA undergoes several posttranslational modifications, such as glycosylation of Asn 302, phosphorylation on Ser138/303 and fucosylation of Thr18, located in the EGF-like domain.1517

The 6.4 kb pro-uPA gene has been isolated and characterized. The analysis of the exonintron organization relatively to the protease domains strongly suggests that exons encoding single functional domains were exchanged between different genes by recombination events.18 A variety of biological, chemical and physical agents affect the rate of prouPA transcription, thus modulating mRNA and protein levels.19The uPA promoter region is highly responsive to phorbol esters, growth factors, steroid hormones and cytoskeletal changes through the activity of a transcriptional enhancer located about 2 kb upstream of the cap site. Recent data assign to the Ets1 and Ets2 transcription factors the link connecting epidermal growth factor stimuli with activation of uPA and 92 kDa type IV collagenase (MMP9) promoters. This mechanism is likely to facilitate invasion and metastasis in breast cancer.20 Constitutive expression of uPA leading to an highly metastatic phenotype is achieved by increasing mRNA stability in MDAMB231 breast carcinoma cells.21

The Tissue-type Plasminogen Activator (tPA)

Unlike uPA, the 527 amino acid single-chain human tPA is proteolytically active. Plasmin cleavage of Arg275-Ile276 peptide bond yields a disulfide-linked two-chain molecule, which exhibits a 10-50-fold increased activity with respect to the uncleaved form. The N-terminal region contains a “fibronectin-type II” domain, an “EGF-like” domain and two “kringle” domains (Fig. 1). Fucosylation of Thr61, which may be relevant to the tPA clearance process in the liver has also been reported.22 Similarly to uPA, a large catalytic domain is located at the carboxy terminus.

It is generally assumed that the major role of tPA is to degrade fibrin in blood vessels. The activity of tPA is, in fact, greatly stimulated by fibrin which interacts with kringle 2, finger and EGF-like domains. Accordingly, tPA synthesis is induced under ischemic conditions. Also, in tPA-deficient mice, clot lysis is strongly impaired whereas, in uPA-deficient mice, there is an occasional fibrin deposition.23

However, other roles for tPA are emerging from the analysis of nervous system development in mice. In tPA/ mice, cerebellar granule neurons migrate significantly slower than granule neurons from wildtype mice; as a consequence, late arriving neurons are impaired in their synaptic interactions.24

Plasminogen Activator Inhibitors

Plasminogen Activator Inhibitor-type 1 (PAI1)

Specific inhibitors of plasminogen activation belong to the serine protease inhibitor superfamily (SERPINS).

The 379 amino acid plasminogen activator inhibitor-type 1 or PAI1 is the primary inhibitor of plasminogen activators in plasma and in the pericellular matrices. Covalent complex formation occurs between the reactive center loop of PAI1 and the active site serine of uPA or tPA through an ester bond.25 Pro-uPA does not react with PAI1, whereas two-chain active uPA rapidly associates with this inhibitor. However, when two-chain uPA is phosphorylated on Ser138/303, its capability to form complexes with PAI1 is strongly reduced, although the catalytic ability to activate plasminogen is unchanged.26

The physiologically relevant and stable form of PAI1 is in complex with vitronectin, a glycoprotein occurring in plasma and in the extracellular matrices. This association, which stabilizes the inhibitor in a reactive conformation, involves the N-terminal somatomedin B domain of vitronectin (amino acids 144). Interestingly, the latter domain overlaps with the binding site of vitronectin to the αvβ3 integrin. Therefore, PAI1 may prevent vitronectin binding to vitronectin receptor, thereby interfering with vitronectin receptor-dependent effects, such as smooth muscle cell migration.27 Because of its ability to associate with vitronectin, PAI1 may also prevent vitronectin binding to the urokinase receptor, thereby inducing cell release from vitronectin substrates.28

These previously unrecognized interactions are independent of PAI1 capability to function as a protease inhibitor. However, it is conceivable that active uPA may regulate integrin-dependent effects by complexing PAI1, thereby releasing vitronectin which can bind to vitronectin receptor. As a consequence, the level of tree PAI-1 can affect the biological outcome/s of vitronectin/vitronectin receptor in a particular context.

Plasminogen Activator Inhibitor-type 2 (PAI2)

Plasminogen activator inhibitor 2 or PAI2 is a 47-kDa single chain protein. This inhibitor blocks uPA and, less efficiently, tPA, although not as fast as PAI1, raising the possibility that PAI2 may have some yet unidentified roles. The physical localization of PAI2 is peculiar, as this molecule exists in a 60-kDa glycosylated secreted and in a 47-kDa cytosolic forms, both consisting in 415 amino acids devoid of an N-terminal signal peptide sequence. These two N-glycosylated forms of PAI2 are generated by facultative translocation.29

The role of intracellular PAI2, which accounts for most of the produced inhibitor, still remains to be elucidated. Recent data show that it may play a role in macrophage protection from TNFa-mediated apoptosis. Although PAI2 ability to inhibit uPA and tPA is poorer than that exhibited by PAI1, extracellular PAI2 is considered and inhibitor of plasmin generation both in blood vessels as well as in the ECM. Like PAI1, extracellular PAI2 binds to receptorbound uPA; however, uPAPAI2 complexes are rapidly cleaved into a 70-kDa fragment, which is endocytosed or released and a 22-kDa species which remains on the cell surface preventing the binding of intact uPA.30

Plasminogen Activator Receptors

Receptors for Plasminogen and for tPA

The study of the plasminogen activator/plasmin system has revealed that the proteolytic components interact with cell surface through residues not directly involved in the catalytic mechanism.

