uPAR Promoter Research Articles

SuPAR: A newer biomarker of systemic chronic inflammation

Migraine is a chronic neurological disorder. Inflammation has a key role in migraine pathophysiology. Urokinase plasminogen activator receptor (uPAR) directly involves in inflammatory conditions by facilitating migration of inflammatory cells to different tissues. The aim of this study was to investigate whether uPAR rs344781, common genetic polymorphism in the uPAR promoter region, might be associated with migraine without aura susceptibility in an Iranian population.We enrolled 103 newly diagnosed patients with migraine and 100 healthy controls. Peripheral blood sample was used for DNA extraction and uPAR rs344781 gene polymorphism was determined. Patients filled HIT-6 as a tool to evaluate headache severity.The genotype frequency of uPAR is significantly different between migraine patients and control subjects. Heterozygote genotype (AG) was statistically more frequent in the patients than the controls (P = 0.001; OR = 2.67, 95% CI = 1.51–4.7). Also G allele was more frequent in the patients. Total HIT-6 score was not significantly different between heterozygote and homozygote patients (55.50 ± 2.22 vs. 49.60 ± 3.68 respectively, P = 0.075).In conclusion, our study showed a significant association between uPAR rs344781 gene promoter polymorphism and migraine without aura susceptibility but not with headache severity.

PBK overexpression promotes metastasis of hepatocellular carcinoma via activating ETV4-uPAR signaling pathway.

Invasion and metastasis are the predominant causes of lethal outcomes in patients with hepatocellular carcinoma (HCC). However, the molecular mechanism underlying the invasive or metastatic process are still insufficiently understood. Here, we first integrated several public databases and identified a novel protein kinase, PDZ-binding kinase (PBK) that was frequently upregulated and correlated with poor prognosis in patients with HCC. Gain- or loss-of-function analysis revealed that PBK promoted migration and invasion of HCC cells both in vitro and in vivo. Mechanistically, PBK enhanced uPAR expression by activating its promoter activity. Chromatin immunoprecipitation (ChIP) assay showed that ETV4 directly bound to the core region of uPAR promoter while PBK could enhance the binding of ETV4 to uPAR promoter. In orthotopic mouse model, PBK knockdown markedly inhibited the lung metastasis of HCC cells, while this effect was significantly restored by uPAR overexpression. Finally, there was a positive correlation between PBK and uPAR, ETV4 and uPAR in HCC clinical samples. Collectively, these findings revealed that PBK acted as a crucial kinase by promoting invasion and migration via the ETV4-uPAR signaling pathway, and it therefore could be a promising diagnostic biomarker and therapeutic target for HCC metastasis.

A NOTCH-sensitive uPAR-regulated oncolytic adenovirus effectively suppresses pancreatic tumor growth and triggers synergistic anticancer effects with gemcitabine and nab-paclitaxel.

Notch signaling pathway is an embryonic program that becomes reactivated in pancreatic cancer and contributes to cancer stem cell (CSC) maintenance. We explored the concept of oncolytic adenoviral activity in response to Notch activation signaling, in the context of a chimeric promoter with uPAR regulatory sequences, as a strategy to drive its activity in neoplastic and CSC. We explored the advantages of a chemo-virotherapy approach based on synergistic combinations. Regulatory sequences recognized by the transcriptional factor CSL upstream a minimal uPAR promoter were engineered in adenoviral vectors and in the oncolytic adenovirus AdNuPARmE1A. Viral response to Notch signaling, and viral potency in cell lines and pancreatic cancer stem cells (PCSC) was tested. Preclinical toxicity and antitumor efficacy in xenografts and Patient-derived xenografts (PDX) mouse models was evaluated, as unimodal or in combination with gemcitabine+nab-paclitaxel. Mechanistic studies were conducted to explore the synergism of combined therapies.We demonstrate that CSL-binding site optimized-engineered sequences respond to Notch activation in AdNuPARmLuc and AdNuPARmE1A. AdNuPARmE1A showed strong lytic effects in pancreatic cancer cell lines and PCSC. AdNuPARmE1A displayed attenuated activity in normal tissues, but robust antitumor effects in xenograft and PDX models, leading to a reduced capacity of treated tumors to form tumorspheres. Chemo-virotherapy treatment enlarged therapeutic response in both tumor models. Synergistic effects of the combination resulted from viral sensitization of apoptotic cell death triggered by chemotherapy.In summary we present a novel effective oncolytic adenovirus, AdNuPARmE1A that reduces PCSC and presents synergistic effects with gemcitabine and nab-paclitaxel, supporting further clinical development.

