[Frontiers in Bioscience 14, 4575-4587, January 1, 2009]
The urokinase receptor as an entertainer of signal transduction
Silvia D'Alessio1, Francesco Blasi1,2
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TABLE OF CONTENTS
1. ABSTRACT
The serine-protease urokinase (uPA) and its specific membrane receptor uPAR controls matrix degradation through the conversion of plasminogen into plasmin and play a crucial role in a number of biological processes including local fibrinolysis, inflammation, angiogenesis, matrix remodelling during wound healing, tumor invasion and metastasis. Most of the cellular responses modulated by the uPA/uPAR system, including migration, cellular adhesion, differentiation, proliferation and apoptosis require transmembrane signaling, which is mediated by direct contacts of uPAR with a variety of extracellular proteins and membrane receptors, such as integrins, EGF receptor, high molecular weight kininogen, caveolin and the G-protein-coupled receptor FPRL1. As a result of these interactions, uPAR activates intracellular signalling molecules such as tyrosine- and serine-protein kinases, Src, focal adhesion kinase (FAK), Rac, extracellular-signal-regulated kinase (ERK)/mitogen- activated protein kinase (MAPK) and JAK/STAT, being part of a large "signalosome" interacting with several molecules on both the outside and inside of the cell. This review is focused on the biochemistry of the pathways affected by uPAR and its partners.
2. INTRODUCTION
Urokinase plasminogen activator (uPA) is a serine protease whose major substrate is the zymogen plasminogen which is cleaved and activated to form plasmin. By activating plasminogen, uPA is at the top of a proteolytic cascade that ends up in the cleavage, degradation, sometimes activation of a myriad of proteins, including other proteases. Unlike other proteases uPA has a specific, high affinity receptor, uPAR that allows its localization (and the localization of its proteolytic activity) at the cell surface. Through their extracellular proteolysis activity, uPA and uPAR are regulators of many cell functions like adhesion, proliferation, chemotaxis, neutrophil priming for oxidant production and cytokine release, functions which can contribute to the development, implantation, angiogenesis, inflammation and metastasis of tumors (1). Levels of components of the uPA/uPAR system correlates with metastatic potential of cell lines in vitro and with tumor progression and patient survival in vivo; indeed, overexpression of uPA/uPAR was found to be significant in several human tumors including leukemias, tumors of the breast, lung, bladder, colon, liver, pleura, pancreas and brain (2-12). For this reason, inhibition of one or more of the components of this system is an attractive target for anti-cancer therapy (13). Specific antagonists that suppress binding of uPA to uPAR have also been shown to inhibit cell-surface plasminogen activation, tumor growth and angiogenesis both in vitro and in vivo models (14-17).
The uPA/uPAR system is made up of the serine protease uPA, its cell membrane-associated receptor (uPAR), a substrate (plasminogen) and the plasminogen activator inhibitors (PAI-1 and PAI-2) (18, 19). Human uPAR is a 335 aminoacids-long polypeptide, which during the cell surface sorting is post-translationally modified, losing an aminoterminal signal peptide and a carboxyterminal GPI-anchor peptide and being processed for GPI anchoring. In addition, the protein is extensively glycosylated. The mature uPAR protein consists of three homologous cysteine-rich repeats of about 90 amino acids each (Domain I, II and III). Cell surface uPAR is found both as full length (DIDIIDIII) as well as two domains derivative (DIIDIII) having lost the amino-terminal domain. Both forms can also be found in the serum, urine and other body liquids. Their function is largely unknown. The formation of a full length soluble suPAR (sDIDIIDIII) is caused either by a proteolytic cleavage close to the GPI anchor or to the hydrolysis of the GPI-anchor by a phospholipase (20, 21). This process is referred to as uPAR shedding. The second type is a proteolytic cleavage in the linker region connecting DI and DII and results in the release of the D1 fragment from the rest of the receptor. These cleavages change the biochemical properties of uPAR completely, probably facilitating appearance of previously hidden epitopes on the surface of the molecule further broadening the spectrum of uPAR interactions. In fact, different conformations of uPAR are able to interact with different proteins, e.g. the interaction with FPR receptors appears to require cleavage of uPAR between DI and DII at position 84 (22). Uncleavable uPAR mutants still binding uPA can differentially interact with different trans-membrane proteins, such as integrins and the EGFR (23). Thus, cleavage of uPAR appear to be an important physiologic/pathologic event.
