The expression of urokinase-type plasminogen activator system in upper tract urothelial carcinoma and its prognostic value after radical nephroureterectomy.

BACKGROUND: To evaluate the expression pattern and prognostic role of the urokinase-type plasminogen activator (uPA) system in patients who underwent radical nephroureterectomy (RNU) for nonmetastatic upper tract urothelial carcinoma (UTUC). METHODS: A total of 732 patients who were treated with RNU for clinically nonmetastatic UTUC comprised our analytical cohort. Immunohistochemical staining of uPA, uPA receptor (uPAR) and uPA inhibitor (PAI-1) was performed using Murine IgG1 monoclonal antibodies. Outcomes of interest were recurrence-free survival, cancer-specific survival, and overall survival. RESULTS: The median age of the patients was 69.8 years and 56.6% of them were males. Overall, overexpression of uPA, uPAR, and PAI-1 was observed in 292 (39.9%), 346 (47.3%), and 345 (47.1%) patients, respectively. The uPA system components showed a statistically significant association with adverse clinicopathologic features such as lymphovascular invasion, multifocality, sessile tumors, and advanced pathologic stage (P < 0.01). On multivariable models, higher pathologic tumor stage, multifocality, and lymph node involvement were associated with RFS, OS, and CSS, but not the overexpression of uPA, uPAR, or PAR-1. In patients with organ-confined disease (≤pT2N0), however, uPA was significantly associated with RFS (hazard ratio [HR]: 2.04, 95% confidence interval [CI]: 1.21-3.43), OS (HR: 1.59, 95% CI:1.08-2.24) and CSS (HR: 2.55, 95% CI:1.44-4.52). uPA improved the predictive accuracy of a standard post-RNU model for all 3 endpoints, in organ-confined disease, by a prognostically significant margin. CONCLUSIONS: Overexpression of uPA system components was associated with adverse clinicopathologic characteristics and survival outcomes on the univariable, but not multivariable analyses. uPA expression was an independent predictor of survival outcomes in patients with organ-confined disease. While the clinical value of the uPA system remains limited in this cohort, further studies are needed to identify a marker or constellation of markers of high predictive value to help in counseling and treatment planning of UTUC patients.

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

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

Circulating uPA as a potential prognostic biomarker for resectable esophageal squamous cell carcinoma.

Previous research showed that the 4 genes of matrix metallopeptidase 9 (MMP9), cyto-keratin 20 (CK20), cyto-keratin 19 (CK19) and urokinase type plasminogen activator (uPA) are detectable in the peripheral blood. All the 4 genes are related to tumor invasion and metastasis. However, whether their expression is associated with clinicopathologic factors and the prognosis of patients with esophageal squamous cell carcinoma (ESCC) is still confused. Expression levels of MMP9, CK20, CK19, and uPA were evaluated by quantificational real-time polymerase chain reaction (qRT-PCR) in peripheral blood of 205 ESCC patients who received radical resection. The cut-off value was 1000 copy numbers. Their impacts on clinicopathologic factors and survival were investigated. The uPA expression positively correlated with gender (P = .046) and tumor size (P = .046). Meanwhile, CK19 expression positively correlated with tumor size (P = .029), vascular invasion (P = .024), and CK20 expression positively correlated with tumor size (P = .035) and degrees of differentiation (P = .032). Moreover, the overexpression of MMP9 has a correlation with postoperative radiotherapy (P = .041) and chemotherapy (P = .012). Among the 4 genes, only uPA is a prognostic indicator for disease-free survival and overall survival both in univariate analysis and multivariate analysis (P = .015). This study suggests that circulating uPA mRNA in peripheral blood can serve as a potential unfavorable prognosis biomarker in ESCC. Further perspective, multi-center and large-scale study is still needed.

Identification of the key genes associated with neuropathic pain.

