High Levels Of uPA Research Articles

MTA2 knockdown suppresses human osteosarcoma metastasis by inhibiting uPA expression.

The relationship between metastasis-associated protein 2 (MTA2) overexpression and tumor growth and metastasis has been extensively studied in a variety of tumor cells but not in human osteosarcoma cells. This study aims to elucidate the clinical significance, underlying molecular mechanisms, and biological functions of MTA2 in human osteosarcoma in vitro and in vivo. Our results show that MTA2 was elevated in osteosarcoma cell lines and osteosarcoma tissues and was associated with tumor stage and overall survival of osteosarcoma patients. Knockdown of MTA2 inhibited osteosarcoma cell migration and invasion by reducing the expression of urokinase-type plasminogen activator (uPA). Bioinformatic analysis demonstrated that high levels of uPA in human osteosarcoma tissues correlated positively with MTA2 expression. Furthermore, treatment with recombinant human uPA (Rh-uPA) caused significant restoration of OS cell migration and invasion in MTA2 knockdown osteosarcoma cells. We found that ERK1/2 depletion increased the expression of uPA, facilitating osteosarcoma cell migration and invasion. Finally, MTA2 depletion significantly reduced tumor metastasis and the formation of lung nodules in vivo. Overall, our study suggests that MTA2 knockdown suppresses osteosarcoma cell metastasis by decreasing uPA expression via ERK signaling. This finding provides new insight into potential treatment strategies against osteosarcoma metastasis by targeting MTA2.

MMP-11 expression in early luminal breast cancer: associations with clinical, MRI, pathological characteristics, and disease-free survival

BackgroundEarly hormone-positive breast cancers typically have favorable outcomes, yet long-term surveillance is crucial due to the risk of late recurrences. While many studies associate MMP-11 expression with poor prognosis in breast cancer, few focus on early-stage cases. This study explores MMP-11 as an early prognostic marker in hormone-positive breast cancers.MethodsIn this retrospective study, 228 women with early hormone-positive invasive ductal carcinoma, treated surgically between 2011 and 2016, were included. MMP-11 expression was measured by immunohistochemistry, and its association with clinical and MRI data was analyzed.ResultsAmong the patients (aged 31–89, median 60, with average tumor size of 15.7 mm), MMP-11 staining was observed in half of the cases. This positivity correlated with higher uPA levels and tumor grade but not with nodal status or size. Furthermore, MMP-11 positivity showed specific associations with MRI features. Over a follow-up period of 6.5 years, only 12 oncological events occurred. Disease-free survival was linked to Ki67 and MMP-11.ConclusionMMP-11, primarily present in tumor-surrounding stromal cells, correlates with tumor grade and uPA levels. MMP-11 immunohistochemical score demonstrates a suggestive trend in association with disease-free survival, independent of Ki67 and other traditional prognostic factors. This highlights the potential of MMP-11 as a valuable marker in managing early hormone-positive breast cancer.

QSAR modelling to predict structural features of certain sulfonamide as Urokinase-type Plasminogen Activator inhibitors

The serine hydrolase family includes serine proteases. It is essential for hydrolyzing protein peptide bonds and breaking them. The urokinase-type plasminogen activator (uPA) selectively binds to the uPAR on numerous cell types, including cancer cells. Pericellular proteolysis of cell-bound proteins requires this interaction. High uPA and uPAR levels regularly worsen cancer prognoses. Thus, small chemical active-site inhibitors that block uPA may diminish cancer cell invasion and metastasis. In compliance with Organization for Economic Corporation and Development guidelines, this research performed a complete Quantitative structure activity relationship analysis of sulfonamide compounds as Urokinase-type Plasminogen Activator inhibitors. Py-Descriptors were used for this investigation. PyDescriptor uses PyMOL standards and idioms to calculate 11,145 simple molecular descriptors. This plugin calculates molecular descriptors irrespective of molecular representation properties like atom numbering or labelling, spatial reference frame, translational and rotational invariance, etc. The investigation sought to find essential and hidden structural characteristics that regulate sulfonamide-type drugs' Urokinase-type Plasminogen Activator Inhibitory action. Twenty-eight sulfonamide chemicals are used in the Quantitative structure activity relationship study to generate statistically robust and highly predictive univariate and multivariate models. All models were thoroughly evaluated and meet several statistical parameter thresholds (e.g., R2 =0.9259–0.9280, Q2Loo= 0.8579–0.8558, Q2LMO= 0.8013–0.7865). The analysis reveal that occurance of ring carbon atoms exactly at 3 A0 from carbon atom, number of negatively charged atoms from sulphur atoms within 5 bonds, presence of hydrogen atom exactly at 3 bonds from donar atoms, presence of carbon atom exactly at 4 A0 from donar atom, presence of acceptor atom exactly at 5 A0 from sulphur atom and sum of partial charges of lipo atoms within 6 bonds from sulphur atom are important pharmacophoric features for Urokinase-type Plasminogen Activator Inhibition binding affinity. Thus, the developed Quantitative structure activity relationship study has an equilibrium of quantitative and qualitative tactics. The results could be useful for future optimizations of sulfonamide analogues.

