Expression of urokinase-type plasminogen activator system in non-metastatic prostate cancer.

PURPOSE: To investigate the prognostic role of expression of urokinase-type plasminogen activator system members, such as urokinase-type activator (uPA), uPA-receptor (uPAR), and plasminogen activator inhibitor-1 (PAI-1), in patients treated with radical prostatectomy (RP) for prostate cancer (PCa). METHODS: Immunohistochemical staining for uPA system was performed on a tissue microarray of specimens from 3121 patients who underwent RP. Cox regression analyses were performed to investigate the association of overexpression of these markers alone or in combination with biochemical recurrence (BCR). Decision curve analysis was used to assess the clinical impact of these markers. RESULTS: uPA, uPAR, and PAI-1 were overexpressed in 1012 (32.4%), 1271 (40.7%), and 1311 (42%) patients, respectively. uPA overexpression was associated with all clinicopathologic characteristics of biologically aggressive PCa. On multivariable analysis, uPA, uPAR, and PAI-1 overexpression were all three associated with BCR (HR: 1.75, p < 0.01, HR: 1.22, p = 0.01 and HR: 1.20, p = 0.03, respectively). Moreover, the probability of BCR increased incrementally with increasing cumulative number of overexpressed markers. Decision curve analysis showed that addition of uPA, uPAR, and PAI-1 resulted in a net benefit compared to a base model comparing standard clinicopathologic features across the entire threshold probability range. In subgroup analyses, overexpression of all three markers remained associated with BCR in patients with favorable pathologic characteristics. CONCLUSION: Overexpression of uPA, uPAR, and PAI-1 in PCa tissue were each associated with worse BCR. Additionally, overexpression of all three markers is informative even in patients with favorable pathologic characteristics potentially helping clinical decision-making regarding adjuvant therapy and/or intensified follow-up.

Changes in podocyte TRPC channels evoked by plasma and sera from patients with recurrent FSGS and by putative glomerular permeability factors.

Primary forms of focal and segmental glomerulosclerosis (FSGS) are driven by circulating factors that cause dysfunction or loss podocytes. Rare genetic forms of FSGS can be caused by mutations in TRPC6, which encodes a Ca2+-permeable cationic channel expressed in mesangial cells and podocytes; and NPHS2, which encodes podocin, a TRPC6-binding protein expressed in podocyte slit diaphragm domains. Here we observed that exposing immortalized mouse podocytes to serum or plasma from recurrent FSGS patients for 24h increased the steady-state cell-surface abundance of TRPC6, accompanied by an increase in currents through endogenous TRPC6 channels evoked by a hypoosmotic stretch stimulus. These effects were mimicked by the soluble urokinase receptor (suPAR) and by tumor necrosis factor (TNF), circulating factors implicated in nephrotic syndromes. Most but not all of the recurrent FSGS plasma samples that we examined also caused a loss of podocin over a period of several hours. The loss of podocin was also seen following exposure to suPAR but not TNF. However, TNF increased the effects of suPAR on TRPC6 and podocin, and TNF and suPAR are required for the full effects of one of the recurrent FSGS plasma samples. The actions of FSGS plasma, suPAR and TNF on surface abundance of TRPC6 were blocked by cilengitide, an inhibitor of αvß3-integrin signaling. These data suggest that primary FSGS is a heterogeneous condition mediated by multiple circulating factors, and support TRPC6 and αvß3-integrin as potential therapeutic targets.

The NuRD chromatin-remodeling enzyme CHD4 promotes embryonic vascular integrity by transcriptionally regulating extracellular matrix proteolysis.

