uPA Levels Research Articles

Abstract 5187: Urokinase plasminogen activator receptor (uPAR) and integrin α5β1 regulate plasminogen activator inhibitor-1 (PAI-1) activity in medulloblastoma cells

Abstract The urokinase plasminogen activator (uPA) system is a dynamic extracellular protease system that regulates both proteolytic and non-proteolytic events associated with cancer progression. In earlier studies, we have demonstrated that radiation-induced cell adhesion was associated with uPAR expression and downregulation of uPAR effectively inhibited radiation-induced cell adhesion of medulloblastoma cells. Herein, we further investigated the impact of uPAR downregulation on plasminogen activator inhibitor (PAI-1), a key regulator component of the uPA system, and its role in cell adhesion. With radiation treatment, the levels of uPAR and PAI-1 increased in D283 and UW228 cells. Surprisingly, knockdown of uPAR elevated the levels of PAI-1. The increase in PAI-1 levels in uPAR-knocked down cells was regulated by activation of cJUN/CREB signaling molecules. Moreover, studies show that the reduced interaction between uPAR and integrin α5β1 plays a critical role in PAI-1 release into the extracellular matrix, thereby enhancing the cell detachment process. Incubation of medulloblastoma cells with Tiplaxtinin-PAI-039, a potent PAI-1 activity inhibitor, increased the association of uPAR with integrin α5β1 and cell adhesion. Interestingly, prolonged inhibition of PAI-1 activity adversely affected cell viability; we were able to revert this effect by exogenously supplementing cells with recombinant PAI-1. Moreover r-PAI-1 showed no significant effect on cell adhesion whereas it increased the association of uPAR with integrin α5β1. Expression of full-length (FL) uPAR elevated uPA and PAI-1 levels both at the transcriptional and the translational levels. These increased PAI-1 levels play a pivotal role in regulating uPA activity by binding and translocating integrin α5β1 towards the uPA/uPAR complex. Taken together, the results of the present study lead us to conclude that levels of uPAR and integrin α5β1, and their association on the cell surface dictate the function of PAI-1 to maintain extracellular homeostasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5187. doi:1538-7445.AM2012-5187

Abstract 5184: Expression profiling of ECM proteins in advanced carcinoma of uterine cervix

Abstract Background Carcinoma of uterine cervix accounts for most common malignancy in Indian women. Migration is one of the important processes fundamental to cell invasion and dissemination. Role of extracellular matrix (ECM) proteins is also very important in this crucial process and cannot be ignored. ECM proteins are involved in cell migration, tissue remodeling, angiogenesis and cell adhesion. Fibulin-1 is cysteine-rich, calcium-binding extracellular matrix and plasma protein that has been implicated as playing a role in tumour progression by modulating cell morphology, growth, adhesion, and motility. Increased plasminogen activator (PA) secretion has been observed in malignant cells and tissue and PA is thought to be involved in the processes of tumorigenesis, cancer invasion and metastasis. Laminins are important for proliferation, differentiation, growth, migration & angiogenesis. Besides the structural function of nidogens, it has long been suggested that nidogens play a role in cell attachment and tumor angiogenesis. Objectives This study was aimed to investigate the expression and significance of ECM proteins (Fibulin-1, uPA, Laminin and Nidogen) in advanced carcinoma of uterine cervix. Material & Methods 40 patients of cancer cervix stage IIIb were included in this study. Circulatory levels of Fibulin-1, Laminin and Nidogen were measured using high sensitivity ELISA kits. uPA activity was measured by activity assay kit. The levels of biochemical markers were compared with 20 healthy females, taken as control. mRNA levels of these molecules were further assessed by Real Time RT-PCR. Data was analyzed statistically. Results Statistical significant (p < 0.001) increase was observed in Fibulin-1, uPA and Laminin levels and found to be significantly correlated with each other whereas Nidogen evels were insignificantly increased as compared to controls. Increased mRNA expression for all these molecules has also been observed in the isolated PBMC by qRT-PCR except nidogen (statistically insignificant increase). Conclusion The elevated serum/plasma levels and increased quantitative expression of these ECM proteins suggests their involvement in the pathogenesis of the disease. This study suggests that rise in ECM proteins might contribute to the complicated and unregulated process of metastasis in cancer of cervix. The association of extracellular matrix proteins and the prognosis of the disease may lead to have a better approach in treating the cervical cancer patients with a combination therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5184. doi:1538-7445.AM2012-5184

Abstract 679: Retrospective evaluation of the BRCA1 - PARP pathway in breast cancer tissues