Hypoxia inducible factor-1 mediates upregulation of urokinase-type plasminogen activator receptor gene transcription during hypoxia in cervical cancer cells.

Hypoxia occurs during development of cervical cancer and is considered to correlate with its invasion. Hypoxia mediates tumor cells to have more invasive property in a variety of cancers. Urokinase plasminogen activator receptor (uPAR) which mediates invasion is considered to be induced by hypoxia. We sought to determine the regulators of uPAR expression during hypoxia in cervical cancer. We showed that cervical cancer cell lines, CaSki and CA, were more invasive under hypoxic condition (1% O2) than under normoxic condition (20% O2) by invasion assays. Using western blot analysis, hypoxia enhanced the endogenous hypoxia-inducible factor (HIF)-1α and uPAR protein expression. uPAR mRNA level was also upregulated by hypoxia using real-time RT-PCR. Overexpression of HIF-1α which is induced by hypoxia activated the transcriptional activity of the uPAR promoter by luciferase assays. HIF-1 protein bound the putative HIF-1 response element on the uPAR promoter using electrophoretic mobility shift analysis, and additional luciferase assays show that this is essential for uPAR transactivation by HIF-1. HIF-1 overexpression enhanced the endogenous uPAR expression and introduction of siRNA for HIF-1α diminishes uPAR expression during hypoxia. These results indicate the upregulation of uPAR by hypoxia in cervical cancer cells is mediated through HIF-1. In cervical cancer tissues, we also demonstrated that uPAR protein expression was detected in cervical cancer but not in normal cervix or cervical intraepithelial neoplasia (CIN) by immunohistopathological staining. Our results provide evidence that regulation of uPAR expression by HIF-1 represents a mechanism for cervical cancer invasion during hypoxia.

AduPARE1A and gemcitabine combined treatment trigger synergistic antitumor effects in pancreatic cancer through NF-κB mediated uPAR activation.

BackgroundCombined treatment of oncolytic adenoviruses with chemotherapeutic agents is foreseen as a therapeutic option for cancer. Here we have investigated the potential to use gemcitabine in combination with the oncolytic adenovirus AduPARE1A to treat pancreatic cancer and evaluate the underlying mechanism.MethodsWe treated pancreatic cancer cell lines BxPC-3 and PANC-1 with AduPARE1A and gemcitabine individually or in combination and analyzed cell viability, combination index, apoptosis and viral production. We also investigated the effects of the combination on tumor growth and mice survival in two xenograft models. Furthermore, we analyzed uPAR promoter activity from different uPAR-controlled adenovirus and studied NF-κB mediated effects.ResultsSynergistic cell killing from the combination AduPARE1A/Gemcitabine was observed in BxPC-3 and PANC-1 cells. Moreover, the combination treatment produced therapeutic benefits over either individual modality in two mouse models bearing orthotopic tumors, showing reduced tumor progression and significant prolonged mouse survival. Mechanistic studies showed that the synergistic cell death was not due to an increase in viral replication but occurred through an enhancement of apoptotic cell death. Gemcitabine stimulation increased the transcription of uPAR-controlled transgenes through the induction of NF-κB acting on the uPAR promoter. Interestingly, NF-κB gemcitabine-mediated induction of AduPAR adenoviruses interfered with the activation of NF-κB regulated genes, probably as a result of an intracellular competition for NF-κB DNA binding. Consequently, AduPARE1A infection sensitized cells to gemcitabine-induced apoptosis in the combined treatment.ConclusionsThese data highlights the potential of the combination as a treatment modality for pancreatic cancer patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0413-2) contains supplementary material, which is available to authorized users.