In addition to directing extracellular proteolysis, uPAR is a genuine signalling receptor. Indeed, uPAR knock-out mice while not showing any evidence of deficient fibrinolysis, are deficient in a series of signalling pathways. Most of the cellular responses modulated by the uPA/uPAR system, including migration, cellular adhesion, differentiation and proliferation require transmembrane signaling, which has been reported to be mediated by direct contacts of uPAR with a variety of extracellular proteins and membrane receptors, such as integrins, EGF receptor, high molecular weight kininogen, caveolin and the G-protein-coupled receptor FPRL1 (24, 25). Besides, the results of uPAR structure analysis strongly support the hypothesis that uPAR is a molecule capable of establishing multiple contacts, since it shows a large outer surface necessary for various binding sites (26-29). As a result, uPAR activates intracellular signalling molecules such as tyrosine- and serine-protein kinases, Src, focal adhesion kinase (FAK), Rac, extracellular-signal-regulated kinase (ERK)/mitogen- activated protein kinase (MAPK) and JAK/STAT, leading to a predominantly migratory and adhesive, but also, in various cells, proliferative and more recently apoptotic response. In this review, we will confine ourselves to the biochemistry of the pathways affected by uPAR and its partners.
3. MAP KINASE PATHWAY
MAPKs are known to be key elements of signal transduction chains leading to the activation of early immediate genes (30-32). In many cancers, as breast cancer, the mitogen-activated protein kinases, extracellular signal-regulated kinase ERK-1 and ERK-2, are frequently hyperexpressed and exhibit increased activity (33). This is important because activated ERKs control many processes that are central to cancer progression, including cell growth, apoptosis and cell migration (34). Activated ERK may also promote cancer cell invasion by upregulating expression of proteinases and associated receptors that are involved in this process, including uPA and its cell-surface receptor uPAR (35-37). Most of the cellular responses modulated by the uPA/uPAR system, including migration, cellular adhesion, differentiation, proliferation and apoptosis (24) require in fact the activation of ERK1/2. In the context of tumor proliferation, the best-characterized pathway has been described by Aguirre-Ghiso and colleagues (38). They describe a uPAR-dependent mechanism by which the majority of tumor cells modulate the activity ratio between the proliferation inducer ERK (39) and the negative growh regulator p38 (40). Based on the study of 10 different cell lines, their results show how uPAR and α5β1 activate the EGFR in a EGF-independent but FAK-dependent manner (41), generate high ERK and low p38 activity necessary for the in vivo growth of cancer cells. A positive loop is activated in which ERK activity transactivates uPAR and uPA expression (35, 41-43). Besides, high uPAR level, by activating α5β1 maintains high ERK activity (41, 44). Sustained ERK phosphorylation allows then for nuclear localization and subsequent stabilization of c-Fos and other immediate early genes, which are necessary for S-phase entry (45-47).
The EGFR has been implicated in signalling from uPAR to ERK, as well; however, previous studies suggest that the EGFR is not essential (23, 48). In fact, in its absence, alternative co-receptors function to activate the Ras-ERK pathway (23) but when it is expressed, the EGFR assumes a dominant role and becomes essential for uPA-initiated signalling to ERK, without influencing the kinetics of ERK activation, thus promoting cell proliferation (48). Because uPAR is linked to the cell surface by a GPI anchor, it is generally assumed that uPAR signals as part of a multiprotein signalling-receptor complex (MSRC). In support of this hypothesis, soluble human uPAR (SuPAR) has been shown to activate cell signalling similarly to uPA (49-52). Jo et al. (52) demonstrated, for example, that human SuPAR may activate or inhibit ERK phosphorylation, depending on the state of the autocrine uPA-uPAR signalling system. They demonstrated for the first time that SuPAR may antagonize cancer progression by direct, uPA-independent effects on cell signalling. These data support a model in which uncleaved SuPAR functions as a partial agonist that triggers cell signalling but not as effectively as membrane-anchored uPAR-uPA complex. In A1 MEFs and HEK293 cells, which lack uPAR, SuPAR find no competition and thus activates ERK. By contrast, in cells that have a highly activated autocrine signalling system, such as MDA-MB 231 breast cancer cells or MEFs2, SuPAR inhibits ERK activation and consequently, cell growth and Matrigel invasion. Since murine uPA does not bind human uPAR or suPAR (53), this precludes the alternative model in which SuPAR inhibits ERK activation by binding uPA produced endogenously by the MEFs2 (54).
