Role of urokinase receptor in tumor progression and development.

Elevated level of urokinase receptor (uPAR) is detected in various aggressive cancer types and is closely associated with poor prognosis of cancers. Binding of uPA to uPAR triggers the conversion of plasminogen to plasmin and the subsequent activation of metalloproteinases. These events confer tumor cells with the capability to degrade the components of the surrounding extracellular matrix, thus contributing to tumor cell invasion and metastasis. uPA-uPAR interaction also elicits signals that stimulate cell proliferation/survival and the expression of tumor-promoting genes, thus assisting tumor development. In addition to its interaction with uPA, uPAR also interacts with vitronectin and this interaction promotes cancer metastasis by activating Rac and stimulating cell migration. Although underlying mechanisms are yet to be fully elucidated, uPAR has been shown to facilitate epithelial-mesenchymal transition (EMT) and induce cancer stem cell-like properties in breast cancer cells. The fact that uPAR lacks intracellular domain suggests that its signaling must be mediated through its co-receptors. Indeed, uPAR interacts with diverse transmembrane proteins including integrins, ENDO180, G protein-coupled receptors and growth factor receptors in cancer cells and these interactions are proven to be critical for the role of uPAR in tumorigenesis. Inhibitory peptide that prevents uPA-uPAR interaction has shown the promise to prolong patients' survival in the early stage of clinical trial. The importance of uPAR's co-receptor in uPAR's tumor-promoting effects implicate that anti-cancer therapeutic agents may also be developed by disrupting the interactions between uPAR and its functional partners.

Introduction

Tumors are result of uncontrolled proliferation of cells in different organs. In order for primary tumor to metastasize to distant organs, tumor cells must undergo a multistage process that includes detachment of tumor cells from primary tumors, cell migration and invasion through the degradation of extracellular matrix (ECM), intravasation into the bloodstream, extravasation from the circulation and colonization in a distant organ 1. Urokinase receptor (uPAR) plays a critical role in cancer metastasis by facilitating various steps of cancer metastasis. Elevated level of uPAR is often detected in aggressive tumor types and is associated with poor patient survival 2, 3. Studies from various experimental tumor models demonstrate that inhibiting uPAR expression or interfering uPA-uPAR partnership suppresses the progression of various cancer types 4. Inhibitory peptide that prevents uPA-uPAR interaction has been tested in clinical trials that have shown promising result of prolonging survival of patients with metastatic ovarian cancers 5. This review intends to give an overview on the current knowledge about the role of uPAR in cancer progression. Figure 1 describes the tumor biology of uPAR in nutshell.

Figure 1

Role of uPAR in tumor progression and metastasis. Binding of uPA to uPAR facilitates uPA activation and subsequent initiation of protease cascade, which in turn results in the degradation of extracellular matrix proteins and tumor cells invasion. Binding of uPA and vitronectin also promotes cell adhesion and cell migration. The action of uPA-uPAR/vitronectin is terminated through its interaction with PAI-1/PN-1 that triggers the internalization of uPAR complex. In addition, uPAR also interacts with various cell surface receptors such as integrins, growth factor receptors and ENDO180. These interactions activate diverse signaling pathways including FAK, Src, MAPK and PI3K, leading to EMT, cell proliferation, cell migration and expression of pro-cancer genes. Together, uPAR plays an essential role in tumor progression and metastasis.

General features of uPAR

Human uPAR gene is located at chromosome 19q13 and encodes a 335-aminoacid protein. uPAR protein includes an N-terminal 22-aminoacid secretory signal peptide and a C-terminal 30-aminoacid region that acts as the signal for addition of the glycosyl phosphatidylinositol (GPI) anchor 6. The mature uPAR is highly glycosylated and anchored to the cell surface through a GPI 7, 8. uPAR is a member of the lymphocyte antigen (Ly-6)/uPAR protein family and characterized by three similar functional domains - D1, D2, and D3 that are connected by bi-sulfide bridges 7. In addition to uPA, vitronectin is anther ligand that can bind uPAR. Since binding sites for uPA and vitronectin are distinct, uPAR can bind both ligands simultaneously 9, 10. One of the unique features of uPAR is that uPAR also exists in soluble form (suPAR) that is generated by the release of entire protein moiety from GPI anchor through proteolytic cleavage. The production of suPAR is believed to be a regulatory mechanism to reduce the number of uPAR on the cell surface. Additionally, both uPAR and suPAR can be cleaved in the linker region between D1 and D2 domains by uPA 11, plasmin 12 and matrix metalloproteinases (MMPs) 13 to produce D1 fragment and D2-D3 fragments 14-17. The cleaved suPAR possesses the ability to disrupt both uPA-uPAR and vitronectin-uPAR interactions, thus acting as an inhibitor of plasminogen activation at the cell surface 18, 19. The formation of suPAR and the cleavage at D1-D2 linker of uPAR are regarded as two post-transcriptional modifications that can control global uPAR cell surface expression and activity 20.

