uPA Levels Research Articles

Effects of Aging and Respiratory Syncytial Virus Infection on the uPA/Plasmin System in Pulmonary Fibrosis

RationaleIdiopathic pulmonary fibrosis (IPF) is a serious disease often resulting in death. Aging, infection, pulmonary type 2 (Th2) immune responses and impaired fibrolytic activity play central roles in IPF. We hypothesize that aging and viral infection reduces lung urokinase (uPA) levels and increases uPA inhibitor PAI‐1 and that this leads to altered immune responses and TGF‐beta signaling, resulting in worsened fibrosis.MethodsWe compared the severity of bleomycin‐induced pulmonary fibrosis (BLM‐IPF) in mice deficient for uPA (uPA‐/‐) and its receptor (uPAR‐/‐) to wild type (WT) young and aged mice in the presence or absence of RSV infection.ResultsWT mice have diminished uPA expression following infection and BLM challenge, uPA activity levels and uPA total antigen levels diminished in the serum and lung tissues of infected BLM challenged WT aged mice compared to young mice. Whereas the PAI‐1 expression was significantly higher in uPA‐/‐ or uPAR‐/‐ aged BLM challenged mice compared to the WT mice. uPA or uPAR deficient viral infected and BLM treated mice showed increased fibrosis compared to WT as shown by hydroxyproline, total lung collagen, lung histology, and increased TGF‐beta expression. Lung T cells from infected uPA‐/‐ or uPAR‐/‐ aged mice produced lower levels of Th1, Th17 and higher levels of Th2 cytokines compared to WT mice.ConclusionsThis novel study provided both in vivo and in vitro data that showed how age‐infection‐related changes in the uPA/plasmin system impact the development of IPF. Supported by a VA Merit Review Grant (MRG.)

Role of G Protein‐Coupled Receptor Kinase 2 in TNFα Signaling in Colon Epithelial Cells

G protein-coupled receptor kinases (GRKs) compose a 7-member family of serine/threonine protein kinases that are critical in the regulation of GPCR lifecycle. In addition to GPCR phosphorylation, GRKs have been shown to have a large interactome and can play a critical role in cell signaling pathways as well as in consequent cell physiological processes. We wanted to test the hypothesis that GRK2 plays an essential role in colon epithelial cell biology in response to TNFα. For this we used colon epithelial cell line (SW480) that were transfected with either control or GRK2 siRNA. The cells were treated with TNFα and assessed for various cell physiological processes including wound healing, proliferation, apoptosis, and gene expression. Our results demonstrate that GRK2 has a positive role basally in the rate of wound closure although the addition of TNFα restored that rate in the siGRK2 group to control levels. Interestingly, upon examining the levels of various gene transcripts, we observed that matrix metalloproteinases (MMP) 7 and 9 as well as urokinase plasminogen activator (uPa) (proteins involved in cell migration) were markedly upregulated in GRK2 knockdown cells. To assess the mechanism by which GRK2 affects these physiological processes, we examined MAPK and NFkB pathways following TNFα treatment. We found that knockdown of GRK2 significantly inhibits TNFa-induced IkBa phosphorylation whereas ERK2 phosphorylation was significantly enhanced. These data suggest that GRK2 may play a critical role in cellular migration and/or wound healing by modulating MMP7, 9 and uPA levels potentially via differential regulation of MAPK and NFkB signaling pathways. Together, our results provide a novel role for GRK2 in TNFa signaling in colon epithelial cells. Supported by NIH Grant.

Selection and characterization of camelid nanobodies towards urokinase-type plasminogen activator

Urokinase-type plasminogen activator (uPA) is a trypsin-like serine protease that plays a vital role in extracellular conversion of inactive plasminogen into catalytically active plasmin. Activated plasmin facilitates the release of several proteolytic enzymes, which control processes like pericellular proteolysis and remodeling of ECM. uPA and the receptor uPAR, are overexpressed in a number of malignant tumours and uPA/uPAR play major roles in adhesion, migration, invasion and metastasis of cancer cells. Elevated levels of uPA have been reported as a risk biomarker for disease relapse, increased cancer malignancy and poor survival prognosis. For these reasons uPA is considered an important target for anticancer drug therapy.In this study we isolated two camel single domain antibodies (nanobodies) from a naïve library by phage display. The nanobody sequences were sequence-optimized for Escherichia coli expression, cloned into the pET22-B(+) vector system, expressed in BL-21 cells and purified from the periplasmic fraction by IMAC. ELISA tests demonstrated that the purified nanobodies were specific for uPA when tested towards other trypsin-like serine proteases. The apparent affinities of the nanobodies were determined by competitive ELISA to 80nM and 522nM, respectively. The best binder did not inhibit uPA (nAb-C3), however the lowest affinity binder (nAb-C8) was able to inhibit the uPA-mediated cleavage of the substrate S-2444.The results validate the naïve library as a resource for retrieval of relevant lead molecules and the novel uPA-nanobodies can be useful pharmacological tools to study uPA structure–function relationships.