The extracellular matrix (ECM) supports vascular integrity during embryonic development. Proteolytic degradation of ECM components is required for angiogenesis, but excessive ECM proteolysis causes blood vessel fragility and hemorrhage. Little is understood about how ECM proteolysis is transcriptionally regulated during embryonic vascular development. We now show that the NuRD ATP-dependent chromatin-remodeling complex promotes vascular integrity by preventing excessive ECM proteolysis in vivo. Mice lacking endothelial CHD4--a catalytic subunit of NuRD complexes--died at midgestation from vascular rupture. ECM components surrounding rupture-prone vessels in Chd4 mutants were significantly downregulated prior to embryonic lethality. Using qPCR arrays, we found two critical mediators of ECM stability misregulated in mutant endothelial cells: the urokinase-type plasminogen activator receptor (uPAR or Plaur) was upregulated, and thrombospondin-1 (Thbs1) was downregulated. Chromatin immunoprecipitation assays showed that CHD4-containing NuRD complexes directly bound the promoters of these genes in endothelial cells. uPAR and THBS1 respectively promote and inhibit activation of the potent ECM protease plasmin, and we detected increased plasmin activity around rupture-prone vessels in Chd4 mutants. We rescued ECM components and vascular rupture in Chd4 mutants by genetically reducing urokinase (uPA or Plau), which cooperates with uPAR to activate plasmin. Our findings provide a novel mechanism by which a chromatin-remodeling enzyme regulates ECM stability to maintain vascular integrity during embryonic development.

In vivo anti-metastatic effects of uPAR retargeted measles virus in syngeneic and xenograft models of mammary cancer.

The urokinase receptor (uPAR) plays a critical role in breast cancer (BC) progression and metastases and is a validated target for novel therapies. The current study investigates the effects of MV-uPA, an oncolytic measles virus fully retargeted against uPAR in syngeneic and xenograft BC metastases models. In vitro replication and cytotoxicity of MVs retargeted against human (MV-h-uPA) or mouse (MV-m-uPA) uPAR were assessed in human and murine cancer and non-cancer mammary epithelial cells. The in vivo effects of species-specific uPAR retargeted MVs were assessed in syngeneic and xenograft models of experimental metastases, established by intravenous administration of luciferase expressing 4T1 or MDA-MD-231 cells. Metastases progression was assessed by in vivo bioluminescence imaging. Tumor targeting was evaluated by qRT-PCR of MV-N, rescue of viable viral particles, and immunostaining of MV particles in lungs from tumor bearing mice. In vitro, MV-h-uPA and MV-m-uPA selectively infected, replicated, and induced cytotoxicity in cancer compared to non-cancer cells in a species-specific manner. In vivo, MV-m-uPA delayed 4T1 lung metastases progression and prolonged survival. These effects were associated with identification of viable viral particles, viral RNA, and detection of MV-N by immunostaining from lung tissues in treated mice. In the human MDA-MB-231 metastases model, intravenous administration of MV-h-uPA markedly inhibited metastases progression and significantly improved survival, compared to controls. No significant treatment-related toxicity was observed in treated mice. The above preclinical findings strongly suggest that uPAR retargeted measles virotherapy is a novel and feasible systemic therapy strategy against metastatic breast cancer.

Macrophages of M1 phenotype have properties that influence lung cancer cell progression.

Stromal macrophages of different phenotypes can contribute to the expression of proteins that affects metastasis such as urokinase-type plasminogen activator (uPA), its receptor uPAR, and plasminogen activator inhibitor-1 (PAI-1), but knowledge of how essential their contribution is in comparison to the cancer cells in small cell lung cancer (SCLC) and lung squamous cell carcinoma (SCC) is lacking. The expression of uPA, uPAR, and PAI-1 and of the matrix metalloproteinases (MMP)-2 and MMP-9 were studied in human macrophages of M1 and M2 phenotype and compared to a lung SCC (NCI-H520) and a SCLC (NCI-H69) cell line. Effects of treatment with conditioned media (CM) from M1 and M2 macrophages on the expression of these genes in H520 and H69 cells as well as effects on the cell growth were investigated. In addition, data on the stromal macrophages immunoreactivity of uPAR, MMP-2, and MMP-9 in a few SCC and SCLC biopsies was included. uPAR, MMP-2, and MMP-9 were confirmed in stromal cells including macrophages in the SCC and SCLC biopsies. In vitro, both macrophage phenotypes expressed considerably higher mRNA levels of uPA, uPAR, PAI-1, and MMP-9 compared to the cancer cell lines, and regarding uPAR, the highest level was found in the M1 macrophage phenotype. Furthermore, M1 CM treatment not only induced an upregulation of PAI-1 in both H520 and H69 cells but also inhibited cell growth in both cell lines, giving M1 macrophages both tumor-promoting and tumor-killing potential.