Abstract Purpose: Poly(adenosine diphosphate-ribose) polymerase (PARP) and BRCA1 plays a key role in DNA repair and cellular stress response. Inhibitors of PARP show promising clinical activity in metastatic, triple-negative or BRCA-mutated breast cancer. However, a comprehensive analysis of the PARP activity / BRCA1 methylation profile in non-BRCA1 mutated tumors has not yet been assessed. Methods: We investigated cytosolic PARP activity and BRCA1 methylation profile in 155 surgical tumour samples from patients treated in our institution between January 2006 and November 2009 and evaluated their statistical association with classical breast cancer predictive and prognostic factors. DNA methylation patterns in the CpG islands of BRCA1 gene promoter were determined by a methylation specific PCR distinguishing unmethylated from methylated alleles in a given gene on the basis of sequence changes produced following bisulfite treatment of DNA. Cytosolic PARP activity was evaluated using a commercially available assay kit. Results: A total of 155 BC patients (50 HER-2 positive, 57 hormone receptors positive (HR+) / HER-2 negative and 48 triple negative tumors) were considered eligible for this study. Median age was 54 years (range 29-75 years). Only one tumor was classified as SBR score I and SBR Tubule formation score 1. No tumor was SBR Nuclear pleomorphism score 1. Considering these results, SBR scores I and II and SBR Tubule formation scores 1 and 2 were grouped for statistical analyses. Mean PARP activity (U/mg of cytosolic protein) was 12.2 (standard deviation 17.02), with a median of 7.0 (range 1.0 to 114.2). The only clearly associated parameter was SBR Mitotic count score. Bisulfite treatment was successfully performed for the whole 155 specimen. Eighteen tumors presented an hypermethylation of the BRCA1 gene promoter. The hypermethylation status was significantly associated with UPA and PAI-1 levels, SBR grade, SBR Mitotic count score, ER, PR and HER-2 negative status, and then conversely triple negative (ER, PR and HER-2 negative) profile. Twenty nine percent of the triple negative tumors presented and hypermethylated status, comparing to 5 and 2% for HR+/HER-2- and HER-2+ tumors respectively. No statistical association was found between a hypermethylation status and the PARP cytosolic activity. Conclusion: Cytosolic PARP activity is equally represented in TN, HER2+ and SBR II and III HR+ breast cancers. The only parameter clearly associated with the cytosolic PARP activity was the SBR Mitotic count score. In contrast, hypermethylation of the BRCA1 gene promoter appears restricted to a subgroup of TN tumors expressing an aggressive phenotype. No statistical association was found between a hypermethylation status and the PARP cytosolic activity. These results could allow a more accurate stratification of patients without BRCA1 famillial mutation in the future PARP inhibitors clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 679. doi:1538-7445.AM2012-679

Abstract 136: Suppression of the uPA/uPAR system causes the overexpression of miR124 and suppresses stemness in glioma stem cells

Abstract The invasiveness and destructiveness of malignant neoplasms in the central nervous system are of great clinical importance. Higher-grade tumors, such as glioblastomas, are associated with poor prognosis, and patients’ average survival is only 8 to 12 months after chemotherapy and/or radiotherapy. This poor prognosis reflects the resistance of tumor cells to radiation and cytotoxic agents as well as the difficulty in achieving total tumor resection. Highly infiltrative gliomas are known to overexpress both uPA and uPAR. MicroRNAs (miRNAs) are essential post-transcriptional regulators known to determine cell identity and fate and are also known to be involved not only in development and differentiation but also in glioma progression. Acquisition of EMT has also recently been linked to stem cell phenotypes which is mediated by microRNAs. However, the molecular mechanism underlying EMT regulation still remains elusive. In the present study, we used glioma initiating cells (GICs), which show stem cell-like character, and determined whether their stemness can be suppressed by targeting the uPA/uPAR system. We raised glioma GICs from U87MG and 4910 glioma xenograft cells and observed that GICs expressed 2- to 3-fold increased levels of uPA and uPAR when compared to non-GIC. Further, the simultaneous downregulation of both uPA and uPAR suppressed GIC from establishing intracranial tumors in nude mice. Further, to understand whether this suppression of GIC involved microRNAs, we profiled miR expression in U87 cells and observed that miR124 and miR200a were significantly overexpressed. Using miRanda analysis, we determined that miR124 targets the regulation of Lhx-2 and Lhx-2 is over expressed (3- to 4-fold) in GIC when compared to non-GIC. To determine whether this suppression of Lhx-2 by downregulation of uPA and uPAR is mediated via miR124, we overexpressed miR124 in U87MG and 4910 glioma xenograft GICs and observed that miR124-overexpressed cells failed to establish intracranial tumors in nude mice. Our results demonstrate that the uPA/uPAR system may be involved in GIC maintenance via suppression of miR124. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 136. doi:1538-7445.AM2012-136

Abstract 3441: Silencing glucosylceramide synthase using MBO-asGCS eliminates tumor metastasis

Abstract Disseminated metastasis is often observed in cancer patients fail in chemotherapy. Tumor metastasis is known to rely on the interaction of cancer cells and the extracellular matrix components but the molecular mechanism by which drug resistance promotes tumor metastasis is still not clear. Glucosylceramide synthase (GCS) that is a limiting enzyme for ceramide glycosylation and regulates glycosphingolipid (GSL) synthesis can cause drug resistance. Hence we report that GCS is a determinant of metastatic properties of drug-resistant cancer cells, and silencing GCS eliminates tumor metastasis. Our recent study found that the metastatic potency of drug-resistant cancer cells depended on the overexpression of GCS. Compared with drug-sensitive MCF-7 cells, the wound healing, matrigel attachment and MMP-9 activity significantly was 1.5 fold faster, 3 fold more and 2 folds higher respectively in the drug resistant MCF-7/Dox that also had 39-fold higher GCS protein level and 2-fold higher GCS enzyme activity. Introduction of GCS gene enhanced the metastatic potency of MCF-7 cells, because its matrigel attachment and invasion potential significantly increased by 2-fold and 1.5-fold after transfection. In contrast, silencing GCS by using mixed-backbone oligonucleotide against GCS (MBO-asGCS, 100 nM) substantially diminished the metastatic potency of MCF-7/Dox cells, as the wound healing, matrigel attachment and invasion reduced by 2-fold, 1.5-fold and 3-fold respectively. MBO-asGCS treatments eliminated lung metastasis of MCF-7/Dox tumors. Further mechanistic studies disclosed that silencing GCS by MBO-asGCS decreased its expression and enzyme activity and also decreased the levels of globo-series glycosphingolipids (GSLs) in GSL-enriched microdomain located on the plasma membrane. Sequentially, silencing GCS decreased the expression levels of uPA, MMP-9, VEGF and FGF-2 that actively mediated the migration and invasion of cancer cells. This study thus indicates that GCS is determinant for the metastatic potency of drug-resistant cancer cells and silencing GCS by using MBO-asGCS is an effective approach to target the metastasis of tumor with drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3441. doi:1538-7445.AM2012-3441