These results were confirmed in xenograft animal model experiments, where SuPAR reduces the growth and the metastasis of MDA-MB 231 and OV-MZ-6#8 ovarian cancer cells (55, 56).
Cleavage of SuPAR by proteinases increases its signalling agonist activity and reverses its inhibitory effects on growth and invasion. Thus, proteolytic cleavage represents a molecular switch that neutralizes the anticancer activity of SuPAR.
In terms of cell migration and invasion, Mirshahi et al. many years ago demonstrated that uPA/uPAR-stimulated ovarian cell motility depends on tyrosine kinase activation (57). In 1999, using a transfection strategy that included dominant-negative and costitutively active Ras and MEK mutants, Nguyen et al. showed that uPA promotes cell migration, in an integrin-selective manner, by initiating a uPAR-dependent signalling cascade in which Ras, MEK, ERK and Myosin light chain kinase (MLCK) serve as essential downstream effectors (58). In these cells uPA-induced ERK activation is highly transient; however, the effects of uPA on cellular migration are sustained (59). Degryse et al. (2001) also showed that pro-uPA might promote a direct interaction between uPAR and integrins, modulating their function and this stimulates phosphorylation and nuclear translocation of ERK inducing rat smooth muscle cell (RSMC) migration, a pathway which differs from the one induced by Vitronectin (VN) (60).
Other reports have shown that uPAR might underlie a MEK/ERK-dependent signalling mechanism in cancers (i.e. ovarian, breast, melanoma, hepatocarcinoma) (58, 61-63) and an ERK-dependent signalling event via uPAR drove motility through polarized lamellipodia extension in colon cancer cells (64).
Since it is well known that at least two forms of uPAR are present on the cell surface (full length and cleaved uPAR), each specifically interacting with one or more transmembrane proteins, Mazzieri et al. (2006) exploited an uPAR mutant (hcr, human cleavage resistant) to dissect the pathways involved in uPA-induced cell migration. Both wild-type and hcr-uPAR are able to mediate uPA-induced migration, are costitutively associated with the EGFR, and associate with α3β1 integrin upon uPA binding. However they engage different pathways in response to uPA. Wt-uPAR requires both integrins and FPRL1 to mediate uPA-induced migration, and association of wt-uPAR to α3β1 results in uPAR cleavage and ERK activation. On the contrary, hcr-uPAR doesn't activate ERK, but it activates an alternative pathway engaging different trans-membrane receptors. uPAR can thus signal through several types of trans-membrane receptors upon "activation" by several ligands and/or upon cleavage by different proteases.
A recent evidence shows a close relationship between the uPA/uPAR system and cell sensitivity to programmed cell death (65). In this context the anti-apoptotic ability of uPAR may be due, at least in part, to its ability to activate the Ras-ERK signalling pathway in many different cell types. In MDA-MB-231 breast cancer cells cultured in the presence of anti-uPA antibodies that block the binding of uPA to uPAR, the level of phosphorylated ERK decreases substantially and apoptosis is promoted, showing that endogenous uPA is a major determinant of ERK activation and protection from apoptosis (66). Activated ERK was also necessary to maintain uPA and uPAR expression. This positive-feedback loop may in fact be critical in determining the aggressive nature of MDA-MB-231 cells. The ability of uPAR and ERK to function in a positive feedback loop and to suppress apoptosis represents a novel mechanism whereby the uPA-uPAR system may promote cancer progression.