uPAR expression and its diagnostic significance in cancer

Under normal condition, cells and tissues exhibit limited uPAR expression. However, uPAR expression is greatly elevated in the processes of tissue remodeling, injury/wound healing and inflammation/immune response. For example, uPAR is highly expressed in gestational tissues during embryo implantation/placental development 21, 22 and in migrating keratinocytes at the edge of wounds 23. High level of uPAR is also observed during the process of leukocyte activation and differentiation 24, 25. Moreover, aberrant uPAR expression is frequently detected in pathological conditions. For instance, uPAR is readily seen in kidney during chronic proteinuric disease 26 and in the central nervous system following ischemia or trauma 27.

Unlike normal tissues, uPAR level is constitutively increased in majority of cancer types including solid tumors, leukemia and lymphomas as well as in tumor-associated stromal cells such as fibroblasts and macrophages 28-30. For instance, overexpression of uPAR is observed in a significant portion of individual cells from primary tumors and in circulating malignant cells from patients with advanced breast cancers 31. Importantly, increased levels of uPAR mRNA and protein in tumor tissue extracts are associated with poor prognosis of wide spectrum of malignancies such as colon, lung, gastric and breast 32, 33. In breast cancer, elevated uPAR expression is used as an independent prognostic marker of shortened relapse-free survival 34-36. In gastric cancer, high uPAR level in primary tumors is the indicator of aggressive cancer type 37, 38.

Additionally, intact as well as cleaved forms of suPAR are found at high levels in blood, urine, and ascite of cancer patients with aggressive cancer types and their levels are frequently correlated with poor prognosis 39, 40. For example, elevated suPAR level is detected in the urine of patients with clinically high-risk pancreatic ductal adenocarcinoma 41 and metastatic ovarian carcinoma 11. The convenience of detecting secreted antigens clearly implicates that suPAR is a better biomarker than the membrane-bound uPAR 17, 42. The fact that D2-D3 fragment of suPAR is generated by tumor progression-associated uPA, plasmin and MMPs further suggest that D2-D3 fragment is a more accurate prognostic indicator than entire suPAR 18.

Regulation of uPAR expression in cancer

The promoter of uPAR gene contains binding sites for transcription factors such as activator protein 1 (AP1), PEA3/Ets, specificity protein 1 (SP1) and AP2 which mediate either induced or sustained uPAR transcription in cancer cells 43-45. Hypoxia transcriptionally upregulates uPAR expression in cancer cells through the direct binding of hypoxia-induced factor 1α (HIF1α) to a hypoxia responsive element (HRE) in the uPAR promoter 46, 47. Post-transcription regulation also contributes to the level of uPAR in cancer cells. For example, RNA binding protein Hu antigen R and heterogeneous nuclear ribonucleoprotein C stabilize uPAR mRNA by directly binding to the AU-rich element (ARE) in the 3'-untranslated region (3'-UTR) of the uPAR mRNA 48, 49. In the contrary, tumor suppressor protein p53 accelerates uPAR mRNA degradation through binding to a 37-nucleotide element in the 3'-UTR of uPAR mRNA 50. Recent studies also indicate the role of microRNA in uPAR expression. For example, uPAR level was significantly enhanced by miR-10b in glioma cells and this miRNA-10b-induced uPAR upregulation was mediated by suppressing the expression of HOXD10, a negative regulator of uPAR 51, 52.