Antitumor effect and mechanism of action of a tumor-targeting recombinant human tumor necrosis factor-α fusion protein mediated by urokinase.

The aim of this study was to investigate the effect of the tumor‑targeting recombinant human tumor necrosis factor (rhTNF)‑α fusion protein mediated by urokinase on Sl80 tumor‑bearing mice, as well as to explore its mechanisms of action. Furthermore, the study aimed to observe the effect of the protein on liver and kidney function. rhTNF‑α fusion protein prokaryotic expression vectors were constructed using genetic engineering techniques, and were introduced into Escherichia coli. Expression of the fusion protein was induced, and it was then separated and purified in order to determine its cytotoxic activity on L929 cells. Kunming mice were randomly divided into four groups after being inoculated with S180 tumor cells. The groups were then injected with saline (control group, groupS), or saline with 0.1µg/ml fusion protein (low dose group, groupL), 0.2µg/ml fusion protein (middle dose group, groupM) or 0.3µg/ml (high dose group, groupH). The mice were sacrificed after 12 days and liver [mg/kg; (liver weight/body weight)x1,000] and kidney [mg/kg; (kidney weight/body weight)x1,000] indices, tumor weight, the percentage reduction in mean tumor size, and the levels of alanine transaminase (ALT), albumin (ALB), creatinine (Cr) and blood urea nitrogen (BUN) in each group of mice were determined. In addition, the levels of urokinase‑type plasminogen activator (uPA), the expression of bcl‑2, bax and vascular endothelial growth factor (VEGF), and the percentage of apoptotic cells were measured with an enzyme‑linked immunosorbent assay, streptavidin‑biotin complex of immunohistochemistry and terminal deoxynucleotidyl transferase‑mediated dUTP nick end labeling, respectively. The fusion protein significantly inhibited the growth of S180 tumor cells invivo in a dose‑dependent manner. With an increase in the dose of fusion protein, ALT, uPA, bcl‑2 and VEGF levels decreased, and ALB levels increased. However, liver and kidney indices and bax expression were not significantly altered. Cr and BUN levels did not change significantly in the low and middle dose groups, but did increase in the high dose group. Compared with the control group, the percentage of apoptotic cells in the high‑dose group was significantly higher. In conclusion, the fusion protein significantly inhibited S180 tumor growth in a mouse model, possibly by reducing the levels of uPA, bcl‑2 and VEGF. There was a mildly toxic effect on the kidneys with the high dose, but a protective effect in the liver.

Inhibition of tumor growth by β-elemene through downregulation of the expression of uPA, uPAR, MMP-2, and MMP-9 in a murine intraocular melanoma model.

This paper explores the underlying mechanism through which β-elemene inhibits the growth of intraocular melanoma in a mouse model. C57BL/6J mice were administered a subretinal injection of B16F10 melanoma cells and divided into two groups: treatment and control. The treatment group was administered β-elemene through an intravitreal injection and the control group was injected with a blank emulsion. After 21 days of continuous treatment, tumor masses were removed and weighed. The mRNA expression levels of the urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), matrix metalloproteinase (MMP)-2, and MMP-9 were assayed by real-time PCR, and the protein expression levels of uPA, uPAR, MMP-2, and MMP-9 were assayed by immunocytochemistry and western blotting. Tumor size was inhibited by β-elemene in the treatment group, and the expressions of uPA, uPAR, MMP-2, and MMP-9 were all downregulated at both the mRNA and the protein level compared with the control group. In a mouse model of intraocular melanoma, β-elemene inhibits tumor growth by downregulating the expression of uPA, uPAR, MMP-2, and MMP-9.

Effect of lentivirus-mediated uPA silencing on the proliferation and apoptosis of chondrocytes and the expression of MMPs.