4. FOCAL ADHESION KINASE SIGNALLING
FAK is a cytoplasmic tyrosine kinase involved in the transduction of signals generated by cell matrix contacts and is overexpressed in several human cancers. It localizes to focal adhesions and becomes tyrosine phosphorylated in response to integrin-derived signals for motility, survival and proliferation (67, 68). Yebra et al. (69) demonstrated an association between uPAR and β1 integrin in the cytoskeletal fraction of a LNCaP human prostate carcinoma cell line, that depends on the presence of uPA. This findings suggests that uPA binding to uPAR induces either a conformational change or a change in the lateral mobility of uPAR so it can physically associate with α5β1, leading to enhanced FAK and p130Cas tyrosine phosphorylation and enhanced cell migration by increasing turnover of focal adhesion contacts (69). Nguyen et al. in 2000 also showed that binding of uPA to uPAR can stimulate the Ras/ERK signalling pathway and migration of MCF-7 breast cancer cells by a mechanism that requires FAK, Src and Shc (70). Most importantly Aguirre Ghiso in 2002 showed one of the first attempts of testing the role of FAK in signal transduction induced by activating association of uPAR with integrin and its effect on epithelial tumor growth in vivo. He explored the role of FAK in regulating tumorigenicity of human carcinoma cells, HEp3, which is dependent on uPAR- α5β1-integrin association (41, 71). Active FAK is an important mediator of uPAR-regulated tumorigenicity of HEp3 cells and interruption of FAK mitogenic signalling either through down-regulation of uPAR or by expression of a FAK related non-kinase (FRNK), known to have a dominant negative function, can force human carcinoma cells into dormancy (72). These results lend functional significance to the finding of frequent overexpression of FAK and uPAR in tumors from different origin, indicating that uPAR-mediated active FAK may enable tumor cells to activate more efficiently survival and mitogenic signals derived from the ECM, providing a growth advantage at the primary or metastatic growth site.
A role for caveolin, a non-integrin membrane protein, and uPAR in integrin-mediated adhesion and signalling has also been shown (73-75). Many data indicate that uPAR is localized in caveolae and forms a stable complex with caveolin (76-79). Caveolin and uPAR may in fact operate within adhesion sites to organize kinase-rich lipid domains in proximity to integrins, promoting efficient signal transduction. Lipid rafts seem to be important for src-kinase signalling and for the GP130 mediated pathway: upon clustering of uPAR, activation of JAK occurred, followed by STAT phosphorylation and redistribution from the caveolae to the nucleus (80).
5. JAK/STAT- AND Src-FAMILY SIGNALLING PATHWAY
Kinases of the Janus kinase (JAK)-family were found to be associated with uPAR, in several cell types. One example is the kidney tumor epithelial cell line TCL-598 in which uPAR was found to be associated with JAK1 and STAT proteins in detergent-insoluble membrane fractions, as revealed by coimmunoprecipitation (80). Upon clustering of uPA/uPAR complex by a monoclonal antibody, JAK1 associates with uPAR, which in turn leads to STAT1 phosphorylation, dimerization, nuclear translocation, specific binding to the DNA interferon-gamma activation site (81) or interferon-stimulated response elements (ISREs), and gene activation (80). Similar findings were reported by Dumler et al. showing that uPA binding to its receptor induces the JAK/STAT pathway, thereby regulating migration of smooth muscle cells (82, 83). In this case uPAR was found to co-localize with JAK1 and Tyk2 in the leading edge of the migrating human aortic smooth muscle cells, while JAK2, JAK3 and the Src-PTKs remained mobile in the plane of the plasma membrane. This result links uPAR to a known signalling pathway mainly utilized by cytokines. Thereby, most likely the glycoprotein (GP) gp130 might be the transmembrane adapter for this signal transduction pathway.
More recent data show for the first time that uPA leads to activation of STAT3, independent of its catalytic activity but dependent on its interaction with uPAR, leading to DNA synthesis in lung epithelial cells (84). Jo et al. instead demonstrate that in Chinese hamster ovary (CHO)-K1 cells, EGFR supports uPA mitogenic activity by recruiting and activating STAT5b downstream of uPAR. They support a model in which STAT5b and ERK function independently, but in a complementary manner, to promote cell growth and that the composition of the uPAR multiprotein signalling-receptor complex (MSRC) is critical in determining cellular response to uPA (85).
Concerning regulation of transcriptional activity, induction of the c-fos gene has also been shown as a consequence of uPAR activation (86, 87), also indicating involvement of STAT1 in signal transduction.
