The levels of soluble urokinase plasminogen activator receptor (suPAR) in saliva are influenced by acute stress.

Although elevations in systemic suPAR levels have been associated with inflammatory conditions and with exposure to life stress and adversity, it is not yet clear whether acute psychological stress influences suPAR levels, either systemically and/or in saliva. The aim of this study was to investigate whether salivary suPAR levels are increased following exposure to acute psychological stress. Healthy subjects, aged 18-40 years, completed a laboratory psychological stressor and provided saliva samples before and after the stress test (60 min apart). Levels of suPAR as well as those of cytokines increased in the post-stress samples (all ps < .001). Baseline and post-stress IL-1ß and TNF-α as well as post-stress IL-6 correlated significantly with suPAR (all ps < .01), but IL-10 and baseline IL-6 did not. These results show that suPAR levels in saliva are stress-reactive and suggest a potential application as stress biomarkers in saliva, particularly given the advantage of easily detectable concentrations.

The prognostic value of the urokinase-plasminogen activator system (uPA) in bladder cancer patients treated with radical cystectomy (RC).

PURPOSE: Urokinase-plasminogen activator (uPA), its receptor (uPAR), and the plasmin-activator inhibitor type 1 (PAI-1) have been associated with oncologic outcomes in various malignancies and could help identify bladder cancer (BC) patients treated with radical cystectomy (RC) who are likely to benefit from intensification of therapy to prevent disease progression. Our aim was to assess the value of uPA, uPAR, and PAI-1 for prognosticating survival outcomes of patients treated with RC for BC. MATERIALS AND METHODS: Tumor specimens from 272 consecutive patients treated with RC for advanced BC were assessed with immunohistochemical staining for uPA, uPAR, and PAI-1. Overexpression was assessed by pathological image analysis. Kaplan-Meier estimates and multivariable Cox-regression were used to analyze survival. Harrell's C-index was used to assess for clinical impact of the uPA system. RESULTS: uPA, uPAR, and PAI-1 were overexpressed in 48.2%, 51.1%, and 52.2% of patients, respectively. uPA overexpression was associated with lymphovascular invasion (P = 0.034) and nodal status (P = 0.013); PAI-1 overexpression was associated with primary muscle-invasive BC (P = 0.015) and lymphovascular invasion (P = 0.024). uPA, uPAR, and the number of overexpressed markers were all 3 significantly associated with shorter overall recurrence-free-, distant recurrence-free-, and cancer-specific survival. In multivariable analyses, uPA overexpression remained associated with shorter recurrence-free survival (hazard ratio [HR] = 1.79; P = 0.036) in the entire cohort, in patients without lymph node metastasis (HR = 1.98; P = 0.018) and those with nonorgan-confined disease (HR = 1.98; P = 0.022). uPAR overexpression was associated with shorter recurrence-free survival in patients without lymph node metastasis (HR = 2.01; P = 0.021) and those with organ-confined disease (HR = 4.11; P = 0.037). CONCLUSION: Members of the uPA system are associated with features of biologically aggressive BC and oncologic outcomes. However, their value beyond currently available information remains limited.

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.

The uPAR System as a Potential Therapeutic Target in the Diseased Eye.

Dysregulation of vascular networks is characteristic of eye diseases associated with retinal cell degeneration and visual loss. Visual impairment is also the consequence of photoreceptor degeneration in inherited eye diseases with a major inflammatory component, but without angiogenic profile. Among the pathways with high impact on vascular/degenerative diseases of the eye, a central role is played by a system formed by the ligand urokinase-type plasminogen activator (uPA) and its receptor uPAR. The uPAR system, although extensively investigated in tumors, still remains a key issue in vascular diseases of the eye and even less studied in inherited retinal pathologies such as retinitis pigmantosa (RP). Its spectrum of action has been extended far beyond a classical pro-angiogenic function and has emerged as a central actor in inflammation. Preclinical studies in more prevalent eye diseases characterized by neovascular formation, as in retinopathy of prematurity, wet macular degeneration and rubeosis iridis or vasopermeability excess as in diabetic retinopathy, suggest a critical role of increased uPAR signaling indicating the potentiality of its modulation to counteract neovessel formation and microvascular dysfunction. The additional observation that the uPAR system plays a major role in RP by limiting the inflammatory cascade triggered by rod degeneration rises further questions about its role in the diseased eye.