Plasminogen binds to cells with low affinity and high capacity via its “kringle” domains which recognize carboxyterminal lysines of proteins exposed on cell surface. Following binding to membrane receptors, plasminogen is activated more efficiently; bound plasmin exhibits an increased enzymatic activity and is protected from inhibition by a2-antiplasmin and a2-macroglobulin.31,32

Functional evidence for specific and saturable binding sites for tPA, involved in cell surface plasmin generation has been recently obtained in endothelial cells. High affinity binding of tPA on vascular smooth muscle cells occurs by a novel mechanism involving the serine protease domain of tPA and leading to the stimulation of cell-associated plasminogen activation.33 Saturable high affinity binding sites for tPA on rat and human hepatoma cells which mediate internalization and degradation of the bound protease by a PAI1 and mannose receptor-independent mechanism. Clearance of tPA occurs through low density lipoprotein receptor protein (LRP) which plays an important role in the clearance of circulating tPA, thus regulating plasma fibrinolytic activity.34 The role of specific noncatalytic regions in this recognition/internalization process is documented by the inhibition of rat hepatocytes clearance by the recombinant tPA “finger/“EGF-like” regions.22

The Urokinase Receptor (uPAR)

The interaction of uPA with cell surface has received considerable attention over the past 15 years. The initial studies showed that uPA binds specifically and with high affinity to human blood monocytes and to monocyte-like U937 cells.35 Urokinase ability to become membrane-associated is retained by the purified amino-terminal fragment of uPA (ATF, residues 1135) of uPA. Interestingly, uPA receptors (uPARs) are subjected to a 10- to 20-fold differentiation-dependent increase in U937 myelomonocytic cells.36 Binding to uPAR occurs through the “EGFlike” domain of uPA, with a Kd in the nanomolar range.37 In particular, Lys23, Tyr24, Phe25, IIe28, and Trp30 in the B loop are important determinants for uPA binding.38

The 313 residues uPAR molecule becomes membrane-associated with a concurrent removal of a 21 amino acid signal peptide and is processed to add a glycosyl phosphatidylinositol (glycosylPtdIns) (GPI) anchor to the carboxy-terminal region which targets it to cell membrane.39 Site-directed mutagenesis of the uPAR carboxy-terminal region indicated that Gly283 is the likely attachment site.40 The uPAR includes 28 cysteine residues arranged in three homologous repeats and can be assigned to the Ly6 superfamily which includes CD59 and a variety of elapid snake venom toxins.41 The uPAR exhibits a three-domain structure: the amino-terminal D1 domain which directly contacts uPA, the linker D2 domain and the carboxy-terminal D3 domain which maintains the ligand receptor high affinity interaction. Upon binding to uPA, uPAR undergoes a conformational change that uncovers the linker region between D1 and 2 that has a potent chemotactic activity. This conformational change can be mimicked in vitro by enzymatic processing of a soluble uPAR with chimotrypsin, that exposes a chemotactic epitope (residues 8892, SRSRY). The cleaved suPAR and the isolated chemotactic epitope exhibit a strong chemotactic activity in the subnanomolar range.42,43

D2 and D3 domains are involved in a high affinity interaction with the matrix protein vitronectin which is promoted by simultaneous binding of uPA or ATF.44 However, efficient binding to vitronectin only occurs with intact uPAR.45 The ability of uPAR to form ternary complexes with ATF and also vitronectin has been observed in cell lines, such as MCF7 breast carcinoma or HT1080 fibrosarcoma cells and in membranes from breast ductal carcinoma specimens.46 Recent findings uncovered the possibility that uPAR may interact with a cleaved form of kininogen in a zinc-dependent manner, through D2 and D3 domains. This interaction may underlie uPAR ability to promote kallikrein-dependent cell surface plasmin generation.47

In the GPI-anchored form, unoccupied uPAR is relatively mobile. Upon ligation with uPA, uPARs stably cluster at focal adhesions and cell-cell contacts.48 This property is likely to play an important role in concentrating cell-surface proteolysis at the leading edge of migrating cells, as shown in monocytes.49

By virtue of its N-terminal signal peptide, anchorless uPAR can be secreted in the extracellular milieu. Several mechanisms may account for the lack of uPAR anchoring. Alternative splicing generates a protein lacking the carboxy-terminal amino acidic sequence for GPI anchor attachment. This mRNA variant is expressed in different human cell lines and tissues and upregulated by phorbol ester in A549 cells.50