Besides the JAK-family, src-kinases can also associate with uPAR in several cell types (81, 88, 89). c-Src, normally localized to endosomal membranes, is redistributed to focal adhesions upon cell activation, where it regulates cell adhesion and migration (90-94). Fazioli et al. demonstrated that peptides derived from the linker region between the first and the second domains of uPAR, which contain the sequence SRSRY, activate the Src-family tyrosine kinase, p56/p59hck, and demonstrate chemotactic activity, similarly to uPA (81). In smooth muscle cells, uPA induces the sub-cellular relocalization of c-Src to the plasma membrane, preferentially toward the leading-edge of migration (95). uPA also causes cytoskeletal reorganization in c-Src+/+ but not in c-Src-/- fibroblasts (95). However, in endothelial cells, uPA activates ERK by a pathway that is not affected by a general antagonist of Src family kinases (96). Nguyen et al., on the contrary, support a model in which c-Src is necessary for uPA-induced ERK activation and MCF-7 cell migration, together with FAK, Shc and Ras, which demonstrates for the first time a link between uPAR and Ras (70). Although they cannot precisely define the relationship of these factors in activating the Ras-ERK pathway, it is significant that the uPAR-initiated pathway shares many similarities with pathways that link integrins to ERK (97-101).
In a more recent study, experiments were conducted by Monaghan-Benson et al., to address the mechanism of uPAR regulation of matrix assembly. This group has previously demonstrated that treatment of fibroblasts with the uPAR ligand, P25, results in an increase in the activation of the β1 integrin and a 35-fold increase in fibronectin matrix assembly (102). Lately, they demonstrated that ligation of uPAR with P25 causes a Src-dependent transactivation of the EGFR and promotes the formation of EGFR-β1 integrin complexes. Both Src kinase and EGFR are required for the uPAR-dependent increase in β1 integrin activation and fibronectin matrix assembly (103). These studies suggest that both uPAR and EGFR may represent novel targets for the regulation of fibronectin matrix deposition under conditions where dysregulated fibronectin deposition may contribute to pathological conditions such as tumor survival and tissue fibrosis.
6. INTRACELLULAR CALCIUM MOBILIZATION
As a GPI-anchored protein, uPAR has no direct link to signalling elements inside the cells (104). This could be circumvented by uPAR using other proteins as signal transduction devices, and precisely this mechanism has been demonstrated for uPA-induced Ca2+ fluxes in neutrophils, in which CR3 (Mac-1; CD11b/CD18), a β2 integrin, serves as the partner protein (105). However, few years later Sitrin et al., demonstrated that uPAR aggregation of human promyelocytic cell line U937 and human monocytes, initiates phosphoinositide hydrolysis and subsequent Ca2+ mobilization by mechanism that are not strictly dependent on associated uPA or CR3 (106). At the same time Christow et al. using patch-clamp techniques showed that uPA binding to uPAR stimulates Ca2+-activated K+ channels via induction of inositol 1,4,5-triphosphate (Ins (1,4,5)P3) formation and the liberation of Ca2+ from internal stores by G-protein- and phospholipase C-dependent mechanism (107). The release of Ca2+ , as indicated by FRET analysis, could be linked also to a direct interaction of uPAR with L-selectin (CD62L), an adhesion protein that participates in the initial stages of leukocyte rolling on endothelial cells (106). Essentially all chemokine receptors induce the mobilization of Ca2+ from intracellular stores, and FPRL1, which likely mediates uPA signaling, is not an exception (108). However, uPA and uPAR are different, as clustering of uPAR must occur before uPA can induce the mobilization of Ca2+ (106).
