uPA Levels Research Articles

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.

Decreased Hepatocyte Growth Factor (HGF) and Gamma Aminobutyric Acid (GABA) in Individuals with Obsessive-Compulsive Disorder (OCD)

IntroductionThere is support for the role of gamma aminobutyric acid (GABA) in the etiology of mood disorders. Recent research has shown that hepatocyte growth factor (HGF) modulates GABAergic inhibition and seizure susceptibility. This study was designed to determine and correlate plasma levels of HGF and GABA as well as symptom severity in individuals with obsessive-compulsive disorder (OCD).Subjects and methodsPlasma from 15 individuals with OCD (9 males, 6 females;, mean age 38.7 years) and 17 neurotypical controls (10 males, 7 females; mean age 35.2 years) was assessed for HGF, GABA, urokinase plasminogen activator (uPA), and urokinase plasminogen activator receptor (uPAR) concentration using enzyme-linked immunosorbest assays ELISAs. Symptom severity was assessed in these OCD individuals and compared with HGF and GABA concentrations.ResultsIn this preliminary study, individuals with OCD had significantly decreased HGF levels, decreased plasma levels of GABA and decreased uPA. We found that both uPA and uPAR levels correlate with HGF. Both low uPA and low uPAR levels correlate with high symptom severity in individuals with OCD. Low GABA levels in OCD individuals also correlate with high symptom severity.DiscussionThese results demonstrate a preliminary association between HGF, GABA, uPA levels, and OCD and suggest that plasma GABA and uPA levels are related to symptom severity in individuals with OCD.

Urokinase-type plasminogen activator receptor as a predictor of poor outcome in patients with systemic inflammatory response syndrome

BACKGROUND:Urokinase-type plasminogen activator (uPA) and urokinase-type plasminogen activator receptor (uPAR) are known as important factors, which mediate a variety of functions in terms of vascular homeostasis, inflammation and tissue repair. However, their role in systemic inflammatory response syndrome (SIRS) has been less well studied. This study aimed to test the hypothesis that the abnormalities of fibrinolysis and degradation of extracellular matrix mediated by uPA and uPAR are directly related to the patients with SIRS. We therefore analyzed their role and clinicopathological significance in patients with SIRS.METHODS:A case-control study was conducted with 85 patients who were divided into two groups according to the diagnostic criteria of SIRS: SIRS group (n=50) and non-SIRS group (n=35). The SIRS group was divided into MODS group (n=26) and non-MODS group (n=24) by their severity, and survival group (n=35) and non-survival group (n=15) by their prognosis. Another 30 healthy adults served as normal controls. uPA and uPAR in plasma were detected by commercial enzyme-linked immunosorbent assay (ELISA) kits.RESULTS:The plasma level of uPA was lower in the SIRS group than in the non-SIRS group and controls (P<0.001 and P<0.001). It was lower in sepsis patients and the MODS group than in the non-sepsis patients and the non-MODS patients (all P<0.05). However, there was no difference in uPA level between survivors and non-survivors (P>0.05). The plasma level of uPAR increased in the SIRS group compared with the non-SIRS group and controls (P<0.001 and P<0.001). There was a significant elevation of uPAR in sepsis patients, MODS patients and non-survivors as compared with non-sepsis patients, non-MODS patients and survivors respectively (all P<0.05). Plasma uPAR levels were positively correlated with APACHE II score (r=0.575, P<0.001) and SOFA score (r=0.349, P=0.013). AUCs for the prediction of SIRS mortality were 0.67 and 0.51, respectively, for uPA and uPAR.CONCLUSION:uPAR could be a predictor of poor outcome in patients with SIRS.

Skip Regulates TGF- β 1-Induced Extracellular Matrix Degrading Proteases Expression in Human PC-3 Prostate Cancer Cells.