Overexpression of ETV4 is associated with poor prognosis in prostate cancer: involvement of uPA/uPAR and MMPs.

ETS gene fusions involving ERG, ETV1, ETV4, ETV5, and FLI1 define a distinct class of prostate cancer (PCa), and this might have a bearing on diagnosis, prognosis, and rational therapeutic targeting. In the current study, we focused on the clinicopathological significance of ETV4 in Chinese PCa patients and the mechanisms whereby ETV4 overexpression mediates tumor invasion in the prostate. Overall, ETV4 overexpression was identified in 30.4 % (45/148) of PCa cases by immunohistochemistry. Accordingly, ETV4 was rearranged in only 1.6 % (2/128) of PCa patients. Clinically, ETV4 overexpression was significantly correlated with Gleason score (P = 0.045) and pathological tumor stage (P = 0.041). Multivariate Cox regression analysis indicated that ETV4 is an unfavorable independent prognostic factor (P = 0.040). Functional studies further showed that small interfering RNA (siRNA) knockdown of ETV4 significantly decreases proliferation and invasion of PC-3 cell and partially reverses epithelial-mesenchymal transition in vitro. Notably, ETV4 knockdown significantly downregulated expression of urokinase plasminogen activator (uPA) and its receptor (uPAR) at messenger RNA (mRNA) and protein levels. Chromatin immunoprecipitation assay demonstrated that ETV4 regulates uPA expression through direct binding to its promoter region. Additionally, ETV4 knockdown was also observed to significantly inhibit expression of matrix metalloproteinase (MMP)-2 and MMP-9. In conclusion, for the first time, our study suggested that ETV4 is an independent poor prognostic factor in Chinese PCa patients. Silencing of ETV4 suppresses invasion of PCa cells by inhibiting the expression of uPA/uPAR as well as MMPs. Further studies will be needed to determine whether ETV4 could be regarded as a potential target for the management and prevention of PCa.

Subpopulations of uPAR+ contribute to vasculogenic mimicry and metastasis in large cell lung cancer.

The urokinase plasminogen activator receptor (uPAR) is closely associated with poor prognosis in various aggressive cancers including large-cell lung cancer (LCLC). Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks involving the blood supply in early tumor formation. We demonstrate the statistically positive correlation of uPAR expression with VM formation, metastasis, and poor prognosis of LCLC patients. uPAR(+) cells sorted from the LCLC H460 cell line show higher invasion, migration capacity, and tube structure formation capability on Matrigel compared with uPAR(-) cells. uPAR(+) tumor cells highly expressed vimentin and VE-cadherin; the epithelial marker E-cadherin was low expressed. Higher EMT-regulated protein twist and snail expressions were also observed in these cells. uPAR(+) cells injected subcutaneously into nude mice markedly increased tumor growth, induced VM formation and liver metastasis; by contrast, uPAR(-) cells did not. The data suggest that uPAR expression may predict VM formation, tumor metastasis and poorer prognosis of LCLC patients. The uPAR gene may be used as a novel therapeutic target for inhibiting angiogenesis and metastasis in LCLC.

Elongation factor-2 kinase regulates TG2/ß1 integrin/Src/uPAR pathway and epithelial-mesenchymal transition mediating pancreatic cancer cells invasion.