The lentivirus-mediated uPA interference in the proliferation, apoptosis, and secretion of osteoarthritic chondrocytes was examined in this study. Cells were obtained from the cartilage tissues of New Zealand white rabbits. They were cultured with interleukin (IL)-1β (10 ng/mL) for 24 h and then divided into three groups: uPA-siRNA group (cells transfected with uPA-siRNA lentiviruses), blank control group (untreated cells), and negative control group (cells transfected with empty vectors). Western blotting and real-time quantitative reverse transcription-PCR (RT-QPCR) were performed to detect the protein and mRNA expression levels of uPA, MMP-1, MMP-3, MMP-9, MMP-10, MMP-13 and MMP-14 in osteoarthritic chondrocytes. Cell Counting Kit-8, flow cytometry, and colony formation assay were used to examine the proliferation and apoptosis of chondrocytes. The results showed that after uPA-siRNA transfection, the protein and mRNA expression levels of uPA, MMP-1, MMP-3, MMP-9, MMP-10, MMP-13, and MMP-14 were significantly decreased (P<0.05 for MMP-1, MMP-9, MMP-10 and MMP-14, P<0.01 for uPA, MMP-3 and MMP-13). Cell proliferation and colony formation rate were significantly higher and the cell apoptosis rate was significantly lower in uPA-siRNA group than in control groups (P<0.01). The proportion of cells in G0/G1 phase was markedly increased and that in the S phase decreased, and the cell cycle was arrested at the G1/S phase in the control group. In the uPA-siRNA group, the proportion of cells in the S phase was significantly increased, resulting in a different proportion of cells in cell cycle phase (P<0.01). It was suggested that the down-regulation of uPA gene could inhibit the expression of MMPs protein and cell apoptosis, increase the proliferation and colony formation of osteoarthritic chondrocytes.

Urokinase is a negative modulator of Egf-dependent proliferation and motility in the two breast cancer cell lines MCF-7 and MDA-MB-231.

The epidermal growth factor receptor (EGFR) is involved in the regulation of various cellular processes and dysregulation of its signalling plays a critical role in the etiology of a variety of malignancies like breast cancer. At the same time, elevated levels of urokinase (uPA), its receptor uPAR, and other components of the plasminogen activation system are found to be correlated with a poor prognosis in breast cancer. Interestingly, EGFR appears to participate in transducing the signal generated upon binding of uPA to uPAR. However, whether uPA signalling would thereby interfere with ligand-driven EGFR signalling was not described before. Therefore, it was the aim of the present study to investigate the combined effects of uPA and EGF in the low invasive and high invasive breast adenocarcinoma cell lines MCF-7 and MDA-MB-231, respectively. Simultaneous exposure of cells to both signals negatively affected ERK1/2 and AKT activation whereas positive effects on p38 and Src kinase phosphorylation were noted in both cell lines. Furthermore, uPA attenuated the mitogenic effect of EGF on cellular proliferation, invasion and motility in both MCF-7 and MDA-MB-231 cells. Experiments with the uPA amino terminal fragment (ATF) revealed that the negative effects of uPA were independent from its protease activity. Together, these data suggest that enhanced levels of uPA in breast cancer modulate the mitogenic effects of EGF and thus, this knowledge may help to better understand breast cancer pathogenesis as well as to develop new therapeutic options.

Serum level of Urokinase Plasminogen Activator (uPA) Correlates with the Survival of Patients with Pancreatic Ductal Adenocarcinoma (PDAC)

Background: Urokinase plasminogen activator (uPA) is a serine protease which transforms inactive plasminogen into active plasmin. UPA plays an important role in neoplasm progression, cell growth and metastases through degradation of proteins in basement membranes and extracellular matrix. The aim of the study was the analysis of serum uPA concentration in patients suffering from pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis (CP) in order to determine its possible diagnostic and prognostic value. Methods: A study group involved 90 patients: 40 patients with PDAC, 30 patients with CP and 20 healthy individuals. UPA serum concentration was evaluated with ELISA. Results: We observed the threefold increase of uPA serum concentration in patients with PDAC (3,23 ng/ml), twofold increase of uPA serum concentration in patients with CP (2,18 ng/ml) compared to the control group (1,01 ng/ ml) (PDAC vs CP p<0,01; PDAC vs control p<0,01; CP vs control p<0,01). We revealed significant positive correlation between uPA serum level and CA19-9 (r=0.305 p<0,05) in all analyzed groups. We also found the significant correlation between serum uPA concentration and the survival time. Higher uPA concentration was observed in patients with shorter survival time (r=-0,391; p<0,05). Significant differences were present between uPA levels lower and greater than 2 ng/ml and the patients survival time (p<0,05). Conclusions: The presented results confirm the negative prognostic role of high serum uPA concentration in pancreatic cancer patients. The significant positive correlation between uPA serum concentration and CA19-9 provides new insights into the potential role of uPA in pancreatic cancer diagnosis.