Soluble urokinase-type plasminogen activator receptor (suPAR) and interleukin-6 levels in hyperemesis gravidarum.

BACKGROUND: The aim was to compare serum soluble urokinase-type plasminogen activator receptor (suPAR) levels as well as interleukin-6 levels (IL-6) in pregnant women with hyperemesis gravidarum (HG) and asymptomatic pregnant women. METHODS: Our study population consists of voluntary first trimester-pregnant women who applied to the outpatient clinic of the department of obstetrics and gynecology of Ankara Ataturk Training and Research Hospital. Between February and May 2016, 60 pregnant women were included in our prospective study. Serum suPAR and IL-6 levels were evaluated with the ELISA method. Twenty-nine pregnant women with HG and 31 asymptomatic pregnant women were included in the study. RESULTS: Serum suPAR level in the HG group was measured as 0.36 ± 0.56 ng/ml, whereas this level in the healthy pregnant control group was measured as 0.15 ± 0.15 ng/ml (p < 0.05). The interleukin-6 level in the HG group was 5.69 ± 2.16 pg/ml, whereas in the control group it was measured as 3.88 ± 0.28 pg/ml (p < 0.05). CONCLUSION: Serum suPAR and IL-6 levels proved to be high in the HG group. It is likely that suPAR could play a role in the etiopathogenesis of hyperemesis gravidarum.

Structured models of cell migration incorporating molecular binding processes.

The dynamic interplay between collective cell movement and the various molecules involved in the accompanying cell signalling mechanisms plays a crucial role in many biological processes including normal tissue development and pathological scenarios such as wound healing and cancer. Information about the various structures embedded within these processes allows a detailed exploration of the binding of molecular species to cell-surface receptors within the evolving cell population. In this paper we establish a general spatio-temporal-structural framework that enables the description of molecular binding to cell membranes coupled with the cell population dynamics. We first provide a general theoretical description for this approach and then illustrate it with three examples arising from cancer invasion.

Alpha-enolase (ENO1) controls alpha v/beta 3 integrin expression and regulates pancreatic cancer adhesion, invasion, and metastasis.

BACKGROUND: We have previously shown that in pancreatic ductal adenocarcinoma (PDA) cells, the glycolytic enzyme alpha-enolase (ENO1) also acts as a plasminogen receptor and promotes invasion and metastasis formation. Moreover, ENO1 silencing in PDA cells induces oxidative stress, senescence and profoundly modifies PDA cell metabolism. Although anti-ENO1 antibody inhibits PDA cell migration and invasion, little is known about the role of ENO1 in regulating cell-cell and cell-matrix contacts. We therefore investigated the effect of ENO1 silencing on the modulation of cell morphology, adhesion to matrix substrates, cell invasiveness, and metastatic ability. METHODS: The membrane and cytoskeleton modifications that occurred in ENO1-silenced (shENO1) PDA cells were investigated by a combination of confocal microscopy and atomic force microscopy (AFM). The effect of ENO1 silencing was then evaluated by phenotypic and functional experiments to identify the role of ENO1 in adhesion, migration, and invasion, as well as in senescence and apoptosis. The experimental results were then validated in a mouse model. RESULTS: We observed a significant increase in the roughness of the cell membrane due to ENO1 silencing, a feature associated with an impaired ability to migrate and invade, along with a significant downregulation of proteins involved in cell-cell and cell-matrix adhesion, including alpha v/beta 3 integrin in shENO1 PDA cells. These changes impaired the ability of shENO1 cells to adhere to Collagen I and IV and Fibronectin and caused an increase in RGD-independent adhesion to vitronectin (VN) via urokinase plasminogen activator receptor (uPAR). Binding of uPAR to VN triggers integrin-mediated signals, which result in ERK1-2 and RAC activation, accumulation of ROS, and senescence. In shENO1 cancer cells, the use of an anti-uPAR antibody caused significant reduction of ROS production and senescence. Overall, a decrease of in vitro and in vivo cell migration and invasion of shENO1 PDA cells was observed. CONCLUSION: These data demonstrate that ENO1 promotes PDA survival, migration, and metastasis through cooperation with integrins and uPAR.