Pancreatic ductal adenocarcinoma is one of the lethal cancers with extensive local tumour invasion, metastasis, early systemic dissemination and poorest prognosis. Thus, understanding the mechanisms regulating invasion/metastasis and epithelial-mesenchymal transition (EMT), is the key for developing effective therapeutic strategies for pancreatic cancer (PaCa). Eukaryotic elongation factor-2 kinase (eEF-2K) is an atypical kinase that we found to be highly up-regulated in PaCa cells. However, its role in PaCa invasion/progression remains unknown. Here, we investigated the role of eEF-2K in cellular invasion, and we found that down-regulation of eEF-2K, by siRNA or rottlerin, displays impairment of PaCa cells invasion/migration, with significant decreases in the expression of tissue transglutaminase (TG2), the multifunctional enzyme implicated in regulation of cell attachment, motility and survival. These events were associated with reductions in ß1 integrin/uPAR/MMP-2 expressions as well as decrease in Src activity. Furthermore, inhibition of eEF-2K/TG2 axis suppresses the EMT, as demonstrated by the modulation of the zinc finger transcription factors, ZEB1/Snail, and the tight junction proteins, claudins. Importantly, while eEF-2K silencing recapitulates the rottlerin-induced inhibition of invasion and correlated events, eEF-2K overexpression, by lentivirus-based expression system, suppresses such rottlerin effects and potentiates PaCa cells invasion/migration capability. Collectively, our results show, for the first time, that eEF-2K is involved in regulation of the invasive phenotype of PaCa cells through promoting a new signalling pathway, which is mediated by TG2/ß1 integrin/Src/uPAR/MMP-2, and the induction of EMT biomarkers which enhance cancer cell motility and metastatic potential. Thus, eEF-2K could represent a novel potential therapeutic target in pancreatic cancer.

Expression of uPAR in tumor-associated stromal cells is associated with colorectal cancer patient prognosis: a TMA study.

BACKGROUND: The receptor for urokinase-type plasminogen activator (uPAR) is associated with cancer development and progression. Within the tumor microenvironment uPAR is expressed by malignant cells as well as tumor-associated stromal cells. However, the contribution of uPAR expression in these stromal cells to malignancy and patient survival in colorectal cancer is still unclear. This study compares the association of uPAR expression in both colorectal tumor-associated stromal cells and neoplastic cells with clinico-pathological characteristics and patient survival using tissue micro arrays (TMA). METHODS: Immunohistochemical staining of uPAR expression was performed on tumor tissue from 262 colorectal cancer patients. Kaplan-Meier, log rank, and uni- and multivariate Cox's regression analyses were used to calculate associations between uPAR expression and patient survival. RESULTS: In the colorectal tumor-associated stromal microenvironment, uPAR is expressed in macrophages, (neoangiogenic) endothelial cells and myofibroblasts. uPAR expression in tumor-associated stromal cells and neoplastic cells (and both combined) were negatively associated with overall survival (OS) and Disease Free Survival (DFS). Uni- and multivariate Cox's regression analysis for combined uPAR expression in tumor-associated stromal and neoplastic cells showed significant and independent negative associations with OS and DFS. Only uPAR expression in tumor-associated stromal cells showed independent significance in the uni- and multivariate analysis for DFS. CONCLUSION: This study demonstrates a significant independent negative association between colorectal cancer patient survival and uPAR expression in especially tumor-associated stromal cells.

Regulation of u-PAR gene expression by H2A.Z is modulated by the MEK-ERK/AP-1 pathway.

The urokinase receptor (u-PAR) which is largely regulated at the transcriptional level has been implicated in tumor progression. In this study, we explored the epigenetic regulation of u-PAR and showed that the histone variant H2A.Z negatively regulates its expression in multiple cell lines. Chromatin immunoprecipitation assays revealed that H2A.Z was enriched at previously characterized u-PAR-regulatory regions (promoter and a downstream enhancer) and dissociates upon activation of gene expression by phorbol ester (PMA). Using specific chemical and dominant negative expression constructs, we show that the MEK-ERK signaling pathway terminating at AP-1 transcription factors intersects with the epigenetic control of u-PAR expression by H2A.Z. Furthermore, we demonstrate that two other AP-1 targets (MMP9 gene and miR-21 microRNA) are also H2A.Z regulated. In conclusion, our work demonstrates that (i) the expression of two genes and a microRNA all implicated in tumor progression are directly regulated by H2A.Z and (ii) MEK-ERK signaling terminating at AP-1 intersects with the epigenetic control of target gene expression by H2A.Z.

DJ-1 promotes invasion and metastasis of pancreatic cancer cells by activating SRC/ERK/uPA.

