Upregulation Of uPA Research Articles

Abstract 5156: Is the formation of Stroma AReactive Invasion Front Areas in colorectal cancer driven by characteristic extracellular matrix reorganization processes based on the plasmin/plasminogen system

Abstract Background: As patient stratification still needs to be refined in colorectal cancer (CRC), we established SARIFA (Stroma AReactive Invasion Front Areas) as a novel H&E-based prognostic biomarker that is defined by a direct tumor-adipocyte interaction with a lack of desmoplastic reaction. Even though we could prove the high prognostic relevance of SARIFA now in multiple cohorts in gastric, colorectal, and pancreatic cancer, as well as could link an upregulation of lipid metabolism and immune alterations to this specific aggressive tumor biology, it remains an open question how SARIFAs are formed mechanistically. Materials/Methods: Based on the results of our recently established SARIFA-scores in the TCGA-CRC (TCGA-COAD & TCGA-READ cohorts; SARIFA-positive n=69, SARIFA-negative n=138) and the corresponding differential gene expression analysis, which are part of a previous study, protein levels (in ng/mL) measured by commercially available uPA and PAI1 enzyme-linked immunosorbent assays (ELISAs) were compared between SARIFA-positive and SARIFA-negative CRC patients (SARIFA-positive n=35, SARIFA-negative n=62). Additionally, immunohistochemistry for FABP4 as a key player in lipid metabolism was performed. Moreover, spatial transcriptomics (digital spatial profiling, DSP) was performed with tumor and stroma section segmentation on a subset of cases. Results: In TCGA-CRC, SERPINE1 (PAI1), as well as PLAU (uPA), are both significantly upregulated on mRNA level (LFC: 1.14 and 0.74, respectively; both adjusted p-values ≤0.001). Logically, SERPINE1 and PLAU are highly expressed in mesenchymal CMS4 CRCs, which overlap with SARIFA-positivity. This is reflected by an upregulation of matrix metalloproteinases in predicted functional protein networks (STRING analysis). In our local cohort, we could now validate this upregulation of uPA in SARIFA-positive CRCs on protein level (p=0.0027). For PAI1, a trend towards higher protein levels in SARIFA-positive CRCs could be observed (p=0.209). Moreover, SARIFA-positive CRCs show a higher number of tumor buds (p=0.013) as well as increased FABP4 expression at the invasion front (p<0.001) in our local cohort. In DSP analysis, the stroma fraction of SARIFA-positive CRCs showed significant changes compared to SARIFA-negative cases. Conclusion: Our current data shows that the formation of SARIFAs and the consecutive direct tumor-adipocyte interaction with a tumor-promoting increase in lipid metabolism may rely on alterations in the plasmin/plasminogen system, which could be a novel potential drug target in the aggressive subset of SARIFA-positive CRCs. Citation Format: Bianca Grosser, Nic G. Reitsam, Florian Sommer, Jochen Hardt, Bruno Märkl. Is the formation of Stroma AReactive Invasion Front Areas in colorectal cancer driven by characteristic extracellular matrix reorganization processes based on the plasmin/plasminogen system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5156.

Epigallocatechin gallate effect on Interleukin-1 and MMP-9 expression as Pseudomonas aeruginosa keratitis adjuvant therapy

Link of Video Abstract: https://youtu.be/Ja9klXNlw5A Background: Keratitis Pseudomonas aeruginosa is the most frequently occurring eye infection. It is a rapidly developing condition that can lead to ocular infections (endophthalmitis) and corneal perforation resulting in vision loss. Although prompt administration of antibiotics can effectively kill the bacteria, the corneal damage caused by toxins persists. The following inflammatory process results in corneal thinning, fibrosis, and eventual perforation. Methods: This study is a literature review to support future experimental studies which will be conducted to examine the expression of IL-1b and MMP-9 on pseudomonas keratitis after administration of EGCG. Results: Interleukin-1ß is a proinflammatory cytokine secreted by various immune cells in response to acute and chronic inflammation. At the same time, matrix metalloproteinase (MMP) is a group of proteolytic enzymes involved in the pathophysiology of various ocular surface diseases. Epigallocatechin gallate (EGCG) is the major polyphenol compound in green tea, produced from the Camellia sinensis plant. Recent studies suggest that EGCG has anti-inflammatory and antioxidant effects on various cell types, including the cornea. EGCG inhibits IL-1β, which induces the activation of the NF-kB signaling pathway, which may explain the decrease in uPA expression and the reduction of collagen degradation by corneal fibroblasts. EGCG inhibits the activation of pro-MMP 9 produced by corneal fibroblasts in response to IL-1β stimulation, and this effect is mediated by the inhibition of uPA upregulation without a significant impact on pro-MMP 9 expressions. Conclusion: Epigallocatechin gallate (EGCG) in Camellia sinensis extract has the potential as an alternative adjuvant therapy for bacterial keratitis because it has antibacterial, anti-inflammatory and antioxidant effects.