Seahorse-derived peptide suppresses invasive migration of HT1080 fibrosarcoma cells by competing with intracellular α-enolase for plasminogen binding and inhibiting uPA-mediated activation of plasminogen

α-Enolase is a glycolytic enzyme and a surface receptor for plasminogen. α-Enolase-bound plasminogen promotes tumor cell invasion and cancer metastasis by activating plasmin and consequently degrading the extracellular matrix degradation. Therefore, α-enolase and plasminogen are novel targets for cancer therapy. We found that the amino acid sequence of a peptide purified from enzymatic hydrolysates of seahorse has striking similarities to that of α-enolase. In this study, we report that this peptide competes with cellular α-enolase for plasminogen binding and suppresses urokinase plasminogen activator (uPA)-mediated activation of plasminogen, which results in decreased invasive migration of HT1080 fibrosarcoma cells. In addition, the peptide treatment decreased the expression levels of uPA compared to that of untreated controls. These results provide new insight into the mechanism by which the seahorse-derived peptide suppresses invasive properties of human cancer cells. Our findings suggest that this peptide could emerge as a potential therapeutic agent for cancer. [BMB Reports 2014; 47(12): 691-696]

Prognostic impact of urokinase-type plasminogen activator system components in clear cell renal cell carcinoma patients without distant metastasis.

BackgroundMembers of the urokinase-type plasminogen activator (uPA) system including uPA, its receptor uPAR and the plasminogen activator inhibitor 1 (PAI-1) play an important role in tumour invasion and progression in a variety of tumour types. Since the majority of clear cell renal cell carcinoma (ccRCC) shows distant metastasis at time of diagnosis or later, the interplay of uPA, uPAR and PAI-1 might be of importance in this process determining the patients’ outcome.MethodsCorresponding pairs of malignant and non-malignant renal tissue specimens were obtained from 112 ccRCC patients without distant metastasis who underwent tumour nephrectomy. Tissue extracts prepared from fresh-frozen tissue samples by detergent extraction were used for the determination of antigen levels of uPA, uPAR and PAI-1 by ELISA. Antigen levels were normalised to protein concentrations and expressed as ng per mg of total protein.ResultsAntigen levels of uPA, uPAR, and PAI-1 correlated with each other in the malignant tissue specimens (rs=0.51-0.65; all P<0.001). Antigen levels of uPA system components were significantly higher in tissue extracts of non-organ confined tumours (pT3+4) compared to organ-confined tumours (pT1+2; all P<0.05). Significantly elevated levels of uPAR and PAI-1 were also observed in high grade ccRCC. When using median antigen levels as cut-off points, all three uPA system factors were significant predictors for disease-specific survival (DSS) in univariate Cox’s regression analyses. High levels of uPA and uPAR remained independent predictors for DSS with HR=2.86 (95% CI 1.07-7.67, P=0.037) and HR=4.70 (95% CI 1.51-14.6, P=0.008), respectively, in multivariate Cox’s regression analyses. A combination of high antigen levels of uPA and/or uPAR further improved the prediction of DSS in multivariate analysis (HR=14.5, 95% CI 1.88-111.1, P=0.010). Moreover, high uPA and/or uPAR levels defined a patient subgroup of high risk for tumour-related death in ccRCC patients with organ-confined disease (pT1+2) (HR=9.83, 95% CI 1.21-79.6, P=0.032).ConclusionsHigh levels of uPA and uPAR in tumour tissue extracts are associated with a significantly shorter DSS of ccRCC patients without distant metastases.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-974) contains supplementary material, which is available to authorized users.

Plasma-soluble urokinase-type plasminogen activator receptor levels are associated with clinical and pathological activities in lupus nephritis: a large cohort study from China.

In this study, we detected plasma urokinase plasminogen activator (uPA) and soluble urokinase-type plasminogen activator receptor (uPAR) levels in Chinese lupus nephritis patients from a large cohort. The associations between plasma uPA and soluble uPAR and clinico-pathological characteristics were further analyzed. The levels of plasma uPA and soluble uPAR were detected by ELISA in 202 patients with active lupus nephritis, 17 systemic lupus erythematosus (SLE) patients without renal involvement and 21 normal controls. There were no significant differences in the levels of the average plasma uPA among the lupus nephritis group, non-renal SLE group and normal control group (p = 0.129). The plasma-soluble uPAR level in the lupus nephritis group was significantly higher than that in the non-renal involvement SLE group (p = 0.004) and that in normal controls (p < 0.001). The plasma uPAR levels were positively associated with SLEDAI scores (r = 0.215, p = 0.007). In renal pathological data, there was significant difference of plasma-soluble uPAR levels among various pathological classes, which was the highest in the class IV group (p = 0.012). The level of plasma-soluble uPAR was found to be a risk factor for long-term renal outcomes in lupus nephritis by univariate survival analysis (p = 0.013, HR = 6.326, 95% CI: 1.466-27.298). Our study showed that the significantly increased plasma levels of soluble uPAR could be found in active lupus nephritis, and they were associated with some clinico-pathological features. Its involvement in the pathogenesis of lupus nephritis warrants further study.