The urokinase-type plasminogen activator and inhibitors in resectable lung adenocarcinoma

BackgroundThe urokinase-type plasminogen activator (uPA) system is closely related to the occurrence and progression of cancer in many aspects. Previous studies demonstrated that the conclusions about the prognosis value of uPA, plasminogen activator inhibitor 1 (PAI-1) and plasminogen activator inhibitor 2 (PAI-2) in lung cancer are controversial, so this study was performed for the exploration of the predictive effect of uPA, PAI-1 and PAI-2 on the overall survival (OS) of resectable pulmonary adenocarcinoma patients. MethodsUPA, PAI-1 and PAI-2 expression levels were assayed by immunohistochemical staining based on tissue microarray (TMA) that is composed of formalin-fixed paraffin-embedded specimens from 84 resectable lung adenocarcinoma patients from July 2004 to June 2009. The relationship of IHC, mRNA expression levels of three molecules were investigated respectively. The three molecules’ relationship with clinicopathologic parameters and OS was explored by Chi-square, Kaplan-Meier, and Cox regression analyses. The Cancer Genome Atlas (TCGA) database was used to analyze differential gene expressions of RNA-sequencing data of pulmonary adenocarcinoma and normal tissues, and Kaplan-Meier methods were adopted to confirm the prognostic value of uPA, PAI-1 and PAI-2 in resectable lung adenocarcinoma in TCGA database and the R package MethylMix was used to conduct an analysis integrating methylation data and gene expression of RNA-sequencing data based on TCGA. ResultsUPA, PAI-1 and PAI-2 had much higher IHC expression levels in tumor than those in the normal tissues (uPA, Z = -10.511; PAI-1, Z = -4.836; PAI-2, Z = -6.794; all P < 0.0001). High DNA methylation level of gene uPA resulted in the decrease of its expression. In addition, expression level of PAI-2 was positively associated with tumor size (χ2 = 8.372, P = 0.004). Multivariate analyses showed TNM stage III was an independent adverse prognostic factor (hazard ratio = 3.736, 95 % confidence interval = 1.097–12.72, P = 0.035). Kaplan-Meier method revealed that uPA, PAI-1 and PAI-2 expression levels were not related to the OS for 84 resectable lung adenocarcinoma patients. According to TCGA data, PAI-1 expression level was identified as a potential adverse predictor for prognosis of resectable lung adenocarcinoma (Gehan-Breslow-Wilcoxon test, P = 0.025). ConclusionsOur data show that, the expression levels of uPA, PAI-1 and PAI-2 are significantly up-regulated in resectable lung adenocarcinoma. Besides, this study highlights PAI-1 as a latent adverse prognostic factor in resectable adenocarcinoma of lung.

Deficiency of Urokinase Plasminogen Activator May Impair β Cells Regeneration and Insulin Secretion in Type 2 Diabetes Mellitus.

Background: The relationship between urokinase-type plasminogen activator (uPA) and the development of type 2 diabetes mellitus (T2DM) was investigated in the study by using mice and cell models, as well as patients with T2DM. Methods: In mice models, wild-type and uPA knockout (uPA-/-) BALB/c mice were used for induction of T2DM. In cell models, insulin secretion rate and β cell proliferation were assessed in normal and high glucose after treating uPA siRNA, uPA, or anti-uPA antibody. In our clinical study, patients with T2DM received an oral glucose-tolerance test, and the relationship between uPA and insulin secretion was assessed. Results: Insulin particles and insulin secretion were mildly restored one month after induction in wild-type mice, but not in uPA-/- mice. In cell models, insulin secretion rate and cell proliferation declined in high glucose after uPA silencing either by siRNA or by anti-uPA antibody. After treatment with uPA, β cell proliferation increased in normal glucose. In clinical study, patients with T2DM and higher uPA levels had better ability of insulin secretion than those with lower uPA levels. Conclusion: uPA may play a substantial role in insulin secretion, β cell regeneration, and progressive development of T2DM. Supplementation of uPA might be a novel approach for prevention and treatment of T2DM in the future.