An anchorless uPAR variant is secreted from blood leukocytes affected by the stem cell disorder paroxysmal nocturnal hemoglobinuria (PNH), due to a defect in the synthesis of GPI anchors. Unlike normal leukocytes, the PNH-affected cells do not express surface uPARs, although they contain apparently normal levels of uPAR-specific mRNA. In this context, uPAR is found in plasma as well as in the conditioned medium from cultured PNH leukocytes.51 A full-length soluble uPAR missing the GPI region is also found in ascites and serum of patients with ovarian carcinoma.52 However, in this case uPAR release is likely to be catalyzed by a cellular phospholipase D, which cleaves GPI anchors.53 Short forms of soluble uPAR also exist in vivo as a result of limited proteolytic cleavage. Fragments corresponding to the uPAR domains D1 and D2+D3 have recently been isolated from human urine.54

Cell Surface-Associated Plasminogen Activation

The concept of pericellular proteolysis through the specific association of plasminogen activation components to the cell surface has received extensive experimental support. The uPAR is a high affinity site for prouPA, which is the major form of the enzyme in cells, tissues, and body fluids. After secretion, pro-uPA may become associated to uPARs expressed by the same cell, in an autocrine manner.55 Alternatively, a paracrine interaction would involve a cooperation between uPA-producing and uPAR-bearing cells. This model has been confirmed in vivo: a four-fold stimulation of chick embryo chorioallantoic membrane invasion by uPAR expressing mouse L-cells is observed following cocultures with uPA-producing L-cells.56

Regardless the producing cell, membrane-bound pro-uPA may be converted by plasmin or, possibly, other proteolytic enzymes to two-chain uPA, not significantly affecting the binding parameters and not inducing uPA release.57 Moreover, while bound to uPAR through its amino-terminal moiety, the carboxy-terminal catalytic domain of uPA retains full catalytic activity. In particular, kinetic studies have shown that receptor-bound uPA exhibits a 40-fold lower Km than soluble uPA and a concurrent 6-fold reduction in kcat, thus resulting in an overall increase of catalytic efficiency.58 Since plasminogen can become membrane bound, the occurrence of receptors for plasminogen and uPA on the same cell results in the formation of surface-associated plasmin.59 This machinery generates broad-spectrum proteolytic activity which is restricted to cell surface and protected by circulating inhibitors, such as a2-antiplasmin.

Unlike bound plasmin, uPAR-bound uPA can still interact with the inhibitor PAI1, which is therefore able to inhibit plasmin formation. Then, the uPAPAI1 complex bound to uPAR is internalized and degraded.60 This clearance process occurs through direct binding of uPAR D3 domain to low density lipoprotein receptor (LRP) and can be blocked by purified recombinant D3 domain.61

All these data, taken together, suggest the existence of an uPA cycle that can be summarized as follows: after synthesis pro-uPA is secreted, bound to the receptor and activated to two-chain uPA. On the membrane, uPA can activate surface-bound plasminogen to produce surface-associated plasmin. However, in the presence of PAI1, uPA activity is inhibited and plasmin generation interrupted, while the uPA-PAI1 complexes are internalized and degraded. Therefore, PAI1 blocks uPA activity and also causes its degradation.

It is beyond doubt that in culture, cell surface is the site of a powerful proteolytic activity. In vivo, cell surface uPA is proven to be relevant, at least in one case. Mice overexpressing either uPA or uPAR in basal epidermis and hair follicles do not exhibit detectable alterations. However, the combined overexpression of both uPA and uPAR resulted in an extensive alopecia, as a consequence of hair follicles' involution, epidermal thickening and subepidermal blisters. These findings show that uPA and uPAR act synergistically in promoting pathogenic extracellular proteolysis in vivo and confirm the importance of uPAR in directing surface-associated proteolysis.62

Biological Role of the uPA/uPA Receptor System

uPAR and Cell Adhesion

Early evidence pointed to a role of uPAR in the enhancement of myelomonocytic cell adhesion. If anchorage-independent nondifferentiated U937 cells are incubated for 20 h with transforming growth factor type β-1 (TGF-β), 1,25(OH)2-vitamin D3 and, subsequently, with nanomolar concentrations of diisopropyl fluorophosphate-inactivated urokinase (DFPuPA), they become adherent within minutes.63

The acquisition of uPA-induced adherence is accompanied by dramatic cytoskeletal changes and by a rapid inhibition of p56/59(hck) and p55(fgr) tyrosine kinases. This transient p56/59(hck) downregulation is required for adherence, as this process is inhibited by the expression of a constitutively active p56/59(hck) variant. Ligand-independent adherence can also result from the expression of a kinase-defective p56/59(hck) variant, confirming that p56/59(hck) downmodulation increases adherence and that this switch is controlled by uPA.64