Proteolytic function and endocytosis/internalization of uPAR in cancer

A critical role of uPAR in cancer progression is its involvement in proteolysis of ECM. Binding of pro-uPA, a zymogen of uPA to uPAR triggers the conversion of pro-uPA to active uPA. uPAR-bound uPA subsequently converts plasminogen to active plasmin that degrades ECM/basement membrane and releases active MMPs, thereby facilitating cancer cell invasion and metastasis 53, 54. Reciprocally, active plasmin can cleave and activate pro-uPA 55, exhibiting a positive feedback loop of uPA-plasmin cascade in cancer cells 56. Recently-solved crystal structure of uPA-uPAR complex reveals that uPAR recognizes an N-terminal growth-factor-like domain (GFD) of uPA where all three D domains of uPAR are packed closely and form a unique cone-shaped cavity at the center 10, 57.

The proteolytic functions of uPAR are negatively regulated by plasminogen activator inhibitor-1 (PAI-1), PAI-2 and protease nextin-1(PN-1) 58, 59. Both PAI-1 and PN-1 can bind to uPA-uPAR complex and such bindings trigger direct interaction between D3 of uPAR and low-density lipoprotein receptor-related protein (LRP-1) on plasma membrane. The entire complex (PAI-1:uPA:uPAR:LRP1) is internalized via clathrin-coated vesicles and trafficked together to the early endosomes where uPA:PA-1 and uPAR are dissociated 60. uPA and PAI-1/PN-1 are eventually degraded in the lysozome while uPAR and LRP-1 are recycled back from the endocytic compartment to the plasma membrane 60-63. Alternatively, uPAR can also be internalized through its interaction with uPAR-associated protein (uPARAP)/endocytic receptor 180 (ENDO180), a member of mannose 6-phosphate (Man-6-P)/insulin-like growth factor-II receptor family, in a clathrin-dependent manner 64. ENDO180 is a constitutively recycling endocytic receptor 65, 66 and uPAR-ENDO180 interaction delivers uPAR into the lysosomes for degradation 64, 67. Interestingly, a recent study also shows that uPAR can be constitutively internalized without uPA in an LRP-1 and clathrin-independent manner 68. However, such endocytosed uPAR is only detected in early endosomes and does not reach lysosomes 68.

Internalization and recycling of uPAR have a complex role in uPAR function. Its internalization is apparently essential for clearing uPA:PAI-1:uPAR complex and decreasing the amount of cell surface uPAR available, thereby inhibiting diverse uPAR-mediated actions including proteolysis 69, 70. On the contrary, its internalization and recycling can facilitate uPAR-mediated action by redistributing unoccupied uPAR on the cell surface 69, 70. In addition, the internalization of uPAR also dissociates uPAR from its co-receptors including matrix-engaged integrins, thereby abrogating the pertinent downstream signaling 58. Clearly, endocytosis and recycling are key events that regulate the level and distribution of uPAR along the plasma membrane, thus controlling uPAR functions including proteolysis 69, 70.

Non-proteolytic function of uPAR in cancer

Many tumor-promoting effects of uPAR occur independently from the proteolytic function. For example, knockdown of uPAR suppresses the phosphorylation of FAK, p38MAPK, JNK and ERK1/2, signaling molecules in the Ras-activated signaling pathways, leading to the inhibition in cell migration and angiogenesis in glioma 71. Notch 1 signaling can cross talk with ERK, NF-κB and PI3-K/AKT/mTOR signaling pathways 72 and impact cancer invasion and angiogenesis 73. However, how Notch 1 achieves this is not clearly understood. In glioblasoma cells, downregulating uPAR abolishes in vitro invasion and in vivo tumor development by suppressing Notch 1-pertinent gene expression and signaling events 74. This study implicates that uPAR could be the potential functional link between Notch1 and tumorigenicity. Although whether non-proteolytic function of uPAR requires uPA-uPAR interaction remains to be answered, it is clear that uPAR can promote tumor progression independent of its proteolytic function 75.

With the aid of human breast cancer MDA-MB-468 cell line that exhibits epithelial cell phenotype, uPAR was found to promote epithelial-mesenchymal transition (EMT) under hypoxic condition through the activation of diverse signaling molecules including ERK, PI3K/Akt, Src and Rac1 76, 77. However, uPAR-induced EMT is reversible by reoxygenation, preventing uPA-uPAR interaction or inhibiting the activities of PI3K, Src and ERK 76. In contrast, breast cancer cell line MDA-MB-231 that displays mesenchymal cell morphology expresses high level of uPAR. However, the mesenchymal morphology of MDA-MB-231 cells requires the presence of uPAR because their phenotype alters upon the knockdown of uPAR 76. Again, uPAR-induced EMT is independent of the proteolytic function of uPAR.