The Mechanisms Mediated by α7 Acetylcholine Nicotinic Receptors May Contribute to Peripheral Nerve Regeneration.

Due to the microenvironment created by Schwann cell (SC) activity, peripheral nerve fibers are able to regenerate. Inflammation is the first response to nerve damage and the removal of cellular and myelin debris is essential in preventing the persistence of the local inflammation that may negatively affect nerve regeneration. Acetylcholine (ACh) is one of the neurotransmitters involved in the modulation of inflammation through the activity of its receptors, belonging to both the muscarinic and nicotinic classes. In this report, we evaluated the expression of α7 nicotinic acetylcholine receptors (nAChRs) in rat sciatic nerve, particularly in SCs, after peripheral nerve injury. α7 nAChRs are absent in sciatic nerve immediately after dissection, but their expression is significantly enhanced in SCs after 24 h in cultured sciatic nerve segments or in the presence of the proinflammatory neuropeptide Bradykinin (BK). Moreover, we found that activation of α7 nAChRs with the selective partial agonist ICH3 causes a decreased expression of c-Jun and an upregulation of uPA, MMP2 and MMP9 activity. In addition, ICH3 treatment inhibits IL-6 transcript level expression as well as the cytokine release. These results suggest that ACh, probably released from regenerating axons or by SC themselves, may actively promote through α7 nAChRs activation an anti-inflammatory microenvironment that contributes to better improving the peripheral nerve regeneration.

Targeting uPA-uPAR interaction to improve intestinal epithelial barrier integrity in inflammatory bowel disease.

SummaryBackgroundLoss of intestinal epithelial barrier integrity is a critical component of Inflammatory Bowel Disease (IBD) pathogenesis. Co-expression regulation of ligand-receptor pairs in IBD mucosa has not been systematically studied. Targeting ligand-receptor pairs which are induced in IBD mucosa and function in intestinal epithelial barrier integrity may provide novel therapeutics for IBD.MethodsWe performed transcriptomic meta-analysis on public IBD datasets combined with cell surface protein-protein-interaction (PPI) databases. We explored primary human/mouse intestinal organoids and Caco-2 cells for expression and function studies of uPA-uPAR (prime hits from the meta-analysis). Epithelial barrier integrity was measured by Trans-Epithelial Electrical Resistance (TEER), FITC-Dextran permeability and tight junction assessment. Genetic (CRISPR, siRNA and KO mice) and pharmacological (small molecules, neutralizing antibody and peptide inhibitors) approaches were applied. Mice deficient of uPAR were studied using the Dextran Sulfate Sodium (DSS)-induced colitis model.FindingsThe IBD ligand-receptor meta-analysis led to the discovery of a coordinated upregulation of uPA and uPAR in IBD mucosa. Both genes were significantly upregulated during epithelial barrier breakdown in primary intestinal organoids and decreased during barrier formation. Genetic inhibition of uPAR or uPA, or pharmacologically blocking uPA-uPAR interaction protects against cytokine-induced barrier breakdown. Deficiency of uPAR in epithelial cells leads to enhanced EGF/EGFR signalling, a known regulator of epithelial homeostasis and repair. Mice deficient of uPAR display improved intestinal barrier function in vitro and during DSS-induced colitis in vivo.InterpretationOur findings suggest that blocking uPA-uPAR interaction via pharmacological agents protects the epithelial barrier from inflammation-induced damage, indicating a potential therapeutic target for IBD.FundingThe study was funded by Boehringer Ingelheim.

TLR3 promotes hepatocyte proliferation after partial hepatectomy by stimulating uPA expression and the release of tissue-bound HGF.