Cadmium induces urokinase-type plasminogen activator receptor expression and the cell invasiveness of human gastric cancer cells via the ERK-1/2, NF-κB, and AP-1 signaling pathways.

Cadmium exposure has been linked to human cancers, including stomach cancer. In this study, the effects of cadmium on urokinase-type plasminogen activator receptor (uPAR) expression in human gastric cancer cells and the underlying signal transduction pathways were investigated. Cadmium induced uPAR expression in a time- and concentration-dependent manner. Cadmium also induced uPAR promoter activity. Additionally, cadmium induced the activation of extracellular signal regulated kinase-1/2 (ERK-1/2), p38 mitogen-activated protein kinase (MAPK), and the activation of c-Jun amino terminal kinase (JNK). A specific inhibitor of MEK-1 (PD98059) inhibited cadmium-induced uPAR expression, while JNK and p38 MAPK inhibitors did not. Expression vectors encoding dominant-negative MEK-1 (pMCL-K97M) also prevented cadmium-induced uPAR promoter activity. Site-directed mutagenesis and electrophoretic mobility shift studies showed that sites for the transcription factors nuclear factor (NF)-κB and activator protein-1 (AP-1) were involved in cadmium-induced uPAR transcription. Suppression of the cadmium-induced uPAR promoter activity by a mutated-type NF-κB-inducing kinase and I-κB and an AP-1 decoy oligonucleotide confirmed that the activation of NF-κB and AP-1 are essential for cadmium-induced uPAR upregulation. Cells pretreated with cadmium showed markedly enhanced invasiveness and this effect was partially abrogated by uPAR-neutralizing antibodies and by inhibitors of ERK-1/2, NF-κB, and AP-1. These results suggest that cadmium induces uPAR expression via ERK-1/2, NF-κB, and AP-1 signaling pathways and, in turn, stimulates cell invasiveness in human gastric cancer AGS cells.

FOXM1c Promotes Pancreatic Cancer Epithelial-to-Mesenchymal Transition and Metastasis via Upregulation of Expression of the Urokinase Plasminogen Activator System

The transcription factor Forkhead box M1 (FOXM1) plays important roles in the formation of several human tumors, including pancreatic cancer. However, the molecular mechanisms by which FOXM1 promotes pancreatic tumor epithelial-to-mesenchymal transition (EMT) and metastasis are unknown. The effect of altered expression of FOXM1 and urokinase-type plasminogen activator receptor (uPAR) on EMT and metastasis was examined using animal models of pancreatic cancer. Also, the underlying mechanisms of altered pancreatic cancer invasion and metastasis were analyzed using in vitro molecular biology assays. Finally, the clinical relevance of dysregulated FOXM1/uPAR signaling was investigated using pancreatic tumor and normal pancreatic tissue specimens. Pancreatic tumor specimens and cell lines predominantly overexpressed the FOXM1 isoform FOXM1c. FOXM1c overexpression promoted EMT in and migration, invasion, and metastasis of pancreatic cancer cells, whereas downregulation of FOXM1 expression inhibited these processes. The level of FOXM1 expression correlated directly with that of uPAR expression in pancreatic cancer cell lines and tumor specimens. Moreover, FOXM1c overexpression upregulated uPAR expression in pancreatic cancer cells, whereas inhibition of FOXM1 expression suppressed uPAR expression. Furthermore, transfection of FOXM1c into pancreatic cancer cells directly activated the uPAR promoter, whereas inhibition of FOXM1 expression by FOXM1 siRNA suppressed its activation in these cells. Finally, we identified an FOXM1-binding site in the uPAR promoter and demonstrated that FOXM1 protein bound directly to it. Deletion mutation of this site significantly attenuated uPAR promoter activity. Our findings demonstrated that FOXM1c contributes to pancreatic cancer development and progression by enhancing uPAR gene transcription, and thus, tumor EMT and metastasis.