7. Rho-FAMILY GTPase AND VITRONECTIN (VN)-INDUCED SIGNALLING PATHWAY
An additional complication to the role of uPAR in cell adhesion and motility is its ability to bind VN. Binding of multimeric or surface-absorbed forms of VN to uPAR has been demonstrated both in vitro with purified components and in vivo where the uPAR-VN interaction mediates cellular adhesion of cytokine-stimulated monocytes as well as uPAR-transfected HEK293 and erythroid progenitor cells (79, 109-112). uPAR-mediated cell adhesion to Vn does not always depend on receptor occupancy as several transfected cell lines, which do not produce uPA, still adhere strongly to VN in a uPAR-dependent manner (79, 113-115). However at physiological expression levels, uPAR-dependent cell adhesion to Vn requires uPA binding (112, 115, 116). Lately Madsen et al., showed that a direct uPAR-Vn interaction is required for ERK1/2 activation, as Vn binding-deficient uPAR mutants displayed levels of active ERK1/2 comparable to those of mock-transfected cells (117). Interestingly, uPA binding to uPAR also leads to ERK1/2 activation in different experimental systems (59, 71), suggesting that both overexpression of the receptor and ligand binding induces the same signal transduction pathway (s), possibly through a common molecular mechanism. However, it appears likely that uPA binding may actually induce "Vn signaling" by stimulating uPAR binding to matrix Vn. In support of this possibility there is a strict correlation between the ability of pro-uPA to promote Vn binding and to induce ERK1/2-activation and changes in cell morphology (117). The interactions of uPAR with components normally associated with cytoskeletal structures such as integrins and extracellular matrix molecules and its co-localization with integrins and cytoskeletal components such as vinculin at sites of cell-matrix contact (118-122) suggest that its role in cell motility may involve regulation of the actin cytoskeleton. It has been demonstrated years ago that the interaction between cell surface uPAR and ECM Vn causes a potent induction of actin cytoskeleton rearrangement and cell motility (60) by a mechanism which requires Rac-activation (123).
In the regulation of the actin cytoskeleton, small GTPases of the Rho-family play a pivotal role and the best characterized members of this family are Rho, Rac and Cdc42. Kjoller et al. showed that upon uPAR binding to VN, RhoA and Cdc42 are not involved in actin reorganization, while this process resulted to be Rac-dependent and accompanied by an increase in Rac-mediated Swiss 3T3 cell motility (113). However, independently from VN, uPAR was found to activate Rac and regulate lamella/ruffling activity in Hct-116 and BE colon carcinoma cells in an ERK-dependent manner (64). Besides, other studies strongly suggests that the small GTPases RhoA and Rac1 may be important downstream mediators of the uPAR/Tyk2/PI3-K signaling pathway in human vascular smooth muscle cells (124).
8. OTHER SIGNALLING MOLECULES
Several signaling molecules were found to cooperate with uPAR, including vinculin, alpha-actinin, actin (125), PKC (126) and PI3-K (124, 127, 128). In the last case, the positive correlation between uPAR expression level and activation of the PI3K-Akt-dependent anti-apoptotic pathway is suggested by the finding that glioblastoma cells bearing an antisense to uPA exhibit a reduced level of phosphorylated PI3K and Akt as well as impaired migration and survival (124, 127, 128). It has been shown that uPAR itself, other than concentrating uPA proteolytic activity on cell surface and being a mediator of most ligand-dependent effects on growth, motility and apoptosis, could be an anti-apoptotic factor (66). The reduced levels of active PI3K/Akt and ERK1/s in uPAR-deficient cells indicate that uPAR may modulate the survival/apoptosis ratio through the control of crucial signaling cascades.
Several years ago two groups demonstrated that the DII-DIII fragment of uPAR was involved in binding to the cation-independent, mannose 6-phosphate/insulin-like growth factor-II (M6P/IGF-II) receptor (129, 130). This binding is not affected by uPA or mannose-6-phosphate and leads to internalization of uPAR in lysosomes (130). Whether association of uPAR with M6P/IGF-II receptor has only clearing function or might contribute to signal transduction is not yet understood. The M6P/IGF-II receptor interaction with uPAR, however, seems to be involved in the plasmin-dependent generation of TGF-β and, thereby, indirectly in signal transduction via uPA/uPAR (129).
9. THE uPAR-INDUCED SIGNALLING PATHWAYS AS TARGET FOR ANTI-CANCER THERAPY
Researchers that have used either antisense or siRNA technologies for the successful in vivo downregulation of uPAR in various cancers have concurrently tested these same technologies in in vitro biological assays. Evaluation of the results of these in vitro assays reveals that downregulation of uPAR has lead, in most cases, to inhibition of invasion (14, 131-135), migration (131, 133), adhesion (131) and proliferation (14, 132, 134). In addition, reduced uPAR levels lead to inhibition of tumor-induced angiogenesis (132) and ECM degradation (136, 137).