A major hallmark of pancreatic ductal adenocarcinoma (PDAC) is extensive local tumor invasion and early systemic dissemination. DJ-1 has been shown to prevent cell death via the Akt pathway, thereby playing an important role in cancer progression and Parkinson's disease development. Here, we investigated the role of DJ-1 in tumor invasion and metastasis of pancreatic cancer and showed that DJ-1 is upregulated in 68.5% of pancreatic cancer specimens, correlated with tumor stage and predictive of short overall survival. Knockdown of DJ-1 expression in two PDAC cell lines reduced cell migration and invasion potential in vitro and inhibited metastasis in vivo. Knockdown of DJ-1 led to cytoskeleton disruption and diminished urokinase plasminogen activator (uPA) activity and expression, without affecting plasminogen activator inhibitor-1 and uPA receptor (uPAR) expression. All these effects were reversed by restoration of DJ-1 expression. In determining the pathway through which DJ-1 regulated cell migration and invasion, DJ-1 was found not to regulate Akt phosphorylation. Rather, it promoted extracellular signal-regulated kinase (ERK) and SRC phosphorylation. Inhibition of the ERK pathway in PDAC mimicked the effects of DJ-1 on cell migration, invasion, actin cytoskeleton and uPA/uPAR system and abolished the effects on promoting PDAC cell invasion and migration. These data represent the first identification of an important function of DJ-1, which is to regulate the invasion and metastasis properties of PDAC through the ERK/uPA cascade.

Urokinase receptor is necessary for bacterial defense against pneumonia-derived septic melioidosis by facilitating phagocytosis.

Urokinase receptor (urokinase-type plasminogen activator receptor [uPAR], CD87), a GPI-anchored protein, is considered to play an important role in inflammation and fibrinolysis. The Gram-negative bacterium Burkholderia pseudomallei is able to survive and replicate within leukocytes and causes melioidosis, an important cause of pneumonia-derived community-acquired sepsis in Southeast Asia. In this study, we investigated the expression and function of uPAR both in patients with septic melioidosis and in a murine model of experimental melioidosis. uPAR mRNA and surface expression was increased in patients with septic melioidosis in/on both peripheral blood monocytes and granulocytes as well as in the pulmonary compartment during experimental pneumonia-derived melioidosis in mice. uPAR-deficient mice intranasally infected with B. pseudomallei showed an enhanced growth and dissemination of B. pseudomallei when compared with wild-type mice, corresponding with increased pulmonary and hepatic inflammation. uPAR knockout mice demonstrated significantly reduced neutrophil migration toward the pulmonary compartment after inoculation with B. pseudomallei. Further in vitro experiments showed that uPAR-deficient macrophages and granulocytes display a markedly impaired phagocytosis of B. pseudomallei. Additional studies showed that uPAR deficiency did not influence hemostatic and fibrinolytic responses during severe melioidosis. These data suggest that uPAR is crucially involved in the host defense against sepsis caused by B. pseudomallei by facilitating the migration of neutrophils toward the primary site of infection and subsequently facilitating the phagocytosis of B. pseudomallei.

Expression patterns of uPAR, TF and EGFR and their potential as targets for molecular imaging in oropharyngeal squamous cell carcinoma.

The clinical introduction of molecular imaging for the management of oropharyngeal squamous cell carcinoma (OPSCC) relies on the identification of relevant cancer­specific biomarkers. The application of three membrane­bound receptors, namely urokinase­type plasminogen activator receptor (uPAR), tissue factor (TF) and EGFR have been previously explored for targeted imaging and therapeutic strategies in a broad range of solid cancers. The present study aimed to investigate the expression patterns of uPAR, EGFR and TF by immunohistochemistry (IHC) to evaluate their potential for targeted imaging and prognostic value in OPSCC. In a retrospective cohort of 93 patients with primary OPSCC, who were balanced into the 45 human papillomavirus (HPV)­positive and 48 HPV­negative groups, the IHC­determined expression profiles of uPAR, TF and EGFR in large biopsy or tumor resection specimens were analyzed. Using the follow­up data, overall survival (OS) and recurrence­free survival were measured. Specifically, associations between survival outcome, biomarker expression and clinicopathological factors were examined using Cox proportional hazards model and log­rank test following Kaplan­Meier statistics. After comparing the expression pattern of biomarkers within the tumor compartment with that in the adjacent normal tissues, uPAR and TF exhibited a highly tumor­specific expression pattern, whereas EGFR showed a homogeneous expression within the tumor compartment as well as a consistent expression in the normal mucosal epithelium and salivary gland tissues. The positive expression rate of uPAR, TF and EGFR in the tumors was 98.9, 76.3 and 98.9%, respectively. No statistically significant association between biomarker expression and survival outcome could be detected. Higher uPAR expression levels had a trend towards reduced OS according to results from univariate analysis (P=0.07; hazard ratio=2.01; 95% CI=0.92­4.37). Taken together, these results suggest that uPAR, TF and EGFR may be suitable targets for molecular imaging and therapy in OPSCC. In particular, uPAR may be an attractive target owing to their high positive expression rates in tumors and a highly tumor­specific expression pattern.