TLR3 is implicated in anti-viral immune responses, but may also act as a sensor of tissue damage in the absence of infection. Here, we provide evidence for an essential role of TLR3 in liver regeneration after an acute loss of tissue due to partial hepatectomy. Mice lacking TLR3 had a severe and sustained defect in the restoration of liver tissue with reduced liver-to-body weight ratios even after an extended recovery period of 2weeks. Hepatocyte cell cycle progression into S phase was impaired in TLR3-deficient mice. Mechanistic analyses revealed that TLR3-deficient mice had markedly reduced systemic levels of active HGF, but had increased amounts of inactive tissue-bound HGF. Importantly, expression of uPA, which orchestrates the processing and release of HGF from the hepatic extracellular matrix, was reduced in regenerating livers of TLR3-deficient mice. In addition, expression of the HGF maturation factor HGFAC was transiently diminished in TLR3-deficient mice. In vitro, engagement of TLR3 directly stimulated expression of uPA by hepatic stellate cells. Thus, TLR3 supports liver regeneration through upregulation of uPA, which promotes the release of preformed HGF from extracellular matrix stores.

Urokinase Plasminogen Activator Overexpression Reverses Established Lung Fibrosis.

Impaired plasminogen activation (PA) is causally related to the development of lung fibrosis. Prior studies demonstrate that enhanced PA in the lung limits the severity of scarring following injury and in vitro studies indicate that PA promotes matrix degradation and fibroblast apoptosis. These findings led us to hypothesize that increased PA in an in vivo model would enhance the resolution of established lung fibrosis in conjunction with increased myofibroblast apoptosis. Transgenic C57BL/6 mice with doxycycline inducible lung-specific urokinase plasminogen activator (uPA) expression or littermate controls were treated (day 0) with bleomycin or saline. Doxycycline was initiated on days 1, 9, 14, or 21. Lung fibrosis, stiffness, apoptosis, epithelial barrier integrity, and inflammation were assessed. Protection from fibrosis with uPA upregulation from day 1 through day 28 was associated with reduced parenchymal stiffness as determined by atomic force microscopy. Initiation of uPA expression beginning in the late inflammatory or the early fibrotic phase reduced stiffness and fibrosis at day 28. Induction of uPA activity in mice with established fibrosis decreased lung collagen and lung stiffness while increasing myofibroblast apoptosis. Upregulation of uPA did not alter lung inflammation but was associated with improved epithelial cell homeostasis. Restoring intrapulmonary PA activity diminishes lung fibrogenesis and enhances the resolution of established lung fibrosis. This PA-mediated resolution is associated with increased myofibroblast apoptosis and improved epithelial cell homeostasis. These studies support the potential capacity of the lung to resolve existing scar in murine models.

Inhibition by Epigallocatechin Gallate of IL-1-Induced Urokinase-Type Plasminogen Activator Expression and Collagen Degradation by Corneal Fibroblasts.

The proinflammatory cytokine interleukin (IL)-1 is implicated in corneal ulceration and promotes collagen degradation by corneal fibroblasts cultured in a three-dimensional (3D) collagen gel. Epigallocatechin-3-gallate (EGCG), the principal polyphenol in extracts of green tea, has various beneficial health effects, some of which appear to be mediated through direct or indirect inhibition of protease activity. We therefore examined the effect of EGCG on IL-1β-induced collagen degradation by corneal fibroblasts embedded in a collagen gel. Human corneal fibroblasts were cultured in a type I collagen gel. Collagen degradation was assessed by measurement of hydroxyproline in acid hydrolysates of culture supernatants. The expression of urokinase-type plasminogen activator (uPA) was examined by real-time and RT-PCR analysis and by fibrin zymography, and that of the collagenase matrix metalloproteinase 1 (MMP1) was detected by immunoblot analysis. EGCG inhibited IL-1β-induced, plasminogen-dependent collagen degradation by corneal fibroblasts in a concentration-dependent manner. It also attenuated the IL-1β-induced expression of uPA at both mRNA and protein levels. EGCG inhibited the IL-1β-induced conversion of exogenous plasminogen to plasmin as well as the plasminogen-dependent activation of pro-MMP1 in the 3D cultures without a substantial effect on pro-MMP1 abundance. EGCG inhibits IL-1β-induced collagen degradation by corneal fibroblasts, with this effect likely being mediated by suppression of the upregulation of uPA, the uPA-mediated conversion of plasminogen to plasmin, and the plasmin-mediated activation of pro-MMP1. EGCG thus warrants further investigation as a potential treatment for corneal ulcer.