Inhibitory effect of emodin on migration, invasion and metastasis of human breast cancer MDA-MB-231 cells in vitro and in vivo.

In breast cancer, metastasis is the main reason for patient mortality. In the present study, we used breast cancer MDA-MB-231 cells and a mouse xenograft model to demonstrate the effect of emodin on the migration, invasion and metastasis of human breast cancer MDA-MB-231 cells and the related mechanisms. In vitro, wound healing and Transwell assays showed that emodin dose-dependently inhibited the migration and invasion of MDA-MB-231 cells. Enzyme-linked immunosorbent assay (ELISA) showed that emodin decreased the secretion of MMP-2 and MMP-9. Western blot analysis showed that emodin downregulated the expression levels of MMP-2, MMP-9, uPA and uPAR as well as p38 inhibitor SB203580 and ERK inhibitor PD980559, even though TIMP-1 and TIMP-2 were not obviously changed in the MDA-MB-231 cells. Furthermore, emodin inhibited the activity of p38 and ERK1/2 in the MDA-MB-231 cells. In vivo, emodin inhibited lung metastasis in mice bearing the breast cancer MDA-MB-231 xenografts with no obvious changes in body weight, liver and kidney functions. These results indicated that emodin inhibited the lung metastasis of human breast cancer in a mouse xenograft model, and inhibited the invasion of MDA-MB-231 cells associated with the downregulation of MMP-2, MMP-9, uPA and uPAR expression as well as decreased activity of p38 and ERK.

Inhibitory effect of ethanol extract of Ocimum sanctum on osteopontin mediated metastasis of NCI-H460 non-small cell lung cancer cells.

BackgroundOsteopontin (OPN) is one of important molecular targets in cancer progression, metastasis as a calcium-binding, extracellular-matrix-associated protein of the small integrin-binding ligand and, N-linked glycoprotein. In the present study, anti-metastatic mechanism of ethanol extracts of Ocimum sanctum (EEOS) was elucidated on OPN enhanced metastasis in NCI-H460 non- small cell lung cancer cells.MethodsCell viability was measured by MTT assay. Adhesion and invasion assays were carried out to see that EEOS inhibited cell adhesion and invasion in OPN treated and non-treated NCI-H 460 cells. RT-PCR was used to determine the mRNA levels of uPA, uPAR, and EGFR.ResultsEEOS significantly inhibited cell adhesion and invasion in OPN treated and non treated NCI-H460 cells, though EEOS did not show any toxicity up to 200 μg/ml. EEOS effectively attenuated the expression of OPN and CD44 and also OPN activated the expression of CD44 in NCI-H460 cells. In addition, EEOS effectively suppressed the expression of phosphatidylinositide 3-kinases (PI3K) and cyclooxygenase 2 (COX-2) and the phosphorylation of Akt at protein level in OPN treated NCI-H460 cells. Also, EEOS significantly attenuated the expression of urokinase plasminogen activator (uPA), its receptor (uPAR) and epidermal growth factor receptor (EGFR) at mRNA level and reduced vascular endothelial growth factor (VEGF) production and MMP-9 activity in OPN treated NCI-H460 cells. Furthermore, PI3K/Akt inhibitor LY294002 enhanced anti-metastatic potential of EEOS to attenuate the expression of uPA and MMP-9 in OPN treated NCI-H 460 cells.ConclusionOverall, our findings suggest that anti-metastatic mechanism of EEOS is mediated by inhibition of PI3K/Akt in OPN treated NCI-H460 non-small cell lung cancer cells.

Anti-metastatic effect of Smilax china L. extract on MDA-MB-231 cells.