Purpose. To determine whether Ski-interacting protein (SKIP) regulates TGF-β1-stimulated expression of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-9 (MMP-9), and uPA Inhibitor (PAI-1) in the androgen-independent human prostate cancer cell model. Materials and Methods. PC-3 prostate cancer cell line was used. The role of SKIP was evaluated using synthetic small interference RNA (siRNA) compounds. The expression of uPA, MMP-9, and PAI-1 was evaluated by zymography assays, RT-PCR, and promoter transactivation analysis. Results. In PC-3 cells TGF-β1 treatment stimulated uPA, PAI-1, and MMP-9 expressions. The knockdown of SKIP in PC-3 cells enhanced the basal level of uPA, and TGF-β1 treatment inhibited uPA production. Both PAI-1 and MMP-9 production levels were increased in response to TGF-β1. The ectopic expression of SKIP inhibited both TGF-β1-induced uPA and MMP-9 promoter transactivation, while PAI-1 promoter response to the factor was unaffected. Conclusions. SKIP regulates the expression of uPA, PAI-1, and MMP-9 stimulated by TGF-β1 in PC-3 cells. Thus, SKIP is implicated in the regulation of extracellular matrix degradation and can therefore be suggested as a novel therapeutic target in prostate cancer treatment.

Effects of aerosolized earthworm fibrinolytic enzyme on bleomycin induced pulmonary fibrosis in rats

To explore the effects of aerosolized earthworm fibrinolytic enzyme (EFE) on bleomycin-induced pulmonary fibrosis in rats. A total of 72 male SD rats were divided randomly into 3 groups of bleomycin (BLM) group with intratracheal BLM (5 mg/kg), control group with the same dose of normal saline, then after both receiving aerosolization of normal saline once daily instead of EFE, EFE group with EFE (2500 U/kg) by aerosolization once daily after BLM instillation. Lung histopathology, immunohistochemistry for transforming growth factor β(1) (TGF-β(1)), lung hydroxyproline contents, levels of urokinase PA (uPA), tissue plasminogen activator (tPA) and PA inhibitor 1 (PAI-1) in lung and blood were observed at Days 7, 14 and 28 of experiment, respectively. Compared with BLM group, pulmonary fibrosis improved and the TGF-β(1) expression of lung tissue decreased (P < 0.01). Hydroxyproline content of lung tissue decreased in EFE group compared with BLM group ((5.8 ± 2.5) vs (9.6 ± 1.3), (6.7 ± 1.4) vs (9.7 ± 1.5), (7.5 ± 1.2) vs (9.7 ± 1.4) mg/L, P < 0.01). Compared with BLM group, the uPA levels of lung were elevated in EFE group at Days 7 and 14 ((1.04 ± 0.36) vs (0.72 ± 0.11), (0.90 ± 0.09) vs (0.75 ± 0.08) µg/L, P < 0.05). Moreover, the plasma levels uPA of increased at Days 14 and 28 ((0.32 ± 0.04) vs (0.25 ± 0.02), (0.36 ± 0.05) vs (0.28 ± 0.04) µg/L, P < 0.05). Consistently, compared with BLM group, the tPA levels of lung increased in EFE group ((4.70 ± 0.87) vs (3.01 ± 0.62), (5.72 ± 0.37) vs (3.00 ± 0.51), (6.73 ± 1.12) vs (3.18 ± 0.38) µg/L, P < 0.01) and the plasma levels of tPA also increased ((3.40 ± 0.36) vs (1.79 ± 0.38), (3.17 ± 0.37) vs (2.18 ± 0.17), (3.85 ± 0.56) vs (2.80 ± 1.06) µg/L, P < 0.01). However, compared with BLM group, the PAI-1 levels of lung decreased in EFE group ((6.04 ± 0.81) vs (8.52 ± 1.01), (6.78 ± 0.81) vs (9.81 ± 1.73), (7.63 + 0.99) vs (11.44 ± 2.54), P < 0.05) and the plasma levels of PAI-1 also decreased in EFE group ((4.82 ± 0.42) vs (6.89 ± 0.84), (5.73 ± 0.40) vs (7.30 ± 1.09), (5.64 ± 0.87) vs (7.98 ± 1.10) µg/L, P < 0.05). Earthworm fibrinolytic enzyme may decrease bleomycin-induced pulmonary fibrosis and TGF-β(1) expression while increasing fibrinolytic activation. And fibrinolytic strategies are probably useful for the therapy of fibrotic lung diseases.

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.