Evidence that vitronectin is a potent migration-enhancing factor for cancer cells chaperoned by fibrinogen: a novel view of the metastasis of cancer cells to low-fibrinogen lymphatics and body cavities.

Diluted (1%) plasma induces migration of malignant cell lines much more strongly than potent pro-metastatic factors. To characterize the factor(s) present in diluted plasma responsible for this phenomenon we performed i) heat inactivation, ii) dialysis, iii) proteinase K treatment, and iv) molecular size filtration studies. We found that this remarkable pro-migratory activity of diluted normal plasma is associated with a ~50-100-kD protein that interacts with GαI protein-coupled receptors and activates p42/44 MAPK and AKT signaling in target cells. Since this pro-migratory activity of 1% plasma decreases at higher plasma concentrations (> 20%), but is retained in serum, we hypothesized that fibrinogen may be involved as a chaperone of the protein(s). To identify the pro-migratory protein(s) present in diluted plasma and fibrinogen-depleted serum, we performed gel filtration and hydrophobic interaction chromatography followed by mass spectrometry analysis. We identified several putative protein candidates that were further tested in in vitro experiments. We found that this pro-migratory factor chaperoned by fibrinogen is vitronectin, which activates uPAR, and that this effect can be inhibited by fibrinogen. These results provide a novel mechanism for the metastasis of cancer cells to lymphatics and body cavities, in which the concentration of fibrinogen is low, and thus suggests that free vitronectin stimulates migration of tumor cells.

FAP Promotes Immunosuppression by Cancer-Associated Fibroblasts in the Tumor Microenvironment via STAT3-CCL2 Signaling.

Cancer-associated fibroblasts (CAF) are components of the tumor microenvironment whose contributions to malignant progression are not fully understood. Here, we show that the fibroblast activation protein (FAP) triggers induction of a CAF subset with an inflammatory phenotype directed by STAT3 activation and inflammation-associated expression signature marked by CCL2 upregulation. Enforcing FAP expression in normal fibroblasts was sufficient to endow them with an inflammatory phenotype similar to FAP(+)CAFs. We identified FAP as a persistent activator of fibroblastic STAT3 through a uPAR-dependent FAK-Src-JAK2 signaling pathway. In a murine liver tumor model, we found that FAP(+)CAFs were a major source of CCL2 and that fibroblastic STAT3-CCL2 signaling in this setting promoted tumor growth by enhancing recruitment of myeloid-derived suppressor cells (MDSC). The CCL2 receptor CCR2 was expressed on circulating MDSCs in tumor-bearing subjects and FAP(+)CAF-mediated tumor promotion and MDSC recruitment was abrogated in Ccr2-deficient mice. Clinically, we observed a positive correlation between stromal expression of FAP, p-STAT3, and CCL2 in human intrahepatic cholangiocarcinoma, a highly aggressive liver cancer with dense desmoplastic stroma, where elevated levels of stromal FAP predicted a poor survival outcome. Taken together, our results showed how FAP-STAT3-CCL2 signaling in CAFs was sufficient to program an inflammatory component of the tumor microenvironment, which may have particular significance in desmoplasia-associated cancers. Cancer Res; 76(14); 4124-35. ©2016 AACR.

Role of mesenchymal stem cell-derived fibrinolytic factor in tissue regeneration and cancer progression.

