uPAR Proteins Research Articles

Evaluation of New Cardiac Damage Biomarkers in Polytrauma: GDF-15, HFABP and uPAR for Predicting Patient Outcomes.

Background: Polytrauma is one of the leading mortality factors in younger patients, and in particular, the presence of cardiac damage correlates with a poor prognosis. Currently, troponin T is the gold standard, although troponin is limited as a biomarker. Therefore, there is a need for new biomarkers of cardiac damage early after trauma. Methods: Polytraumatized patients (ISS ≥ 16) were divided into two groups: those with cardiac damage (troponin T > 50 pg/mL, n = 37) and those without cardiac damage (troponin T < 12 pg/mL, n = 32) on admission to the hospital. Patients' plasma was collected in the emergency room 24 h after trauma, and plasma from healthy volunteers (n = 10) was sampled. The plasma was analyzed for the expression of HFABP, GDF-15 and uPAR proteins, as well as miR-21, miR-29, miR-34, miR-122, miR-125b, miR-133, miR-194, miR-204, and miR-155. Results were correlated with patients' outcomes. Results: HFABP, uPAR, and GDF-15 were increased in polytraumatized patients with cardiac damage (p < 0.001) with a need for catecholamines. HFABP was increased in non-survivors. Analysis of systemic miRNA concentrations showed a significant increase in miR-133 (p < 0.01) and miR-21 (p < 0.05) in patients with cardiac damage. Conclusion: All tested plasma proteins, miR-133, and miR-21 were found to reflect the cardiac damage in polytrauma patients. GDF-15 and HFABP were shown to strongly correlate with patients' outcomes.

A Dual-Reporter Platform for Screening Tumor-Targeted Extracellular Vesicles.

Extracellular vesicle (EV)-mediated transfer of biomolecules plays an essential role in intercellular communication and may improve targeted drug delivery. In the past decade, various approaches to EV surface modification for targeting specific cells or tissues have been proposed, including genetic engineering of parental cells or postproduction EV engineering. However, due to technical limitations, targeting moieties of engineered EVs have not been thoroughly characterized. Here, we report the bioluminescence resonance energy transfer (BRET) EV reporter, PalmReNL-based dual-reporter platform for characterizing the cellular uptake of tumor-homing peptide (THP)-engineered EVs, targeting PDL1, uPAR, or EGFR proteins expressed in MDA-MB-231 breast cancer cells, simultaneously by bioluminescence measurement and fluorescence microscopy. Bioluminescence analysis of cellular EV uptake revealed the highest binding efficiency of uPAR-targeted EVs, whereas PDL1-targeted EVs showed slower cellular uptake. EVs engineered with two known EGFR-binding peptides via lipid nanoprobes did not increase cellular uptake, indicating that designs of EGFR-binding peptide conjugation to the EV surface are critical for functional EV engineering. Fluorescence analysis of cellular EV uptake allowed us to track individual PalmReNL-EVs bearing THPs in recipient cells. These results demonstrate that the PalmReNL-based EV assay platform can be a foundation for high-throughput screening of tumor-targeted EVs.

Structural Diversity and Dynamics of Human Three-Finger Proteins Acting on Nicotinic Acetylcholine Receptors.

Ly-6/uPAR or three-finger proteins (TFPs) contain a disulfide-stabilized β-structural core and three protruding loops (fingers). In mammals, TFPs have been found in epithelium and the nervous, endocrine, reproductive, and immune systems. Here, using heteronuclear NMR, we determined the three-dimensional (3D) structure and backbone dynamics of the epithelial secreted protein SLURP-1 and soluble domains of GPI-anchored TFPs from the brain (Lynx2, Lypd6, Lypd6b) acting on nicotinic acetylcholine receptors (nAChRs). Results were compared with the data about human TFPs Lynx1 and SLURP-2 and snake α-neurotoxins WTX and NTII. Two different topologies of the β-structure were revealed: one large antiparallel β-sheet in Lypd6 and Lypd6b, and two β-sheets in other proteins. α-Helical segments were found in the loops I/III of Lynx2, Lypd6, and Lypd6b. Differences in the surface distribution of charged and hydrophobic groups indicated significant differences in a mode of TFPs/nAChR interactions. TFPs showed significant conformational plasticity: the loops were highly mobile at picosecond-nanosecond timescale, while the β-structural regions demonstrated microsecond-millisecond motions. SLURP-1 had the largest plasticity and characterized by the unordered loops II/III and cis-trans isomerization of the Tyr39-Pro40 bond. In conclusion, plasticity could be an important feature of TFPs adapting their structures for optimal interaction with the different conformational states of nAChRs.