Quantification of uPA in breast tumour tissue extracts by microarray immunoassay: Comparison with ELISA technology

The urokinase-type plasminogen activator (uPA) and PA inhibitor 1 (PAI-1) play important roles in breast cancer metastasis through cell migration and invasion. They are clinically applicable prognostic and predictive markers. High levels of uPA and PAI-1 are associated with high risk of recurrence and adjuvant chemotherapy provides substantial benefit for this breast cancer population. The current sole validated method for quantifying uPA level in breast tumour tissue is ELISA assay. It requires 50–300mg of fresh or frozen tissue, which is the main limitation for routine use. In this study, we evaluated the performances of customized antibody microarray to quantify uPA concentration from reduced extraction solution of breast tumour tissue and compared it with standard ELISA kit. We firstly optimized the elaboration of customized antibody microarray in order to sensitively detect and quantify uPA standard solutions. In the best conditions, we analysed uPA concentration in 16 cytosolic extracts from breast tumour tissue. Results showed that our customized antibody microarray could correctly quantify uPA concentration while consuming 100 times less volume of tumour tissue extraction solution than ELISA. Our antibody microarray is a powerful and promising tool for the miniaturization of the immunoassay quantification of uPA from breast tumour tissue extracts.

Serum levels of urokinase-type plasminogen activator in healthy dogs and oncologic canine patients.

Aim:Urokinase plasminogen activator (uPA) has been scarcely studied in veterinary oncology. The aim of this study was to determine the uPA serum concentrations in healthy and oncologic canine patients and to investigate its potential value as a tumor biomarker.Materials and Methods:Serum uPA concentrations of healthy and oncologic canine patients were measured by enzyme-linked immunosorbent assay. Their relationships with the dogs’ health status and tumor characteristics were analyzed through ANOVA and independent t-test.Results:There were no significant differences between mean serum values (±standard deviation) of healthy dogs (0.19±0.13 ng/ml) and oncologic canine patients (0.22±0.33 ng/ml), or between dogs with benign or malignant tumors, and with or without metastases, although the latter tended to show higher uPA serum levels.Conclusion:This is the first study describing the uPA serum levels in dogs. Although its results do not support uPA as a tumor biomarker, higher uPA levels in dogs with metastatic neoplasms may reflect the role of the enzyme in tumor progression.

Prognostic impact of the inclusion of uPA/PAI-1 for adjuvant treatment decision-making in ER+/Her2- pN0 early breast cancers.

Intermediate-risk early breast cancer (EBC) is a heterogeneous group in which adjuvant chemotherapy decision proves to be difficult. Clinical and pathological criteria are sometimes insufficient to determine the best therapeutic options, and validated biomarkers such as uPA/PAI-1, are needed to contribute to the decision-making. The objective of this study was to evaluate the clinical outcome of an unselected ER+/HER2- pN0 EBC cohort of patients in whom the routine clinical decision process included a prospective uPA/PAI-1 determination. This monocentric retrospective study included 520 patients who underwent curative surgery in our institute between 2006 and 2011. Adjuvant therapeutic strategy was decided based on clinical-pathological data, altogether with a routine prospective determination of uPA/PAI-1 tumor levels using fresh, extemporaneously sampled tissue. We evaluated the correlation between uPA/PAI-1 levels, clinical-pathological variables, and the patient's outcome (relapse-free survival, RFS, and overall survival, OS). Median follow-up was 5.4years. The 5- and 10-year RFS rates were ,respectively, 95 and 89%, and the five-year OS rate was 96.3%. Forty percent of tumors had low uPA/PAI-1 levels. Seventy-five percent of patients with low uPA/PAI-1 levels did not receive chemotherapy, when 25% did. Sixty percent of patients with high uPA and/or PAI-1 levels received chemotherapy, while 40% did not. No statistical significant correlation was found between the uPA/PAI-1 levels and RFS or OS. The personalization of the patients' treatment using uPA/PAI-1 tumor levels allows the reversion of the well-known poor prognostic impact of high uPA/PAI-1 levels and strongly supports the use of this biomarker in clinical practice.