ERK1/2 mediates the lipopolysaccharide-induced upregulation of FGF-2, uPA, MMP-2, MMP-9 and cellular migration in cardiac fibroblasts

Myocardial fibrosis is a critical event during septic shock. Upregulation in the fibrosis signaling cascade proteins such as fibroblast growth factor (FGF), urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA) and activation of matrix metalloproteinases (MMPs) are widely associated with the development of myocardial infarction, dilated cardiomyopathy, cardiac fibrosis and heart failure. However, evidences suggest that the common upstream mediators of fibrosis cascade play little role in cardiac fibrosis induced by LPS; further, it is unknown if LPS directly triggers the expressions and/or activity of FGF-2, uPA, tPA, MMP-2 and MMP-9 in cardiac fibroblasts. In the present study, we treated primary cultures of cardiac fibroblasts with LPS to explore whether LPS upregulates FGF-2, uPA, tPA, MMP-2, MMP-9 and enhance cellular migration. Further the precise molecular and cellular mechanisms behind these LPS induced responses were identified. Inhibition assays on MAPKs using U0126 (ERK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), SP600125 (JNK1/2 inhibitor), CsA (calcineurin inhibitor) and QNZ (NFκB inhibitor) show that LPS-induced upregulation of FGF-2, uPA, MMP-2 and MMP-9 in cardiac fibroblasts was mediated through ERK1/2 signaling. Collectively, our results provide a link between LPS-induced cardiac dysfunction and ERK1/2 signaling pathway and thereby implies ERK1/2 as a possible target to regulate LPS induced upregulation of FGF-2, uPA, MMP-2, MMP-9 and cellular migration in cardiac fibroblasts.

Upregulation of P2Y2R, Active uPA, and PAI-1 Are Essential Components of Hantavirus Cardiopulmonary Syndrome.

Sin Nombre virus (SNV) causes hantavirus cardiopulmonary pulmonary syndrome (HCPS) with the loss of pulmonary vascular endothelial integrity, and pulmonary edema without causing cytopathic effects on the vascular endothelium. HCPS is associated primarily with a dysregulated immune response. We previously found occult signs of hemostatic imbalance in the form of a sharp >30–100 fold increase in the expression of plasminogen activator inhibitor type 1 (PAI-1), in serial blood plasma draws of terminal stage-patients. However, the mechanism of the increase in PAI-1 remains unclear. PAI-1 is a primary inhibitor of fibrinolysis caused by tissue plasminogen activator (tPA) and urokinase plasminogen activator plasma (uPA). Here, we investigate factors that contribute to PAI-1 upregulation during HCPS. Using zymography, we found evidence of PAI-1-refractory uPA activity and no tPA activity in plasma samples drawn from HCPS patients. The sole prevalence of uPA activity suggested that severe inflammation drove PAI-1 activity. We have recently reported that the P2Y2 receptor (P2Y2R) mediates SNV infectivity by interacting in cis with β3 integrins, which activates the latter during infection. P2Y2R is a known effector for several biological processes relevant to HCPS pathogenesis, such as upregulation of tissue factor (TF), a primary initiator of the coagulation cascade, stimulating vascular permeability and leukocyte homing to sites of infection. As P2Y2R is prone to upregulation under conditions of inflammation, we compared the expression level of P2Y2R in formalin fixed tissues of HCPS decedents using a TaqMan assay and immunohistochemistry. Our TaqMan results show that the expression of P2Y2R is upregulated significantly in HCPS cases compared to non- HCPS controls (P < 0.001). Immunohistochemistry showed that lung macrophages were the primary reservoir of high and coincident localization of P2Y2R, uPA, PAI-1, and TF antigens. We also observed increased staining for SNV antigens in the same tissue segments where P2Y2R expression was upregulated. Conversely, sections of low P2Y2R expression showed weak manifestations of macrophages, SNV, PAI-1, and TF. Coincident localization of P2Y2R and PAI-1 on macrophage deposits suggests an inflammation-dependent mechanism of increasing pro-coagulant activity in HCPS in the absence of tissue injury.