Elastin-derived peptides increase invasive capacities of lung cancer cells by post-transcriptional regulation of MMP-2 and uPA

Elastin-rich lung extracellular matrix is largely remodeled during tumor invasion. Elastin degradation produces peptides displaying a wide range of biological activities. These elastin derived peptides (EP) interact with the elastin receptor complex (ERC) but also bind to α(V)β(3) integrin and galectin-3. In this study, we explored the role of EP and their receptors in tumor progression of lung carcinomas. Non-invasive and invasive lung tumor cell lines were incubated in presence of kappa-elastin (κE) or with synthetic peptides displaying receptor-specific sequences (VGVAPG, GRKRK, AGVPGLGVG and AGVPGFGAG). Modified Boyden chamber assays revealed an increased invasive capacity of invasive cells induced by κE. EP treatment had no effect on cell proliferation but zymography analysis revealed an increase of pro-MMP-2 and uPA levels in the conditioned media of treated cells. Moreover, the active form of MMP-2 was increased in invasive cells. Interestingly, this regulation was not observed at the mRNA level and actinomycin D was unable to inhibit κE effects. We also observed that the regulation of proteases protein level following κE treatment was an early process detectable after 1 h. All these effects could not be inhibited by lactose and V14, two ERC antagonists, or by blocking antibodies against α(V)β(3) integrin and galectin-3. Finally, VGVAPG and GRKRK failed to reproduce κE effects whereas nonapeptides partially mimicked them. These results demonstrate that treatment with EP up-regulates invasiveness of lung tumor cells via the release of proteolytic enzymes. This modulation involves post-transcriptional mechanisms and a nonapeptide-receptor different from the ERC, α(V)β(3) integrin and galectin-3.

Expression of Urokinase‐Type Plasminogen Activator (uPA), its Receptor (uPAR), and Inhibitor (PAI‐1) in Human Breast Carcinomas and Their Clinical Relevance

Serine proteases convert plasminogen to plasmin which is involved in tissue remodeling under physiologic and pathophysiologic conditions, including breast carcinoma invasion and progression. Both urokinase-type plasminogen activator (uPA) and pro-uPA associate with uPA receptor (uPAR) on target cells, where plasminogen activator inhibitors (e.g., PAI-1) may modulate their activities. Expression levels of these factors were compared in breast carcinomas relative to patient characteristics, carcinoma features, and clinical outcome. uPA, uPAR, and PAI-1 were quantified by enzyme-linked immunosorbent assay (ELISA) in extracts of 226 biopsies while estrogen receptor (ER) and progestin receptor (PR) were determined by enzyme immunoassay (EIA) or radio-ligand binding. Each set of assays contained a novel reference specimen with known quantities of each of these five analytes. Levels in ng/mg protein of these biomarkers exhibited ranges: uPA (0-12.3); uPAR (0-19.5); PAI-1 (0-91.2). When considered independently, expression of uPA, uPAR, or PAI-1 was unrelated to patient age or menopausal status. Although no correlation was observed between each analyte with stage, grade, or ER/PR status, levels appeared to differ with pathology and nodal status. A dendrogram from hierarchical clustering of uPA, uPAR, and PAI-1 levels in 106 specimens revealed three clusters of breast cancer patients. Kaplan-Meier analyses of uPA, uPAR, and PAI-1 indicated a correlation with overall survival (OS), suggesting collective examination of these biomarkers is useful in predicting clinical outcome of breast cancer.

P016 Stimulation of lymphatic function via VEGFR-3 as a novel therapy for chronic experimental intestinal inflammation

influencing the development of inflammatory and immune responses. Aim of the present study is to evaluate the expression and function of uPA-uPAR system in experimental models of colitis. Methods: The dextran sulfate sodium (DSS) and trinitrobenzene sulfonic acid (TNBS) were used as models of colitis. Expression of uPAR and uPA in healthy and colitic mice was assessed by Real-time. uPAR expression changes were further confirmed by western-blot analyses. Specificity of uPAR expression was investigated by both confocal microscopy and flow cytometry. The functional role of uPAR was examined in uPAR knock out (KO) mice. Colitis disease activity index (DAI) was calculated and intestinal damage evaluated by both endoscopy and histology. Results: The expression levels of uPA and uPAR increased during both DSS and TNBS treatment, as measured by Real-Time (4 6 fold increase for uPAR, p < 0.01 and 2 6 fold increase for uPA, p < 0.01). Western Blot studies confirmed uPAR changes in colitic mice (6 fold increase, p < 0.01). Confocal microscopy and FACS analysis identified CD68+ cells as the major cell type expressing high levels of uPAR. KO mice displayed higher weight loss and increased DAI (p < 0.01, at day 8 and 9 for DSS model; p < 0.05 at day 3 and 4 for TNBS model), compared to wild-type littermates. Histological and endoscopic score were significantly higher in KO mice (p < 0.05). Conclusions: Our findings reveal the uPA-uPAR system as a new component involved in controlling inflammation. In particular, uPAR expression is up-regulated during colitis by CD68+ macrophage and its genetic deletion leads to detrimental inflammation. uPAR seems to play a protective role in the development of experimental colitis and modulation of the uPAuPAR system could offer a new pathway to control intestinal inflammation.