Relating GPI-Anchored Ly6 Proteins uPAR and CD59 to Viral Infection.

The Ly6 (lymphocyte antigen-6)/uPAR (urokinase-type plasminogen activator receptor) superfamily protein is a group of molecules that share limited sequence homology but conserved three-fingered structures. Despite diverse cellular functions, such as in regulating host immunity, cell adhesion, and migration, the physiological roles of these factors in vivo remain poorly characterized. Notably, increasing research has focused on the interplays between Ly6/uPAR proteins and viral pathogens, the results of which have provided new insight into viral entry and virus–host interactions. While LY6E (lymphocyte antigen 6 family member E), one key member of the Ly6E/uPAR-family proteins, has been extensively studied, other members have not been well characterized. Here, we summarize current knowledge of Ly6/uPAR proteins related to viral infection, with a focus on uPAR and CD59. Our goal is to provide an up-to-date view of the Ly6/uPAR-family proteins and associated virus–host interaction and viral pathogenesis.

Podocin and uPAR are good biomarkers in cases of Focal and segmental glomerulosclerosis in pediatric renal biopsies.

There are controversies whether Minimal Change Disease (MCD) and Focal and Segmental Glomerulosclerosis (FSGS) are distinct glomerular lesions or different manifestations within the same spectrum of diseases. The uPAR (urokinase-type plasminogen activator receptor) and some slit diaphragm proteins may be altered in FSGS glomeruli and may function as biomarkers of the disease in renal biopsies. Thus, this study aims to evaluate the diagnostic potential of uPAR and glomerular proteins for differentiation between MCD and FSGS in renal pediatric biopsy. Renal biopsies from 50 children between 2 and 18 years old were selected, with diagnosis of MCD (n = 29) and FSGS (n = 21). Control group consisted of pediatric autopsies (n = 15) from patients younger than 18 years old, with no evidences of renal dysfunction. In situ expressions of WT1, nephrin, podocin and uPAR were evaluated by immunoperoxidase technique. Renal biopsy of patients with MCD and FSGS expressed fewer WT1 (p≤0.0001, F = 19.35) and nephrin (p<0.0001; H = 21.54) than patients in the control group. FSGS patients expressed fewer podocin than control (p<0.0359, H = 6.655). FSGS cases expressed more uPAR than each of control and MCD (p = 0.0019; H = 12.57) and there was a positive and significant correlation between nephrin and podocin (p = 0.0026, rS = 0.6502) in these cases. Podocin had sensitivity of 73.3% and specificity of 86.7% (p = 0.0068) and uPAR had sensitivity of 78.9% and specificity of 73.3% (p = 0.0040) for diagnosis of FSGS patients. The main limitation of the study is the limited number of cases due to the difficulty in performing biopsy in pediatric patients. Podocin and uPAR are good markers for FSGS and differentiate these cases from MCD, reinforcing the theory of distinct glomerular diseases. These findings suggest that podocin and uPAR can be used as biomarkers in the routine analysis of renal biopsies in cases of podocytopathies when the lesion (sclerosis) is not sampled.

C-Src is in the effector pathway linking uPAR and podocyte injury.