Tissue regeneration during wound healing or cancer growth and progression depends on the establishment of a cellular microenvironment. Mesenchymal stem cells (MSC) are part of this cellular microenvironment, where they functionally modulate cell homing, angiogenesis, and immune modulation. MSC recruitment involves detachment of these cells from their niche, and finally MSC migration into their preferred niches; the wounded area, the tumor bed, and the BM, just to name a few. During this recruitment phase, focal proteolysis disrupts the extracellular matrix (ECM) architecture, breaks cell-matrix interactions with receptors, and integrins, and causes the release of bioactive fragments from ECM molecules. MSC produce a broad array of proteases, promoting remodeling of the surrounding ECM through proteolytic mechanisms. The fibrinolytic system, with its main player plasmin, plays a crucial role in cell migration, growth factor bioavailability, and the regulation of other protease systems during inflammation, tissue regeneration, and cancer. Key components of the fibrinolytic cascade, including the urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1), are expressed in MSC. This review will introduce general functional properties of the fibrinolytic system, which go beyond its known function of fibrin clot dissolution (fibrinolysis). We will focus on the role of the fibrinolytic system for MSC biology, summarizing our current understanding of the role of the fibrinolytic system for MSC recruitment and the functional consequences for tissue regeneration and cancer. Aspects of MSC origin, maintenance, and the mechanisms by which these cells contribute to altered protease activity in the microenvironment under normal and pathological conditions will also be discussed.

Hepatic Overexpression of Soluble Urokinase Receptor (uPAR) Suppresses Diet-Induced Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient (LDLR-/-) Mice.

OBJECTIVE: Atherosclerosis, a chronic inflammatory disease, arises from metabolic disorders and is driven by inappropriate recruitment and proliferation of monocytes / macrophages and vascular smooth-muscle-cells. The receptor for the urokinase-type plasminogen activator (uPAR, Plaur) regulates the proteolytic activation of plasminogen. It is also a coactivator of integrins and facilitates leukocyte-endothelial interactions and vascular smooth-muscle-cell migration. The role of uPAR in atherogenesis remains elusive. METHODS AND RESULTS: We generated C57Bl6/J low-density lipoprotein receptor (LDL) and uPAR double knockout (uPAR-/-/LDLR-/-) mice to test the role of uPAR in two distinct atherosclerosis models. In LDLR-/- mice, hepatic overexpression following hydrodynamic transfection of soluble uPAR that competes with endogenous membrane-bound uPAR was performed as an interventional strategy. Aortic root atherosclerotic lesions induced by feeding a high-fat diet were smaller and comprised less macrophages and vascular smooth-muscle-cells in double knockout mice and animals overexpressing soluble uPAR when compared to controls. In contrast, lesion size, lipid-, macrophage-, and vascular smooth muscle cell content of guide-wire-induced intima lesions in the carotid artery were not affected by uPAR deficiency. Adhesion of uPAR-/--macrophages to TNFα-stimulated endothelial cells was decreased in vitro accompanied by reduced VCAM-1 expression on primary endothelial cells. Hepatic overexpression of soluble full-length murine uPAR in LDLR-/- mice led to a reduction of diet-induced atherosclerotic lesion formation and monocyte recruitment into plaques. Ex vivo incubation with soluble uPAR protein also inhibited adhesion of macrophages to TNFα-stimulated endothelial cells in vitro. CONCLUSION: uPAR-deficiency as well as competitive soluble uPAR reduced diet-promoted but not guide-wire induced atherosclerotic lesions in mice by preventing monocyte recruitment and vascular smooth-muscle-cell infiltration. Soluble uPAR may represent a therapeutic tool for the modulation of hyperlipidemia-associated atherosclerotic lesion formation.

Spontaneous lung and lymph node metastasis in transgenic breast cancer is independent of the urokinase receptor uPAR.

Urokinase-type plasminogen activator (uPA) is an extracellular protease that plays a pivotal role in tumor progression. uPA activity is spatially restricted by its anchorage to high-affinity uPA receptors (uPAR) at the cell surface. High tumor tissue expression of uPA and uPAR is associated with poor prognosis in lung, breast, and colon cancer patients in clinical studies. Genetic deficiency of uPA leads to a significant reduction in metastases in the murine transgenic MMTV-PyMT breast cancer model, demonstrating a causal role for uPA in cancer dissemination. To investigate the role of uPAR in cancer progression, we analyze the effect of uPAR deficiency in the same cancer model. uPAR is predominantly expressed in stromal cells in the mouse primary tumors, similar to human breast cancer. In a cohort of MMTV-PyMT mice [uPAR-deficient (n = 31) or wild type controls (n = 33)], tumorigenesis, tumor growth, and tumor histopathology were not significantly affected by uPAR deficiency. Lung and lymph node metastases were also not significantly affected by uPAR deficiency, in contrast to the significant reduction seen in uPA-deficient mice. Taken together, our data show that the genetic absence of uPAR does not influence the outcome of the MMTV-PyMT cancer model.