Exposure of myelomonocytic cells and other cell types, such as melanoma cells, to uPA causes a specific increase of cell adhesion to vitronectin.65,66 Although the underlying mechanisms of uPA-induced adhesion have not been fully elucidated, a reasonable possibility is that engaged uPAR may directly bind matrix vitronectin, as shown with recombinant molecules in vitro.45 This hypothesis is also supported by the occurrence of ternary complexes with uPAR, vitronectin and uPA in breast cancer cell membranes.46 Recent data also indicate that vitronectin binding to uPAR initiates a p130Cas/Rac-dependent signaling pathway controlling actin polymerization state.67 However, direct binding of uPAR to vitronectin does not provide a satisfactory explanation of the uPA ability to enhance general cell adhesion. For example, in freshly isolated monocytes, urokinase receptors (CD87) form complexes with the β2-integrin complement receptor 3 (CR3, CD11b/CD18) and this association increases adhesion of CR3 to fibrinogen. Conversely, adhesion of monocytes to fibrinogen promotes CR3uPAR association at the monocyte/macrophage ventral surface.68

Recent evidence suggests that uPAR may be a dynamic regulator of integrin function, possibly, through “lateral” interactions. First of all, uPAR is associated in large molecular complexes with various integrins.69 Secondly, the physical interaction uPAR/activated integrin can be reproduced in vitro and disrupted in vivo by a specific peptide homologous to integrin sequences.70 Functional consequences of this dissociation include the inhibition of integrin-dependent spreading and migration.71 Finally, the concept that uPAR signals through integrin activation is fully supported in a variety of systems, in which anti-integrins antibodies inhibit uPA-dependent signaling. Among the most significant examples linking uPAR association with integrins to the biological outcomes is the activation of α5β1-integrin by uPAR in Hep3 human carcinoma cells which generates a persistently high level of active extracellular signal-regulated kinase1 (ERK1) necessary for tumor growth in vivo. Disruption of uPAR-a5b1 complexes with peptides or antibodies inhibits the fibronectin-dependent ERK1 activation, thereby reducing tumorigenicity.72 A further level of complexity is added by the recent finding that uPAR is a true integrin ligand in both soluble and GPI-anchored forms. In fact, it specifically binds to integrins on adjacent cells, suggesting that uPAR-integrin binding may also mediate cell-cell interaction.73

uPAR and Cell Migration

As shown over a decade ago, both uPA native molecule and the ATF are true chemoattractants in the Boyden system, a two-compartment device for testing random and directional cell migration. The involvement of uPAR is supported by the finding that ATF-induced endothelial cell translocation is impaired by antibodies which inhibit ligand-receptor interaction.74 Inactivation of uPA does not affect its chemotactic ability, whereas the reduction of uPAR expression with an antisense oligonucleotide strongly inhibits chemotaxis, showing that uPAR is required for migration.75

Since uPAR expression is widespread, a variety of cell lines and primary cells respond to nanomolar concentrations of uPA, either DFP-inactivated or lacking the catalytic domain, by increasing their motility.76 In vivo, the important role of uPAR in directing cell locomotion emerges from the analysis of uPAR-deficient mice which exhibit a reduced neutrophil recruitment in response to P. aeruginosa pneumonia as compared to control mice.77 Interestingly, the impaired neutrophil migration in uPAR-deficient mice is not due to the disruption of uPAR-mediated proteolysis, but to uPAR occupancy.78

Although uPA-directed chemotaxis is dependent on uPAR, the underlying events may be far more complexes than expected. For example, uPA phosphorylated on Ser138/303 binds to uPAR with unchanged affinity, but it is unable to elicit cell migration. This suggests that binding to uPAR is not sufficient for uPA-dependent cell mobilization.16 It is beyond any doubt that cell response to uPA involves the functional and physical association of uPAR with integrin receptors. In breast carcinomas, uPAR physically associates with αvβ5 vitronectin receptor and this association leads to a functional interaction of these receptors. In breast cancer cells, anti-αvβ5 antibodies inhibit uPA-dependent migration, showing that vitronectin receptor is required for uPA signaling.79 In rat smooth muscle cells, migration and cytoskeletal rearrangements are inhibited by anti-uPAR or anti-αvβ3 vitronectin receptor antibodies, suggesting that a functional association between these receptors is required to elicit both responses.80

Migration is a complex process which involves the dynamic participation of cytoskeletal structural and regulatory components. THP1 macrophage-like cells migrating along a chemotactic ATF gradient undergo a transient activation of the p56/p59hck Src family tyrosine kinase. Under similar conditions, uPAR physically associates with p56/p59hck.42 A general role of p56/p59hck in migration can be envisaged, as expression of a constitutively active p56/p59hck variant enhances U937 monocyte-like cell migration in a ligand-independent manner.64