The role of urokinase-type plasminogen activator receptor (uPAR) in kidney physiology and pathology has attracted considerable attention. The protein uPAR has dual functions: as a key regulator of plasmin generation and a component of the innate immune system. In the current issue, Wei and colleagues describe a transgenic mouse expressing Plaur RNA in glomerular podocytes. The mice manifested podocyte injury, including c-Src phosphorylation, proteinuria, and focal segmental glomerulosclerosis (FSGS). Plaur-transgenic mice on a β3 integrin-deficient background were protected from podocyte injury. Renal biopsies from subjects with FSGS, but not those with other glomerular diseases, manifested increased c-Src phosphorylation in podocytes. These findings suggest a novel injury mechanism in FSGS, with possible implications for new treatment strategies.

Diagnostic accuracy of combinations of serological biomarkers for identifying clinical tuberculosis.

Confirmation of tuberculosis (TB) cases in endemic TB settings is a challenge; obtaining fast and cheap, though accurate, diagnostic tools such as biomarkers is thus urgently needed to enable the early detection of TB. This paper evaluates the diagnostic accuracy of combinations of host serological biomarkers for identifying TB. Enzyme-linked immunosorbent assays (ELISA) were used on 70 Venezuelan Creole individuals for evaluating host biomarkers (i.e. CXCL9, sCD14, MMP9 and uPAR proteins) and anti-synthetic peptides covering certain Mycobacterium tuberculosis (Mtb) ESAT-6 (P-12033, P-12034 and P-12037) and Ag85A (P-29878) antigen sequences. The target population consisted of adults having active TB (ATB, n = 28), the tuberculin skin test positive (TST+) or individuals with latent TB infection (LTB, n = 28) and TST- or control subjects (CTRL, n = 14). Receiver operator curve (ROC) analysis revealed good biosignature discriminative ability for 5 serological biomarkers; the accuracy of 3 combinations had a good discriminative ability for diagnosing TB. Anti-P-12034/uPAR detected TB with 96.7% sensitivity and 86.0% specificity, followed by anti-P-12033/uPAR having 96.7% sensitivity and 81.4% specificity. Anti-P-29878/MMP9 had the highest sensitivity (100%), but low specificity (52.17%). Biomarker combinations did not prove efficacious for identifying incipient subclinical TST+TB- subjects at high-risk for TB. The anti-P-12034/uPAR combination could be useful for identifying clinical TB patients. Such an approach holds promise for further validation.

Probing Cholesterol-Dependence of Integrin-Urokinase Receptor Complex Formation using Confocal Dual-Color Fluorescence Intensity Analysis

The modern view of a plasma membrane is that of a complex supramolecular assembly of notable lipid/protein heterogeneity, in which the local lipid environment may have a profound impact on membrane protein distribution and function. Yet, the underlying mechanisms of lipid-mediated regulation of membrane protein functionality remain elusive, in part due to the limitations of existing methodologies and the often small size and transient nature of lipid/protein heterogeneities in cellular membranes. Exemplary, we still have rather limited knowledge about the significance of lipid composition in hetero-protein complex formation in such membrane systems. To address this fascinating, but poorly understood topic, here we introduce a single molecule-sensitive confocal experimental strategy, comprised of dual-color fluorescence peak analysis and photon counting histogram (PCH) method, which not only allows the detection of hetero-protein complexes in well-defined lipid environments, but also provides accurate insight into the composition of such complexes in the membrane. By applying the described methodology, here we demonstrate the formation of complexes of αvβ3 integrins and GPI-anchored urokinase plasminogen activator receptors (uPAR) in cholesterol-containing model lipid mixtures. PCH analysis of fluorescence intensity signals of complexes confirms furthermore that αvβ3-uPAR complexes are comprised of one integrin and two uPAR proteins. Remarkably, formation of comparable hetero-protein complexes is largely suppressed in a cholesterol-free lipid composition, highlighting the importance of cholesterol in αvβ3-uPAR complex formation. Our results are intriguing in light of the already established significance of cholesterol in integrin function in cellular membranes.