Recent study has also revealed functional connection between uPAR and cancer stem cell (CSC)-like properties. Forced expression of uPAR was shown to promote the emergence of a CD24-/CD44+ phenotype, the characteristic of CSCs, and the increase in the number of cell surface integrin subunits β1/CD29 and α6/CD49f, marker of mammary gland stem cells in human breast cancer cell lines MCF-7 and MDA-MB-468 78. These uPAR-overexpressing cells were also found to exhibit significantly greater tumor initiation and growth in severe combined immunodeficient (SCID) mice 78. Interestingly, uPAR-induced CSC-like properties in MDA-MB-468 cells are associated with EMT but were independent of EMT in MCF7 cells. These findings indicate that uPAR is capable of inducing CSC-like properties in breast cancer cells, either concomitantly with or separately from EMT 78.

uPAR functions through its interaction with vitronectin

Vitronectin can bind uPAR in the absence of uPA, although its binding is enhanced by concurrent uPA-uPAR interaction. Vitronectin-uPAR interaction is also unaffected by vitronectin-αv integrin interaction 79, 80, and this can explained by crystal structures of ternary complex of uPA-uPAR-vitronectin. In the ternary complex of uPA-uPAR-vitronectin, the GFD of uPA occupies the central cavity of the uPAR whereas N-terminal somatomedin B (SMB) domain of vitronectin binds to D1 domain and D1-D2 linker which are on the outer side of the central cavity of uPAR, thus there is no direct interaction between uPA and vitronectin in this ternary complex 10, 57, 81. Similar to uPA-uPAR complex, PAI-1 also interacts with vitronectin-uPAR complex through the SBD of vitronectin 82. The binding site of PAI-1 on vitronectin overlaps with those on uPAR and αvβ3 integrin, and hence PAI-1 competes with both uPAR and αvβ3 integrin for vitronectin binding 83. Vitronectin-uPAR interaction promotes cell adhesion of various cell types 9, 79 and the adhesion is further increased by MRJ, a uPAR-interacting heat shock protein, in breast cancer cells 84. MRJ-increased adhesion involves the D1 of uPAR because it is sensitive to anti-uPAR D1 domain antibody 84. Therefore, uPAR not only plays a role in invasion and metastasis, but also in the attachment and colonization of cancer cells in the distant organs.

uPAR and its transmembrane co-receptors

uPAR being a GPI anchored cell surface protein, requires co-receptors to relay its downstream signals. Integrins, GPCRs and growth factor receptors are found to physically interact with uPAR and are assumed to serve as the co-receptors of uPAR. Among them, integrins are the most studied and are considered as the most significant co-receptors associated with uPAR signaling 85, 86. The interaction of uPAR with integrins was originally found in uPAR immunoprecipitates of human monocytes 87, and αMβ2 (MAC1) being the first reported uPAR-interacting integrin 88. uPAR-αMβ2 integrin interaction is capable of simultaneously increasing the binding of αMβ2 integrin to its ligand fibrinogen and to promote adhesion to vitronectin in an uPA-independent mechanism 89-91.

In addition to αMβ2 integrin, uPAR can also interact with α5β1, α3β1, αvβ3 and αvβ5 integrins. Binding of fibronectin to α5β1 integrin induces FAK phosphorylation and activates Ras-ERK signaling pathway. These two events are greatly enhanced by uPAR- α5β1 interaction 92, 93. Similarly, uPAR-α3β1 integrin interaction further enhances Src activity induced by the binding of laminin to α3β1 integrin 85, 94. In human lung cancer cells, the interaction between uPAR-α5β1 integrin transactivates EGFR in an FAK-dependent mechanism, leading to the activation of Erk signaling pathway 92, 95. Co-immunoprecipitation experiment also shows that EGFR directly interacts with α5β1 integrin and this interaction is enhanced by uPAR expression, suggesting that uPAR regulates EGFR-α5β1 integrin interaction 92, 95. EGFR-mediated activation of Erk signaling pathway is essential for cell proliferation driven by uPAR-α5β1 integrin interaction because enhanced proliferation is abrogated by EGFR kinase inhibitor or downregulating uPAR expression 92, 95. EGFR activation appears to be specific for events driven by the uPAR-α5β1 integrin interaction because EGFR inhibitor does not affect other cellular events initiated by uPAR signaling 96.