Cancer metastases are not always cured by chemotherapy. Conventional and alternative drugs, including Chinese herbal remedies, have been developed to target metastatic cancer cells. Smilax china L. (SCL), a member of the Smilacaceae family, exerts anti-inflammatory, detoxification and anti-cancer effects. However, the effect of SCL on breast cancer cell metastasis and the underlying mechanisms are yet to be elucidated. The aim of this study was to investigate the effect of a SCL ethanol extract (SCLE) on the proliferation and migration of MDA-MB-231 human breast cancer cells, as well as the expression of urokinase plasminogen activator (uPA), uPA receptor (uPAR) and tissue inhibitors of metalloproteinases (TIMPs). Cell proliferation was assessed using the Cell Counting Kit‑8 and cell migration was determined by wound healing assay. Quantitative polymerase chain reaction was performed to quantify the mRNA levels of uPA, uPAR and TIMPs. SCLE markedly inhibited the proliferation and migration of MDA-MB-231 cells, and reduced the mRNA levels of the extracellular matrix (ECM) degradation-associated molecules uPA, uPAR. By contrast, SCLE significantly increased the mRNA levels of TIMP1 and TIMP2. These findings show that SCLE exerts an anti-metastatic effect on human breast cancer cells, which may involve the modulation of ECM degradation.

Abstract 39: Expression of uPA-system members correlate with the clinico-pathological parameters of PCa patients

Abstract Background: Members of the urokinase-type plasminogen activator (uPA) system play an important role in tumor progression and angiogenesis in different human malignancies including breast, kidney, colon, stomach and prostate cancer (PCa). The objective of this study was to determine the mRNA expression and protein levels of uPA system components in tissue specimens and serum samples, respectively, from patients with PCa, and to assess their potential association with clinic-pathological parameters and overall survival (OS). Material and methods: mRNA levels of uPA, its receptor (uPAR) and its inhibitor type 1 (PAI-1) were analyzed in corresponding malignant and adjacent non-malignant prostate tissue specimens from 132 PCa patients. Relative mRNA expression levels were determined by quantitative PCR and normalized to the reference gene TATA-box-binding protein. Pre-operative serum samples from 81 of the 132 PCa patients and 36 patients with benign prostatic hyperplasia (BPH) were analyzed for antigen levels of uPA system members by ELISA. Results: mRNA levels of the uPA system components displayed significant correlations with each other in the tumor tissues (uPA vs. uPAR: rs = 0.550; uPA vs. PAI-1: rs = 0.552; uPAR vs. PAI-1: rs = 0.643; all P &amp;lt; 0.001). A significantly decreased uPA mRNA expression in PCa tissue compared to the corresponding non-malignant tissue specimens was detected (data not shown). High mRNA expression of uPA and PAI-1 was significantly associated with a high Gleason score (P = 0.001 and P = 0.007, respectively; Fig. 2). Antigen levels of uPA and soluble uPAR (suPAR) in serum correlated weakly, but significantly, with each other (rs = 0.382, P &amp;lt; 0.001). A significant association was found between elevated suPAR concentrations in serum and poor OS of PCa patients (P = 0.022, log rank test). In multivariate Cox's regression analysis (adj. for age, lymph node status, tumor stage and Gleason sum) a high suPAR level was associated with a 7.12-fold increased risk of death (P=0.027). Conclusion: The observed association of high suPAR levels with poor survival of PCa patients is consistent with published data on patients afflicted with other types of solid cancer and strongly suggests a general prognostic impact of uPAR antigen levels in serum of cancer patients. Citation Format: Omar Al-Janabi, Helge Taubert, Andrea Lohse-Fischer, Michael Fröhner, Sven Wach, Robert Stöhr, Bastian Keck, Max Burger, Wolf Wieland, Kati Erdmann, Manfred P. Wirth, Bernd Wullich, Gustavo Baretton, Viktor Magdolen, Matthias Kotzsch, Susanne Füssel. Expression of uPA-system members correlate with the clinico-pathological parameters of PCa patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 39. doi:10.1158/1538-7445.AM2014-39