Association between promoter region of the uPAR (rs344781) gene polymorphism in genetic susceptibility to migraine without aura in three Iranian hospitals

IntroductionMigraine is a chronic neurological disorder. Inflammation has a key role in migraine pathophysiology. Urokinase plasminogen activator receptor (uPAR) directly involves in inflammatory conditions by facilitating migration of inflammatory cells to different tissues. The aim of this study was to investigate whether uPAR rs344781, common genetic polymorphism in the uPAR promoter region, might be associated with migraine without aura susceptibility in an Iranian population. MethodsWe enrolled 103 newly diagnosed patients with migraine and 100 healthy controls. Peripheral blood sample was used for DNA extraction and uPAR rs344781 gene polymorphism was determined. Patients filled HIT-6 as a tool to evaluate headache severity. ResultsThe genotype frequency of uPAR is significantly different between migraine patients and control subjects. Heterozygote genotype (AG) was statistically more frequent in the patients than the controls (P=0.001; OR=2.67, 95% CI=1.51–4.7). Also G allele was more frequent in the patients. Total HIT-6 score was not significantly different between heterozygote and homozygote patients (55.50±2.22 vs. 49.60±3.68 respectively, P=0.075). ConclusionIn conclusion, our study showed a significant association between uPAR rs344781 gene promoter polymorphism and migraine without aura susceptibility but not with headache severity.

Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H2O2) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H2O2 increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells.

MSP-induced RON activation upregulates uPAR expression and cell invasiveness via MAPK, AP-1 and NF- B signals in gastric cancer cells

Overexpression of recepteur d'Origine nantais (RON) and urokinase plasminogen activator receptor (uPAR) have been observed in human gastric cancers. However, the interaction between RON and uPAR in gastric cancer is unclear. The present study investigated the effect of macrophage-stimulating protein (MSP, the RON ligand) on uPAR expression and the underlying signal pathways in human gastric cancer AGS cells. uPAR messenger RNA expression was induced by MSP in a time- and concentration-dependent manner. MSP also induced uPAR promoter activity. The introduction of RON-specific small interfering RNA (siRNA) significantly affected the MSP-induced uPAR transcription. Deleted and site-directed mutagenesis studies demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the MSP-induced uPAR expression. Studies with expression vectors encoding mutated-type NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the MSP-induced uPAR expression. In addition, MSP induced the activation of extracellular signal-regulated kinase-1/2 (Erk-1/2), c-Jun amino terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Dominant-negative mutants (K97M and TAM67) and specific inhibitors of Erk-1/2 and JNK were able to suppress the MSP-induced uPAR expression. AGS cells pretreated with MSP showed a remarkably enhanced invasiveness, which was partially abrogated by siRNA-targeted RON and uPAR-neutralizing antibodies. The above results suggest that MSP induces uPAR expression via MAPK, AP-1 and NF-κB signaling pathways and, in turn, stimulates cell invasiveness in human gastric cancer AGS cells.

Molecular networks implicated in speech-related disorders: FOXP2 regulates the SRPX2/uPAR complex

It is a challenge to identify the molecular networks contributing to the neural basis of human speech. Mutations in transcription factor FOXP2 cause difficulties mastering fluent speech (developmental verbal dyspraxia, DVD), whereas mutations of sushi-repeat protein SRPX2 lead to epilepsy of the rolandic (sylvian) speech areas, with DVD or with bilateral perisylvian polymicrogyria. Pathophysiological mechanisms driven by SRPX2 involve modified interaction with the plasminogen activator receptor (uPAR). Independent chromatin-immunoprecipitation microarray screening has identified the uPAR gene promoter as a potential target site bound by FOXP2. Here, we directly tested for the existence of a transcriptional regulatory network between human FOXP2 and the SRPX2/uPAR complex. In silico searches followed by gel retardation assays identified specific efficient FOXP2-binding sites in each of the promoter regions of SRPX2 and uPAR. In FOXP2-transfected cells, significant decreases were observed in the amounts of both SRPX2 (43.6%) and uPAR (38.6%) native transcripts. Luciferase reporter assays demonstrated that FOXP2 expression yielded a marked inhibition of SRPX2 (80.2%) and uPAR (77.5%) promoter activity. A mutant FOXP2 that causes DVD (p.R553H) failed to bind to SRPX2 and uPAR target sites and showed impaired down-regulation of SRPX2 and uPAR promoter activity. In a patient with polymicrogyria of the left rolandic operculum, a novel FOXP2 mutation (p.M406T) was found in the leucine-zipper (dimerization) domain. p.M406T partially impaired the FOXP2 regulation of SRPX2 promoter activity, whereas that of the uPAR promoter remained unchanged. Together with recently described FOXP2-CNTNAP2 and SRPX2/uPAR links, the FOXP2-SRPX2/uPAR network provides exciting insights into molecular pathways underlying speech-related disorders.