Neuropathic pain is a chronic pain state associated with multiple etiologies that results in considerable social and economic burden. The identification of key genes associated with neuropathic pain is important for the development of novel therapies. Therefore, the present study downloaded the gene expression profile GSE15041 from the Gene Expression Omnibus database. The unverified gene chip was removed and the microarray data was normalized following quality control. The limma package in R was used to screen the differentially expressed genes (DEGs), followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Furthermore, a protein­protein interaction (PPI) network based on the identified DEGs was constructed to select hub proteins, and reverse transcription­quantitative polymerase chain reaction was performed to detect the expression of these proteins in a mouse model of neuropathic pain. In total, 86 common DEGs were identified. DEGs were significantly enriched in ̔extracellular space̓ and KEGG pathway enrichment analysis demonstrated that the DEGs were significantly enriched in inflammatory diseases and the mitogen­activated protein kinase signaling pathway. The PPI network consisted of 27 nodes (proteins) and 47 PPI edges (interactions). Interleukin (IL)­6, transcription factor AP­1 (c­Jun) and urikinase­type plasminogen activator (Plau) were identified as hub proteins and key genes in neuropathic pain. The mRNA expression of these hub proteins was significantly increased in the neuropathic pain model, compared with the sham group. IL­6, c­Jun, and Plau may be involved in development of neuropathic pain and further research investigating the exact role of these key genes is required.

Aeroallergen Der p 2 promotes motility of human non-small cell lung cancer cells via toll-like receptor-mediated up-regulation of urokinase-type plasminogen activator and integrin/focal adhesion kinase signaling.

House dust mite (HDM) allergens are one of the major causes leading to respiratory hypersensitiveness and airway remodeling. Here we hypothesized that a major HDM allergen Der p 2 could increase cell motility and invasiveness of non-small cell lung cancer (NSCLC) cells. Our results showed that low dose (1 and 3 µg/mL) recombinant Der p 2 protein (DP2) enhanced the migration and invasiveness of human NSCLC cell A549, H1299 and CL1-5, but nonsignificantly altered their growth. Further investigation revealed that integrin αV level was increased and its downstream signaling including focal adhesion kinase (FAK) and paxillin were activated in A549 cells exposed to DP2. In parallel, DP2 also activated the FAK-associated signaling effectors such as Src, phosphatidyl inositol 3-kinase (PI3K), AKT, p38 mitogen-activated protein kinase (P38), extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). Our findings also revealed that DP2 increased expression level of urokinase type plasminogen-activated kinase (uPA) and uPA receptor (uPAR), and subsequently enhanced the binding of uPAR to integrin αV. Moreover, the involvement of toll-like receptor 2/4 (TLR2/4)-triggered ERK1/2 activation in the increased expression of uPA and uPAR was also demonstrated. Collectively, these findings indicate that DP2 can enhance cell motility and invasiveness of NSCLC cells, attributing to TLR2/4-ERK1/2 activation, increased uPA and uPAR expression, enhanced binding of uPAR to integrin αV, and the consequent FAK signaling cascades. Thus, we suggest that DP2 may exacerbate NSCLC via promoting metastatic ability of carcinoma cell.

Optimization of construct design and fermentation strategy for the production of bioactive ATF-SAP, a saporin based anti-tumoral uPAR-targeted chimera.

BACKGROUND: The big challenge in any anti-tumor therapeutic approach is represented by the development of drugs selectively acting on the target with limited side effects, that exploit the unique characteristics of malignant cells. The urokinase (urokinase-type plasminogen activator, uPA) and its receptor uPAR have been identified as preferential target candidates since they play a key role in the evolution of neoplasms and are associated with neoplasm aggressiveness and poor clinical outcome in several different tumor types. RESULTS: To selectively target uPAR over-expressing cancer cells, we prepared a set of chimeric proteins (ATF-SAP) formed by the human amino terminal fragments (ATF) of uPA and the plant ribosome inactivating protein saporin (SAP). Codon-usage optimization was used to increase the expression levels of the chimera in the methylotrophic yeast Pichia pastoris. We then moved the bioprocess to bioreactors and demonstrated that the fed-batch production of the recombinant protein can be successfully achieved, obtaining homogeneous discrete batches of the desired constructs. We also determined the cytotoxic activity of the obtained batch of ATF-SAP which was specifically cytotoxic for U937 leukemia cells, while another construct containing a catalytically inactive mutant form of SAP showed no activity. CONCLUSION: Our results demonstrate that the uPAR-targeted, saporin-based recombinant fusion ATF-SAP can be produced in a fed-batch fermentation with full retention of the molecules selective cytotoxicity and hence therapeutic potential.