Gemcitabine treatment induces endoplasmic reticular (ER) stress and subsequently upregulates urokinase plasminogen activator (uPA) to block mitochondrial-dependent apoptosis in Panc-1 cancer stem-like cells (CSCs).

BackgroundPancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor survival rates. The presence of cancer stem-like cells (CSCs) is believed to be among the underlying reasons for the aggressiveness of PDAC, which contributes to chemoresistance and recurrence. However, the mechanisms that induce chemoresistance and inhibit apoptosis remain largely unknown.MethodsWe used serum-free medium to enrich CSCs from panc-1 human pancreatic cancer cells and performed sphere formation testing, flow cytometry, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and semi-quantitative western blotting to confirm the stemness of panc-1 CSCs. Hallmarks of endoplasmic reticulum (ER) stress, including IRE1, PERK, ATF4, ATF6α, GRP78 and uPA expression, were detected after gemcitabine treatment. Effects of gemcitabine-induced uPA expression on cell invasion, sphere formation, colony formation and gemcitabine sensitivity were detected. Electrophoretic mobility shift assays (EMSAs) and RNA-immunoprecipitation (RIP) were performed to detect interaction between the uPA mRNA 3’-UTR and mutant p53-R273H expressed by panc-1 CSCs. The effects of upregulated uPA by gemcitabine on apoptosis were detected by Annexin V-FITC/PI staining, and the impact of uPA on small molecule CP-31398-restored mutant p53 transcriptional activity was measured by a luciferase reporter assay.ResultsEnriched panc-1 CSCs expressing high levels of CD44 and CD133 also produced significantly higher amounts of Oct4 and Nanog. Compared with panc-1 cells, panc-1 CSCs presented chemoresistance to gemcitabine. ER stress gene detections demonstrated effects of gemcitabine-induced ER stress on both the pro-apoptotic and pro-survival branches. ER stress-induced ATF6α upregulated level of uPA by transcriptionally activating GRP78. Gemcitabine-induced uPA promoted invasion, sphere formation and colony formation and attenuated apoptosis induced by gemcitabine in panc-1 CSCs, depending on interaction with mutant p53-R273H. Upregulation of uPA abolished CP-31398-mediated restoration of mutant p53 transcriptional activity in panc-1 CSCs.ConclusionGemcitabine treatment induced ER stress and promoted mutant p53-R273H stabilization via transcriptionally activated uPA which may contribute to chemoresistance to gemcitabine. Notably, upregulation of uPA by gemcitabine treatment may lead to the failure of CP-31398; thus, a novel strategy for modulating mutant p53 function needs to be developed.

Attenuation of the LPS-induced, ERK-mediated upregulation of fibrosis-related factors FGF-2, uPA, MMP-2, and MMP-9 by Carthamus tinctorius L in cardiomyoblasts.

Severe and potentially fatal hypotension and cardiac contractile dysfunction are common symptoms in patients with sepsis. LPS was previously found to dramatically upregulate expression of fibrosis-related factors FGF-2, uPA, MMP-2, and MMP-9 in primary cardiac fibroblasts. MMPs are capable of denaturing and degrading fibrillar collagens and other components of the extracellular matrix (ECM). Studies have shown that dysregulation of expression of MMPs is associated with development of myocardial extracellular matrix remodeling and cardiac fibrosis, which contribute to progression of heart failure. In this study, H9c2 cells and cardiac fibroblasts were divided into five treatment groups: control, LPS (1 μg/mL) and three concentrations of FCEtOH (Carthami Flos ethanolic extract) (31.25, 62.5, and 125 μg/mL). Phosphorylation of ERK-1/2 was observed to be rapidly induced upon treatment with LPS. In contrast, it was significantly suppressed by the administration of FCEtOH (125 μg/mL). Effects of FCEtOH on LPS-induced MMP-2 and MMP-9 expression in H9c2 cells occurred directly through ERK1/2 were determined. H9c2 cells were therefore pretreated with EGF-R to activate ERK pathway. Both protein levels of MMP-2 and MMP-9 and immunefluorescent signals of MMP-9 were significantly enhanced by EGFR. In contrast, MMP-2 and MMP-9 were significantly reduced after FCEtOH administration. Based on these findings, the authors concluded that FCEtOH elicits a protective effect against LPS-induced cardio-fibrosis through the ERK1/2 pathway. Carthamus tinctorius L may potentially serve as a cardio-protective agent against LPS- induced cardiac fibrosis. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 754-763, 2017.