Selective Suicide Gene Therapy of Colon Cancer Exploiting the Urokinase Plasminogen Activator Receptor Promoter

Colon cancer is the third and fourth most prevalent cancer among Iranian men and women, respectively. Suicide gene therapy is one of the alternative therapeutic modalities for cancer. The application of specific promoters for therapeutic genes should decrease the adverse effects of this modality. The combined aims of this study were to design a specific suicide gene therapy construct for colon cancer and study its effect in distinct representatives of transformed and nontransformed cells. The KRAS oncogene signaling pathway is one of the most important signaling pathways activated in colon cancer; therefore, we inserted the urokinase plasminogen activator receptor (uPAR; PLAUR gene) promoter as one of the upregulated promoters by this pathway upstream of a suicide gene (thymidine kinase [TK]) and a reporter gene (beta-galactosidase, beta-gal [LacZ]). This promoter is a natural combination of different motifs responsive to the RAS signaling pathway, such as the transcription factors AP1 (FOS/JUN), SP1, SP3, and AP2alpha, and nuclear factor kappa B (NFkappaB). The reporter plasmid under the control of the uPAR promoter (PUCUPARLacZ) had the ability to express beta-gal in colon cancer cells (human colon adenocarcinoma [SW480] and human colorectal carcinoma [HCT116] cell lines), while it could not express beta-gal in nontransformed human umbilical vein endothelial cells (HUVEC) and normal colon cells. After confirming the ability of pUCUPARTK (suicide plasmid) to express TK in SW480 and HCT116 cells by real-time PCR, cytotoxicity assays showed that pUCUPARTK decreased the viability of these cells in the presence of ganciclovir 20 and 40 microg/mL (and higher), respectively. Although M30 CytoDEATH antibody could not detect a significant rate of apoptosis induced by ganciclovir in pUCUPARTK-transfected HCT116 cells, the percentage of stained cells was marked in comparison with untreated cells. While this antibody could detect apoptosis in HCT116 cell line transfected with positive control plasmid, it could not detect apoptosis in SW480 cells transfected with the same positive control. This discrepancy could be attributed to the different mechanisms of TK/ganciclovir-induced apoptosis in tumor protein p53 (TP53)-expressing (HCT116) and -deficient (SW480) cells. Annexin-propidium iodide staining could detect apoptosis in treated, pUCUPARTK-transfected SW480 and HCT116 cells. This study showed that the uPAR promoter can be considered as a suitable candidate for specific suicide gene therapy of colon cancer and probably other cancers in which the RAS signaling pathway is involved in their carcinogenesis process.

Urokinase receptor, MMP‐1 and MMP‐9 are markers to differentiate prognosis, adenoma and carcinoma in thyroid malignancies

The identification of high-risk patients with thyroid cancer and the preoperative differentiation between follicular adenoma and carcinoma remain clinically challenging. Our study was conducted to analyze whether the quantification of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator receptor (u-PAR) and transcription factor binding to the u-PAR promoter improve prognostic predictability and differential diagnosis of thyroid tumors. Tumor/normal tissue was collected from 69 prospectively followed patients with thyroid carcinomas (papillary, medullary, follicular and anaplastic, PTC, MTC, FTC and ATC) or follicular adenomas. U-PAR, MMP-1, MMP-7 and MMP-9 amounts were determined by ELISA, and transcription factor binding was determined by electrophoretic mobility shift assay. Binding of transcription factors to the u-PAR promoter was observed, but not associated with u-PAR expression. Carcinomas except MTC expressed significantly more u-PAR/MMPs than adenomas/normal tissues, this being associated with advanced pT- or M-stages. MMP-1 and MMP-9 were significantly higher in follicular carcinomas than in adenomas. In carcinomas, high u-PAR-gene expression correlated significantly with high MMP-9, the latter being associated with MMP-7 in normal tissues. Poor survival in differentiated tumors was associated in trend (p = 0.07); poor survival of all patients (p = 0.043) and especially of patients with carcinomas of follicular origin (including ATC), but not medullary carcinomas, were significantly associated with high u-PAR-protein (p = 0.015). Quantification of u-PAR is of prognostic relevance in thyroid carcinomas of non-c-cell origin, and u-PAR in part may be regulated nontranscriptionally in thyroid cancers. This is the first study to suggest MMP-1/-9 as significant differentiation markers between follicular adenoma and follicular carcinoma.