The mRNA Expression of Various Angiogenesis-Related Genes in Pediatric Sarcomas and Nonmalignant Lesions of Tissue

For better understanding cancer pathogenesis and searching a potential target for antineoplastic therapy, the authors have studied mRNA expression profile in tissues from 39 children with histological confirmed malignant sarcomas and from 23 patients with bone and soft tissue nonmalignant lesions. mRNA levels of Angiogenesis-related genes VEGFA (including isoforms of 121, 165, 189), VEGFC, VEGFR-1, VEGFR-2, VEGFR-3, HIF-1α, TF, TFPI-1, TFPI-2, uPA, PAI-1 in pediatric specimens were examined using quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR). uPA, HIF-1α, VEGFR-1, VEGFR-2, VEGFR-3, and VEGFC mRNA levels from nonmalignant tissue were significantly higher than those from cancer tissue. On the other hand, isoform VEGFA121 and VEGFA165 and ratio VEGFA165/189 mRNA levels in cancer were higher in comparison with nonmalignant tissue. There was a strong correlation between VEGFA165 and VEGFA189 mRNA expression levels both in cancer tissue and in nonmalignant tissue. In grade 4 tumors in comparison with grade 2 tumors, there was a reduced VEGFA165/189 ratio. Moreover, TFPI-1 and TFPI-2 mRNA levels were significantly lower in sarcomas than in nonmalignant lesions and TFPI-2 was significantly lower in grade 4 tumors than in grade 2. The present data suggested that mRNA overexpression of angiogenesis-related genes is not a prerogative of malignant tissues. The authors supposed that in pediatric bone and soft tissue pathology, high expression of mRNAs of some angiogenesis-related genes may be associated with inflammation and physiological angiogenesis rather than with the development of a malignant tumor. The authors showed the importance of VEGFA121 and/or VEGFA165 and VEGFA165/189 isoform ratio in pediatric sarcomas neoangiogenesis and TFPI-2 for tumors grade 4.

High level of urokinase plasminogen activator contributes to cholangiocarcinoma invasion and metastasis.

To investigate the role of urokinase plasminogen activator (uPA) in cholangiocarcinoma (CCA) invasion and its correlation with clinicopathological parameters. uPA expression in CCA tissue was determined by immunohistochemistry. The level of uPA from two CCA cell lines (HuCCA-1 and KKU-M213) and a non-cancer immortalized cholangiocyte cell line (H69) was monitored by plasminogen-gelatin zymography and western blotting, whereas that of plasminogen activator inhibitor type 1 (PAI-1) protein and uPA receptor (uPAR) mRNA was monitored by western blotting and quantitative real-time reverse transcriptase polymerase chain reaction, respectively. Two independent methods were employed to suppress uPA function: a synthetic uPA inhibitor (B428) and silencing of uPA gene expression using siRNA. In vitro invasion of the uPA-disrupted cells was assessed by Matrigel-coated Transwell assay. The immunohistochemical study showed that 75.3% (131/174) of CCA tissues expressed uPA. High uPA expression was correlated with lymphatic invasion and metastasis of CCA patients. Plasminogen-gelatin zymography of the conditioned media and cell-surface eluates showed that both CCA cell lines, but not H69, expressed both secreted and membrane-bound forms of uPA. Although the two CCA cell lines, HuCCA-1 and KKU-M213, expressed a relatively high level of uPA and uPAR, the latter exhibited a much lower degree of in vitro invasiveness, correlating with a high expression of PAI-1 in the latter, but not in the former. Suppressing uPA function with a specific uPA inhibitor, B428, or with siRNA against uPA reduced in vitro invasiveness of KKU-M213 cells, demonstrating the requirement for uPA in the invasiveness of CCA cells. Therefore, our in vivo and in vitro studies suggest that uPA is an important requirement for the invasion process of CCA. uPA expression correlates with lymphatic invasion and metastasis in vivo and is required for CCA cell invasion in vitro, suggesting its potential as a therapeutic target.