Upregulation of circulating cytokeratin 20, urokinase plasminogen activator and C-reactive protein is associated with poor prognosis in gastric cancer.

Gastric cancer is one of the most common types of cancer, with a high mortality rate. The aim of this study was to investigate the role of several key molecules, including cytokeratin (CK) 19 and CK20, urokinase plasminogen activator (uPA), C-reactive protein (CRP) and matrix metalloproteinase (MMP)-9, which are involved in cancer invasion and metastasis, in order to determine whether they may be considered as novel prognostic factors for gastric cancer. Peripheral blood was collected from 165 patients with gastric adenocarcinoma who underwent curative surgical resection at Zhejiang Cancer Hospital (Hangzhou, China) between 2010 and 2011. The mRNA levels of CK19, CK20, uPA and MMP-9 were detected by reverse transcription-quantitative polymerase chain reaction. The protein expression of CRP was measured by immunoturbidimetry. The Students t-test was used in the univariate analyses and the Kaplan-Meier method was used to analyze the survival curves. The relative mRNA expression of CK19 and MMP-9 was not found to be significantly associated with gender, age or cancer stage, whereas that of CK20 and uPA was associated with gastric cancer stage: The low-expression group was associated with early-stage and the high-expression group with more advanced-stage disease (P<0.05). The CRP protein level was associated with gender and cancer stage: The low-expression group was predominantly associated with male gender and early-stage disease, whereas the high-expression group was associated with female gender and advanced-stage disease (P<0.05). The expression of CK19, CK20, uPA and CRP, but not MMP-9, was negatively associated with overall survival (OS): The OS rate in the high-expression groups was significantly lower compared with that in the low-expression groups (P<0.05). In conclusion, the upregulation of CK20, uPA and CRP was found to be a negative prognostic factor for gastric cancer.

CARF (Collaborator of ARF) overexpression in p53-deficient cells promotes carcinogenesis

Collaborator of ARF (CARF), initially identified as a binding partner of ARF (Alternate Reading Frame), has been shown to activate ARF–p53 pathway by multiple ways including stabilization of ARF and p53 tumor suppressor proteins, and transcriptional repression of a p53 antagonist, HDM2. Level of CARF expression was shown to determine fate of cells. Whereas its knockdown caused apoptosis, its over- and super-expressions caused senescence and increase in malignant properties of cancer cells, respectively, and were closely linked to increase and decrease in p53 activity. Using p53-compromised cancer cells, we demonstrate that CARF induces growth arrest when wild type p53 is present and in p53-absence, it promotes carcinogenesis. Biochemical analyses on CARF-induced molecular signaling revealed that in p53-null cells, it caused transcriptional repression of p21WAF1 leading to increase in CDK4, CDK6, pRb and E2F1 resulting in continued cell cycle progression. Furthermore, it instigated increase in migration and invasion of cancer cells that was marked by upregulation of MMP2, MMP3, MMP9, uPA, several interleukins and VEGF expression. Consistent with these findings, we found that human clinical samples of epithelial and glial cancers (frequently marked by loss of p53 function) possessed high level of CARF expression showing a relationship with cancer aggressiveness. The data demonstrated that CARF could be considered as a diagnostic marker and a therapeutic target in p53-compromised malignancies.

Involvement of urokinase in cigarette smoke extract-induced epithelial–mesenchymal transition in human small airway epithelial cells

Urokinase-type plasminogen activator (uPA) augments inflammation and tissue remodeling during lung injury and repair. The uPA expression in small airway epithelium of chronic obstructive pulmonary disease (COPD) increases. Epithelial–mesenchymal transition (EMT) is important in the small airway fibrosis of COPD. This study shows the uPA regulation in cigarette smoke extract (CSE)-induced EMT in human small airway epithelial cell lines (HSAEpiCs). uPA is overexpressed in the small airway epithelium of COPD patients and CSE-treated cell lines. Furthermore, uPA expression correlated with vimentin expression in the small airway epithelium of COPD patients. uPA inhibition blocks CSE-induced EMT by reversing E-cadherin and α-catenin expression and retarding the induction of N-cadherin and vimentin, resulting in reduction in migration. uPA overexpression in HSAEpiC cells also promotes EMT and migration. EMT is partly reversed in uPA-overexpressing HSAEpiC cells through the silencing expression of uPA receptor. In conclusion, this study provides new insights into the contribution of uPA upregulation to EMT associated with small airway remodeling in COPD.