Chronic Resveratrol Treatment Inhibits MRC5 Fibroblast SASP-Related Protumoral Effects on Melanoma Cells.

Cellular senescence is related to organismal aging and is observed after DNA damaging cancer therapies, that induce tumor-suppressive modifications, but it is characterized by a strong increase in secreted factors, termed the "senescence-associated secretory phenotype" (SASP). Particularly, SASP from stroma senescent fibroblasts creates a cancer-favoring microenvironment, providing targets for anti-cancer interventions. In the present article, chronic treatment (5 weeks) with 5 µM resveratrol has been used to modulate senescence-related protumoral features of MRC5 fibroblasts, reducing SASP-related interleukins IL1α, IL1β, IL6, and IL8; transforming-growth-factor-β (TGFβ); matrix metallo-proteinases MMP3 and MMP2; urokinase plasminogen activator (uPA); receptor proteins uPAR, IL6R, insulin growth factor receptor-1 (IGF-1R), TGFβ-R2, and CXCR4. The cellular nuclear-factor-kB (NF-kB) protein level was also reduced, confirming its role in the induction of SASP. Resveratrol pretreated MRC5 fibroblasts were resistant to activation by TGFβ. Resveratrol treatment of senescent MRC5 induced the production of conditioned media (CM) which counteracted the protumoral effect of senescent CM on A375 and A375-M6 melanoma cell proliferation and invasiveness, and reduced the expression of epithelial-to-mesenchymal transition markers related to malignant features. This experimental approach proposes a treatment that targets the senescent stromal cell phenotype to induce an anti-tumor hosting microenvironment, which is suitable for both preventive and therapeutic purposes.

Heat shock proteins HSP70 and MRJ cooperatively regulate cell adhesion and migration through urokinase receptor.

BackgroundThe urokinase-type plasminogen activator receptor (uPAR) is an important regulator of ECM proteolysis, cell-ECM interactions and cell signaling. uPAR and heat shock proteins HSP70 and MRJ (DNAJB6) have been implicated in tumor growth and metastasis. We have reported recently that MRJ (DNAJB6, a heat shock protein) can interact with uPAR and enhance cell adhesion. Here, we identified another heat shock protein HSP70 as a novel uPAR-interacting protein.MethodsWe performed co-immunoprecipitation in human embryonic kidney (HEK) 293 and colon cancer HCT116 cells as well as immunofluorence assays in HEK293 cells stably transfected with uPAR to investigate the association of suPAR with HSP70/MRJ. To understand the biological functions of the triple complex of suPAR/HSP70/MRJ, we determined whether HSP70 and/or MRJ regulated uPAR-mediated cell invasion, migration, adhesion to vitronectin and MAPK pathway in two pair of human tumor cells (uPAR negative HEK293 cells vs HEK293 cells stably transfected with uPAR and HCT116 cells stably transfected with antisense-uPAR vs HCT116 mock cells transfected with vector only) using transwell assay, wound healing assay, quantitative RT-PCR analyzing mmp2 and mmp9 transcription levels, cell adhesion assay and Western blotting assay.ResultsHSP70 and MRJ formed a triple complex with uPAR and over-expression of MRJ enhanced the interaction between HSP70 and uPAR, while knockdown of MRJ decreased soluble uPAR in HCT116 cells (P < 0.05) and reduced the formation of the triple complex, suggesting that MRJ may act as an uPAR-specific adaptor protein to link uPAR to HSP70. Further experiments showed that knockdown of HSP70 and/or MRJ by siRNA inhibited uPAR-mediated cell adhesion to vitronectin as well as suppressed cell invasion and migration. Knockdown of HSP70 and/or MRJ inhibited expression of invasion related genes mmp2 and mmp9. Finally, HSP70 and/or MRJ up-regulated phosphorylation levels of ERK1/2 and FAK suggesting MAPK pathway was involved. All the biological function experiments in cell level showed an additive effect when HSP70 and MRJ were regulated simultaneously indicating their collaborated regulation effects on uPAR.ConclusionsThese findings may offer a novel insight into the interactions between uPAR and HSP70/MRJ and their functions in cell adhesion and migration may provide more understanding of the roles in regulating cancer metastasis.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-639) contains supplementary material, which is available to authorized users.