Targeting Urokinase and the Transferrin Receptor with Novel, Anti-Mitotic N-Alkylisatin Cytotoxin Conjugates Causes Selective Cancer Cell Death and Reduces Tumor Growth

Tumor-specific delivery of ligand-directed prodrugs can increase the therapeutic window of chemotherapeutics by maintaining efficacy whilst decreasing toxic side effects. We have previously described a series of synthetic N-alkylated isatin cytotoxins that destabilize microtubules and induce apoptosis with 10-fold greater potency than conventional anti-mitotics in vitro. Here, we report the characterization, in vitro cytotoxicity and in vivo efficacy of a lead compound, 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin (N-AI) conjugated via an esterase-labile linker (N-AIE) to two proven targeting ligands, transferrin (Tf) and plasminogen activator inhibitor type 2 (PAI-2/serpinB2). N-AI was released from N-AIE and the targeting ligands Tf/PAI-2 in an esterase-dependent manner at 37 C and both Tf- and PAI-2-N-AIE conjugates were stable at physiological pH. Human cancer cell lines which vary in their expression levels of Tf receptor (TfR/CD71) and PAI-2 target, receptor bound urokinase (uPA) selectively internalized the conjugates. Tf-N-AIE was up to 24 times more active than the free drug and showed clear selectivity patterns based on TfR levels. PAI-2-N-AIE showed equivalent activity compared to the parent drug and strong selectivity patterns for uPA levels. In preliminary in vivo experiments, the PAI-2- and Tf-N-AIE conjugates were efficacious at 1/20(th) and 1/10(th) of the dose of the free N-AI, respectively, in a metastatic, orthotopic human breast tumor xenograft mouse model. Thus, this strategy specifically delivers and concentrates a novel class of isatin-based, tubulin destabilizing agents to tumors in vivo and warrants further detailed preclinical investigation.

P2-11-08: Clinical Value of Combination Assay for Quantitative Determination of Cancer Biomarkers C2P and uPA/PAI-1 for Disease Recurrence Prediction of Early Breast Cancer Patients.

Abstract [Background] C2P is an assay measuring specific activities (SA; kinase activity compensated by its protein expression) of cyclin-dependent kinases CDK1 and CDK2 by examining a small piece of fresh-frozen tumor tissue. We reported previously that the C2P risk score given by CDK1SA and CDK2SA is a potent prognostic factor in node-negative breast cancer patients. Likewise, a uPA/PAI-1 ELISA test (FEMTELLE®, American Diagnostica Inc. Stamford, CT) quantitatively determines uPA (urokinase-type plasminogen activator) and PAI-1 (plasminogen activator inhibitor type-1) antigen levels in tumor tissue extracts, to identify patients with high or low risk of disease recurrence of node-negative breast cancer patients. These cancer biomarkers are recommended by the ASCO at the highest level of evidence (LOE-1) for therapy decision making in node-negative breast cancer patients. From the biological point of view, the above two assays can be placed into two categories: tumor cell proliferation for C2P and tumor cell invasion for uPA/PAI-1. This fact led us to examine the concept of combination of the two assays to better select breast cancer patients at risk. [Results] Fifty-nine cases of frozen primary breast cancer tissues were subjected to the C2P assay in a blinded manner. Twenty-one cases (40%) were categorized into “high risk”, 7 cases (14%) into “intermediate risk”, and 24 cases (46%) into “low risk”. Seven cases were judged as “not informative”. The uPA/PAI-1 results and clinical information (26: recurrent cases, 30: non-recurrent cases, 1: stage IV, 2: unknown) were provided by the TUM tumor bank. uPA/PAI-1 risk categories of 36 cases (61%) were classified “high” and 23 cases (39%) categorized “low”. C2P and uPA/PAI-1 showed statistically significant correlation to histological grading (Pearson correlation coefficient; 0.45 and 0.40, respectively). No significant correlation was observed between C2P and uPA/PAI-1. By Kaplan-Meier analysis for disease-free survival, in cases treated with endocrine therapy only, both C2P and uPA/PAI-1 showed a reproducible trend to the respective claimed performances as prognostic factors. In the combination analysis of the two parameters, where low/low was judged as “low” and the others as “high”, 11 cases (24%) were categorized into “low” and 34 cases (76%) into “high”. The sensitivity and negative predictive value for disease recurrence were 90% (19/20) and 91% (10/11), respectively. Strong statistical significance was observed between the risk categories by the log-rank test; p=0.0089, and also by Cox proportional hazards regression analysis; HR=9.18, p=0.032. By multivariate analysis, also including tumor size and nodal status, the CP2 uPA/PAI-1 combination evolved as a significant, statistically independent parameter (HR: 6.51, p&amp;lt;0.01). [Conclusion] In this investigative study, the significance of the combination concept was strongly suggested for early breast cancer patients treated with endocrine therapy only. Yet, prior to implementation in the clinical setting, the practical performance of the combination assay should be validated by investigating an independent patient cohort. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-11-08.

Apigenin attenuates insulin-like growth factor-I signaling in an autochthonous mouse prostate cancer model.

Deregulation of IGF signaling plays an important role in prostate cancer and contributes to invasion and metastasis. We determined the effect of apigenin, a plant flavone, on IGF signaling and its downstream targets in TRAMP mice. Mice received p.o. apigenin at 20 and 50 μg/day dose for 20 weeks. ELISA, Western blotting and immunohistochemistry were performed to examine the IGF-axis and its regulated pathway in response to apigenin intake. Increased serum levels of IGF-I, VEGF, uPA and concomitant decrease in IGFBP-3 were observed; p-Akt (Ser473), p-ERK1 (T202/Y204) and p-ERK2 (T185/Y187) expression increased in the dorso-lateral prostate of TRAMP mice during the course of cancer progression as a function of age. P.o. administration of apigenin resulted in substantial reduction in the levels of IGF-I and increase in the levels of IGFBP-3 in the serum and the dorso-lateral prostate. This modulation of IGF/IGFBP-3 was associated with an inhibition of p-Akt and p-ERK1/2. Apigenin intake resulted in marked inhibition of VEGF, uPA, MMP-2 and MMP-9 which coincided with tumor growth inhibition and complete absence of metastasis in TRAMP mice. Our results indicate that apigenin effectively suppressed prostate cancer progression in TRAMP mice by attenuating IGF-I/IGFBP-3 signaling and inhibiting angiogenesis and metastasis.