Urokinase-Type Plasminogen Activator Receptor Transcriptionally Controlled Adenoviruses Eradicate Pancreatic Tumors and Liver Metastasis in Mouse Models

Treatment options for pancreatic cancer have shown limited success mainly owing to poor selectivity for pancreatic tumor tissue and to a lack of activity in the tumor. In this study, we describe the ability of the urokinase-type plasminogen activator receptor (uPAR) promoter to efficiently and selectively target pancreatic tumors and metastases, which enables the successful management of pancreatic cancer. We have generated a replication-defective reporter adenovirus, AduPARLuc, and a conditionally replicating adenovirus, AduPARE1A, and we have studied the selectivity and antitumoral efficacy in pancreatic tumors and metastases. Toxicity was studied on intravascular delivery. We demonstrate that the uPAR promoter is highly active in pancreatic tumors but very weak in normal tissues. Tumor specificity is evidenced by a 100-fold increase in the tumor-to-liver ratio and by selective targeting of liver metastases (P < .001). Importantly, the AduPARE1A maintains the oncolytic activity of the wild-type virus, with reduced toxicity, and exhibits significant antitumoral activity (25% tumor eradication) and prolonged survival in pancreatic xenograft models (P < .0001). Furthermore, upon intravascular delivery, we demonstrate complete eradication of liver metastasis in 33% of mice, improving median survival (P = 5.43 x 10(-5)). The antitumoral selective activity of AduPARE1A shows the potential of uPAR promoter-based therapies in pancreatic cancer treatment.

Cetuximab attenuates metastasis and u-PAR expression in non-small cell lung cancer: u-PAR and E-cadherin are novel biomarkers of cetuximab sensitivity.

Cetuximab, which blocks ligand binding to epidermal growth factor receptor (EGFR), is currently being studied as a novel treatment for non-small cell lung cancer (NSCLC). However, its mechanisms of action toward metastasis, and markers of drug sensitivity, have not been fully elucidated. This study was conducted to (a) determine the effect of Cetuximab on invasion and NSCLC-metastasis; (b) investigate urokinase-type plasminogen activator receptor (u-PAR), a major molecule promoting invasion and metastasis, as a target molecule; (c) delineate molecular mediators of Cetuximab-induced metastasis inhibition; and (d) identify biomarkers of drug sensitivity in NSCLC. Cetuximab treatment resulted in reduced growth and Matrigel invasion of H1395 and A549 NSCLC cell lines, in parallel with reduced u-PAR mRNA and protein. u-PAR down-regulation was brought about by suppressing the binding of JunD and c-Jun to u-PAR promoter motif -190/-171 in vivo, and an inhibition of MAP/ERK kinase signaling. Furthermore, Cetuximab inhibited NSCLC proliferation and metastasis to distant organs in vivo as indicated by the chicken embryo metastasis assay. Low E-cadherin and high u-PAR, but not EGFR, was associated with resistance to Cetuximab in seven NSCLC cell lines. Furthermore, siRNA knockdown of u-PAR led to a resensitization to Cetuximab. Moreover, low E-cadherin and high u-PAR was found in 63% of resected tumor tissues of NSCLC patients progressing under Cetuximab therapy. This is the first study to show u-PAR as a target and marker of sensitivity to Cetuximab, and to delineate novel mechanisms leading to metastasis suppression of NSCLC by Cetuximab.