Nuclear Translocation of Hand-1 Acts as a Molecular Switch to Regulate Vascular Radiosensitivity in Medulloblastoma Tumors: The Protein uPAR Is a Cytoplasmic Sequestration Factor for Hand-1

Urokinase-type plasminogen activator receptor (uPAR) is overexpressed in the tumor-stromal invasive microenvironment in many human cancers, including medulloblastoma. The role of uPAR in tumor progression and angiogenesis has been well characterized. Previously, in medulloblastoma cells, we showed that ionizing radiation (IR)-induced uPAR is a potent activator of cancer stem cell (CSC)-like properties and is associated with various transcription factors that are involved during embryonic development and cancer. In the present study, we show that uPAR protein acts as a cytoplasmic sequestration factor for a novel basic helix-loop-helix transcription factor, Hand-1. The Hand-1 protein plays an essential role in the differentiation of trophoblast giant cells and cardiac morphogenesis, and yet its precise cellular function and its contribution to cancer remain mostly unknown. We also observed that the Hand-1 protein is upregulated in uPAR short hairpin RNA-treated medulloblastoma cells and accompanies sustained cell growth and angiogenesis. Furthermore, IR-induced uPAR overexpression negatively regulates Hand-1 activity and results in the stabilization of angiogenesis-promoting molecules, including hypoxia-inducible factor-1α. Finally, uPAR overexpression and its association with Hand-1 after IR treatment indicate that uPAR is capable of regulating Hand-1 and that uPAR has a role in the process of IR-induced tumor angiogenesis.

The role of fibrinolytic genes and proteins in the development of allograft vascular disease

We have previously shown that lack of plasminogen activator inhibitor-1 (PAI-1) expression in donor tissue greatly increases intimal proliferation (IP) after allogeneic transplantation. We sought to determine the relative role of PAI-1 and other fibrinolytic proteins in the development of IP. We used an abdominal aortic transplant model in mice to investigate IP in 3 groups of 6 recipients. In the isograft group, CBA/J strain mice were donors and recipients, donors for allograft group were C57BL/6J mice, and for the allograft/knockout group, C57BL/6J PAI-1 knockout mice. All groups received weekly injections of anti-CD8/CD4 monoclonal antibodies. IP was calculated at 50 days, and sections were analyzed for fibrinolytic proteins, messenger RNA (mRNA) and PAI-1 activity using immunohistochemistry (IHC), in situ hybridization (ISH), reverse transcription-polymerase chain reaction (RT-PCR), and Western blot analysis. Significantly more IP developed in the allograft/knockout group vs the isograft (p < 0.001) and the allograft groups (p = 0.003). There was marked intimal expression of tissue plasminogen activator (tPA), urokinase PA (uPA), and uPA receptor (uPAR) proteins and mRNA in the allograft and allograft/knockout groups vs the isograft group. Allografts also showed significant intimal staining for PAI-1 protein and mRNA. RT-PCR demonstrated a stepwise increase in profibrinolytic protein mRNA from isograft to allograft to allograft/knockout groups, particularly uPA (p = 0.02) and uPAR (p = 0.016). Western blot data showed complementary findings. PAI-1 activity was persistently present in isograft and allograft animals, only. Intimas in allograft and allograft/knockout groups were primarily smooth muscle cells. PAI-1 reduces IP by limiting smooth muscle cell activity, with little change in matrix composition likely by modulating profibrinolytic protein expression.