Gastrin stimulates expression of plasminogen activator inhibitor-1 in gastric epithelial cells.

Plasminogen activator inhibitor (PAI)-1 is associated with cancer progression, fibrosis and thrombosis. It is expressed in the stomach but the mechanisms controlling its expression there, and its biological role, are uncertain. We sought to define the role of gastrin in regulating PAI-1 expression and to determine the relevance for gastrin-stimulated cell migration and invasion. In gastric biopsies from subjects with elevated plasma gastrin, the abundances of PAI-1, urokinase plasminogen activator (uPA), and uPA receptor (uPAR) mRNAs measured by quantitative PCR were increased compared with subjects with plasma concentrations in the reference range. In patients with hypergastrinemia due to autoimmune chronic atrophic gastritis, there was increased abundance of PAI-1, uPA, and uPAR mRNAs that was reduced by octreotide or antrectomy. Immunohistochemistry revealed localization of PAI-1 to parietal cells and enterochromaffin-like cells in micronodular neuroendocrine tumors in hypergastrinemic subjects. Transcriptional mechanisms were studied by using a PAI-1-luciferase promoter-reporter construct transfected into AGS-G(R) cells. There was time- and concentration-dependent increase of PAI-1-luciferase expression in response to gastrin that was reversed by inhibitors of the PKC and MAPK pathways. In Boyden chamber assays, recombinant PAI-1 inhibited gastrin-stimulated AGS-G(R) cell migration and invasion, and small interfering RNA treatment increased responses to gastrin. We conclude that elevated plasma gastrin concentrations are associated with increased expression of gastric PAI-1, which may act to restrain gastrin-stimulated cell migration and invasion.

Differences in integrin expression and signaling within human breast cancer cells.

BACKGROUND: Integrins are used as prognostic indicators in breast cancer. Following engagement with extracellular matrix proteins, their signaling influences numerous cellular processes including migration, proliferation, and death. Integrin signaling varies between cell types through differential expression of integrin subunits, and changes within a given cell upon exposure to a cell agonist or through changes in its surroundings. These variations in signaling can profoundly affect the phenotypic, tumorogenecity and metastatic properties of cancer cells. In the present study, we investigated if there were differences in the expression of integrins, integrin structures, and integrin co-receptors within three breast cancer cells and if these differences effected integrin signaling. METHODS: Expression of integrins, urokinase receptor and vascular endothelial cell growth factor receptor (VEGFR) in metastatic MDA-MB-435 and MDA-MB-231, non-metastatic MCF7 and non-breast cancer Hek-293 cells was measured by flow cytometry. Cell adhesion was assessed using collagen, fibrinogen, fibronectin and vitronectin coated plates. Changes in kinase levels following PMA stimulation, and cell adhesion-induced activation of kinases were determined by western blot analysis. Distribution of actin stress fibers and focal adhesions was assessed by immunocytochemistry. RESULTS: All cells expressed αv integrins, while high ß5 and αvß5 expression was restricted to the cancer cells and high ß3 and αvß3 expression was restricted to MDA-MB-435 cells. The two metastatic cells were the least adhesive, but all cells adhered well to most proteins in the absence of PMA. All proliferating cells expressed activated pSrc, but only proliferating metastatic cells expressed high pMEK levels. PMA treatment resulted in time-dependent changes in activated kinase levels, and only MDA-MB-231 cells constitutively expressed high levels of activated pMEK. MDA-MB-435 cells formed more stress fibers and focal adhesions and only exhibited adhesion-induced activation of pMEK and pFAK. All cells expressed the urokinase receptor, but MCF7 cells had markedly higher VEGFR expression. Adhesion induced differential expression of pFAK, pMEK and pERK. CONCLUSIONS: This study demonstrates that breast cancers vary in their expression of integrins, their capacity to form focal adhesion and to signal through integrins. These differences likely contribute to phenotypic variations between cancer lines and account for some of the heterogeneity of breast cancer.