ATF-dependent stimulation of MCF7 breast carcinoma cell migration results in the activation of ERK1 and ERK2 kinases. Responses to uPA and ATF are blocked when the cells are pretreated with PD098059, an inhibitor of mitogen-activated protein kinase kinase.81 A p130Cas/Rac-dependent cascade leading to actin reorganization and increased cell motility is activated by vitronectin binding to uPAR and may play a role at sites where vitronectin and uPAR are coexpressed.67

uPAR and Cell Growth

The biological outcomes of the uPA/uPAR interaction are not just related to cell migration, adhesion and actin polymerization state. In the human melanoma cell line GUBSB, inhibition of receptor-bound uPA by specific anti-uPA antibodies reduces cell proliferation, suggesting that cell growth is constantly stimulated by uPAR occupancy, in an autocrine fashion.82 Additional information is provided by a recent report confirming that ATF is a mitogen for melanoma cells in culture through a yet unidentified membrane-associated mediator of uPA-dependent signal transduction.83 In vivo, it has been recognized that uPA favors the formation and growth of melanomas, as described later in this chapter. Growth stimulatory effects have been observed also in human SaOS2 osteosarcoma cells exposed to ATF. In this case, fucosylation of Thr18 within the “EGFlike” domain seems to be required for eliciting this response.17

In vivo, a 70% reduction of the uPAR level in the human carcinoma HEp3 cells inoculated into chicken chorioallantoic membrane, while not affecting growth in culture, induces a state of tumor dormancy. The observed G(0)/G(1) arrest may be due to scarce uPA/uPAR/ αvβ5 complex formation and, consequently, insufficient ERK1 activity needed for tumor growth in vivo.84

uPAR Signaling Mediators

It is generally assumed that GPI-anchored proteins initiate intracellular signaling through the interaction with transmembrane receptors. In this respect, receptor lateral mobility may be essential to the uPAR signaling mechanism.48 Further evidence of the uPAR ability to move to specific cell sites has been obtained by autoradiographic detection of receptor-bound uPA at the leading edge of migrating human monocytes.49

A relationship between uPAR aggregation and signaling initiation is suggested by the increase in Ca2+ influx and induction of adherence in polymorphonuclear neutrophils crosslinked to anti-uPAR antibodies, which induce uPAR clustering.85 Circumstantial evidence that ligand-dependent uPAR aggregation is required for signaling emerges from the study of uPA phosphorylated on Ser138/303 or carrying Glu138/303, which are neither chemotactic nor proadhesive. These nonsignaling uPA forms are also unable to induce uPAR clustering in U937 monocyte-like cells, as shown by confocal microscopy.16

In most cases, GPI-anchored proteins are localized in lipid-enriched membrane microdomains known as lipid rafts which contains various proteins, including caveolin.86,87 In particular, the latter protein is known to enhance the extent of uPAR-mediated cell responses and is also found in complexes with uPAR, β1 integrins and Src family kinases.88 Similar complexes have been detected in leukocytes, in which uPAR associates with p60fyn, p53/56lyn, p58/64hck, and p59fgr tyrosine kinases, as well as with the integrins LFA1 and CD11b/CD18.69 As discussed earlier, uPAR capability to associate directly to integrin receptors has received broad experimental support. These findings are consistent with the inhibition of uPAR signaling by anti-integrin blocking antibodies in a variety of cells. Interestingly, unlike vitronectin-dependent signaling, uPA-dependent signaling requires protein kinase C in both MCF7 and HT1080 cell lines. This finding, together with the evidence that anti-αvβ5 antibodies block uPA-dependent signaling suggests that uPA directs cytoskeletal rearrangements and cell migration by altering αvβ5 signaling specificity.79

All these observations support a model in which mobile uPAR complexed with caveolin and signaling molecules dynamically associates to ligand-clustered integrins, thereby activating signaling. The complex array of uPAR partners and signaling mediators may be tissue-specific or unique to a particular cell differentiation stage (Fig. 2).

Figure 2. Model for the urokinase-activated, catalytic-independent signaling cascade leading to cell migration, adhesion and growth.

Figure 2

Model for the urokinase-activated, catalytic-independent signaling cascade leading to cell migration, adhesion and growth. A. The non-engaged uPAR is located in lipid-enriched membrane microdomains together with caveolin, Src kinases, focal adhesion kinase (more...)

Novel associations between uPAR and other membrane receptors leading to functional effects have been described. For example, L-selectin mediates uPAR-dependent Ca(2+) mobilization in polymorphonuclear neutrophils.89 The existence of a link between G-proteins and uPAR signaling is suggested by the finding that the pro-adhesive ability of pertussis toxin on differentiating U937 cells is prevented by anti-uPAR antibodies.90 In rat smooth muscle cells, uPA-dependent chemotaxis correlates with a dramatic reorganization of actin cytoskeleton and adhesion plaques: these effects are sensitive to pertussis toxin, thereby supporting the involvement of G proteins.91

Consistently with uPAR ability to direct cytoskeletal rearrangements and cell migration, focal adhesion kinase (pp125FAK) and paxillin are tyrosine phosphorylated in bovine aortic endothelial cells exposed to uPA.92 Mobilization of MCF7 breast cancer cells initiated by single-chain uPA or ATF requires the activation of a signaling cascade which includes Ras, MEK, ERK1 and myosin light chain kinase (MLCK).93