Suppressive effects of a proton beam on tumor growth and lung metastasis through the inhibition of metastatic gene expression in 4T1 orthotopic breast cancer model.

A proton beam is a next generation tool to treat intractable cancer. Although the therapeutic effects of a proton beam are well known, the effect on tumor metastasis is not fully described. Here, we investigated the effects of a proton beam on metastasis in highly invasive 4T1 murine breast cancer cells and their orthotopic breast cancer model. Cells were irradiated with 2, 4, 8 or 16 Gy proton beam, and changes in cell proliferation, survival, and migration were observed by MTT, colony forming and wound healing assays. 4T1 breast cancer cell-implanted BALB/c mice were established and the animals were randomly divided into 4 groups when tumor size reached 200 mm3. Breast tumors were selectively irradiated with 10, 20 or 30 Gy proton beam. Breast tumor sizes were measured twice a week, and breast tumor and lung tissues were pathologically observed. Metastasis-regulating gene expression was assessed with quantitative RT-PCR. A proton beam dose-dependently decreased cell proliferation, survival and migration in 4T1 murine breast cancer cells. Also, growth of breast tumors in the 4T1 orthotopic breast cancer model was significantly suppressed by proton beam irradiation without significant change of body weight. Furthermore, fewer tumor nodules metastasized from breast tumor into lung in mice irradiated with 30 Gy proton beam, but not with 10 and 20 Gy, than in control. We observed correspondingly lower expression levels of urokinase plasminogen activator (uPA), uPA receptor, cyclooxygenase (COX)-2, and vascular endothelial growth factor (VEGF), which are important factors in cancer metastasis, in breast tumor irradiated with 30 Gy proton beam. Proton beam irradiation did not affect expressions of matrix metalloproteinase (MMP)-9 and MMP-2. Taken together, the data suggest that, although proton beam therapy is an effective tool for breast cancer treatment, a suitable dose is necessary to prevent metastasis-linked relapse and poor prognosis.

High glucose and insulin enhance uPA expression, ROS formation and invasiveness in breast cancer-derived cells.

BACKGROUND: Accumulating evidence indicates that type 2 diabetes is associated with an increased risk to develop breast cancer. This risk has been attributed to hyperglycemia, hyperinsulinemia and chronic inflammation. As yet, however, the mechanisms underlying this association are poorly understood. Here, we studied the effect of high glucose and insulin on breast cancer-derived cell proliferation, migration, epithelial-mesenchymal transition (EMT) and invasiveness, as well as its relationship to reactive oxygen species (ROS) production and the plasminogen activation system. METHODS: MDA-MB-231 cell proliferation, migration and invasion were assessed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), scratch-wound and matrigel transwell assays, respectively. ROS production was determined using 2' 7'-dichlorodihydrofluorescein diacetate. The expression of E-cadherin, vimentin, fibronectin, urokinase plasminogen activator (uPA), its receptor (uPAR) and its inhibitor (PAI-1) were assessed using qRT-PCR and/or Western blotting assays, respectively. uPA activity was determined using gel zymography. RESULTS: We found that high glucose stimulated MDA-MB-231 cell proliferation, migration and invasion, together with an increased expression of mesenchymal markers (i.e., vimentin and fibronectin). These effects were further enhanced by the simultaneous administration of insulin. In both cases, the invasion and growth responses were found to be associated with an increased expression of uPA, uPAR and PAI-1, as well as an increase in active uPA. An osmolality effect of high glucose was excluded by using mannitol at an equimolar concentration. We also found that all changes induced by high glucose and insulin were attenuated by the anti-oxidant N-acetylcysteine (NAC) and, thus, depended on ROS production. CONCLUSIONS: From our data we conclude that hyperglycemia and hyperinsulinemia can promote breast cancer cell proliferation, migration and invasion. We found that these features were associated with increased expression of the mesenchymal markers vimentin and fibronectin, as well as increased uPA expression and activation through a mechanism mediated by ROS.