Oncogenic NRAS has multiple effects on the malignant phenotype of human melanoma cells cultured in vitro

Activating mutations in the NRAS gene, which occur predominantly in codon 61 (Q61R, Q61K) are among the most common genetic events in malignant melanoma. NRAS protein with oncogenic codon 61 mutations may therefore be good therapeutic targets. In the present study, we used gene expression profiling as a method for global characterization of gene expression alterations that resulted from treatment of melanoma cells with siRNA specifically targeting NRAS(Q61R). Sixteen probe sets representing 15 unique genes were identified whose expression was significantly altered by siRNA against NRAS(Q61R) in 2 melanoma cell lines. The genes with altered expression are involved in several functions, including modulation of cell growth, invasion and migration. The results suggest that downregulation of cyclin E2 and cyclin D1 and also upregulation of the negative cell-cycle regulator HBP1 in NRAS(Q61R) knockdown cells contribute to the inhibition of cell proliferation. Furthermore, suppression of oncogenic NRAS results in reduced migration and invasion, which is accompanied by downregulation of EphA2 (a receptor tyrosine kinase), uPAR (urokinase receptor) and cytoskeleton proteins such as leupaxin, paxillin and vinculin. These studies support the concept that suppression of oncogenic NRAS by siRNA can induce growth arrest and inhibit invasion of human melanoma cells by modulating the levels of these gene products.

PATE Gene Clusters Code for Multiple, Secreted TFP/Ly-6/uPAR Proteins That Are Expressed in Reproductive and Neuron-rich Tissues and Possess Neuromodulatory Activity

We report here syntenic loci in humans and mice incorporating gene clusters coding for secreted proteins each comprising 10 cysteine residues. These conform to three-fingered protein/Ly-6/urokinase-type plasminogen activator receptor (uPAR) domains that shape three-fingered proteins (TFPs). The founding gene is PATE, expressed primarily in prostate and less in testis. We have identified additional human PATE-like genes (PATE-M, PATE-DJ, and PATE-B) that co-localize with the PATE locus, code for novel secreted PATE-like proteins, and show selective expression in prostate and/or testis. Anti-PATE-B-specific antibodies demonstrated the presence of PATE-B in the region of the sperm acrosome and at high levels on malignant prostatic epithelial cells. The syntenic mouse Pate-like locus encompasses 14 active genes coding for secreted proteins, which are all, except for Pate-P and Pate-Q, expressed primarily in prostate and/or testis. Pate-P and Pate-Q are expressed solely in placental tissue. Castration up-regulates prostate expression of mouse Pate-B and Pate-E, whereas testosterone ablates this induced expression. The sequence similarity between TFP/Ly-6/uPAR proteins that modulate activity of nicotinic acetylcholine receptors and the PATE (Pate)-like proteins stimulated us to see whether these proteins possess analogous activity. Pharmacological studies showed significant modulation of the nicotinic acetylcholines by the PATE-B, Pate-C, and Pate-P proteins. In concert with these findings, certain PATE (Pate)-like genes were extensively expressed in neuron-rich tissues. Taken together, our findings indicate that in addition to participation of the PATE (Pate)-like genes in functions related to fertility and reproduction, some of them likely act as important modulators of neural transmission.

Monomer–dimer dynamics and distribution of GPI-anchored uPAR are determined by cell surface protein assemblies

To search for functional links between glycosylphosphatidylinositol (GPI) protein monomer–oligomer exchange and membrane dynamics and confinement, we studied urokinase plasminogen activator (uPA) receptor (uPAR), a GPI receptor involved in the regulation of cell adhesion, migration, and proliferation. Using a functionally active fluorescent protein–uPAR in live cells, we analyzed the effect that extracellular matrix proteins and uPAR ligands have on uPAR dynamics and dimerization at the cell membrane. Vitronectin directs the recruitment of dimers and slows down the diffusion of the receptors at the basal membrane. The commitment to uPA–plasminogen activator inhibitor type 1–mediated endocytosis and recycling modifies uPAR diffusion and induces an exchange between uPAR monomers and dimers. This exchange is fully reversible. The data demonstrate that cell surface protein assemblies are important in regulating the dynamics and localization of uPAR at the cell membrane and the exchange of monomers and dimers. These results also provide a strong rationale for dynamic studies of GPI-anchored molecules in live cells at steady state and in the absence of cross-linker/clustering agents.