Other effects include activation of the Janus kinases Jak1 and Tyk2 which mediate uPA-induced activation of transcription in human vascular smooth muscle cells. In particular, Tyk2 triggers a signaling cascade leading to phosphatidylinositol 3-kinase (PI3K) activation. Inhibition of Tyk2 or PI3K prevents uPA-dependent migration.94 Exposure of HT1080 cells to catalytically inactive uPA results in cfos mRNA stimulation and PAI2 induction, possibly through AP1 transcriptional activator.95 Finally, in breast carcinoma cells uPAR engagement activates a signaling cascade resulting in a rapid upregulation of the transcriptional factor Sp1 binding activity, which, in turn, may upregulate uPAR levels.96

Plasminogen Activators and Tissue Remodeling

In the emerging picture, plasminogen activators participate in a wide spectrum of biological events, including the remodeling of the normal surrounding tissue induced by cancer cells, as well as the nonneoplastic tissue involution and regeneration processes. Urokinase-type plasminogen activator expression is induced in the mouse mammary gland during development and postlactational involution. Results confirming the important role of plasmin have been obtained in plasminogen-deficient mice which are impaired in the lactational differentiation and mammary gland remodeling during involution.97 Increasing evidence shows that plasmin activity is not just involved in the degradation processes but is also required for tissue regeneration. In acute liver injury, induced by carbon tetrachloride intoxication, mice with targeted disruption of uPA gene exhibit a remarkable delay in the repair process. Interestingly, tPA-deficient mice are slightly defective in the hepatic regeneration. In vivo experiments have assessed that the accumulation of fibrin, fibronectin and necrotic cells within injured areas are responsible for the delayed regeneration process.98 A severe regeneration defect is observed in uPA deficient mice with experimentally damaged skeletal muscle.99 Similarly to the previous case, tPA-deficient mice are indistinguishable from controls, suggesting that uPA is selectively involved in the regeneration processes. In the remodeling processes, the plasminogen/plasmin system acts together with the metalloproteases system: this possibility is supported by the requirement for both plasminogen deficiency and metalloprotease inhibition to prevent the wound healing process.8

Plasminogen Activators and Invasion in Animal Models

A reasonable hypothesis is that permanent alterations in the proteolytic balance of malignant cells may contribute to tumor dissemination. Early evidence showed that inhibition of plasminogen activation may prevent tumor metastasis in animal models. In the first report, human carcinoma HEp3 cell were allowed to grow on the chorioallantoic membrane and metastasize to the chicken embryo. Inoculation of anti-uPA antibodies delayed the onset of pulmonary metastases.100 Similar results were obtained in mice inoculated with B16 melanoma cells which are impaired in their ability to metastasize following preincubation with inhibitory anti-uPA immunoglobulins.101 More recently, the analysis of tumor growth in mice with targeted gene disruptions has provided new information on the role of individual plasminogen activation/plasmin components. In plasminogen-deficient mice, expressing polyoma middle T antigen, the occurrence and the number of pulmonary metastases are significantly reduced as compared to wild-type controls, indicating that tumor dissemination is enhanced by plasmin-dependent matrix degradation.102 Less obvious is the effect of uPA deficiency on tumor growth and dissemination: chemically induced melanocytic neoplasms in wild-type mice progress to melanomas, whereas the uPA-/- mice are not susceptible to melanoma induction. This suggests that uPA contributes to malignant progression, possibly by decreasing the liberation and availability of growth factors such as basic fibroblast growth factor.103

The absence of uPAR also negatively affects tumor growth, as shown by the reduced in vivo tumorigenesis of human carcinoma (HEp3) cells bearing a 70% reduced surface uPARs. If these receptor-deficient cells are inoculated in the chorioallantoic membrane of the chick embryo, they enter a state of dormancy, characterized by survival without progressive growth. This seems to be attained through regulation of the balance between the mitogenic extracellular regulated kinase ERK1 and the apoptotic/growth suppressive stressactivated protein kinase 2, p38(MAPK).72 An antisense approach to reduce surface uPARs of the highly metastatic HCT116 colon carcinoma cells resulted in an impairment of cell ability to degrade the surrounding matrix. In mice, the artificial downregulation of uPAR strongly reduces the number of pulmonary metastases following intravenous injection of HCT116 cells.104