Human urokinase-type plasminogen activator gene-modified bone marrow-derived mesenchymal stem cells attenuate liver fibrosis in rats by down-regulating the Wnt signaling pathway.

AIM: To evaluate the therapeutic effects of bone marrow-derived mesenchymal stem cells (BMSCs) with human urokinase-type plasminogen activator (uPA) on liver fibrosis, and to investigate the mechanism of gene therapy. METHODS: BMSCs transfected with adenovirus-mediated human urokinase plasminogen activator (Ad-uPA) were transplanted into rats with CCl4-induced liver fibrosis. All rats were sacrificed after 8 wk, and their serum and liver tissue were collected for biochemical, histopathologic, and molecular analyzes. The degree of liver fibrosis was assessed by hematoxylin and eosin or Masson's staining. Western blot and quantitative reverse transcription-polymerase chain reaction were used to determine protein and mRNA expression levels. RESULTS: Serum levels of alanine aminotransferase, aminotransferase, total bilirubin, hyaluronic acid, laminin, and procollagen type III were markedly decreased, whereas the levels of serum albumin were increased by uPA gene modified BMSCs treatment. Histopathology revealed that chronic CCl4-treatment resulted in significant fibrosis while uPA gene modified BMSCs treatment significantly reversed fibrosis. By quantitatively analysing the fibrosis area of liver tissue using Masson staining in different groups of animals, we found that model animals with CCl4-induced liver fibrosis had the largest fibrotic area (16.69% ± 1.30%), while fibrotic area was significantly decreased by BMSCs treatment (12.38% ± 2.27%) and was further reduced by uPA-BMSCs treatment (8.31% ± 1.21%). Both protein and mRNA expression of ß-catenin, Wnt4 and Wnt5a was down-regulated in liver tissues following uPA gene modified BMSCs treatment when compared with the model animals. CONCLUSION: Transplantation of uPA gene modified BMSCs suppressed liver fibrosis and ameliorated liver function and may be a new approach to treating liver fibrosis. Furthermore, treatment with uPA gene modified BMSCs also resulted in a decrease in expression of molecules of the Wnt signaling pathway.

Resveratrol inhibits hypoxia-driven ROS-induced invasive and migratory ability of pancreatic cancer cells via suppression of the Hedgehog signaling pathway.

A hypoxic microenvironment is commonly found in the central region of solid tumors, including pancreatic cancer. Our previous study revealed that resveratrol plays an important role in suppressing the proliferation and EMT of pancreatic cancer cells. However, whether resveratrol could suppress hypoxia-induced cancer progression and the underlying mechanisms have not been fully elucidated. The aim of the present study was to evaluate whether resveratrol affects hypoxia-induced reactive oxygen species (ROS) production and the activation of the Hedgehog (Hh) signaling pathway as well as the invasion of pancreatic cancer. The human pancreatic cancer cell lines, BxPC-3 and Panc-1, were subjected to a hypoxic condition and three different concentrations of resveratrol. The intracellular ROS were determined using 2,7-dichlorodihydrofluorecein diacetate. Wound healing and Transwell invasion assays were used to detect the migratory and invasive potential of the cancer cells. Metastatic-related and Hh signaling-related factors were detected by qRT-PCR and western blot analysis. Immunofluorescence staining was used to test the nuclear translocation of GLI1. The results showed that the hypoxia-induced production of ROS was decreased by resveratrol in a concentration-dependent manner. Resveratrol significantly inhibited the hypoxia-stimulated invasion and migration of pancreatic cancer cells. Resveratrol inhibited hypoxia-induced HIF-1α protein expression. Resveratrol also suppressed hypoxia­induced expression of metastatic-related factors, uPA and MMP2. In addition, resveratrol markedly inhibited hypoxia-mediated activation of the Hh signaling pathway. Furthermore, the antioxidant N-acetylcysteine (NAC) significantly suppressed the invasive and migratory ability of pancreatic cancer cells during hypoxia. Taken together, these data indicate that resveratrol plays an important role in suppressing hypoxia-driven ROS-induced pancreatic cancer progression by inhibiting the Hh signaling pathway, providing evidence that resveratrol may be a potential candidate for the chemoprevention of cancer.