Transcriptional Regulation of Urokinase-type Plasminogen Activator Receptor by Hypoxia-Inducible Factor 1 Is Crucial for Invasion of Pancreatic and Liver Cancer

Angioinvasion is critical for metastasis with urokinase-type plasminogen activator receptor (uPAR) and tumor hypoxia-activated hypoxia-inducible factor 1 (HIF-1) as key players. Transcriptional control of uPAR expression by HIF has never been reported. The aim of the present study, therefore, was to test whether tumor hypoxia-induced HIF expression may be linked to transcriptional activation of uPAR and dependent angioinvasion. We used human pancreatic cancer cells and a model of parental and derived HIF-1β-deficient mouse liver cancer cell lines and performed Northern blot analysis, nuclear runoff assays, electrophoretic mobility shift assay, polymerase chain reaction-generated deletion mutants, luciferase assays, Matrigel invasion assays, and in vivo angioinvasion assays in the chorioallantoic membrane of fertilized chicken eggs. Urokinase-type plasminogen activator receptor promoter analysis resulted in four putative HIF binding sites. Hypoxia strongly induced de novo transcription of uPAR mRNA. With sequential deletion mutants of the uPAR promoter, it was possible to identify one HIF binding site causing a nearly 200-fold increase in luciferase activity. Hypoxia enhanced the number of invading tumor cells in vitro and in vivo. In contrast, HIF-1β-deficient cells failed to upregulate uPAR expression, to activate luciferase activity, and to invade on hypoxia. Taken together, we show for the first time that uPAR is under transcriptional control of HIF and that this is important for hypoxia-induced metastasis.

Abstract A22: HIF-1 mediates up-regulation of urokinase-type activator receptor (uPAR) in cervical cancer cells

Abstract A22 Hypoxia occurs during development of cervical cancer and is considered to correlate with its invasion. Hypoxia induces cancer cells to have more invasive property through the urokinase plasminogen activator receptor (uPAR) expression. We sought to determine the regulator(s) of uPAR expression during hypoxia. According to the invasion assays, cervical cancer cell lines, CaSki and CA, under hypoxic condotion (1% O2) showed more invasive property than those under normoxia. By western blot analysis, hypoxia enhanced the endogenous hypoxia inducible factor (HIF)-1alpha and uPAR protein expression. uPAR mRNA level was also up-regulated by hypoxia using realtime RT-PCR. Overexpression of HIF-1alpha which is induced by hypoxia activated the transcriptional activity of the uPAR promoter by luciferase assays. HIF-1 protein bound the putative HIF-1 response element on the uPAR promoter using the NoShift Transcription Factor Assay kit, and additional luciferase assays show that this is essential for uPAR transactivation by HIF-1. HIF-1 overexpression enhanced the endogenous uPAR expression and introduction of siRNA for HIF-1alpha diminishes uPAR expression during hypoxia. These results indicate the up-regulation of uPAR by hypoxia in cervical cancer cells is mediated through HIF-1. Using cervical cancer cell tissues, we also demonstrated that the level of uPAR mRNA in normal cervix is significantly lower than cervical cancer. Our results provide evidence that regulation of uPAR expression by HIF-1 represents a mechanism for cervical cancer invasion during hypoxia. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A22.

Lysophosphatidic acid promotes cell invasion by up‐regulating the urokinase‐type plasminogen activator receptor in human gastric cancer cells

There is a strong correlation between the overexpression of urokinase-type plasminogen activator receptor (uPAR) and gastric cancer invasion. This study examined the effect of phospholipid lysophosphatidic acid (LPA) on uPAR expression in human gastric cancer AGS cells and the underlying signal transduction pathways. Treating human gastric AGS cells with LPA induced the expression of uPAR mRNA and promoter activity in both a time- and dose-dependent manner. Small interfering RNA targeting for LPA receptors, dominant negative Rho-family GTPase (RhoA, Rac1, and Cdc42) and an expression vector encoding a mutated c-jun (TAM67) partially blocked the LPA-induced uPAR expression. Site-directed mutagenesis and electrophoretic mobility shift studies showed that the transcription factors activation protein-1 (AP-1) and nuclear factor (NF)-kappaB are essential for the LPA-induced uPAR transcription. In addition, AGS cells treated with LPA showed enhanced invasion, which was partially abrogated by the uPAR-neutralizing antibodies and inhibitors of Rho kinase, JNK, and NF-kappaB. This suggests that LPA induces uPAR expression through the LPA receptors, Rho-family GTPase, JNK, AP-1 and NF-kappaB signaling pathways, which in turn stimulates the cell invasiveness of human gastric cancer AGS cells.