Abstract P6-09-28: Prognostic impact of the inclusion of uPA/PAI-1 in ER+/Her2- pN0 early breast cancer adjuvant treatments decision-making

Abstract Purpose: Intermediate-risk early breast cancer (EBC) is a heterogeneous group in which adjuvant chemotherapy decision prove to be difficult. Clinical and pathological criteria might be insufficient to determine the best therapeutic options for patients, and validated biomarkers are urgently needed to help decision making, such as the LOE-I uPA/PAI-1 biomarker. The aim of this study is to evaluate the clinical outcome of a large, unselected, ER+/HER2- pN0 EBC cohort of patients in whom the routine clinical decision process included a prospective uPA/PAI-1 determination. Methods: This monocentric retrospective study included 520 ER+/HER2- pN0/M0 EBC patients who had curative surgery in our center between 2006 and 2011. Adjuvant therapeutic strategy was decided based on clinico-pathological data, altogether with a routine prospective determination of uPA/PAI-1 tumor levels. We evaluated the correlation between uPA/PAI-1 levels, classical clinico-pathological variables and the patient's outcome (relapse free survival, RFS; overall survival, OS). Results: Median age was 54 years (range 27-85). 75% of tumors were classified T1, 25% T2 and above. We found 80.8% of ductal carcinomas, 12.3% of lobular carcinomas and 6.9% of other histological types. SBR grade 1-2 represented 82.5% of our cohort versus 17.5% of grade 3. Mitotic count was &amp;lt;10 in 63.5% of cases, and ≥10 in 36.5%. Peri-tumoral invasion (PVI) was seen in 17% of tumors. Progesterone receptors (PR) were positive in 79.6% of cases. Median follow-up was 5.4 years. 5 and 10 years RFS were respectively 95% and 89% (n=33 local or metastatic relapse). 5 year OS was 96.3%. We found 40% of low uPA/PAI-1 levels, and 60% of high uPA and/or PAI-1 levels. By using uPA/PAI-1 levels in our adjuvant treatment decision-making, 75% of patients with low uPA/PAI-1 levels did not received chemotherapy and 60% of patients with high uPA and/or PAI-1 levels received chemotherapy. 98.3% of our patients received endocrine therapy as adjuvant treatment for at least 5 years. No statistical significant correlation was found between uPA/PAI-1 levels and RFS (p=0.3) or OS (p=0.28). In univariate analysis, tumor size (p=0.048; p=0.0694), histological grade (p=0.007; p=0.0142), PR status (p=0.001; p=0.0002) and mitotic count (p=0.0001; p=0.0001) were statistically correlated with RFS and OS, respectively. Using cox regression model, PR status (p&amp;lt;0.003) and mitotic count (p&amp;lt;0.001) appeared to be strongly correlated with RFS. No changes in significant variables were seen when the analyses were restricted to the grade 2 subgroup (n=339). Conclusion: The individualization of patients' treatment using uPA/PAI-1 tumor levels allows the reversion of the well-known poor prognostic impact of high uPA/PAI-1 levels and strongly support the use of this LOE-I biomarker in clinical practice. PR status and mitotic activity remains independent major prognostic factors in optimally treated patients. The evaluation of the additional impact of multigene signature in this setting needs to be performed. Longer 10-years follow-up needs to be done in this ER+/HER2- subgroup, particularly to evaluate the risk of late relapse after endocrine therapy. Citation Format: Viala M, Alexandre M, Thézenas S, Lamy P-J, Bibeau F, Gutowski M, Colombo P-E, Romieu G, Jacot W, Guiu S. Prognostic impact of the inclusion of uPA/PAI-1 in ER+/Her2- pN0 early breast cancer adjuvant treatments decision-making [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-09-28.

High Expression of Urokinase-Type Plasminogen Activator Is Associated with Lymph Node Metastasis of Invasive Ductal Carcinoma of the Breast.

PurposeIn the present study, we evaluated the levels of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1) by performing immunohistochemical staining to determine whether they were reliable prognostic markers in patients with breast cancer.MethodsDemographic and clinicopathological parameters of 214 patients with invasive ductal carcinoma (IDC) and 80 patients with ductal carcinoma in situ (DCIS) who were diagnosed and treated from 2006 to 2010 were analyzed. Tissue microarray was constructed and immunohistochemical staining was performed for each specimen.ResultsUnivariate analyses showed that age at diagnosis, history of hormone replacement therapy, radiation therapy, skin and chest wall invasion, Paget disease, lymphovascular invasion, estrogen receptor positivity, and triple-negative subtype were significantly associated with patient prognosis (p<0.005). Patients with DCIS showed higher PAI-1 expression than patients with IDC (82.5% and 36.2%, respectively; p=0.012). Lymph node metastasis was more frequent in patients with high uPA levels than in patients with low uPA levels (p=0.001).ConclusionOur results suggested that PAI-1 was involved in tumor progression in the early stages of breast cancer, such as DCIS. In addition, our results suggested that high uPA levels were associated with the lymph node metastasis of IDC.