Immunohistochemical Detection of Urokinase Plasminogen Activator and Urokinase Plasminogen Activator Receptor in Canine Vascular Endothelial Tumours.

Immunohistochemistry was used to assess the expression of urokinase plasminogen activator (uPA) and uPA receptor (uPAR) in 57 canine primary haemangiosarcomas (HSAs), 26 canine cutaneous haemangiomas (HAs) and in control sections of canine cutaneous granulation tissue. The correlation between uPA/uPAR expression and the Ki67 labelling index (LI) was estimated in the HSA and HA tissues. uPA was expressed by 73.2% and 75.0% of splenic HSAs and non-splenic HSAs, respectively. All HSA tissues tested expressed uPAR. Expression of both molecules was significantly higher in HSAs than in cutaneous HAs (3.8% for uPA and 30.7% for uPAR). The average Ki67 LI of the uPA(+)/uPAR(+) HSAs was significantly higher than that of uPA(-)/uPAR(+) HSAs and HA tissues (mean ± SDs 32.8 ± 15.3, 15.2 ± 7.2 and 2.1 ± 0.7, respectively; P <0.05). These results suggest that uPA and uPAR play a significant role in the malignant proliferation of canine HSA, regardless of the primary origin of the tumour.

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

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

[Expression of plasminogen activator system components in the gastric mucosa in portal hypertensive gastropathy].

OBJECTIVE: to study the expression of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) in the gastric mucosal (GM) vascular endothelium and epithelial cells of patients with portal hypertensive gastropathy (PHG) and those with portal hypertension (PH) without signs of PHG as compared to a control group. MATERIAL AND METHODS: GM biopsy specimens from patients with PHG, those with PH without signs of PHG, and controls with the normal gastric mucosa were immunohistochemically examined. RESULTS: Comparison of the expression of uPA in the GM vascular endothelium and epithelial vessels revealed no significant differences in the patient groups. The level of PAI-1 in the GM vessels was statistically significantly higher in the control group than in the groups of patients with PHG and PH without PHG. PAI-1 expression in the GM epithelial cells was significantly more commonly absent in the PHG group than in the control group. An analysis of an uPA and PAI-1 expression ratio showed a statistically significant predominance of the expression of uPA over its inhibitor in the GM vascular endothelium of the patients with PHG and those with PH without PHG as compared to the controls. CONCLUSION: The predominance of uPA over PAI-1 in the GM vessels and epithelial cells can play a role in the development of GM bleeding.

FOXM1-mediated downregulation of uPA and MMP9 by 3,3'-diindolylmethane inhibits migration and invasion of human colorectal cancer cells.

Although 3,3'-diindolylmethane (DIM) has been suggested to reduce the risk of colorectal cancer, the underlying biological mechanism is not clearly understood. In the present study, we investigated the effect of DIM on the migratory and invasive activities of the human colorectal cancer cell lines DLD-1 and HCT116. DIM significantly inhibited the migration and invasion of colorectal cancer cells as assessed by wound healing and Matrigel invasion assays. The migratory ability of the DLD-1 and HCT116 cells was significantly reduced by DIM at 24 and 48 h. DIM also significantly inhibited the invasion rate of the DLD-1 and HCT116 cells in a dose-dependent manner. The mRNA expression levels of urokinase type plasminogen activator (uPA) and matrix metalloprotease 9 (MMP9) were significantly attenuated, whereas expression of E-cadherin mRNA was significantly enhanced, following DIM treatment. DIM also decreased the protein levels of uPA and MMP9, yet significantly increased E-cadherin protein expression. In addition, DIM significantly reduced the mRNA and protein levels of FOXM1 in the DLD-1 and HCT116 cells. Our results suggest that DIM can influence the cell migratory and invasive properties of human colorectal cancer cells and may decrease the invasive capacity of colorectal cancer through downregulation of uPA and MMP9 mediated by suppression of the transcription factor FOXM1.