The prognostic value of the suPARnostic®ELISA in HIV-1 infected individuals is not affected by uPAR promoter polymorphisms

BackgroundHigh blood levels of soluble urokinase Plasminogen Activator Receptor (suPAR) are associated with poor outcomes in human immunodeficiency-1 (HIV-1) infected individuals. Research on the clinical value of suPAR in HIV-1 infection led to the development of the suPARnostic® assay for commercial use in 2006. The aim of this study was to: 1) Evaluate the prognostic value of the new suPARnostic® assay and 2) Determine whether polymorphisms in the active promoter of uPAR influences survival and/or suPAR values in HIV-1 patients who are antiretroviral therapy (ART) naive.MethodsDNA samples were collected retrospectively from 145 Danes infected with HIV-1 with known seroconversion times. In addition, plasma was collected retrospectively from 81 of these participants for use in the suPAR analysis. Survival was analysed using Kaplan Meier analysis.ResultsSurvival was strongly correlated to suPAR levels (p < 0.001). Levels at or above 6 ng/ml were associated with death in 13 of 27 patients within a two-years period; whereas only one of 54 patients with suPAR levels below 6 ng/ml died during this period. We identified two common uPAR promoter polymorphisms: a G to A transition at -118 and an A to G transition at -465 comparative to the transcription start site. These promoter transitions influenced neither suPAR levels nor patient survival.ConclusionPlasma suPAR levels, as measured by the suPARnostic® assay, were strongly predictive of survival in ART-naïve HIV-1 infected patients. Furthermore, plasma suPAR levels were not influenced by uPAR promoter polymorphisms.

Src Induces Urokinase Receptor Gene Expression and Invasion/Intravasation via Activator Protein-1/p-c-Jun in Colorectal Cancer

The urokinase receptor [urokinase plasminogen activator receptor (u-PAR)] promotes invasion and metastasis and is associated with poor patient survival. Recently, it was shown that Src induces u-PAR gene expression via Sp1 bound to the u-PAR promoter region -152/-135. However, u-PAR is regulated by diverse promoter motifs, among them being an essential activator protein-1 (AP-1) motif at -190/-171. Moreover, an in vivo relevance of Src-induced transcriptional regulators of u-PAR-mediated invasion, in particular intravasation, and a relevance in resected patient tumors have not sufficiently been shown. The present study was conducted (a) to investigate if, in particular, AP-1-related transcriptional mediators are required for Src-induced u-PAR-gene expression, (b) to show in vivo relevance of AP-1-mediated Src-induced u-PAR gene expression for invasion/intravasation and for resected tissues from colorectal cancer patients. Src stimulation of the u-PAR promoter deleted for AP-1 region -190/-171 was reduced as compared with the wild-type promoter in cultured colon cancer cells. In gelshifts/chromatin immunoprecipitation, Src-transfected SW480 cells showed an increase of phospho-c-Jun, in addition to JunD and Fra-1, bound to region -190/-171. Src-transfected cells showed a significant increase in c-Jun phosphorylated at Ser(73) and also Ser(63), which was paralleled by increased phospho-c-jun-NH(2)-kinase. Significant decreases of invasion/in vivo intravasation (chorionallantoic membrane model) were observed in Src-overexpressing cells treated with Src inhibitors, u-PAR-small interfering RNA, and dominant negative c-Jun (TAM67). In resected tissues of 20 colorectal cancer patients, a significant correlation between Src activity, AP-1 complexes bound to u-PAR region -190/-171, and advanced pN stage were observed. These data suggest that Src-induced u-PAR gene expression and invasion/intravasation in vivo is also mediated via AP-1 region -190/-171, especially bound with c-Jun phosphorylated at Ser(73/63), and that this pathway is biologically relevant for colorectal cancer patients, suggesting therapeutic potential.