Plasminogen Activator System and Breast Cancer: Potential Role in Therapy Decision Making and Precision Medicine.

Shifting from the historical TNM paradigm to the determination of molecular and genetic subtypes of tumors has been a major improvement to better picture cancerous diseases. The sharper the picture is, the better will be the possibility to develop subsequent strategies, thus achieving higher efficacy and prolonged survival eventually. Recent studies suggest that urokinase-type plasminogen activator (uPA), uPA Receptor (uPAR), and plasmino-gen activator inhibitor-1 (PAI-1) may play a critical role in cancer invasion and metastasis. Consistent with their role in cancer dissemination, high levels of uPA, PAI-1, and uPAR in multiple cancer types correlate with dismal prognosis. In this respect, upfront determination of uPA and PAI-1 as invasion markers has further opened up the possibilities for individualized therapy of breast cancer. Indeed, uPA and PAI-1 could help to classify patients on their risk for metastatic spreading and subsequent relapse, thus helping clinicians in their decision-making process to propose, or not propose, adjuvant therapy. This review covers the implications for cancer diagnosis, prognosis, and therapy of uPA and PAI-1, and therefore how they could be major actors in the development of a precision medicine in breast cancer.

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.

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.

Quebec Platelet Disorder Is Associated With Greater Than Expected Increases In Urokinase Plasminogen Activator In Granulocytes and Monocytes

Myeloid leukocytes produce urokinase plasminogen activator (uPA), an important activator of fibrinolysis. Stimuli such as lipopolysaccharide and formyl-methionyl-leucyl-phenylalanine (LPS/fMLP), increase the expression of PLAU by myeloid leukocytes. We postulated that the basal, and/or stimulus-induced, uPA production by myeloid leukocytes would be increased in Quebec platelet disorder (QPD), a congenital bleeding disorder caused by duplication of PLAU, the uPA gene. In QPD, plasma and urine uPA levels are within the expected range. However, overexpression of PLAU in QPD was found to emerge during megakaryopoiesis and QPD platelets to contain >100-fold increased uPA.We investigated PLAU expression by platelet-free, granulocytes and monocytes that were differentiated in culture, from peripheral blood CD34+ cells, without added thrombopoietin and erythropoietin. QPD and control (C) myeloid cells were harvested on day 7 or 14 of culture (n= 6 samples per subject for each endpoint). Granulocytes and monocytes were isolated by affinity purification. For some experiments, granulocytes from day 14 cultures were treated with: LPS/fMLP; complement and heat activated immunoglobulin (C5a/IgG); thrombin, thrombin plus fibrinogen, or thrombin plus plasma (IIa, IIa/Fg or IIa/plasma). PLAU expression was characterized by real time quantitative polymerase chain reaction and uPA was quantified by enzyme-linked immunosorbent assays. The lysis of plasma clots (optical density endpoint) that had been spiked with day 14 granulocytes or uPA, was assessed with or without added tranexamic acid. Data were expressed as means ± standard error (SEM) and analyzed by Mann-Whitney tests.Higher PLAU expression by QPD myeloid leukocytes was evident in day 7 (23-fold, p = 0.002) and day 14 (13-fold; p = 0.002) myeloid leukocyte cultures, accompanied by increased cellular uPA (day 7, 16-fold increase [pg/106 cells, QPD: 162 ± 11; C: 10.2 ± 0.3, p = 0.002]; day 14, 9-fold increase [pg/106 cells, QPD: 458 ± 8, C: 50.9 ± 2.5, p = 0.002]). In day 14 cultured cells, PLAU expression was higher in QPD granulocytes (57-fold) and monocytes (9-fold)(p values ≤ 0.005), and allele specific analysis confirmed overexpression of PLAU by the disease chromosome. The higher PLAU expression was associated with higher levels of uPA (pg/106 day 14 cultured cells) in QPD granulocytes (170-fold [QPD: 477 ± 33, C: 2.8 ± 0.4, p = 0.005]) and monocytes (5-fold increase [QPD: 208 ± 11; C: 41.5 ± 4.3; p = 0.002]). Like control granulocytes, QPD granulocytes expressed and secreted much more uPA after exposure to immune, infectious and procoagulant stimuli (Table 1). Interestingly, some of these stimuli reduced the differences between QPD and control granulocytes in PLAU expression and secreted uPA (Table 1). In fibrin clots, QPD granulocytes triggered more fibrinolysis than control granulocytes, and release higher levels of uPA. Tranexamic acid inhibited the fibrinolysis-induced by QPD and control granulocytes.Table1Effect of stimuli on PLAU expression and uPA secreted by granulocytesStimulus:noneLPS/fMLPC5a/IgGIIaFg + IIaPlasma + IIaPLAU expression (fold difference, QPD vs. C)773311143P value (QPD vs. C)0.0020.0020.0020.0020.0020.002uPA released (pg/106 cells ± SEM)C4.5 ± 1.28,250 ± 30011,050 ± 7501,300 ± 501,300 ± 1007,250 ± 550QPD33.5 ± 2.615,900 ± 130032,250 ± 90011,000 ± 35014,800 ± 75016,950 ± 800uPA release (fold difference, QPD vs. C)7238112P value (QPD vs. C)0.0020.0020.0020.0020.0020.002We conclude that the greater than expected effect of the QPD PLAU duplication on PLAU expression during myeloid differentiation extends to granulocytes and monocytes. In QPD, PLAU dysregulation was associated with much higher uPA levels in granulocytes than in monocytes. Based on changes to PLAU expression and uPA in response to procoagulant, immune or septic stimuli, we conclude that QPD has greater effects on the basal expression of PLAU by myeloid cells. The upregulation of PLAU expression by granulocytes exposed to thrombin, fibrin clots, immune and septic stimuli, may contribute to the increased fibrinolysis in some pathological conditions. Disclosures:No relevant conflicts of interest to declare.