Abstract 5219: MicroRNA-874 impedes angiogenesis in non-small cell lung cancer

Abstract MicroRNAs (miRs) are short (20-24nt) non-coding RNAs that are involved in post-transcriptional regulation of gene expression in eukaryotes by affecting both the stability and translation of mRNAs. MicroRNAs play crucial roles in normal biological processes and are commonly dysregulated in human diseases. Thus far, miRs have been shown to act as either tumor suppressor genes or oncogenes in driving critical gene expression pathways in cancer stem cells in a wide range of human malignancies, including non-small cell lung cancer (NSCLC).We previously found that miR-874 could suppress tumor growth and metastasis of NSCLC. Since angiogenesis is important for tumor growth and metastasis, in this study, we further investigated the possible roles of miR-874 in tumor angiogenesis. Attenuation of miR-874 expression in histological sections of NSCLC tumors and the CD133 positive population of NSCLC cells (NSCLC-initiating cells; NSCLC-ICs), as compared to its normal counterparts, was observed by in situ hybridization and real time quantitative PCR, respectively. Tumor-conditioned medium from NSCLC-ICs induced significantly high levels of endothelial cell capillary network and neovascularization in vivo; whereas, re-expression of miR-874 in NSCLC-ICs inhibited this capillary network formation ability by suppressing the protein levels of VEGF, Stat3, MMP-2, uPA and PI3K. Further, miR-874-treatment decreased in vivo angiogenesis and pre-established orthotopic tumor growth in nude mice as compared to mock- and control-miR-treatments. Immunohistochemical analysis revealed a significant decrease in the expression of VEGF, Stat3 and microvessel density (CD31 levels) in xenograft tumor sections from miR-874-treated mice. In conclusion, our study highlights the possible anti-angiogenic role of miR-874 in NSCLC-initiating cells and presents a new, hopeful therapeutic target and prognostic marker for NSCLC. Citation Format: Chandramu Chetty, Dilip R. Maddirela, Venkateswara R. Gogineni, Subramanyam Chittivelu, Jasti S. Rao, Bhagavatula Moorthy. MicroRNA-874 impedes angiogenesis in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5219. doi:10.1158/1538-7445.AM2014-5219

Abstract 3001: Angiogenesis-related cytokine secretion pattern in tumor interstitial fluid and its relationship with VEGF expression and metastatic profile

Abstract The tumor microenvironment (TME) contributes significantly to the phenotypic characteristics of cancer. Unraveling the roles of the TME in cancer growth and treatment, as well as mining the TME for treatment strategies is increasingly occupying center-stage. One of the least examined, and yet critically important factors in the TME is the tumor interstitial fluid (IF). This fluid surrounds cancer and stromal cells within the tumor, and contains various cytokines, and nutritional and molecular factors that shape the outcome of nearly all aspects of tumor growth, metastasis, and response to treatment. Here we have characterized IF from human breast cancer xenografts in SCID mice. The IF was obtained from the tumor using a small custom-built diffusion chamber (outer diameter 6.2 mm, inner volume approx. 40 µl) implanted in the subcutaneous space. The chamber was surrounded by 4-6 tumor pieces that were allowed to grow for 4-6 weeks that resulted in the chamber being incorporated within the tumor. Three different human breast carcinoma cell lines were used to analyze tumor IF content: invasive, highly metastatic MDA-MB-231 cells, less invasive MCF-7 cells and MCF-7 cells engineered to overexpress vascular endothelial growth factor (VEGF). After IF, plasma and tumor collection, IF samples were analyzed using a multiple cytokine assay (Proteome Profiler Human Angiogenesis Kit, R &amp; D Systems Inc., Minneapolis MN). With this kit, 55 human angiogenesis-related cytokines were investigated for relative secretion levels. Our results identified differences in cytokine content in tumors derived from these cells. Out of the 55 cytokines tested, 24 were detected in the IF. Of these, more than 80% (20) were detected in MDA-MB-231 tumors only. Additionally, MDA-MB-231 derived tumor IF showed increased levels of TIMP metallopeptidase inhibitor 1 (TIMP-1), urokinase plasminogen activator (uPA) and Serpin E1 compared to MCF-7 cells. As expected, high VEGF levels were detected in the IF of tumors derived from VEGF-overexpressing MCF-7 cells compared to wild type cells. VEGF overexpression also influenced the levels of TIMP-1, Serpin E1 and uPA in the IF. Variations in IF cytokine content of cancers with different metastatic and angiogenic abilities provide further understanding of the TME. Measurement of cytokine content is only the first step towards the correlation of tumor IF cytokine levels with tumor vascular properties and anti-angiogenic treatments. A better understanding of the TME through characterization of the IF may identify new targets for cancer treatment and allow for optimization of therapeutic strategies. Supported by NIH R01CA138264 and R01CA136576. Citation Format: Louis Dore-Savard, Esak Lee, Aleksander S. Popel, Zaver M. Bhujwalla. Angiogenesis-related cytokine secretion pattern in tumor interstitial fluid and its relationship with VEGF expression and metastatic profile. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3001. doi:10.1158/1538-7445.AM2014-3001

Hyperglycemia is associated with lower levels of urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor in wound fluid