Kininogen binding to the surfaces of macrophages

Kinin generation may be initiated on the cell surfaces via a primary kininogen docking which has been characterized for endothelial cells, platelets, neutrophils, astrocytes and smooth muscle cells. In this work we describe the adsorption of biotin-labeled human kininogens by murine RAW 264.7 macrophages and human U-937 monocytes/macrophages. Both cell types strongly bound high molecular mass kininogen (HK) in a zinc–ion dependent manner with the dissociation constants of 9.1 nM and 3.3 nM, respectively, and the binding capacities of 46 fmol and 71 fmol per million of respective cells. The HK binding was quenched by 50% by antibodies against Mac-1, gC1qR and uPAR proteins indicating that these macrophage surface receptors are involved in the HK adsorption. A significant increase of HK binding was observed after cell activation with phorbol myristate acetate. Our results suggest that macrophages, similarly to neutrophils, may supply kininogens to the inflammatory foci to support the local kinin production at these sites.

A doxycycline-inducible urokinase receptor (uPAR) upregulates uPAR activities including resistance to anoikis in human prostate cancer cell lines

BackgroundThe urokinase receptor (uPAR) mediates a diverse array of cellular processes including several events involved in prostate cancer metastasis. Many of these activities are initiated or enhanced by uPAR binding to its proteolytic ligand, urokinase (uPA). Our objective in this study was to generate and test an inducible lentiviral system capable of expressing uPAR and DsRed fluorescent protein in human prostate cancer cell lines.ResultsA DsRed-uPAR fusion construct was inserted into a lentiviral vector. Transduction of human prostate cancer cell lines with this virus and with a virus containing a reverse-tetracycline transactivator (rt-TA) resulted in a stable transgene which induced both uPAR and DsRed proteins in a dose-responsive fashion upon stimulation with doxycycline. Immunoblots and immunofluorescence studies indicated no detectable uPAR expression in non-induced prostate cancer cell lines. Cells with induced-uPAR demonstrated increased cellular adhesion to the matrix substrate vitronectin and increased net cell proliferation compared to uninduced cells. Finally, induced uPAR-expressing prostate cancer cells were resistant to anoikis over an extended time period when grown in suspension.ConclusionThis doxycycline-inducible lentivirus system produces titerable levels of biologically active uPAR in vitro. This tool can be used to dissect cellular events following induction of uPAR in prostate cancer cells.

High Expression of Plasminogen Activator Inhibitor‐2 (PAI‐2) is a Predictor of Improved Survival in Patients with Pancreatic Adenocarcinoma

Recent findings suggest that the urokinase-type plasminogen activator (uPA), its receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1), and -2 (PAI-2) play key roles in cancer invasion. The prognostic value of components of this system is well established in breast cancer. However, little is known of its involvement in pancreatic cancer (PC). Quantitative real-time polymerase chain reaction (Q-RT-PCR) was used on tissue-banked specimens and immunohistochemistry (IHC) on paraffin specimens was used to measure expression of uPA, uPAR, PAI-1, and PAI-2 proteins in 46 PC and 12 cystadenoma specimens. Results were related to survival using Cox's proportional hazards testing. Increased expression of uPA, uPAR, and PAI-1 in PC tissue were independently associated with a higher Union Internationale Contre le Cancer [International Union Against Cancer (UICC)] tumor stage (P < 0.001) and were intercorrelated (P < 0.001). Overexpression of uPAR indicated reduced survival (P = 0.03). Conversely, PAI-2 messenger ribonucleic acid (mRNA) overexpression, which occurred in 21 of 46 tumors, negatively correlated with tumor size (P = 0.008) and survival (P < 0.007) but not with uPA, uPAR, or tumor stage. There was good agreement between PAI-2 mRNA value and IHC score (P < 0.001). Using Cox's stepwise analysis, PAI-2 mRNA value (HR = 0.24; P = 0.001) and UICC tumor stage (HR = 2.014; P = 0.001) independently predicted survival. An IHC score for PAI-2 of 3+ or 4+ also independently predicted improved survival (HR = 2.72; P = 0.025). The uPA/uPAR/PAI-1 system is activated in advanced pancreatic cancer and may account for the tumor's aggressive behavior, whereas PAI-2 expression appears to be independent of uPA/uPAR/PAI-1 and is associated with improved prognosis. Because of its intercorrelation with mRNA expression, PAI-2 IHC may be used as an indicator of survival.