Plasma urokinase antigen and C-reactive protein predict angina recurrence after coronary angioplasty

This study evaluates the predictive value of several biochemical indices of the coagulation-fibrinolysis system, platelet function, and inflammatory state for angina recurrence after successful percutaneous transluminal coronary angioplasty (PTCA). We measured preprocedural and follow-up plasma levels of C-reactive protein (CRP), fibrinogen, and urokinase plasminogen activator antigen (uPA), plasminogen activator inhibitor-1 (PAI-1) activity, tissue plasminogen activator activity, and adenosine diphosphate-induced platelet aggregation in 53 patients with chronic stable angina who underwent successful elective PTCA of single hemodynamically significant lesions in coronary arteries. All patients were followed up for 12 months after PTCA. The Cox proportional hazards model was used to assess the association of variables with angina recurrence rate. At the end of the follow-up, 16 patients had angina recurrence. Among 36 clinical, biochemical, and angiographic variables, the duration of stable angina more than 12 months before PTCA (χ (2) = 5.73; P = 0.02, hazard ratio (HR) 3.7, 95 % confidence interval (CI) 1.26-10.6), high baseline levels of CRP (>7 mg/l) (χ (2) = 8.34; P = 0.004, HR 2.9, 95 % CI 1.4-5.9), uPA antigen baseline (>1 ng/ml) (χ (2) = 17.11; P = 0.0001, HR 11.5, 95 % CI 3.6-36.7) and 48 h after PTCA (χ (2) = 15.73; P = 0.0001, HR 8.8, 95 % CI 3.01-25.96), baseline PAI-1 activity (>18 IU/ml) (χ (2) = 9.37; P = 0.002, HR 7.6, 95 % CI 2.07-27.84) were significant predictors of recurrent angina by univariate analyses. According to stepwise multivariate analyses, only the levels of plasma uPA antigen and serum CRP were shown to be significant independent predictors of angina recurrence (multivariate uPA χ (2) = 8.22, P = 0.004, HR 6.2, 95 % CI 1.78-21.67; CRP χ (2) = 4.09, P = 0.04, HR 1.9, 95 % CI 1.02-3.68). High preprocedural plasma uPA and serum CRP levels are indicative of angina recurrence after successful PTCA, and are valuable for the prognosis of restenosis.

Urokinase Plasminogen Activator and Its Inhibitor Type‐1 as Prognostic Factors in Differentiated Thyroid Carcinoma Patients