Expression of urokinase plasminogen activator and plasminogen activator inhibitor type-1 in ovarian cancer and its clinical significance

The urokinase plasminogen activator system, which consists of urokinase plasminogen activator (uPA), plasminogen activator inhibitor type-1 (PAI-1) and urokinase plasminogen activator receptor (uPAR), plays an important role in tumor invasion and metastasis, and it may be a potential diagnostic biomarker and therapeutic target in cancer. It has been found that the expression of uPA and PAI-1 in ovarian cancer is related to clinical pathologies, while their effects on the biological behavior of tumor cells and their clinical significance are still unknown. In this study, 100 tissue samples (60 samples from malignant tumors, 20 from benign tumors and 20 from controls) and 147 blood samples (49 samples each from patients with malignant tumors, benign tumors and control group, respectively) were analyzed. The positive expression levels of uPA and PAI-1 in the malignant tumor samples and their serum concentrations in the malignant group were all significantly higher than these levels in the benign tumors and controls. In addition, the levels in patients with poorly differentiated and stageIII-IV cancers, cancers with metastases as well as residual tumors >2cm after surgery, were all obviously increased, consistent with their concentrations in serum. The Cox model analysis showed that expression of uPA at the transcription level had significant associations with prognosis. In addition, uPA greatly enhanced the abilities of cell invasion, migration and adhesion through its overexpression in SKOV3 cells. Collectively, our results showed that uPA and PAI-1 play important roles in ovarian cancer development; therefore, their expression in tissues and their concentrations in serum would greatly assist the diagnosis and prediction of the prognosis in ovarian cancer.

The influence of opioid peptides on matrix metalloproteinase-9 and urokinase plasminogen activator expression in three cancer cell lines

Matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) regulate proteolysis of the extracellular matrix (ECM) and as a consequence are involved in a number of physiological and pathological states, including cancer. A crucial feature of cancer progression and metastasis is the disruption of the ECM and spreading of proliferating cancer cells. Over-expression of MMPs and uPA is common for most types of cancers and correlates well with the adverse prognosis. Compounds able to modulate the activity of these proteolytic enzymes may become important agents in cancer therapy. In the present study, we examined the effect of the mu-opioid receptor selective peptide, morphiceptin, and its two synthetic analogs on mRNA and protein levels of MMP-9 and uPA in three human cancer cell lines: MCF-7, HT-29, and SH-SY5Y. Our findings indicate that in all three cell lines morphiceptin and its analogs attenuated MMP-9 expression and secretion and that this effect is not mediated by opioid receptors but is under control of the nitric oxide system. On the other hand, tested opioids up-regulated uPA levels through a mechanism that involved opioid-receptors. Different pathways by which opioid peptides exert their actionin cancer cells can explain their contradictory influence on the level of cancer markers.

Significantly increased concentration of soluble urokinase-type plasminogen activator receptor in the blood of patients with pelvic inflammatory disease

BackgroundTo determine expression levels of urokinase-type plasminogen activator (uPA), soluble urokinase-type plasminogen activator receptor (suPAR), plasminogen activator inhibitor-1 (PAI-1) in plasma and to identify gene polymorphisms specific to patients with pelvic inflammatory disease (PID) and healthy controls. MethodsEnzyme-linked immunosorbent assay and polymerase chain reaction–restriction fragment length polymorphism were used to measure plasma levels and polymorphisms in uPA, suPAR, and PAI-1 among seventy healthy controls and 64 PID patients before and after they received routine treatment protocols. ResultsThe levels of plasma uPA (ng/ml) and soluble suPAR (pg/ml) were significantly increased in PID patients (uPA: 0.57±0.03; suPAR: 1372.04±68.20) when compared to that in normal controls (uPA: 0.55±0.06, p=0.002; suPAR: 1192.46±51.98, p=0.04); moreover, suPAR decreased significantly after treatment when compared to levels noted in the same patients (1220.06±58.14; p=0.003) after they received treatment. The increased expression of suPAR was significantly correlated with WBC counts (r=0.382, p=0.002, n=64) in blood as well as the plasma levels of CRP (r=0.441, p<0.0001, n=64) and uPA (r=0.426, p<0.0001, n=64) of PID patients prior to receiving treatment. ConclusionsIncreased plasma suPAR could be a biological marker for the diagnosis of PID and may reflect a new focus in targeted therapy for pelvic inflammatory disease.