Evaluation of urokinase plasminogen activator and its receptor in different grades of human prostate cancer

Increased expression of urokinase plasminogen activator (uPA) and its receptor (uPAR) has been reported in various malignancies including prostate cancer (CaP). However, their expression in the different grades of CaP remains poorly understood. Here, we use tissue microarrays to examine the expression of uPA and uPAR in different grades of human CaP and to establish the potential of these tumor-associated antigens as candidates for targeted therapy. One hundred twenty paraffin-embedded specimens were selected from patients who underwent radical retropubic prostatectomy or transurethral resection of the prostate for primary untreated CaP and 10 matched lymph node metastases. Monoclonal antibodies #394 and #3936 were used on tissue microarrays with standard immunohistochemistry to examine uPA and uPAR expression, respectively. Overexpression of uPA and uPAR was detected in 53% and 64% of primary CaP tissues, respectively, and in more than 90% of lymph node metastases, but not in normal prostates or benign tissues. Of the uPA and uPAR positive tumors, 76% and 68% were Gleason score 7 or higher, respectively, and most of these tumors also showed stromal staining. The overexpression of uPA and uPAR was highly related to tumor differentiation in patients with CaP. Both uPA and uPAR proteins are candidate therapeutic targets for cancer therapy to control micrometastases and hormone refractory disease in CaP.

Specific immunoassays for detection of intact and cleaved forms of the urokinase receptor.

The cell surface receptor (uPAR) for urokinase plasminogen activator (uPA) is a strong prognostic marker in several types of cancer. uPA cleaves the three-domain protein uPAR(I-III) into two fragments: uPAR(I), which contains domain I; and uPAR(II-III), which contains domains II and III. Established immunoassays measure a combination of uPAR forms. Our aim was to design immunoassays for specific quantification of the individual forms of uPAR. Using appropriate combinations of epitope-mapped monoclonal antibodies (Mabs) for capture and europium-labeled detection Mabs, we designed two-site sandwich time-resolved fluorescence immunoassays (TR-FIAs): TR-FIA 1 to measure uPAR(I-III) alone; TR-FIA 2 to measure both uPAR(I-III) and uPAR(II-III); and TR-FIA 3 to measure uPAR(I). To avoid detection of uPAR(I-III) in TR-FIA 3, we used a combination of the peptide uPAR antagonist AE120 and a domain I antibody, R3. AE120 blocks the binding of R3 to uPAR(I-III). In contrast, AE120 does not interact with liberated domain I and therefore does not interfere with the binding of R3 to uPAR(I). The limits of quantification (CV <20%) determined by adding the proteins to uPAR-depleted plasma were <3 pmol/L in all three assays. The interassay CVs in plasma with added analytes were <11%, and recoveries were between 93% and 105%. Cross-reactivities of purified proteins in the three TR-FIAs were no more than 4%. Studies on chymotrypsin cleavage of uPAR and size-exclusion chromatography of plasma with and without added protein further supported the specificity of the assays. The three novel TR-FIAs accurately quantify uPAR(I-III) alone, uPAR(I-III) together with uPAR(II-III), and uPAR(I), respectively, in biological samples, including plasma, and thus are well suited for studies of the diagnostic and prognostic value of individual uPAR forms in cancer patients.