Dysregulation of C10orf55 Expression in Megakaryocytic Cell Lineage From Quebec Platelet Disorder Individuals

Abstract 2274Quebec platelet disorder (QPD) is a unique and serious bleeding disorder, associated with a gain-of-function defect in fibrinolysis that leads to a delayed onset bleeding after surgery or trauma in affected individuals. It has an autosomal dominant pattern of inheritance and affected individuals typically demonstrate an extensive personal and family history of bleeding. The disorder is caused by a tandem duplication mutation of a 78-kb genomic region located on chromosome 10 that contains PLAU (the gene that encodes urokinase plasminogen activator, uPA) in addition to the gene C10orf55, a gene of unknown function that encodes a transcript with partial complementary regions to PLAU mRNA. While QPD has minimal effects on uPA in urine, plasma and many other cell types, it elevates uPA levels >100 fold in QPD platelets compared to controls, due to overexpression of PLAU during megakaryopoiesis. As C10orf55 expression is postulated to have negative regulatory effects on uPA expression, we investigated if there is evidence of C10orf55 dysregulation in QPD. TaqMan assays were utilized for quantitative real-time RT-PCR analysis of C10orf55 transcripts in QPD and control CD34+ peripheral hematopoietic progenitors, platelets, lymphoblasts and saliva buccal cells (n=4–8 control and patient subjects for each cell type). QPD platelets contained elevated levels of C10orf55 mRNA that were >130 fold higher than in control platelets (p<0.001). Whereas in the other QPD cell types that were examined, the levels of C10orf55 mRNA were within the range expected for one additional copy of this gene, based on transcript levels measured in the control cells. These data demonstrate that the tandem duplication mutation of QPD leads to megakaryocyte specific increases in C10orf55 expression that parallel the pathological changes in PLAU expression during megakaryopoiesis. Although C10orf55 has been postulated to downregulate PLAU expression, due to complementarity of its transcripts, this seems unlikely given our findings. Our study indicates that the pathogenesis of QPD involves megakaryocyte-differentiation specific dysregulation of both genes contained in the 78-kb tandem duplicated locus on chromosome 10. Disclosures:No relevant conflicts of interest to declare.

Binding of Anti-SSA Antibodies to Apoptotic Fetal Cardiocytes Stimulates Urokinase Plasminogen Activator (uPA)/uPA Receptor-Dependent Activation of TGF-β and Potentiates Fibrosis

In congenital heart block (CHB), binding of maternal anti-SSA/Ro Abs to fetal apoptotic cardiocytes impairs their removal by healthy cardiocytes and increases urokinase plasminogen activator (uPA)/uPA receptor (uPAR)-dependent plasmin activation. Because the uPA/uPAR system plays a role in TGF-β activation, we evaluated whether anti-Ro binding to apoptotic cardiocytes enhances plasmin-mediated activation of TGF-β, thereby promoting a profibrosing phenotype. Supernatants from cocultures of healthy cardiocytes and apoptotic cardiocytes bound by IgG from a mother whose child had CHB (apoptotic-CHB-IgG [apo-CHB-IgG]) exhibited significantly increased levels of active TGF-β compared with supernatants from cocultures of healthy cardiocytes and apoptotic cardiocytes preincubated with IgG from a healthy donor. Treatment of the culture medium with anti-TGF-β Ab or TGF-β inhibitor (SB431542) abrogated the luciferase response, thereby confirming TGF-β dependency. Increased uPA levels and activity were present in supernatants generated from cocultures of healthy cardiocytes and apo-CHB-IgG cardiocytes compared with healthy cardiocytes and apoptotic cardiocytes preincubated with IgG from a healthy donor, respectively. Treatment of apo-CHB-IgG cardiocytes with anti-uPAR or anti-uPA Abs or plasmin inhibitor aprotinin prior to coculturing with healthy cardiocytes attenuated TGF-β activation. Supernatants derived from cocultures of healthy cardiocytes and apo-CHB-IgG cardiocytes promoted Smad2 phosphorylation and fibroblast transdifferentiation, as evidenced by increased smooth muscle actin and collagen expression, which decreased when fibroblasts were treated with supernatants from cocultures pretreated with uPAR Abs. These data suggested that binding of anti-Ro Abs to apoptotic cardiocytes triggers TGF-β activation, by virtue of increasing uPAR-dependent uPA activity, thus initiating and amplifying a cascade of events that promotes myofibroblast transdifferentiation and scar.

Abstract PL06-03: Urokinase plasminogen activator system: A multifunctional drug target.