Synthesis and docking studies of novel antitumor benzimidazoles

In this work, the benzimidazole-pyrrole conjugates 6a–h and benzimidazole-tetracycles conjugates 12–14 were prepared. The cytotoxicity of the compounds 3, 4a–h, 6a–h, 8, 10 and 12–14 was tested against lung cancer cell line A549. Compound 6b exhibited higher activity than the bis-benzoxazole natural product (UK-1), the standard. The tested 4g,h, 6a–h, 10 and 12–14 exhibited remarkable cytotoxicity activity against breast cancer cell line MCF-7 with higher activity than tamoxifen. Furthermore, compound 4h was found to be also more potent than doxurubicin. The antitumor promotion activity of synthesized compounds 4g,h, 6a–h, 10 and 12–14 has been estimated by studying their possible inhibitory effects on EBV-EA activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Among the studied compounds, the inhibitory activities of compounds 8, 13 and 14 demonstrated strong inhibitory effects on the Epstein–Barr virus early antigen (EBV-EA) activation without showing any cytotoxicity on the Raji cells and their effects being stronger than that of a representative control, oleanolic acid.Moreover, the molecular docking of the new compounds into plasminogen activator (uPA) receptor has been in correlation with the antitumor activity. All synthesized compounds 3, 4a–h, 6a–h, 8, 10 and 12–14 were docked into same groove of the binding site of the native co-crystalized (4-iodobenzo[b]thiophene-2-carboxamidine) ligand (PDB code:1c5x) for activity explaination. Compounds 4h, 6b and 13, giving the best docking results, were further studied to estimate their effect on the level of uPA using AssayMax human urokinase (uPA) ELISA kit. In case of A549 cell line, compound 6 exhibited similar activity to MMC, and for MCF-7 cell line, compound 4h exhibited similar activity to doxorubicin, in inhibiting the expression of uPA.

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.

Interleukin enhancer-binding factor 3 promotes breast tumor progression by regulating sustained urokinase-type plasminogen activator expression

Sustained urokinase-type plasminogen activator (uPA) expression is detected in aggressive breast tumors. Although uPA can be transiently upregulated by diverse extracellular stimuli, sustained, but not transiently-upregulated uPA expression contributes to breast cancer invasion/metastasis. Unfortunately, how sustained uPA expression is achieved in invasive/metastatic breast cancer cells is unknown. Here, we show that sustained and transiently-upregulated uPA expression are regulated by distinct mechanisms. Using a collection of transcription factor-targeted siRNAs, we discovered that interleukin enhancer binding factor 3 (ILF3) is required for sustained uPA expression. Two discrete mechanisms mediate ILF3 action. The first is that ILF3 activates uPA transcription by binding to the CTGTT sequence in the nucleotides -1,004~-1,000 of the uPA promoter; the second is that ILF3 inhibits the processing of uPA mRNA-targeting pri-miRNAs. Knockdown of ILF3 led to significant reduction in in vitro cell growth/migration/invasion and in vivo breast tumor development. Importantly, immunohistochemistry showed that nuclear ILF3, but not cytoplasmic ILF3 staining correlates with elevated uPA level and higher grades of human breast tumor specimens. Nuclear localization of ILF3 highlights the role of ILF3 in sustained uPA expression as a transcription activator and pri-miRNA processing blocker. In conclusion, this study shows that ILF3 promotes breast tumorigenicity by regulating sustained uPA expression.

Connecting the molecular dots from inaccessible antigen to organ injury in the pathogenesis of cardiac manifestations of neonatal lupus