Urokinase plasminogen activator receptor is expressed in invasive cells in gastric carcinomas from high- and low-risk countries.

Gastric cancer is the second cancer causing death worldwide. Both incidence and mortality rates vary according to geographical regions. The receptor for urokinase plasminogen activator (uPAR) is involved in extracellular matrix degradation by mediating cell surface associated plasminogen activation, and its presence on gastric cancer cells is linked to micro-metastasis and poor prognosis. Immunohistochemical analyses of a set of 44 gastric cancer lesions from Costa Rica showed expression of uPAR in cancer cells in both intestinal subtype (14 of 27) and diffuse subtype (10 of 17). We compared the expression pattern of uPAR in gastric cancers from a high-risk country (Costa Rica) with a low-risk country (Norway). We found uPAR on gastric cancer cells in 24 of 44 cases (54%) from Costa Rica and in 13 of 23 cases (56%) from Norway. uPAR was seen in macrophages and neutrophils in all cases. We also examined the nonneoplastic mucosa and found that uPAR was more frequently seen in epithelial cells located at the luminal edge of the crypts in cases with Helicobacter pylori infection than in similar epithelial cells in noninfected mucosa (p = 0.033; chi(2) = 4.54). In conclusion, the expression of uPAR in cancer cells in more than half of the gastric cancer cases suggests that their uPAR-positivity do not contribute to explain the different mortality rates between the 2 countries, however, the actual prevalence of uPAR-positive cancer cells in the gastric cancers may still provide prognostic information.

Increased expression of urokinase-type plasminogen activator receptor in the frontal cortex of patients with intractable frontal lobe epilepsy.

Urokinase-type plasminogen activator receptor (uPAR) is a glycosyl phosphatidylinositol-anchored protein involved in cell adhesion, proliferation, differentiation, migration, invasion, and tissue repair and remodeling. Our aim was to investigate uPAR expression in the frontal cortex of patients with intractable frontal lobe epilepsy and to explore the possible role of uPAR in intractable epilepsy. Tissue samples were obtained from the frontal cortex of 25 patients who had undergone surgery for intractable epilepsy and 15 histologically normal frontal cortex tissues from patients with orbital frontal lobe severe contusion (the control group). The frontal cortex expression of uPAR was studied by Western blot and immnohistochemistry. Double immunofluorescence was used to determine the expression of uPAR in astrocytes, microglia, and neurons. The normal frontal cortex uPAR protein level was shown to be low. In the brain tissue of patients with intractable epilepsy, the expression of uPAR protein increased dramatically. Based on the results of double immunofluorescence, many uPAR-positive cells are colocalized with the cell soma of NeuN-positive neurons, whereas only a few GFAP- and CD11b-positive cells colocalized with uPAR staining. These findings provide new information pertaining to the epileptogenesis of intractable epilepsy and suggest that increased expression of uPAR in human brain may be associated with human intractable epilepsy.

Activation of the plasma kallikrein-kinin system on human lung epithelial cells.