220 ERK1/2 Mediates Lipopolysaccharide-upregulated FGF-2, UPA, MMP-2, MMP-9 and Cellular Migration in Primary Cardiac Fibroblasts

Upregulation of fibroblast growth factor (FGF), urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA) and matrix metallopeptidases (MMPs) is associated with the development of myocardial infarction (MI), dilated cardiomyopathy, cardiac...

IL-1β-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-1β RNA and protein were confirmed to be upregulated. Then, we investigated the effect of IL-1β induced by HGF on the uPA system, facilitating the migration and invasion of cancer cells in the metastatic process. The role for IL-1β in HGF-induced upregulation of uPA was determined by knockdown of IL-1β with IL-1β shRNA and a chromatin immune precipitation assay. The levels of IL-1β and uPA were upregulated in cells treated with HGF in a dose-dependent manner. HGF-induced upregulation of uPA was suppressed by IL-1β knockdown. HGF enhanced the binding activity of NF-κB to the uPA promoter in control cells, but not in the IL-1β shRNA cells. We confirmed the functional role of HGF inactivation of the uPA promoter by a reporter gene assay. Downregulation of IL-1β using IL-1β shRNA also decreased cell proliferation and in vitro cell invasion. IL-1β stimulated uPA expression through ERK and NF-κB in gastric cancer, which may therefore be promising targets for gastric cancer therapy.

Abstract A096: MT-SP2/TMPRSS4 upregulates uPA gene expression through JNK signaling activation to induce cancer cell invasion

Abstract The invasive nature of tumor cells is critical for cancer metastasis. Urokinase-type plasminogen activator (uPA) is a well-known serine protease involved in normal tissue remodeling, such as wound healing, and in pathological events, such as invasion and metastasis. Increased levels of uPA correlate with invasive properties and poor prognosis in breast, lung, stomach, bladder, colon, prostate and ovarian cancers. MT-SP2/TMPRSS4 is a novel type II transmembrane serine protease that is highly expressed in pancreatic, colon, and other cancer tissues. Previously, we demonstrated that MT-SP2/TMPRSS4 mediates tumor cell invasion, migration, and metastasis. However, the mechanisms by which MT-SP2/TMPRSS4 contributes to invasion are not fully understood. Here, we demonstrated that MT-SP2/TMPRSS4 induced the transcription of the uPA gene through activating the transcription factors Sp1, Sp3, and AP-1 in mainly a JNK-dependent manner and that the induction of uPA was required for MT-SP2/TMPRSS4-mediated cancer cell invasion and signaling events. In addition, the uPA receptor was involved in MT-SP2/TMPRSS4-induced signaling activation and subsequent uPA expression probably through its association with TMPRSS4 on the cell surface. Immunohistochemical analysis showed that uPA expression was significantly correlated with MT-SP2/TMPRSS4 expression in human carcinomas. These observations suggest that MT-SP2/TMPRSS4 is an important regulator of uPA gene expression; the upregulation of uPA by TMPRSS4 contributes to invasion and may represent a novel mechanism for the control of invasion. Citation Format: Yunhee Lee, Hye-Jin Min, Hana Lee, Xue-Feng Zhao, Young-Kyu Park, Semi Kim. MT-SP2/TMPRSS4 upregulates uPA gene expression through JNK signaling activation to induce cancer cell invasion. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A096.

TMPRSS4 upregulates uPA gene expression through JNK signaling activation to induce cancer cell invasion

TMPRSS4 is a novel type II transmembrane serine protease that is highly expressed in pancreatic, thyroid, colon, and other cancer tissues. Previously, we demonstrated that TMPRSS4 mediates tumor cell invasion, migration, and metastasis. However, the mechanisms by which TMPRSS4 contributes to invasion are not fully understood. Here, we demonstrated that TMPRSS4 induced the transcription of the urokinase-type plasminogen activator (uPA) gene through activating the transcription factors Sp1, Sp3, and AP-1 in mainly a JNK-dependent manner and that the induction of uPA was required for TMPRSS4-mediated cancer cell invasion and signaling events. In addition, the uPA receptor was involved in TMPRSS4-induced signaling activation and subsequent uPA expression probably through its association with TMPRSS4 on the cell surface. Immunohistochemical analysis showed that uPA expression was significantly correlated with TMPRSS4 expression in human lung and prostate cancers. These observations suggest that TMPRSS4 is an important regulator of uPA gene expression; the upregulation of uPA by TMPRSS4 contributes to invasion and may represent a novel mechanism for the control of invasion.