[Matrix metalloproteinases 2 and 9, their endogenous regulators, and angiotensin-converting enzyme in cervical squamous cell carcinoma].

OBJECTIVE: to investigate the specific features of the expression of matrix metalloproteinases 2 and 9 (MMP-2, MMP-9), tissue inhibitor of metalloptoteinase 2 (TIMP-2), urokinase-type plasminogen activator (uPA), and angiotensin-converting enzyme (ACE) in cervical squamous cell carcinoma (CSCC). MATERIAL AND METHODS: The samples of tumor tissue and morphologically normal tissue adjacent to the tumor were investigated. Enzymatic assays applying specific substrates, as well as zymographic and immunohistochemical studies were used. RESULTS: The invasive potential of CSCC has been established to be substantially influenced by the increased expression of MMP-9 and uPA and by the decreased expression of TIMP-2, as well as to a lesser extent by a change in MMP-2 expression. MMP-9 may serve as a marker for invasive growth. Enhanced ACE activity in cancer confirms the involvement of this enzyme in tumor progression. The morphologically normal tissue adjacent to the tumor shows the substantial expression of MMP-2 and MMP-9 and in some cases the enhanced activity of uPA and ACE, which makes an additional contribution to the increased invasive potential of tumor. CONCLUSION: The findings are important in understanding the mechanisms of cancer progression and may affect therapeutic strategies for the patient.

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

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

Mental stress in atopic dermatitis--neuronal plasticity and the cholinergic system are affected in atopic dermatitis and in response to acute experimental mental stress in a randomized controlled pilot study.

RATIONALE: In mouse models for atopic dermatitis (AD) hypothalamus pituitary adrenal axis (HPA) dysfunction and neuropeptide-dependent neurogenic inflammation explain stress-aggravated flares to some extent. Lately, cholinergic signaling has emerged as a link between innate and adaptive immunity as well as stress responses in chronic inflammatory diseases. Here we aim to determine in humans the impact of acute stress on neuro-immune interaction as well as on the non-neuronal cholinergic system (NNCS). METHODS: Skin biopsies were obtained from 22 individuals (AD patients and matched healthy control subjects) before and after the Trier social stress test (TSST). To assess neuro-immune interaction, nerve fiber (NF)-density, NF-mast cell contacts and mast cell activation were determined by immunohistomorphometry. To evaluate NNCS effects, expression of secreted mammal Ly-6/urokinase-type plasminogen activator receptor-related protein (SLURP) 1 and 2 (endogenous nicotinic acetylcholine receptor ligands) and their main corresponding receptors were assessed by quantitative RT-PCR. RESULTS: With respect to neuro-immune interaction we found higher numbers of NGF+ dermal NF in lesional compared to non-lesional AD but lower numbers of Gap43+ growing NF at baseline. Mast cell-NF contacts correlated with SCORAD and itch in lesional skin. With respect to the NNCS, nicotinic acetylcholine receptor α7 (α7nAChR) mRNA was significantly lower in lesional AD skin at baseline. After TSST, PGP 9.5+ NF numbers dropped in lesional AD as did their contacts with mast cells. NGF+ NF now correlated with SCORAD and mast cell-NF contacts with itch in non-lesional skin. At the same time, SLURP-2 levels increased in lesional AD skin. CONCLUSIONS: In humans chronic inflammatory and highly acute psycho-emotional stress interact to modulate cutaneous neuro-immune communication and NNCS marker expression. These findings may have consequences for understanding and treatment of chronic inflammatory diseases in the future.

Effect of adenovirus­mediated uPA gene transduction on the fibrinolytic activity of human umbilical vein endothelial cells.