Activation of the tissue kallikrein-kinin system (KKS) plays a major inflammatory role in the lung, but the contribution of the plasma KKS remains unclear. Plasma KKS involves assembly and activation of high molecular weight kininogen (HK) and plasma prekallikrein (PPK) on cell surfaces, resulting in the liberation of the inflammatory peptide, bradykinin (BK), from HK by plasma kallikrein (PK). To this end, we determined the possible contribution of plasma KKS in BK formation using airway epithelium. The HK binding proteins, urokinase plasminogen activator receptor, cytokeratin 1 and gC1qR, were expressed on transformed A549 and BEAS-2B cell lines, as well as on normal lung tissue, but Mac-1 was absent. A549 cells bound FITC-labelled HK, which was only partially inhibited by a combination of antibodies to the HK binding proteins. HK-PPK complex activation on the transformed epithelial cell lines, as well as primary epithelial cells, resulted in PK formation and liberation of BK. HK-PPK activation was inhibited by cysteine, BK and protamine, and by novobiocin, a heat shock protein 90 (HSP90) inhibitor. In summary, lung epithelial cells support the assembly and activation of the plasma KKS by a mechanism dependent on HSP90, and could contribute to KKS-mediated inflammation in lung disease.

Urokinase plasminogen activator receptor-deficient mice demonstrate reduced hyperoxia-induced lung injury.

Patients with respiratory failure often require supplemental oxygen therapy and mechanical ventilation. Although both supportive measures are necessary to guarantee adequate oxygen uptake, they can also cause or worsen lung inflammation and injury. Hyperoxia-induced lung injury is characterized by neutrophil infiltration into the lungs. The urokinase plasminogen activator receptor (uPAR) has been deemed important for leukocyte trafficking. To determine the expression and function of neutrophil uPAR during hyperoxia-induced lung injury, uPAR expression was determined on pulmonary neutrophils of mice exposed to hyperoxia. Hyperoxia exposure (O2>80%) for 4 days elicited a pulmonary inflammatory response as reflected by a profound rise in the number of neutrophils that were recovered from bronchoalveolar lavage fluid and lung cell suspensions, as well as increased bronchoalveolar keratinocyte-derived chemokine, interleukin-6, total protein, and alkaline phosphatase levels. In addition, hyperoxia induced the migration of uPAR-positive granulocytes into lungs from wild-type mice compared with healthy control mice (exposed to room air). uPAR deficiency was associated with diminished neutrophil influx into both lung tissues and bronchoalveolar spaces, which was accompanied by a strong reduction in lung injury. Furthermore, in uPAR(-/-) mice, activation of coagulation was diminished. These data suggest that uPAR plays a detrimental role in hyperoxia-induced lung injury and that uPAR deficiency is associated with diminished neutrophil influx into both lung tissues and bronchoalveolar spaces, accompanied by decreased pulmonary injury.

Plasminogen Receptors in Human Malignancies: Effects on Prognosis and Feasibility as Targets for Drug Development.

The major proteases that constitute the fibrinolysis system are tightly regulated. Protease inhibitors target plasmin, the protease responsible for fibrin degradation, and the proteases that convert plasminogen into plasmin, including tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). A second mechanism by which fibrinolysis is regulated involves exosite interactions, which localize plasminogen and its activators to fibrin, extracellular matrix (ECM) proteins, and cell surfaces. Once plasmin is generated in association with cell surfaces, it may cleave transmembrane proteins, activate growth factors, release growth factors from ECM proteins, remodel ECM, activate metalloproteases, and trigger cell-signaling by cleaving receptors in the Proteaseactivated Receptor (PAR) family. These processes are all implicated in cancer. It is thus not surprising that a family of structurally diverse but functionally similar cell-surface proteins, called Plasminogen Receptors (PlgRs), which increase the catalytic efficiency of plasminogen activation, have received attention for their possible function in cancer and as targets for anticancer drug development. In this review, we consider four previously described PlgRs, including: α-enolase, annexin-A2, Plg-RKT, and cytokeratin-8, in human cancer. To compare the PlgRs, we mined transcriptome profiling data from The Cancer Genome Atlas (TCGA) and searched for correlations between PlgR expression and patient survival. In glioma, the expression of specific PlgRs correlates with tumor grade. In a number of malignancies, including glioblastoma and liver cancer, increased expression of α-enolase or annexin-A2 is associated with an unfavorable prognosis. Whether these correlations reflect the function of PlgRs as receptors for plasminogen or other activities is discussed.