Overexpression of p16(INK4a) in urothelial carcinoma in situ is a marker for MAPK-mediated epithelial-mesenchymal transition but is not related to human papillomavirus infection.

BackgroundThe role of human papillomavirus (HPV) in bladder carcinogenesis remains controversial. Overexpression of p16INK4a, a surrogate marker for infection with oncogenic HPV in other tumours, has been described for urothelial carcinoma in situ (UCIS). Our goal was therefore to evaluate whether overexpression of p16INK4a is associated with HPV infection and to identify mechanisms of p16INK4a upregulation in UCIS.Materials and MethodsIn 60 tissue specimens from a total of 45 patients (UCIS and controls), we performed p16INK4a immunohistochemistry followed by detection and subclassification of HPV DNA. In a subset of samples, we tested for gene amplification of p16INK4a applying fluorescence in situ hybridization (FISH). RAS/MAPK signalling and epithelial-mesenchymal transition (EMT) was assessed using immunohistochemistry. Finally, we transfected urothelial carcinoma cells with KRAS and examined the expression of p16INK4a as well as markers of EMT.ResultsWe found overexpression of p16INK4a in 92.6% of UCIS and in all cervical intraepithelial neoplasia (CIN) controls. In contrast, we detected high-risk HPV DNA in 80% of CIN, but none in UCIS. There was no gene amplification of p16INK4a. High levels of phosphorylated kinases and urokinase plasminogen activator (uPA) and loss of membraneous E-cadherin were detected in UCIS. KRAS transfection of urothelial carcinoma cells led to upregulation of p16INK4a and uPA accompanied by loss of E-cadherin that could be inhibited by application of the kinase-inhibitor Sorafenib.ConclusionsOur results show that overexpression of p16INK4a in UCIS is neither associated with HPV infection nor p16INK4a gene amplification but is a consequence of enhanced RAS/MAPK signalling that promotes EMT, possibly due to Sorafenib-sensitive paracrine secretion of the EMT activator uPA. These findings might open a novel therapeutic option for localized but aggressive urothelial cancer.

Stromal cell-derived factor-1 (SDF-1) enhances cells invasion by αvβ6 integrin-mediated signaling in ovarian cancer

Ovarian carcinoma is a common gynecological malignancy and a great threat to health as a result of metastasis. The chemokine stromal-derived factor (SDF-1) plays multiple roles in tumor pathogenesis. However, the precise molecular mechanism underlying SDF-1-induced ovarian cancer cell invasion is still undefined. αvβ6 integrin is an important factor in tumor progression. Therefore, we speculate that SDF-1-enhanced ovarian cancer cell invasion is related to αvβ6 integrin-mediated signaling. After culturing with SDF-1, an obvious time- and dose-dependent increase in αvβ6 integrin was demonstrated. Furthermore, CXC receptor 4 (CXCR4) was responsible for SDF-1-induced αvβ6 integrin expression. Simultaneously, SDF-1 was found to dramatically enhance extracellular matrix degradation via urokinase-type plasminogen activator (uPA) expression and cell invasion by αvβ6 integrin expression; these reinforce failed to be increased when pretreatment was performed with the CXCR4 inhibitor AMD3100 or anti-αvβ6 integrin antibody, respectively. In addition, αvβ6 integrin induced the phosphorylation of p38 MAPK and PI3 K/Akt, contributing to the up-regulation of uPA, as treatment with the specific inhibitor for p38 mitogen-activated protein kinases (MAPK) (SB203580) or phosphatidylinositol 3-kinase (PI3 K)/Akt (LY294002) strikingly abrogated uPA expression. Taken together, these results demonstrated that SDF-1 enhanced ovarian cancer cell invasion through αvβ6 integrin-mediated uPA expression via the p38 MAPK and PI3 K/Akt pathway. Consequently, our findings will provide a new explanation about how SDF-1 aggravates the pathogenesis of ovarian cancer.