Nicolai Sidenius

The urokinase plasminogen activator system in cancer: recent advances and implication for prognosis and therapy.

Cancer dissemination and metastasis is synonymous with invasive cell migration; a process in which the extracellular matrix (ECM) plays the dual role of the substratum on which the cells move as well as the physical obstacle that the cells have to surpass. To degrade the physical obstacle, which the ECM poses in the direction of migration, cells use proteolytic enzymes capable of degrading the ECM components. A major protease system responsible for ECM degradation is the plasminogen activation system, which generates the potent serine protease plasmin. The subject of this review, the urokinase-type plasminogen activator (uPA) and its receptor (uPAR), plays an impressive range of distinct, but overlapping functions in the process of cancer invasion and metastasis: Firstly, uPA/uPAR promotes extracellular proteolysis by regulating plasminogen activation. Secondly, uPA/uPAR regulates cell/ECM interactions as an adhesion receptor for vitronectin (Vn) and through its capacity to modulate integrin function. Thirdly, uPA/uPAR regulates cell migration as a signal transduction molecule and by its intrinsic chemotactic activity. This review is focused on recent insight into the cancer related biology of the uPA/uPAR system as well as its implications for clinical cancer diagnosis, prognosis and therapy.

Proteolytic cleavage of the urokinase receptor substitutes for the agonist-induced chemotactic effect.

Physiological concentrations of urokinase plasminogen activator (uPA) stimulated a chemotactic response in human monocytic THP‐1 through binding to the urokinase receptor (uPAR). The effect did not require the protease moiety of uPA, as stimulation was achieved also with the N‐terminal fragment (ATF), while the 33 kDa low molecular weight uPA was ineffective. Co‐immunoprecipitation experiments showed association of uPAR with intracellular kinase(s), as demonstrated by in vitro kinase assays. Use of specific antibodies identified p56/p59hck as a kinase associated with uPAR in THP‐1 cell extracts. Upon addition of ATF, p56/p59hck activity was stimulated within 2 min and returned to normal after 30 min. Since uPAR lacks an intracellular domain capable of interacting with intracellular kinase, activation of p56/p59hck must require a transmembrane adaptor. Evidence for this was strongly supported by the finding that a soluble form of uPAR (suPAR) was capable of inducing chemotaxis not only in THP‐1 cells but also in cells lacking endogenous uPAR (IC50, 5 pM). However, activity of suPAR require chymotrypsin cleavage between the N‐terminal domain D1 and D2 + D3. Chymotrypsin‐cleaved suPAR also induced activation of p56/p59hck in THP‐1 cells, with a time course comparable with ATF. Our data show that uPA‐induced signal transduction takes place via uPAR, involves activation of intracellular tyrosine kinase(s) and requires an as yet undefined adaptor capable of connecting the extracellular ligand binding uPAR to intracellular transducer(s).

A urokinase-sensitive region of the human urokinase receptor is responsible for its chemotactic activity.

The role of urokinase‐type plasminogen activator (uPA) and its receptor (uPAR/CD87) in cell migration and invasion is well substantiated. Recently, uPA has been shown to be essential in cell migration, since uPA−/− mice are greatly impaired in inflammatory cell recruitment. We have shown previously that the uPA‐induced chemotaxis requires interaction with and modification of uPAR/CD87, which is the true chemoattracting molecule acting through an unidentified cell surface component which mediates this cell surface chemokine activity. By expressing and testing several uPAR/CD87 variants, we have located and functionally characterized a potent uPAR/CD87 epitope that mimics the effects of the uPA–uPAR interaction. The chemotactic activity lies in the region linking domains 1 and 2, the only protease‐sensitive region of uPAR/CD87, efficiently cleaved by uPA at physiological concentrations. Synthetic peptides carrying this epitope promote chemotaxis and activate p56/p59hck tyrosine kinase. Both chemotaxis and kinase activation are pertussis toxin sensitive, involving a Gi/o protein in the pathway.

The urokinase receptor: Focused cell surface proteolysis, cell adhesion and signaling

uPAR is highly expressed during tissue reorganization, inflammation, and in virtually all human cancers. Since its discovery, in vitro and in vivo models, as well as retrospective clinical studies have shown that over‐expression of components of the uPA/uPAR‐system correlates with increased proliferation, migration, and invasion affecting the malignant phenotype of cancer. uPAR regulates the cells–extracellular matrix interactions promoting its degradation and turnover through the plasminogen activation cascade.

uPAR-induced cell adhesion and migration: vitronectin provides the key

Expression of the membrane receptor uPAR induces profound changes in cell morphology and migration, and its expression correlates with the malignant phenotype of cancers. To identify the molecular interactions essential for uPAR function in these processes, we carried out a complete functional alanine scan of uPAR in HEK293 cells. Of the 255 mutant receptors characterized, 34 failed to induce changes in cell morphology. Remarkably, the molecular defect of all of these mutants was a specific reduction in integrin-independent cell binding to vitronectin. A membrane-tethered plasminogen activator inhibitor-1, which has the same binding site in vitronectin as uPAR, replicated uPAR-induced changes. A direct uPAR–vitronectin interaction is thus both required and sufficient to initiate downstream changes in cell morphology, migration, and signal transduction. Collectively these data demonstrate a novel mechanism by which a cell adhesion molecule lacking inherent signaling capability evokes complex cellular responses by modulating the contact between the cell and the matrix without the requirement for direct lateral protein–protein interactions.