AimsWounds in patients with hyperglycemia show impaired healing. Plasminogen activation is crucial in several overlapping phases of wound healing process. In this study, we aimed i) to compare acute wound fluid in patients with hyperglycemia and normoglycemia, ii) to focus on the elements of plasminogen activation in the wound fluid, and iii) to determine if the acute wound fluid characteristics are associated with surgical site infections. MethodsIn a cohort of 54 patients, a closed suction drain was placed in the wound above the anterior abdominal wall fascia under the skin in order to collect postoperative acute wound fluid samples for 3 following days after colorectal surgery. Patients were classified as normoglycemic (n=25) or hyperglycemic (n=29; 17 with type 2 diabetes and 12 with stress induced hyperglycemia). Surgical site infection was defined according to the Centers for Disease Control criteria. The levels of urokinase-type plasminogen activator (uPA), urokinase-type plasminogen activator receptor (uPAr), plasminogen activator inhibitor-1 (PAI-1), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and fibroblast growth factor-1 (FGF-1) were measured in the wound fluid. ResultsCompared to normoglycemic subjects, patients with hyperglycemia had significantly lower levels of uPA and uPAr in the wound fluid despite similar or even higher circulating levels. There was no significant difference in IL-1β, TNF-α, PAI-1 and FGF-1 levels. In the whole study population, the wound fluid levels of uPA and uPAr were negatively correlated with circulating glucose levels. No difference was detected in the wound fluid characteristics of patients with and without surgical site infection. ConclusionPatients with hyperglycemia exhibit decreased levels of uPA and uPAr in the wound fluid, suggesting a local failure in plasminogen activation at the wound site.

UPA and PAI-1 as biomarkers in breast cancer: validated for clinical use in level-of-evidence-1 studies

Urokinase plasminogen activator (uPA) is an extracellular matrix-degrading protease involved in cancer invasion and metastasis, interacting with plasminogen activator inhibitor-1 (PAI-1), which was originally identified as a blood-derived endogenous fast-acting inhibitor of uPA. At concentrations found in tumor tissue, however, both PAI-1 and uPA promote tumor progression and metastasis. Consistent with the causative role of uPA and PAI-1 in cancer dissemination, several retrospective and prospective studies have shown that elevated levels of uPA and PAI-1 in breast tumor tissue are statistically independent and potent predictors of poor patient outcome, including adverse outcome in the subset of breast cancer patients with lymph node-negative disease. In addition to being prognostic, high levels of uPA and PAI-1 have been shown to predict benefit from adjuvant chemotherapy in patients with early breast cancer. The unique clinical utility of uPA/PAI-1 as prognostic biomarkers in lymph node-negative breast cancer has been confirmed in two independent level-of-evidence-1 studies (that is, in a randomized prospective clinical trial in which the biomarker evaluation was the primary purpose of the trial and in a pooled analysis of individual data from retrospective and prospective studies). Thus, uPA and PAI-1 are among the best validated prognostic biomarkers currently available for lymph node-negative breast cancer, their main utility being the identification of lymph node-negative patients who have HER-2-negative tumors and who can be safely spared the toxicity and costs of adjuvant chemotherapy. Recently, a phase II clinical trial using the low-molecular-weight uPA inhibitor WX-671 reported activity in metastatic breast cancer.

Protease Nexin-1 affects the migration and invasion of C6 glioma cells through the regulation of urokinase Plasminogen Activator and Matrix Metalloproteinase-9/2

Protease Nexin-1 (PN-1) or Serpine2 is a physiological regulator of extracellular proteases as thrombin and urokinase (uPA) in the brain. Besides, PN-1 is also implicated in some human cancers and further identified as a substrate for Matrix Metalloproteinase (MMP)-9, a key enzyme in tumor invasiveness. Our aim was to study the role of PN-1 in the migration and invasive potential of glioma cells, using the rat C6 glioma cell line as stable clones transfected with pAVU6+27 vector expressing PN-1 short-hairpin RNA. We find that PN-1 knockdown enhanced the in vitro migration and invasiveness of C6 cells which also showed a strong gelatinolytic activity by in situ zymography. PN-1 silencing did not alter prothrombin whereas increased uPA, MMP-9 and MMP-2 expression levels and gelatinolytic activity in a conditioned medium from stable C6 cells. Selective inhibitors for MMP-9 (Inhibitor I), MMP-2 (Inhibitor III) or exogenous recombinant PN-1 added to the culture medium of C6 silenced cells restored either the migration and invasive ability or gelatinolytic activity thus validating the specificity of PN-1 silencing strategy. Phosphorylation levels of extracellular signal-related kinases (Erk1/2 and p38 MAPK) involved in MMP-9 and MMP-2 signaling were increased in PN-1 silenced cells. This study shows that PN-1 affects glioma cell migration and invasiveness through the regulation of uPA and MMP-9/2 expression levels which contribute to the degradation of extracellular matrix during tumor invasion.