Only 2% of fetuses exposed to anti-SSA/Ro antibodies have cardiac manifestations of neonatal lupus (cardiac NL, referred to herein as congenital heart block), yet these antibodies are present in >85% of mothers whose fetuses are identified with conduction abnormalities in a structurally normal heart. This disparity implies that the antibodies are necessary but insufficient to cause disease, and that the final pathway to fibrosis may be variable: kept totally in check in most fetuses (normal sinus rhythm), subclinical in others (first-degree block) and fully executed in very few (advanced block). A further challenge to defining the pathology of disease is the intracellular location of the target antigens, thus raising questions regarding accessibility to maternal antibodies. Our research has incorporated three components in the presumed cascade from antibody to tissue injury: apoptosis to provide the antigenic complex; macrophage uptake and secretion to drive inflammation and scarring; and persistence of the myofibroblast to drive replacement of healthy cardiac tissue. The totality of experimental data in the last 2 years supports that the watershed may involve conformational properties of Ro60 that influence its surface translocation to an apoptotic cardiocyte surface, its capacity to bind Y RNA and its effect on the biology of the urokinase plasminogen activator (uPA)/urokinase plasminogen activator receptor (uPAR) system. By exploiting murine fibroblast cell lines transfected with Ro60 modified to alter RNA binding sites, very recent data suggest that ssRNA (probably hY3 RNA) is required for surface translocation during apoptosis. However, the accessibility of Ro60 to cognate maternal antibody may be influenced by additional factors. In this regard, Ro60 expressed on the surface of an apoptotic cardiocyte binds domain V of β2GPI with high affinity and effectively blocks binding of anti-Ro60 antibodies. In an important translational step to humans, significantly lower levels of circulating β2GPI are associated with disease as assessed with umbilical cord blood comparing affected and unaffected fetuses. The importance of intact β2GPI as a protective factor became increasingly evident as we continued our search for the molecular explanation of a critical observation that anti-Ro60 antibodies inhibit the uptake of apoptotic cardiocytes by healthy cardiocytes. Although a counter-receptor for Ro60 on the healthy cardiocyte was considered, experiments revealed that anti-Ro60 binding to Ro60 (opsonization, formation of immune complexes) is associated with a conformational change of uPAR resulting in two functional consequences, one of which is a 'don't eat me' signal to healthy cardiocytes. The other is uPA activation leading to proteolytic cleavage of plasminogen and the generation of plasmin. The latter cleaves β2GPI, which renders it incapable of binding to Ro60 thus favoring the continued formation of immune complexes on the apoptotic surface. Moreover, the generation of plasmin also promotes the activation of TGF-β. Accordingly, the uPAR/uPA system plays a profibrotic role in the early and expansive phases of disease and the levels of uPAR, uPA, and plasminogen are increased in affected fetuses compared with healthy siblings. The potential of ssRNA to perpetuate an inflammatory step via a Toll-like receptor (TLR)7 pathway has been experimentally substantiated by the transdifferentiation of human fetal cardiac fibroblasts to a scarring phenotype following exposure to supernatants generated by incubation of macrophages with surrogate immune complexes comprised of Ro60, hY3 and affinity-purified anti-Ro60 antibody. Further evidence for a macrophage contribution to injury was provided by immunohistochemistry, which demonstrated not only a macrophage infiltrate in three hearts from anti-Ro-exposed fetuses dying with cardiac NL, but the formation of multinucleated giant cells. With regard to the fibroblast, we posit that these cells are themselves a major source of TGF-β and that endothelin-1 is one of the components responsible for the profibrosing effects generated by TLR signaling in the macrophages. The potential pathologic role of TLR signaling prompted the initiation of a case-control study in which we demonstrated that the use of hydroxychloroqine, which inhibits endosomal acidification, decreases the risk of cardiac NL. A prospective study to prevent recurrent disease has just been initiated.

Gelsolin Induces Colorectal Tumor Cell Invasion via Modulation of the Urokinase-Type Plasminogen Activator Cascade

Gelsolin is a cytoskeletal protein which participates in actin filament dynamics and promotes cell motility and plasticity. Although initially regarded as a tumor suppressor, gelsolin expression in certain tumors correlates with poor prognosis and therapy-resistance. In vitro, gelsolin has anti-apoptotic and pro-migratory functions and is critical for invasion of some types of tumor cells. We found that gelsolin was highly expressed at tumor borders infiltrating into adjacent liver tissues, as examined by immunohistochemistry. Although gelsolin contributes to lamellipodia formation in migrating cells, the mechanisms by which it induces tumor invasion are unclear. Gelsolin’s influence on the invasive activity of colorectal cancer cells was investigated using overexpression and small interfering RNA knockdown. We show that gelsolin is required for invasion of colorectal cancer cells through matrigel. Microarray analysis and quantitative PCR indicate that gelsolin overexpression induces the upregulation of invasion-promoting genes in colorectal cancer cells, including the matrix-degrading urokinase-type plasminogen activator (uPA). Conversely, gelsolin knockdown reduces uPA levels, as well as uPA secretion. The enhanced invasiveness of gelsolin-overexpressing cells was attenuated by treatment with function-blocking antibodies to either uPA or its receptor uPAR, indicating that uPA/uPAR activity is crucial for gelsolin-dependent invasion. In summary, our data reveals novel functions of gelsolin in colorectal tumor cell invasion through its modulation of the uPA/uPAR cascade, with potentially important roles in colorectal tumor dissemination to metastatic sites.