Abstract The plasminogen activator system with its components uPA (urokinase-type plasminogen activator), its type 1 inhibitor PAI-1, and the uPA-receptor (uPAR), plays a key role in tumor invasion and metastasis. Elevated levels of one or several of these factors have been associated with tumor aggressiveness and poor disease outcome in a variety of solid tumors such as breast cancer, gynaecological (i.e. ovarian, endometrial or cervical cancer) and urological (i.e prostate, kidney, bladder) malignancies, GI cancers (e.g. stomach, colorectal, pancreas), and many other solid tumors. In primary breast cancer, uPA and PAI-1 have achieved the highest level of evidence (LOE I) for their prognostic impact based on a meta-analysis in more than 8,000 patients as well as the prospective randomised Chemo N0 trial with 10-year follow-up. Low levels of uPA and PAI 1, as measured by ELISA in tumor tissue extracts, are associated with such good outcome that these patients may be spared adjuvant chemotherapy as recommended by international guidelines (AGO, ASCO). Patients with high levels of either or both factors derive enhanced benefit from adjuvant chemotherapy. Two additional prospective clinical trials (NNBC-3, Plan B) using the uPA/PAI-1 assay have been completed in early breast cancer, randomizing about 7,000 patients. Next to its value as a prognostic or predictive cancer biomarker, uPA is obviously also an interesting drug target given its clinical relevance in many solid tumors. Two small synthetic uPA inhibitors (WX-UK-1, WX-671) have successfully completed phase I evaluation. Two phase II trials with the oral compound WX-671 have recently been completed in pancreatic and breast cancer. In locally advanced, inoperable, non-metastatic pancreatic cancer (n=75), a randomised open label trial compared two different WX-671 doses together with gemcitabine vs. gemcitabine alone. Compared to chemotherapy alone, the combination of gemcitabine plus 400 mg WX-671 daily led to an increase in response (3.8 vs. 12.9%), 1 year PFS (16.2 vs. 26.9%) and 1 year OS (33.9 vs. 50.6%) rates. No specific toxicities were observed. In first line metastatic breast cancer, oral WX-671 (200 mg daily) or placebo were administered together with capecitabine (1000mg/m2 BID d1–14 q21) in more than 100 patients. This trial is currently collecting follow-up results. An uPA-derived peptide (Å6) is also being explored as a cancer therapeutic. No safety issue were reported from phase I evaluation. First phase II evidence showed an increase in TTP compared to placebo in ovarian cancer patients; results of a second trial (GOG 170N) are still pending. Taken together, uPA/PAI-1 has a validated clinical utility as prognostic markers in early breast cancer. Given its key role in tumor progression, the uPA system remains an interesting drug target. Early clinical trials interfering with this system have shown good tolerability of the compounds and some very promising clinical data which now need to be validated Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PL06-03.

The expression of uPA and uPAR in patients with systemic inflammatory response syndrome

Objective To test plasma levels of uPA,uPAR,D-dimer,IL-6 and TNF-α,and observe the relationships between uPA,uPAR and D-dimer,IL-6,TNF-αin patients with SIRS.Methods A prospective,clinical case-control study was adopted.Cases were collected from our hospital in January 2008 to January 2010,and all were ＞ 55 years of age.Venous blood samples were collected via routine venipuncture.Eighty-five patients were divided into two groups according to diagnostic criteria of SIRS:SIRS group collected from Intensive Care Unit ( n =50) and non SIRS group collected from medical ward ( n =35).The control group comprised 30 unrelated healthy blood donors who visited the General Health Checkup Division at our hospital.Patients with (1) pregnant women; (2) cancer; (3) died after admitted into ICU in 7 days; (4) after cardiopulmonary resuscitation ; ( 5 ) with previous blood system diseases; (6) patients with SIRS before admitted into ICU were excluded from the study.uPA,uPAR,D-D,IL-6 and TNF-α in blood were detected by commercial enzyme-linked immunosorbent assay (ELISA) kits.The data was analyzed using SPSS version17.0.Data accorded with normal distribution of measurement was expressed as mean ± standard,and analyzed by independent-samples t test; non-normal distribution of measurement data,expressed by median,was analyzed with Mann-Whitney test.Relationships between plasma levels of uPA,uPAR and D-dimer,IL-6 TNF-α were analyzed using Spearman rank correlation test.To compare with blood level of uPA,uPAR,IL-6 and TNF-α in SIRS patients in the application of diagnostic value of MODS,we constructed the receiver operating characteristic curve ( ROC curve) for blood levels of uPA,uPAR,IL-6 and TNF-α in 24 h.Results The plasma levels of uPA,uPAR,D-dimer,IL-6 and TNF-αin patients of SIRS were obviously higher compared with non-SIRS and normal controls ( all P ＜ 0.01 ).Correlation analysis showed that there was positive correlation between uPAR level and IL-6 level (r =0.395,P =0.004) ; there was positive correlation between uPAR level and TNF-αlevel ( r =0.606,P ＜0.01 ).There was no correlation between uPAR levd and D-dimer level ( r =- 0.069,P =0.632).There was no correlation between uPA level and D-dimer,IL-6 or TNF-α ( all p ＞ 0.05).There ROC curve were made based on the abilities of uPAR,D-dimer,IL-6 and TNF-αlevels in 24 hours to diagnose MODS,and the ROC areas under the curves were 0.76,0.58,0.86,0.83 respectively.Conclusions These results demonstrate that uPA and uPAR play a major contributory role in patients with SIRS in the process of coagulation disorders,but the mechanism in SIRS is not the same.uPAR may play a central rolein the development of SIRS to MODS. Key words: Systemic inflammatory response syndrome; Multiple organ dysfunction syndrome; Urokinase type plasminogen activator; Urokinase type plasminogen activator receptor; D-dimer; Interleukin-6; Tumor necrosis factor-alpha; Coagulant function

Microarray-based gene expression profiles to predict ELISA-derived uPA and PAI-1 levels in breast cancer biopsies: A comparison between fresh frozen (FF) and formalin-fixed, paraffin-embedded (FFPE) samples.