The present study aimed to investigate the effect of adenovirus­mediated urokinase­type plasminogen activator (uPA) transduction on uPA expression and fibrinolytic activity in human umbilical vein endothelial cells (HUVECs). Recombinant adenovirus vectors containing the human uPA gene were constructed and transduced into HUVECs. The expression and fibrinolytic activity of uPA was then assessed in HUVECs using western blot analysis, ELISA and colorimetric assay. The experiments were performed in three groups: The ad/uPa (n=3), ad/neg control (n=3) and blank control (n=3) groups. Western blot analysis revealed that uPA protein expression in the HUVECs in the ad/uPa group was significantly increased compared with those in the ad/neg control or blank groups (P<0.01). The uPA protein levels in the supernatant of the three groups were 379.40±2.46, 240.01±1.16 and 256.10±3.04 ng/l, respectively, showing that the uPA protein levels were significantly higher in the supernatant in the ad/uPa group compared with those in the ad/neg control or blank groups. uPA activity was determined using a colorimetric method and was found to be 40238.49±5755 IU/mg in the HUVECs in the ad/uPa group, which was significantly higher than that in the HUVECs in the ad/neg control (6180.03±942.38 IU/mg) or blank groups (3346.06±928.81 IU/mg) (both P<0.01). These findings suggested that transduction of the uPA gene increased uPA protein expression and fibrinolytic activity in HUVECs.

Polyserase-1/TMPRSS9 induces pro-tumor effects in pancreatic cancer cells by activation of pro-uPA.

Polyserase-1/TMPRSS9 is a type II transmembrane serine protease showing a complex molecular architecture characterized by the presence of three tandem serine protease domains in its amino acid sequence. This protease is widely expressed in mouse and human tissues, however, its functional significance is unknown in both normal and pathological conditions. In the present study, we evaluated the possible role of polyserase-1 in cancer progression. First, we showed that polyserase-1 increased the invasive capacities of PANC-1 and SK-PC-3 pancreatic cancer cells. Moreover, the presence of polyserase-1 enhanced anchorage-independent growth and diminished the adhesion capability of PANC-1 cells to different extracellular matrix components. These effects were mediated by the efficient conversion of pro-uPA to active uPA and high phosphorylation levels of ERK detected in the PANC-1 cells expressing exogenous polyserase-1. Collectively, our data suggest that polyserase-1 may be involved in cancer progression and, similarly to what has been proposed for the closely related serine proteases matriptase and TMPRSS4, inhibition of TMPRSS9 activity may contribute to the inhibition of tumor growth.

IL-1ß-stimulated urokinase plasminogen activator expression through NF-κB in gastric cancer after HGF treatment.

The potential of hepatocyte growth factor (HGF) to regulate the expression of urokinase plasminogen activator (uPA) in a gastric cancer cell is not widely acknowledged. To identify the genes associated with the plasminogen activator proteolytic axis by HGF, we used cDNA microarray technology and selected genes upregulated or downregulated in two gastric cell lines (NUGC-3 and MKN-28). First, IL-1ß RNA and protein were confirmed to be upregulated. Then, we investigated the effect of IL-1ß induced by HGF on the uPA system, facilitating the migration and invasion of cancer cells in the metastatic process. The role for IL-1ß in HGF-induced upregulation of uPA was determined by knockdown of IL-1ß with IL-1ß shRNA and a chromatin immune precipitation assay. The levels of IL-1ß and uPA were upregulated in cells treated with HGF in a dose-dependent manner. HGF-induced upregulation of uPA was suppressed by IL-1ß knockdown. HGF enhanced the binding activity of NF-κB to the uPA promoter in control cells, but not in the IL-1ß shRNA cells. We confirmed the functional role of HGF inactivation of the uPA promoter by a reporter gene assay. Downregulation of IL-1ß using IL-1ß shRNA also decreased cell proliferation and in vitro cell invasion. IL-1ß stimulated uPA expression through ERK and NF-κB in gastric cancer, which may therefore be promising targets for gastric cancer therapy.