The idea that PAI1 may not be a rate-limiting step in tumor progression was first suggested by PAI1 overexpression in human malignant tumors and by its correlation with a poor prognosis. Also, in PAI1-deficient mice, local invasion and tumor vascularization of transplanted keratinocytes is impaired; intravenous injection of an adenoviral vector expressing human PAI1 restores both invasion and angiogenesis.105,106 Although it is known that PAI1, beyond its inhibitory role, binds to vitronectin, thus causing its dissociation from vitronectin receptor and interfering with cell migration/adhesion, the resulting balance of these effects in vivo is unpredictable; in any event, the data obtained in knockout animals suggest that PAI1 is required for tumor growth. Some molecular insights have been provided by the analysis of tumor growth in mice bearing single and combined deficiencies of uPA, tPA, uPAR, vitronectin and plasminogen. Interestingly, the data indicate that PAI1 promotes tumor growth by supporting angiogenesis: it is noteworthy that these effects are related to the PAI1 inhibition of proteolytic activity, suggesting that excessive plasmin proteolysis prevents tumor vessels formation.107 Similarly to PAI1, the relationship between PAI2 and tumor invasion is not obvious: mice overexpressing PAI2 are more susceptible to skin carcinogenesis and develop epidermal papillomas. Interestingly, overexpressed PAI2 accumulated predominantly in cells and failed to inhibit extracellular uPA, suggesting that the observed stimulation of tumor progression by PAI2 is independent of uPA inhibition.108

Plasminogen Activators and Human Tumors

Tissue Distribution

Cancer invasion and metastasis is a process requiring an efficient ECM degradation which is accomplished through the activity of several proteolytic cascades, including the plasminogen/plasmin system. Based on these considerations, a positive correlation between malignancy and elevated levels of plasminogen activation/plasmin components would be expected.

Consistent with this possibility, the expression level of uPA and uPAR in the malignant tumors are generally higher than in the normal counterparts; according to the results of a quantitative study, breast carcinomas contain 5 times more uPAR and 19 times more uPA than benign breast lesions.109 In malignant astrocytomas, especially in glioblastomas, uPA activity is significantly higher with respect to normal brain tissues or lowgrade gliomas.110 However, quantitative studies do not provide any information on the site of PAs production. Studies on the tissue distribution of uPA and uPAR have been carried out by immunocytochemistry and in situ hybridization on colon and breast tumor sections. Receptor-bound uPA is detected on the epithelial breast cancer cell membrane, suggesting that surface proteolytic activity contributes to the invasive phenotype.111 In human colon cancer, uPA protein and mRNA are expressed in tumour-infiltrating fibroblast-like cells at the invasive foci whereas uPAR is expressed in cancer cells. Thus, surrounding stromal cells actively contribute to tumor invasion, similarly to tissue remodeling events.112 In breast carcinomas, uPA may be expressed by the epithelial component or, more frequently, by fibroblast-like stromal cells.113 Interestingly, fibroblastic rather than epithelial expression of uPA, uPAR and PAI1 is positively correlated with tumor size and predicts a poor prognosis.114 The latter studies confirm the complex interplay occurring between cancer and surrounding stromal cells, thus supporting an important role of fibroblast-like cells in the generation and regulation of pericellular proteolysis.

Prognosis and Diagnosis

In the treatment of cancer there is a need to select patients at high risk of recurrence for adjuvant therapy. In many cases, lymph node status is insufficient to predict whether these patients will experience a relapse and specific markers would be needed. Because of the well established correlation between the plasminogen/plasmin system and tumor invasion, the molecular components of this system are potentially useful markers of the tumor metastatic potential. Many studies directed to assess the prognostic impact of the plasminogen/plasmin components have been conducted, mostly based on antigen level quantitation in tissue extracts from surgically removed tumors. These values have been subsequently correlated with prognosis in several types of cancers.115 In breast cancer, high levels of uPA were associated to a high risk of recurrence and short survival, suggesting that uPA is a more reliable marker than axillary node status, tumor size and estradiol receptor.116 In a variety of neoplastic conditions, high levels of uPA and uPAR are associated to a poor patient prognosis.117

Vice-versa assessment of tPA expression resulted in the identification of patients with a low probability to relapse in melanomas, as lesions with 51-100% tPA-positive tumour cells are associated to the best prognosis, whereas lesions with 6-50% tPA-positive tumour cells to the worst.118

An accurate measurement of preformed uPA-PAI1 complexes with respect to total uPA and total PAI1 has been performed in tissue extracts from breast cancer patients. Surprisingly, high PAI1 levels are bad prognostic indicators, whereas uPA-PAI1 complex levels predict long recurrence-free survival and overall survival.119 The focus of another study was to assess the level of uPA, uPAR and PAI1, as well as the expressing cells and their relationship with tumor clinical and pathological data. Interestingly, patients with a strong expression of uPA, uPAR and PAI1 in fibroblast-like stromal rather than in tumor cells have a high probability to experience a relapse.114

From a clinical point of view, quantitation of these molecules in blood or in urine rather than in tissue samples is highly desirable both for patients prognosis as well as for follow-up purposes. This seems to be a realistic possibility, as plasma levels of uPA are, in fact, elevated in breast, prostate, head and colon cancer patients, as compared to control plasma samples from healthy donors.120 Furthermore, in serum and ascites of patients with ovarian carcinoma is present the full-length soluble uPAR lacking GPI-anchor. Interestingly, high preoperative levels of suPAR predict a poor outcome. In addition to two-chain and LMW uPA, urine samples from healthy volunteers contain measurable amounts of soluble uPAR (suPAR). Urinary suPAR levels are elevated in patients with different types of cancer. Interestingly, part of the suPAR from urine and tumor tissue extracts is present in a cleaved form.121