As stated earlier, some of the biological functions of uPAR, such as proliferation, are facilitated by the regulation of several different signaling molecules. In an attempt to understand and/or elucidate the involvement of uPAR in downstream signaling pathways, studies have investigated the effect of uPAR downregulation on components of the relevant signaling pathways. D'Alessio et al. (14) reported that melanoma cells exhibited a strong decrease in ERK1/2 activation when an 18mer asODN was used to downregulate uPAR. Using this same asODN for the downregulation of uPAR in prostate cancer cells, Margheri et al. (134) reported a strong decrease of FAK/JNK/Jun phosphorylation (thereby causing a decrease in the activation of the FAK/JNK/Jun pathway). At the same time, the synthesis of cyclins A, B, D1 and D3 was inhibited, and these prostate cancer cells accumulated in the G2 phase of the cell cycle. The downregulation of uPAR by a plasmid construct expressing shRNA for uPAR resulted in significantly reduced levels of the phosphorylated forms of MAPK, ERK and AKT signaling pathway molecules (132). However, the majority of studies applying uPAR downregulation for cancer in vivo failed to identify the perturbed signaling pathways. In any case, different laboratories chose to elucidate effects on different pathways and, although there is an abundance of literature looking at individual pathways in vitro, it is difficult to compare results from separate studies because various parameters, including cell line, passage number, minor technical differences, the antisense sequence, the concentration of constructs, the time-points evaluated, and the way the data are reported, often prevent such comparisons.
10. CONCLUSION: THE uPAR SIGNALOSOME
It is now well established that the uPA/uPAR system is connected to the malignant process of tumor growth and invasion and it is clear that uPAR is part of a large "signalosome" associated and interacting with several proteins on both the outside and inside of the cell. Most of the cellular responses modulated by the uPA/uPAR system, including migration, cellular adhesion, differentiation, proliferation and apoptosis require transmembrane signaling, which cannot be mediated directly by a GPI-anchored protein such as uPAR. For this reason, besides the well-established interactions with uPA and Vn, uPAR has been reported to entertain direct contacts with a variety of extracellular proteins and membrane receptors, such as integrins, EGF receptor, high molecular weight kininogen, caveolin and the G-protein-coupled receptor fMLP-receptors. Besides, the results of uPAR structure analysis (29), strongly support the hypothesis that uPAR is a molecule capable of establishing multiple contacts, since it shows a large outer surface necessary for various binding sites. As a result, uPAR activates intracellular signalling molecules such as tyrosine-, serine-protein kinases and small G proteins, Src, focal adhesion kinase (FAK), Rac, extracellular-signal-regulated kinase (ERK)/mitogen- activated protein kinase (MAPK) and JAK/STAT, leading to a predominantly migratory and adhesive, but also, in various cells, proliferative and more recently apoptotic response. In an attempt to understand and/or elucidate the involvement of uPAR in downstream signaling pathways, studies have investigated the effect of uPAR downregulation on components of the relevant signaling pathways, validating uPAR as an anti-tumor therapeutic target and many studies are still in progress. However, the existence of many redundant cellular processes in nature would suggest that because the uPA/uPAR system is inhibited, other similar systems might become biologically more active and ultimately overrule the inhibition of the uPA/uPAR system. Therefore, combination therapy that includes the uPA/uPAR system among others, and that targets several processes at a time may have a greater chance of success at producing tumor killing while reducing the development of resistance.
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Abbreviations: GPI: glycosylphosphatidylinositol; EGFR: epidermal growth factor receptor; FPRL-1: formyl peptide receptor like 1; MEFs: mouse embryonic fibroblasts; MEK: map-erk kinase; STAT: signal transducer and activators of transcription; PTKs: protein tyrosine kinases; FRET: fluorescence resonance energy transfer; PI3-K: phosphoinositide-3 kinase; AKT: protein kinase B; ODN: oligodeoxynucleotide
Key Words: uPA, uPAR, Signal Transduction, Tumor Invasion, Cell Signalling, Cell Adhesion, Cell Migration, EGFR, integrins, MAPkinases, Review
Send correspondence to: Silvia D'Alessio, Department of Molecular Biology and Functional Genomics, DIBIT, Università Vita-Salute San Raffaele, Via Olgettina 58, 20132 Milan, Italy, Tel: 39 02 2643 4758, Fax: 39 02 2643 4844, E-mail:dalessio.silvia@hsr.it