To investigate the prognostic value of urokinase-type plasminogen activator (uPA) and its inhibitor, type-1 plasminogen activator inhibitor (PAI-1), in differentiated thyroid cancer. Prospective cohort study. University hospital. Cytosolic concentrations of uPA and PAI-1 were determined in 105 patients with differentiated thyroid carcinoma and normal matched tissues using an enzyme-linked immunoassay (ELISA). Both uPA and PAI-1 concentrations were significantly higher in differentiated thyroid tumors (uPA = 0.509 ± 0.767 and PAI-1 = 6.337 ± 6.415 ng/mg) compared to normal tissues (uPA = 0.237 ± 0.051, P < .001; PAI-1 = 2.368 ± 0.418 ng/mg, P < .001). uPA and PAI-1 were significantly higher if extrathyroidal invasion (uPA, P = .015; PAI-1, P < .001) or distant metastasis (PAI-1 P < .001) was present, as well as in tumors whose size exceeded 1 cm in diameter (uPA, P = .002; PAI-1, P = .001). Survival analysis revealed the significant impact of both uPA and PAI-1 on progression-free survival (PFS) (82.22 vs 49.478 months for patients with low and high uPA, respectively, P < .001; 87.068 vs 44.964 months for patients with low and high PAI-1, respectively, P < .001). Univariate analysis showed that gender, tumor size, tumor grade, extrathyroid invasion, local lymph node involvement, distant metastasis, uPA, and PAI-1 were significant predictors of PFS. However, multivariate analysis identified only distant metastasis and tumor tissue uPA and PAI-1 as independent prognostic factors. These findings indicate that high uPA and PAI-1 levels represent independent unfavorable prognostic factors in patients with differentiated thyroid carcinoma.

A central role of plasmin in cardiac injury initiated by fetal exposure to maternal anti-Ro autoantibodies

Cardiac neonatal lupus (cardiac-NL), initiated by surface binding of anti-Ro60 autoantibodies to apoptotic cardiocytes during development, activates the urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) system. Subsequent accumulation of apoptotic cells and plasmin generation facilitates increased binding of anti-Ro60 by disrupting and cleaving circulating β2-glycoprotein I (β2GPI) thereby eliminating its protective effect. The association of soluble levels of components of the uPA/uPAR system with cardiac-NL was examined. Levels of the uPA/uPAR system were assessed by ELISA in cord blood and immunohistological evaluation of autopsies. uPA, uPAR and plasminogen levels were each significantly higher in cord blood from cardiac-NL (n = 35) compared with non-cardiac-NL (n = 26) anti-Ro-exposed neonates: 3.3 ± 0.1 vs 1.9 ± 0.05 ng/ml (P < 0.0001), 6.6 ± 0.3 vs 2.1 ± 0.2 ng/ml (P < 0.0001) and 435 ± 34 vs 220 ± 19 ng/ml (P < 0.0001), respectively. In three twin pairs discordant for cardiac-NL, the twin with cardiac-NL had higher levels of uPA, uPAR and plasminogen than the unaffected twin (3.1 ± 0.1 vs 1.9 ± 0.05 ng/ml; P = 0.0086, 6.2 ± 1.4 vs 2.2 ± 0.7 ng/ml; P = 0.147 and 412 ± 61 vs 260 ± 27 ng/ml; P = 0.152, respectively). Immunohistological evaluation of three hearts from fetuses dying with cardiac-NL revealed macrophages and giant cells expressing uPA and plasminogen in the septal region. Increased soluble uPA, uPAR and plasminogen in cord blood and expression in affected tissue of fetuses with cardiac-NL supports the hypothesis that fetal cardiac injury is in part mediated by plasmin generation initiated by anti-Ro binding to the apoptotic cardiocyte.

Paclitaxel Resistance and Multicellular Spheroid Formation Are Induced by Kallikrein-Related Peptidase 4 in Serous Ovarian Cancer Cells in an Ascites Mimicking Microenvironment

High tumor kallikrein-related-peptidase 4 (KLK4) levels are associated with a poor outcome for women with serous epithelial ovarian cancer (EOC), for which peritoneal dissemination and chemoresistance are key events. To determine the role of KLK4 in these events, we examined KLK4-transfected SKOV-3 and endogenous KLK4 expressing OVCA432 cells in 3-dimensional (3D) suspension culture to mimic the ascites microenvironment. KLK4-SKOV-3 cells formed multicellular aggregates (MCAs) as seen in ascites, as did SKOV-3 cells treated with active KLK4. MCA formation was reduced by treatment with a KLK4 blocking antibody or the selective active site KLK4 sunflower trypsin inhibitor (SFTI-FCQR). KLK4-MCAs formed larger cancer cell foci in mesothelial cell monolayers than those formed by vector and native SKOV-3 cells, suggesting KLK4-MCAs are highly invasive in the peritoneal microenvironment. A high level of KLK4 is expressed by ascitic EOC cells compared to matched primary tumor cells, further supporting its role in the ascitic microenvironment. Interestingly, KLK4 transfected SKOV-3 cells expressed high levels of the KLK4 substrate, urokinase plasminogen activator (uPA), particularly in 3D-suspension, and high levels of both KLK4 and uPA were observed in patient cells taken from ascites. Importantly, the KLK4-MCAs were paclitaxel resistant which was reversed by SFTI-FCQR and to a lesser degree by the general serine protease inhibitor, Aprotinin, suggesting that in addition to uPA, other as yet unidentified substrates of KLK4 must be involved. Nonetheless, these data suggest that KLK4 inhibition, in conjunction with paclitaxel, may improve the outcome for women with serous epithelial ovarian cancer and high KLK4 levels in their tumors.