In acute myeloid leukaemia patients, a high level of plasma suPAR is found, which correlates with a poor response to chemotherapy. Also, suPAR concentration in plasma and urine decreases during chemotherapy treatment and depends on the number of circulating tumor cells.122

Therapy and Antagonists

If cancer dissemination is indeed promoted by unrestrained matrix degradation, one obvious therapeutic approach is to design specific protease inhibitors to be employed as antimetastatic agents.123,124 To this purpose, a novel X-ray crystallography-driven screening technique has been recently employed for the discovery and optimization of a new orally available class of uPA inhibitors for cancer treatment.125

New approaches include specific targeting of the PA components on tumor cell surface for tumor-selective cytotoxins. Considering that most tumor cells overexpress uPAR, they could be killed by a fusion protein between urokinase and saporin (a ribosomeinactivating protein) which is subjected to a PAI1-mediated internalization.126 If tumor cells bear receptor-bound uPA, a mutated anthrax toxin protective antigen in which the furin cleavage site is replaced by sequences cleaved by uPA may be selectively activated on cell surface. This causes internalization of the recombinant cytotoxin, thereby killing the uPAR-expressing tumor cells.127

Although the control of excess proteolytic activity in tumors is desirable, novel therapeutic approaches should take into account the nonproteolytic roles of PAs, as well as uPAR physical and functional association with integrins, leading to cell mobilization. Inhibition of uPAR interaction with integrins by the aid of specific peptides has been successfully attempted in model systems.71,123

Another approach to control malignancy is to reduce uPAR expression level, which results in tumor dormancy, a novel promising anticancer strategy in which tumor cell proliferation is balanced by apoptosis.76 This effect has already been obtained in vivo by the angiogenesis inhibitor angiostatin.13 The knowledge of angiostatin biology is now being transferred to the clinical practice: phase I clinical trials with recombinant angiostatin (kringles 13), as well as with an “angiostatin-cocktail” which induces the conversion of plasminogen to kringle 14 and part of kringle 5 are currently ongoing.128

Naturally occurring inhibitors of uPAR signaling have also been described. As mentioned earlier, serine phosphorylated uPA lacks the motogen ability, although it retains uPAR binding. The formation of such a receptor competitive antagonist is dependent on protein kinase C activity, which regulates the in vivo phosphorylation state of uPA.129

A novel inhibitor of tumor growth and invasion for which the mechanism of action is unknown has recently been described. An 8-mer capped peptide (A6) corresponding to the amino acids 136-143 of uPA inhibited breast cancer cell invasion and endothelial cell migration in a dose-dependent manner in vitro without altering cell doubling time. Intraperitoneal administration of A6 results in a significant inhibition of tumor growth and lymph node metastases development, in several models of breast cancer cell growth and metastasis.130 The combination of A6 and cisplatin efficiently inhibits malignant glioma growth and significantly reduces neovascularization, suggesting a mechanism involving A6-mediated inhibition of endothelial cell motility.131

Conclusions and Perspectives

New concepts are emerging in this field based on the growing molecular knowledge of the plasminogen/plasmin components and their plasmin-independent interactions. However, the in vivo relevance as well as the biological outcome of such molecular events is difficult to predict. Recently, mice with targeted disruption of specific genes highlighted the role of plasminogen/plasmin system components in tumor growth and dissemination, wound healing, tissue regeneration and involution. These apparently contradictory roles of PAs and PAIs can be reconciled with the reasonable assumption that time and space-regulated proteolysis may be required in all cases.

On the other hand, the unexpected finding that the level of PAI1 in tumors is associated to a poor prognosis together with its ability to promote angiogenesis by inhibiting plasminogen activation in mouse models is not unique to the plasminogen activation/plasmin system. Interestingly, high plasma levels of the metalloprotease inhibitor TIMP1 are correlated to advanced disease in colorectal cancer patients.132 Useful information to the interpretation of these findings and the design of new therapeutic strategies may be provided by the effects of the metalloprotease inhibitor batimastat which stimulates the outgrowth of capillary structures by human foreskin microvascular endothelial cells in a 3-dimensional fibrin matrix. It has been proposed that batimastat prevents the cleavage of uPAR D1 domain by MMP12, thereby increasing the number of functional uPARs on endothelial cells and stimulating capillary growth.133

These findings show that it is possible to counteract protease activity in vivo, but also suggest that the uncontrolled use of proteolytic inhibitors in pathological conditions, such as cancer, may lead to undesired effects.

Acknowledgments

I am grateful to F. Blasi, M.V. Carriero and P. Ragno for stimulating discussions on this subject and critically reviewing the manuscript. Most of the work from this group was supported by AIRC (Associazione Italiana per la Ricerca sul Cancro).

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