uPAR-integrin interactions facilitate tumor progression and development by eliciting cell migration, invasion, ECM proteolysis and EMT. Current hypothesis is that uPAR-intergrin integrin interactions achieve it by inducing the expression of genes essential for these events 93, 95, 97. Consistent with this hypothesis, enforced uPAR expression is found to promote tumor formation by enhancing the expression of uPA and MMPs while disrupting uPAR-integrin interaction blocks uPAR-induced tumor-promoting effects 98-100. Moreover, preventing uPAR-integrin interaction was found to suppress Erk activity and diminish the expression of ERK-regulated genes in lung cancer cells, thus forcing these cells into a protracted state of dormancy 98-100.

An early study has reported the interaction between uPAR and uFPRL1/LXA4R that is a G protein-coupled receptor for a number of polypeptides and for the endogenous lipoxin A4 (LXA4). This interaction is apparently required for uPAR-mediated cell migration in monocytic cells 101. More detailed study showed that D2D3 of uPAR moiety is sufficient to stimulate cell migration and it necessitates direct binding of D2D3 to FPRL1/LXA4R because inhibition or desensitization of FPRL1/LXA4R by antibodies or specific ligands specifically prevents D2D3-induced cell migration. In addition, D2D3 binding to FPRL1/LXA4R can be competed away by FPRL1/LXA4R agonists such as chemotactic peptide fMLP 101. This study reveals a unique mechanism for uPAR to induce cell migration that is to serve as a ligand to a chemotactic GPCR.

Coordinated action of uPAR, vitronectin and αvβ3 integrin in tumor cell migration

Vitronectin receptor αvβ3 integrin and uPAR are often co-expressed at high level in aggressive tumors 102. Coimmunoprecipitation showed that αvβ3 integrin is in physical interaction with uPAR in a variety of invasive cancer cells. As distinct sequences in vitronectin mediate its interactions with uPAR and αvβ3 integrin (SBD domain for uPAR and Arg-Gly-Asp sequence for αvβ3 integrin), the ability of vitronectin to facilitate cell migration is believed to require both uPAR and αvβ3 integrin. In fact, vitronectin binding to cells induces the activation of Src kinases. Activated Src subsequently phosphorylates p130Cas and facilitates the formation of p130Cas-CRK complex that enables the activation of Rac, ultimately resulting in membrane protrusion and cell migration. Importantly, uPAR-αvβ3 integrin interaction is indispensible for this vitronectin-induced event 103. Moreover, recent study showed that uPA-uPAR interaction can also activate Rac and stimulate cell migration and both vitronectin and αvβ3 integrin are required for this uPA-induced event 26, 104-106. This finding suggests that uPA-uPAR complex signals through vitronectin-αvβ3 integrin for its tumor-promoting actions.

uPAR as a therapeutic target

The nature of uPAR as a cell surface receptor indicates it as a drug-able target. The important role of uPAR in tumor progression suggests that blocking its pertinent functions can potentially lead to the suppression in tumorigenicity. Moreover, the relatively restricted expression in advanced tumor tissues adds another advantage for uPAR-targeted therapy as such therapy can be expected to be more specific to tumor tissues and thus less toxic to the non-cancerous tissues. Early works have mostly focused on inhibiting the proteolytic activity of uPA with specific inhibitors 107-109 or blocking uPA-uPAR binding with peptides 110, 111. Several recent studies have also generated anti-uPAR antibodies that can block uPAR-mediated downstream signaling and/or activation pathways. Importantly, such antibodies possess the capability to suppress tumor growth and metastasis 112, 113. More recently, strategies are developed to target the interactions between uPAR and its binding partners such as vitronectin and integrins 114. Especially, such agents are now advancing towards clinic evaluation 115.

Conclusion

uPAR is overexpressed in almost all aggressive malignancies and plays an essential role in tumor progression and metastasis. In addition to uPAR's well-established role in proteolysis, recent studies clearly demonstrate that uPAR also functions independently from proteolysis. As co-receptors are essential for these non-proteolytical functions of uPAR, distrupting the interactions between uPAR and its co-receptors represents as an attractive strategy for targeting aggressive malignancies.