Abstract P5-20-01: A randomized double-blind phase II study of the combination of oral WX-671 plus capecitabine vs. capecitabine monotherapy in first-line HER2− negative metastatic breast cancer (MBC).

Abstract Background: uPA and its inhibitor PAI-1 play a key role in tumor invasion, metastasis and tumor growth. High levels of uPA and PAI-1 in breast tumors are statistically significant prognostic factors of disease-free (DFS) and overall survival (OS), which were validated at the highest level of evidence, as well as predictors for benefit of adjuvant chemotherapy. WX-UK1 is an active site competitive inhibitor of uPA with an inhibition constant in the submicromolar range. WX-671 (upamostat) is an oral prodrug of WX-UK1. In preclinical animal tumor models, both WX-UK1 and WX-671 have been shown to reduce the growth rate of implanted tumors, to inhibit invasion, and reduce metastases. This current proof of concept study is designed to substantiate the anti-metastatic properties of upamostat for patients appropriate for first line therapy for MBC. Methods: Female patients aged &amp;gt;18, with HER2 negative MBC appropriate for first line monotherapy with capecitabine, with adequate performance status, organ function, bone marrow reserve without brain metastases were eligible. Patients were randomized in a double-blind fashion to receive upamostat (200mg orally daily for 21 days) plus capecitabine (1000 mg/m2 orally twice daily for 14 days) vs. capecitabine (1000 mg/m2 orally twice daily for 14 days) in 3 week treatment cycles until progressive disease or unacceptable toxicity. The primary endpoint is to evaluate the efficacy of the combination of upamostat plus capecitabine compared to monotherapy as assessed by comparison of progression free survival. The secondary objectives are OS, objective response rates, safety and tolerability, and to assess the pharmacokinetics (PK) of upamostat and capecitabine when combined. Results: Between August 2008 and April 2011,132 patients were enrolled. 17 patients are still receiving treatment. 26% of the patients are characterized as triple negative, 13% as only Estrogen Receptor (ER) positive and 4% as only Progesteron Receptor (PR) positive. 57 % of the patients are ER and PR positive. Conclusions: Progression free survival, response rates and safety will be reported. This abstract is being submitted as a placeholder. A completed abstract will be submitted when the analyses are completed. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-20-01.

In papillary thyroid carcinoma BRAFV600E is associated with increased expression of the urokinase plasminogen activator and its cognate receptor, but not with disease‐free interval

It has been suggested that patients with papillary thyroid cancer (PTC) harbouring the BRAF(V600E) mutation have a worse prognosis. We showed in PTC that high levels of urokinase plasminogen activator (uPA) and its cognate receptor (uPAR) inversely correlate with disease-free interval (DFI). To investigate the effects of BRAF(V600E) on the expression of uPA and uPAR and to evaluate the prognostic relevance of BRAF(V600E) alone or in combination with uPA and uPAR. DESIGN/SETTING/PATIENTS/INTERVENTION: The case study included 91 patients with PTC. All patients underwent thyroidectomy and radioiodine therapy. Follow-up was available for 75 patients. The BRAF(V600E) mutation was analysed by sequencing and mutant allele-specific PCR amplification; uPA and uPAR expression by quantitative RT-PCR. BRAF(V600E) was found in 44 of the 91 patients and associated with older age, but not with high-risk clinicopathological features. Urokinase PA and uPAR mRNA levels were higher in tumour tissues by 9·51±1·30 and 4·64±0·44 fold, respectively, compared to normal matched tissues, being significantly higher in BRAF(V600E) -positive patients. In vitro induction of BRAF(V600E) in PCCL3 cells caused a significant increase in both uPA and uPAR mRNAs. Higher levels of uPA and uPAR correlated with lymph node metastases, TNM stage and disease recurrences. Kaplan-Meier and multivariate analyses demonstrated that uPA and uPAR were associated with shorter DFI, while the BRAF(V600E) was not. In PTC, BRAF(V600E) induces uPA and uPAR expression. The latter, but not BRAF(V600E) , associates with advanced stages and shorter DFI. If confirmed in larger case studies, they may represent reliable prognostic markers for more accurate risk stratification and postoperative decision-making in patients with PTC.