194 Background: The urokinase plasminogen-activator (uPA) and its inhibitor (PAI-1) predict the benefit of node-negative breast cancer patients from chemotherapy. The determination of uPA and PAI-1 need(s) large amounts of tumor tissue. In contrast, microarray-based gene expression profiling (MGEP) requires only small quantities of FF tissue and is applicable to FFPE archival tissue. Identification of MPEG-derived surrogate markers for ELISA-based uPA/PAI-1 may facilitate their analysis together with MPEG-based predictive genes and metagenes. Methods: Three groups of breast cancer biopsies were analysed. Group A: 136 FF tissues from 2008-2009, group B: 85 FF tissues from 2010, group C: 20 independent FFPE tissues. Gene expression was assessed by Agilent 4x44K microarrays. Results: For group A a significant correlation between protein expression of uPA and uPA gene (PLAU) expression (r = 0.627 p &lt; 0.001) and PAI-1 and PAI-1 genes (SERPINE1)(r = 0.281 p = 0.001) was found. Top sets of genes correlated with uPA/PAI-1 protein expression were extracted. For group B additional sets of top correlated genes were identified. The correlation of individual genes with uPA/PAI-1 protein expression was highest for their genes themselves (PLAU: r = 0.694 p &lt; 0.001; SERPINE1 (r = 0.469 p &lt; 0.001). Subsequently consensus gene sets were identified with a good performance for both biopsy sets. Only genes which were significantly correlated with their counterparts in group C were selected, resulting in a final 11-gene model (uPA metagene) and a 6-gene model (PAI-1 metagene). They significantly correlated with uPA/PAI-1 protein expression in all groups of biopsies (uPA: group A: r = 0.621, p &lt; 0.001; group B: r = 0.771, p &lt; 0.001; group C: r = 0.665, p = 0.005; for PAI-1: group A: r = 0.569, p &lt; 0.001; group B: r = 0.687, p &lt; 0.001; group C: r = 0.683, p = 0.008). Conclusions: Reliable MPEG-derived metagenes as surrogate markers for ELISA-based uPA/PAI-1 expression were identified.

Implication of MMP-9 and urokinase plasminogen activator (uPA) in the activation of pro-matrix metalloproteinase (MMP)-13

We examined whether the expression and activation of pro-matrix metalloproteinase (MMP)-1 varies from that of pro-MMP-13 in the joint fluid of osteoarthritis (OA) and rheumatoid arthritis (RA) patients. To do this, joint fluid was collected from 34 RA and 34 OA patients. The collagenase (pro-MMP-1 and MMP-13, total MMP-1, and MMP-13), gelatinase (total MMP-2 and MMP-9), stromelysin (total MMP-3), matrilysin (total MMP-7), uPA, and tissue inhibitor of MMP (TIMP) levels were measured by ELISA. The level of total MMP-1 in RA joint fluids was similar to that of the OA joint fluid. In contrast, the level of total MMP-13 in the RA group was significantly higher than that of the OA group. Among various MMPs (MMP-2, MMP-3, MMP-7, and MMP-9), only MMP-9 was strongly associated with total MMP-13 in both RA and OA. The level of uPA was also strongly associated with MMP-13 in RA but not OA, while the level of TIMP-1 and TIMP-2 was not significantly different between RA and OA. In conclusion, MMP-9 and uPA might be involved in the activation of pro-MMP-13 through unknown mechanisms in arthritic diseases.

Modulation of protein expression levels and DNA methylation status of breast cancer metastasis genes by anthracycline-based chemotherapy and the demethylating agent decitabine

Epigenetic drugs are promising add-ons to cancer treatment; still, adverse effects concerning tumour promotion have been reported occasionally. In this in vitro study, we investigated the effect of combination treatment of decitabine with anthracycline-based chemotherapy [5-fluorouracil plus epirubicine plus cyclophosphamide (FEC)] on viability and metastatic activity of breast cancer cell lines, MDA-MB-231 (estrogen receptor-negative) and MCF-7 (estrogen receptor-positive). The effect of decitabine and its combined treatment with FEC on viability of both cancer cell lines was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide and adenosine triphosphate (ATP) cell survival assays. DNA methylation specific real-time polymerase chain reaction (PCR) (Methylight®) was employed to document the methylation status of the metastasis-relevant urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-I (PAI-1) genes. Additionally, protein expression levels of uPA and PAI-1 were determined using enzyme-linked immunosorbent assays. Invasion capacity of cells was assayed using Matrigel® invasion assay. Decitabine lowered the viability of MCF-7 cells, although MDA-MB-231 cells were not affected. Decitabine did not augment FEC-mediated cytotoxicity in both cell lines. In MCF-7 cells, methylation of the uPA and PAI-1 gene promoter was significantly reduced by decitabine or decitabine plus FEC. Protein levels of uPA and PAI-1 were induced by all treatments. Decitabine significantly induced the invasion capacity of MCF-7 cells, whereas all of the drugs resulted in decreased invasion capacity of MDA-MB-231. Our results suggest differential effects of single-dose decitabine and its combination with FEC on the metastatic capacity and survival of breast cancer cell lines endowed with different metastatic behaviour.