Dimerization controls the lipid raft partitioning of uPAR/CD87 and regulates its biological functions

The urokinase‐type plasminogen activator receptor (uPAR/CD87) is a glycosylphosphatidylinositol‐anchored membrane protein with multiple functions in extracellular proteolysis, cell adhesion, cell migration and proliferation. We now report that cell surface uPAR dimerizes and that dimeric uPAR partitions preferentially to detergent‐resistant lipid rafts. Dimerization of uPAR did not require raft partitioning as the lowering of membrane cholesterol failed to reduce dimerization and as a transmembrane uPAR chimera, which does not partition to lipid rafts, also dimerized efficiently. While uPA bound to uPAR independently of its membrane localization and dimerization status, uPA‐induced uPAR cleavage was strongly accelerated in lipid rafts. In contrast to uPA, the binding of Vn occurred preferentially to raft‐associated dimeric uPAR and was completely blocked by cholesterol depletion.

Shedding and cleavage of the urokinase receptor (uPAR): identification and characterisation of uPAR fragments in vitro and in vivo

Applying a novel, highly specific and sensitive immunoabsorption/Western blotting technique we have identified in vitro in conditioned cell culture medium and in vivo in human urine different soluble forms of the urokinase‐type plasminogen activator receptor (uPAR/CD87). These include the uPAR fragment D2D3 and the never before identified domain 1 (D1) fragment. These forms correspond to fragments previously characterised as biologically active as inducers of chemotaxis and cell adhesion. We find that stimulation of U937 cells is associated with increased uPAR expression, cleavage of surface uPAR, and release of soluble fragments to the culture medium suggesting that monocytes are a source of the circulating and urinary soluble uPAR fragments found in vivo. Our study demonstrates that potentially biologically active uPAR fragments are produced in the human body, indicating a possible function in the regulation of not only proteolysis but also signal transduction related processes.

The interaction between urokinase receptor and vitronectin in cell adhesion and signalling

The extracellular matrix (ECM) is a complex structural entity surrounding and supporting cells present in all tissue and organs. Cell-matrix interactions play fundamental roles during embryonic development, morphogenesis, tissue homoeostasis, wound healing, and tumourigenesis. Cell-matrix communication is kept in balance by physical contact and by transmembrane integrin receptors providing the dynamic link between the extracellular and intracellular environments through bi-directional signalling. The urokinase-type plasminogen activator receptor (uPAR) is a plasma membrane receptor overexpressed during inflammation and in almost all human cancers. One of its functions is to endorse ECM remodelling through the activation of plasminogen and downstream proteases, including matrix-metalloproteases (MMPs). Beside its role in ECM degradation, uPAR modulates cell-matrix contact through a direct engagement with the ECM component, vitronectin (Vn), and by regulating the activity state of integrins thus promoting or inhibiting integrin signalling and integrin-mediated cell adhesion to other ECM components, like fibronectin and collagen. In this review we have centred our attention on the non-proteolytic function of uPAR as a mediator of cell adhesion and downstream signalling.

Urokinase Regulates Vitronectin Binding by Controlling Urokinase Receptor Oligomerization

Adhesion of monocytes to the extracellular matrix is mediated by a direct high affinity interaction between cell-surface urokinase-type plasminogen activator (uPA) receptor (uPAR) and the extracellular matrix protein vitronectin. We demonstrate a tight connection between uPA-regulated uPAR oligomerization and high affinity binding to immobilized vitronectin. We find that binding of soluble uPAR (suPAR) to immobilized vitronectin is strictly ligand-dependent with a linear relationship between the observed binding and the concentration of ligand added. Nevertheless, a comparison of experimentally obtained binding curves to those generated using a simple equilibrium model suggests that the high affinity vitronectin-binding pro-uPA·suPAR complex contains two molecules of suPAR. In co-immunoprecipitation experiments, using different epitope-tagged suPAR molecules, suPAR/suPAR co-immunoprecipitation displayed a similar uPA dose dependence as that observed for vitronectin binding, demonstrating that the high affinity vitronectin-binding complex indeed contains oligomeric suPAR. Structurally, the kringle domain of uPA was found to be critical for the formation of the vitronectin-binding competent complex because the amino-terminal fragment, but not the growth factor-like domain, behaved as a full-length uPA. Our data represent the first demonstration of functional, ligand-induced uPAR oligomerization having extensive implications for glycosylphosphatidylinositol-anchored receptors in general, and for the biology of the uPA/uPAR system in particular.

Domain 1 of the urokinase receptor (uPAR) is required for uPAR-mediated cell binding to vitronectin

In the present paper we have analyzed uPAR‐mediated cellular binding to vitronectin using the murine erythroid progenitor cell line 32D. We show that expression of uPAR in 32D cells promotes cellular binding to vitronectin, but fails to support cell spreading. The strength of binding is correlated to the expression level of uPAR and is strongly stimulated by the presence of uPAR ligands. Using a truncated variant of uPAR lacking domain 1 and by antibody inhibition experiments, we demonstrate that domain 1 plays a crucial role in uPAR‐mediated cellular binding. The failure of the mutant uPAR to promote cellular binding is paralleled by a strong reduction in the affinity for vitronectin in vitro.