PTEN expression in endothelial cells is down-regulated by uPAR to promote angiogenesis.

The tumour suppressor phosphatase and tensin homologue (PTEN), mutated or lost in many human cancers, is a major regulator of angiogenesis. However, the cellular mechanism of PTEN regulation in endothelial cells so far remains elusive. Here, we characterise the urokinase receptor (uPAR, CD87) and its tumour-derived soluble form, suPAR, as a key molecule of regulating PTEN in endothelial cells. We observed uPAR-deficient endothelial cells to express enhanced PTEN mRNA- and protein levels. Consistently, uPAR expression in endogenous negative uPAR cells, down-regulated PTEN and activated the PI3K/Akt pathway. Additionally, we found that integrin adhesion receptors act as trans-membrane signaling partners for uPAR to repress PTEN transcription in a NF-κB-dependent manner. Functional in vitro assays with endothelial cells, derived from uPAR-deficient and PTEN heterozygous crossbred mice, demonstrated the impact of uPAR-dependent PTEN regulation on cell motility and survival. In an in vivo murine angiogenesis model uPAR-deficient PTEN heterozygous animals increased the impaired angiogenic phenotype of uPAR knockout mice and were able to reverse the high invasive potential of PTEN heterozygots. Our data provide first evidence that endogenous as well as exogenous soluble uPAR down-regulated PTEN in endothelial cells to support angiogenesis. The uPAR-induced PTEN regulation might represent a novel target for drug interference, and may lead to the development of new therapeutic strategies in anti-angiogenic treatment.

Role of formic receptors in soluble urokinase receptor-induced human vascular smooth muscle migration.

BACKGROUND: Vascular smooth muscle cell (VSMC) migration in response to urokinase is dependent on binding of the urokinase molecule to the urokinase plasminogen receptor (uPAR) and cleavage of the receptor. The aim of this study was to examine the role of the soluble uPAR (suPAR) in VSMC migration. METHODS: Human VSMCs were cultured in vitro. Linear wound and Boyden microchemotaxis assays of migration were performed in the presence of suPAR. Inhibitors to G-protein signaling and kinase activation were used to study these pathways. Assays were performed for mitogen-activated protein kinase and epidermal growth factor receptor activation. RESULTS: suPAR induced concentration-dependent migration of VSMC, which was G protein-dependent and was blocked by Gαi and Gßγ inhibitors. Removal of the full uPAR molecule by incubation of the cells with a phospholipase did not interfere with this response. suPAR induced ERK1/2, p38(MAPK), and c-Jun N-terminal kinase [JNK] activation in a Gαi/Gßγ-dependent manner, and interruption of these signaling pathways prevented suPAR-mediated migration. suPAR activity was independent of plasmin activity. suPAR did not activate epidermal growth factor receptor. Interruption of the low affinity N-formyl-Met-Leu-Phe receptor (FPRL1) but not high affinity N-formyl-Met-Leu-Phe receptor (FPR) prevented cell migration and activation in response to suPAR. suPAR increased matrix metalloproteinase-2 expression and activity, and this was dependent on the low affinity N-formyl-Met-Leu-Phe receptor (FPRL1) and ERK1/2. CONCLUSIONS: suPAR induces human smooth muscle cell activation and migration independent of the full uPAR through activation of the G protein-coupled receptor FPRL1, which is not linked to the plasminogen activation cascade.

The urokinase plasminogen activator receptor (uPAR) controls macrophage phagocytosis in intestinal inflammation.