Conditioned media obtained from human outer root sheath follicular keratinocyte culture activates signalling pathways that contribute to maintenance of hair‐inducing capacity and increases trichogenicity of cultured dermal cells

Findings from recent studies have demonstrated that hair-inducing capacity (trichogenicity) of cultured dermal cells can be maintained by addition of conditioned media obtained from culture of epidermal keratinocytes. In this study, we investigated the question of whether treatment with human follicular keratinocyte-conditioned media (FKCM) can result in activation of signalling pathways that contribute to trichogenicity and increase the trichogenicity of cultured dermal cells. Through conduct of hair reconstitution assays, we observed that treatment of cells with FKCM resulted in induction of a greater number of hair follicles, compared with control cells. Treatment of dermal cells with FKCM resulted in the activation of BMP and β-catenin signalling pathways. In addition, higher levels of IGFBP-7, IL-8, OPG and uPA were observed in FKCM. Altogether, our data suggest that a patient's own FKCM would be ideal for expansion of the patient's own follicular dermal cells for cell therapy for treatment of hair loss.

35P Adjuvant Chemotherapy in Node-Negative (N0) Breast Cancer: Routine Upa/Pai-1 Determination

ABSTRACT More than 70% of patients with primary node-negative breast cancer are treated with local therapy alone. Biomarkers are used to determine disease risk and, consequently, determine whether or not chemotherapy is needed. Urokinase plaminogen activator (uPA) and its inhibitor plasminogen activator inhibitor 1 (PAI-1) are proteases implicated in invasiveness of tumor. The clinical utility of these markers has been proven on the highest level of evidence (LOE-1) in node-negative breast cancer patients. They are ASCO–recommended cancer biomarkers since 2007 and French Cancer Society (INCA)-recommended since 2009. But clinical routine use is poorly developed. The purpose was to register adjuvant chemotherapy decisions by a multidisciplinary discussion using uPA/PAI-1 concentrations compared to conventional prognostic parameters. The study was performed between January 2010 and December 2011. It included 164 frozen breast cancer tissue for determination of uPA and PAI-1 levels by enzyme-linked immunosorbent assay (ELISA) at Marseille Laboratory. However, the assay required a fresh-frozen tissue sample, which was not always feasible (25% of cases) because of tumor size or technical problems. UPA concentration of ≤3 ng/mg total protein and PAI-1 concentration of ≤14 ng/mg total protein were classified as low risk and one elevated marker classified patient as high risk. Tissues were always excised more than 10 days post breast biopsy and were frozen in a delay of 30 to 60 min. Results were obtained in 10 days post excision. Parameters were prospectively registered: age, menopausal status, tumor size, histological grade, steroid hormone receptor, Her2 overexpression, KI67, vascular embolism. Sixty-six tumors were uPA/PAI-1 negative, 41 uPA + /PAI-1 + , 12 uPA + /PAI-1- and 45 uPA-/PAI-1+. In the first 120 patients, uPA/PAI-1 value was discordant with conventional parameters, with a lower or higher prognostic index in 13 and 40 cases respectively. In some cases, the multidisciplinary decisions do not follow this score because of patient age, HER2 overexpression, grade 1 and limit level of uPA/PAI-1. Clinical decisions will be discussed and clinical routine problems of treatment with implication of the patients explained. Disclosure All authors have declared no conflicts of interest.

Abstract 9: Two Distinct Roles of Plasminogen Activator Inhibitor-2 (PAI) and PAI-1 in Deep Vein Thrombosis