OBJECTIVE: Inflammation plays crucial roles in the pathogenesis of several chronic inflammatory disorders, including Crohn's disease (CD) and UC, the two major forms of IBD. The urokinase plasminogen activator receptor (uPAR) exerts pleiotropic functions over the course of both physiological and pathological processes. uPAR not only has a key role in fibrinolysis but also modulates the development of protective immunity. Additionally, uPAR supports extracellular matrix degradation and regulates cell migration, adhesion and proliferation, thus influencing the development of inflammatory and immune responses. This study aimed to evaluate the role of uPAR in the pathogenesis of IBD. DESIGN: The functional role of uPAR was assessed in established experimental models of colitis. uPAR deficiency effects on cytokine release, polarisation and bacterial phagocytosis were analysed in colonic macrophages. uPAR expression was analysed in surgical specimens collected from normal subjects and patients with IBD. RESULTS: In mice, uPAR expression is positively regulated as colitis progresses. uPAR-KO mice displayed severe inflammation compared with wild-type littermates, as indicated by clinical assessment, endoscopy and colon histology. The absence of uPAR led to an increased production of inflammatory cytokines by macrophages that showed an M1 polarisation and impaired phagocytosis. In human IBD, CD68(+) macrophages derived from the inflamed mucosa expressed low levels of uPAR. CONCLUSIONS: These findings point to uPAR as an essential component of intestinal macrophage functions and unravel a new potential target to control mucosal inflammation in IBD.

Associations of suPAR with lifestyle and cardiometabolic risk factors.

BACKGROUND: Soluble urokinase plasminogen activator receptor (suPAR), a novel indicator of low-grade inflammation, is associated with cardiovascular disease and mortality in the general population, while an unhealthy lifestyle influences inflammatory status. We aimed to explore the relationship of suPAR with lifestyle and cardiometabolic risk factors in a black South African population. DESIGN: This cross-sectional study includes 1068 men and women (56·4 ± 10·1 years) from the North West province who took part in the South African leg of the Prospective Urban and Rural Epidemiology (PURE) study in 2010. Captured data included a detailed lifestyle profile (tobacco use, alcohol consumption, physical activity, psychological and dietary intake status), biochemical analyses (suPAR, C-reactive protein (CRP), glucose and lipids), as well as cardiovascular and anthropometric measurements. RESULTS: In exploratory analyses, we observed positive relationships between suPAR and lifestyle factors, such as tobacco use (P-trend < 0·001), both alcohol consumption (P-trend = 0·001) and γ-glutamyl transferase (GGT) (P-trend < 0·001) and unemployment (P-trend = 0·002). suPAR and CRP correlated significantly (r = 0·23; P < 0·001). These relationships were confirmed in multiple regression analyses as suPAR independently associated with tobacco use (ß = 0·13; P < 0·001), GGT (ß = 0·24; P < 0·001) and unemployment (ß = 0·07; P = 0·039). suPAR did not associate with the cardiometabolic factors glucose, lipids, blood pressure or measures of adiposity. CONCLUSION: suPAR was independently associated with unhealthy lifestyle behaviours, but not with cardiometabolic risk factors suggesting that suPAR, as known predictor of cardiovascular disease and mortality, is augmented by modifiable cardiovascular risk factors. These findings emphasise the need for a healthy lifestyle to decrease the burden of cardiovascular disease in Africans.

FOXM1c promotes pancreatic cancer epithelial-to-mesenchymal transition and metastasis via upregulation of expression of the urokinase plasminogen activator system.

PURPOSE: 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. EXPERIMENTAL DESIGN: 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. RESULTS: 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. CONCLUSIONS: Our findings demonstrated that FOXM1c contributes to pancreatic cancer development and progression by enhancing uPAR gene transcription, and thus, tumor EMT and metastasis.

The soluble urokinase receptor is not a clinical marker for focal segmental glomerulosclerosis.

The soluble urokinase receptor (suPAR) promotes proteinuria and induces focal segmental glomerulosclerosis (FSGS)-like lesions in mice. A serum suPAR concentration cutoff of 3000 pg/ml has been proposed as a clinical biomarker for patients with FSGS. Interestingly, several studies in patients with glomerulopathy found an inverse correlation between the estimated glomerular filtration rate (eGFR) and suPAR. As patients with FSGS present at different eGFRs, we studied the relationship between eGFR and suPAR in a cohort of 476 non-FSGS patients and 54 patients with biopsy-proven idiopathic FSGS. In the non-FSGS patients, eGFR was the strongest significant determinant of suPAR. The proposed cutoff for suPAR in FSGS patients was exceeded in 17%, 39%, and 88% in patients with eGFRs of more than 60, 45-60, and 30-45 ml/min per 1.73 m(2), respectively. In patients with eGFR of <30 ml/min per 1.73 m(2), suPAR exceeded the cutoff in 95% of patients. Levels of suPAR in patients with idiopathic FSGS overlapped with non-FSGS controls and for any given eGFR did not discriminate FSGS cases from non-FSGS controls. In the overall cohort, there was a negative association between idiopathic FSGS and suPAR, and idiopathic FSGS was not an independent predictor of FSGS concentration over 3000 pg/ml. Thus, this study does not support an absolute, eGFR-independent, suPAR concentration cutoff as a biomarker for underlying FSGS pathology and questions the validity of relative, eGFR-dependent suPAR cutoff values.

GEP100 regulates epidermal growth factor-induced MDA-MB-231 breast cancer cell invasion through the activation of Arf6/ERK/uPAR signaling pathway.

GEP100, a guanine nucleotide exchanging factor (GEF) for Arf6, plays a pivotal role in promoting breast cancer cell invasion both in vitro and in vivo. However, the precise mechanism for GEP100-mediated cell invasion is still poorly understood. In this study, we found that down-regulation of endogenous GEP100 in MDA-MB-231 cells significantly inhibited EGF-induced cell invasion, which was rescued by over-expression of ectopic GEP100. EGF increased Arf6 activity, ERK phosphorylation, and uPAR expression in a time dependent manner. Additionally, blocking Arf6 with Arf6 siRNA largely abolished EGF-induced cell invasion. GEP100 siRNA or Arf6 siRNA suppressed EGF-induced ERK activity and uPAR expression. Furthermore, blocking ERK signaling with U0126, a specific inhibitor for MEK, markedly inhibited EGF-induced uPAR expression and consequently cell invasion. Inhibition of uPAR expression by uPAR siRNA also significantly abolished EGF-induced cell invasion. Taken together, this study illustrates that GEP100 regulates an Arf6/ERK/uPAR signaling cascade in EGF-induced breast cancer cell invasion. These findings could provide a rationale for designing new therapies based on inhibition of breast cancer metastasis.

Direct evidence of the importance of vitronectin and its interaction with the urokinase receptor in tumor growth.

Extensive evidence implicates the urokinase plasminogen activator receptor (uPAR) in tumor growth, invasion, and metastasis. Recent studies have substantiated the importance of the interaction between uPAR and the extracellular matrix protein vitronectin (VN) for the signaling activity of the receptor in vitro, however, the possible relevance of this interaction for the activity of uPAR in tumor growth and metastasis has not been assessed. We generated a panel of HEK293 cell lines expressing mouse uPAR (muPAR(WT)), an uPAR mutant specifically deficient in VN binding (muPAR(W32A)), and a truncation variant (muPAR(ΔD1)) deficient in both VN and uPA binding. In vitro cells expressing muPAR(WT) display increased cell adhesion, spreading, migration, and proliferation associated with increased p130Cas and MAPK signaling. Disruption of VN binding or ablation of both VN and uPA binding specifically abrogates these activities of uPAR. When xenografted into SCID (severe combined immunodeficiency) mice, the expression of muPAR(WT), but not muPAR(W32A) or muPAR(ΔD1), accelerates tumor development, demonstrating that VN binding is responsible for the tumor-promoting activity of uPAR in vivo. In an orthotopic xenograft model using MDA-MB-231 cells in RAG1(-/-)/VN(-/-) mice, we document that host deficiency in VN strongly impairs tumor formation. These 2 lines of in vivo experimentation independently demonstrate an important role for VN in tumor growth even if the uPAR dependence of the effect in the MDA-MB-231 model remains to be ascertained.