Objectives: Urokinase-plasminogen activator (uPA) has a vital role in deep vein thrombosis (DVT). While PAI-1 is the main inhibitor of uPA in vivo , PAI-2, found mostly intracellularly, has proven to have more anti-inflammatory and anti-apoptotic than anti-fibrinolytic activities. We explored the hypothesis that PAI-1 and PAI-2 play roles in DVT through either fibrinolytic or inflammatory pathways. Methods: Wild type C57Bl/6 (WT), PAI-2 -/- , and PAI-1 -/- mice underwent surgical caval ligation. Subsequent thrombus was harvested at various time points and measured for thrombus weight (TW) normalized to body weight. Tissue was analyzed by western blot, ELISAs, zymography and flow cytometry. Results: PAI-2 deficiency resulted in unchanged thrombus formation (TW day 4: WT = 1.037 ± .043 mg/g n=17; PAI-2 -/- = 1.028 ± .067 mg/g n=17, p=.91), while it enhanced thrombus resolution (TW day 12: WT = .43 ± .03 mg/g n=13; PAI-2 -/- = .319 ± .021 mg/g, n=16, p=.004). PAI-1 deficiency caused decreased formation (TW day 12: PAI-1 -/- = .901 ± .043 mg/g n=11, p=.045) and enhanced resolution (TW day 12: PAI-1 -/- = .248 ± .011 mg/g n=10, p=.00004). Though total uPA levels in thrombi were similar between groups, active uPA by ELISA was increased by 5 fold in PAI-1 -/- mice and 40 fold in PAI-2 -/- mice compared to WT mice at day 12, indicating increased uPA-mediated fibrinolysis. Exploring other mediators of resolution, we found that PAI-1 and PAI-2 deficiencies lead to contrasting results. By zymography, PAI-1 deficiency caused an early increase in matrix metalloproteinase (MMP) activity that decreased over time. Conversely, PAI-2 deficiency trended towards less early MMP activity that then increased. Using flow cytometry to assess clot cell populations, on day 4, PAI-2 -/- mice had an increase in neutrophils with a decrease in macrophages, whereas PAI-1 -/- mice had an increase in macrophages. Conclusions: This identifies PAI-2 as a novel regulator of venous thrombus resolution and suggests the first context in vivo in which PAI-2 has an anti-fibrinolytic role. Both PAI-1 and PAI-2 deficiencies enhance thrombus resolution, but through diverging mechanisms. Further examination of these pathways may lead to potential therapeutic prospects in accelerating thrombus resolution.

Identification of potential prognostic biomarkers for node-negative breast tumours by proteomic analysis: A multicentric 2004 national PHRC study

We used a 2D-electrophoresis (2-DE) proteomic approach to identify novel biomarkers in node-negative breast cancers. This retrospective study focused on a population of patients with ductal pN0M0 tumours. A subset of patients who developed metastases and in whose tumours were found high levels of uPA and PAI-1 (metastatic relapse, MR: n=20) were compared to another subset in whom no metastatic relapse occurred and whose tumours were found to have low levels of uPA and PAI-1 (no relapse, NR: n=21). We used a 2-DE coupled with MS approach to screen cytosol fractions using two pH-gradient scales, a broad scale (3.0-11.0) and a narrower scale focussing in on a protein rich region (5.0-8.0). This study was conducted on 41 cytosol specimens analyzed in duplicate on two platforms. The differential analysis of more than 2,000 spots in 2-DE gels, obtained on the two platforms, allowed the identification of 13 proteins which were confirmed by western blotting. Two proteins, GPDA and FABP4 were down-regulated in the MR subset whereas all the others were up-regulated. An in silico analysis revealed that GMPS (GUAA), GAPDH (G3P), CFL1 (COF1) and FTL (FRIL), the most informative genes, displayed a proliferation profile (high expression in basal-like, HER2+ and luminal B molecular subtypes). Inversely, similar to FABP4, GPD1 [GPDA] displayed a high expression in luminal A subtype, a profile characteristic of tumour suppressor genes. Despite the small size of our cohort, the 2-DE analysis gave interesting results which were confirmed by the in silico analysis showing that some of the corresponding genes had a strong prognostic impact in breast cancer, mostly because of their link with proliferation: GMPS, GAPDH, FTL and GPD1. A validation phase on a larger cohort is now needed before these biomarkers could be considered for use in clinical practice.

Abstract 929: Repression of CD44 is a key molecular action of a novel Gemini vitamin D BXL0124 to inhibit breast cancer cell invasion induced by HGF/Met signaling

Abstract CD44, a transmembrane glycoprotein, is highly expressed in breast tumors, and the overexpression of CD44 has been associated with poor clinical outcome. CD44 has been known to play a key role in invasive growth of cancer by interacting with extracellular matrix, matrix metalloproteinases (MMPs) and other membrane receptors. In addition, CD44 has been shown to be required for the activation of hepatocyte growth factor (HGF)/Met signaling pathway which is associated with invasive phenotype of human breast cancer. Our previous study demonstrated that a novel Gemini vitamin D BXL0124 repressed the expression of CD44 in MCF10DCIS.com human breast cancer cells and inhibited MCF10DCIS xenograft tumor growth in vivo. In the present study, we investigated the effect of CD44 repression by BXL0124 on HGF/Met mediated invasion in MCF10DCIS cells. The effects of HGF and BXL0124 on the invasive phenotype of MCF10DCIS cells were investigated with invasion assays. To identify key molecules in HGF-induced cancer cell invasion, invasive markers and downstream of HGF/Met signaling pathway were evaluated. The treatment of HGF increased CD44 protein expression and invasive growth of MCF10DCIS cells, which was inhibited by BXL0124 treatment. Stat3 DNA-binding and nuclear translocalization experiments demonstrated that the activation of Stat3, one of the key downstream molecules of HGF/Met signaling pathway, was also significantly decreased by BXL0124 treatment. To further study a specific role of CD44 on breast cancer cell invasion, the effects of CD44 repression were investigated by using MCF10DCIS CD44-knockdown cells in vitro and in vivo. The induction of cancer cell invasion by HGF was blocked when CD44 was knockdown in MCF10DCIS cells. In addition, invasion markers, MMP-9 and uPA, were significantly down-regulated in MCF10DCIS CD44 knockdown cells. For the in vivo study, MCF10DCIS control or CD44-knockdown cells were injected into mammary fat pads in immunodeficient mice. Five weeks later, mammary tumors were collected for evaluating the expression level of invasion markers. The xenograft tumor volume and weight with MCF10DCIS CD44-knockdown cells were significantly smaller by 41% (p&amp;lt;0.01) and 36% (p&amp;lt;0.05), respectively, than those from MCF10DCIS control tumors (n=5). The MCF10DCIS CD44-knockdown tumors also showed markedly decreased protein expression levels of pStat3 and MMP-9. The mRNA expression levels of MMP-9 and uPA were significantly down-regulated in the MCF10DCIS CD44-knockdown tumors. The present study demonstrated the critical role of CD44 in HGF-induced breast cancer cell invasion, suggesting that the repression of CD44 might be a key molecular action of BXL0124 to inhibit breast cancer invasion. 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 929. doi:1538-7445.AM2012-929

Abstract 2407: Snail 1 regulates invasion through uPA-uPAR and MMP-9 signaling in prostate cancer cells

Abstract Epithelial-mesenchymal transition (EMT) is a process by which cancer cells acquire mesenchymal properties, such as induction of N-cadherin, while epithelial-associated genes like E-cadherin are lost. This enables the cells to be more invasive, migratory and metastatic. Factors that can induce EMT include growth factors like transforming growth factor -α (TGF-β) and epidermal growth factor (EGF), and transcription factors like Snail1. Snail1-induced EMT promotes migration and invasion and we hypothesized that this may be mediated by urokinase (uPA) and its receptor (uPAR) activities. uPA, a serine protease, may be activated by integrin engagement to bind to its receptor, uPAR, and initiate a signaling cascade that regulates a variety of biological pathways including invasion. Since Snail1 can induce matrix metalloproteinases (MMP 2 and 9) which are also involved in cell invasion, we also examined MMP activity. Androgen-dependent LNCaP, and 22Rv1 and androgen-independent ARCaP human prostate cancer (PC) cells were stably transfected with empty vector control (Neo) or constitutively active Snail1 which led to increased cell migration and invasion. The levels of Snail1, uPA, and uPAR were measured by Western Blot analysis. uPA activity in conditioned media was measured using the Elisa uPA activity assay kit. Additionally, cells were treated with MAPK inhibitor, UO126, at set time points. Gelatin zymography analysis was utilized to detect MMP-9 activity in the conditioned media. Currently, ARCaP-Snail cells are being transiently transfected with uPAR siRNA to assay for the effects of uPAR knockdown on uPA and MMP activity. The data suggests that overexpression of Snail1 increased uPA and uPAR protein levels, while uPA activity was elevated in conditioned media from LNCaP, 22Rv1 and ARCaP cells. Also, MAPK inhibited MAPK expression in the cell and decreased uPA activity. In addition gelatin zymography showed higher MMP 9 activity in ARCaP-Snail overexpressing cells that was inhibited by MAPK inhibitor. Therefore, Snail-mediated cell migration and invasion in human prostate cancer cells may occur via the regulation of uPA and uPAR activities in addition to MMPs. 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 2407. doi:1538-7445.AM2012-2407