Expression Of Plasminogen Activator Receptor Research Articles

In vivo detection of urokinase-type plasminogen activator receptor (uPAR) expression in arterial atherogenesis using [64Cu]Cu-DOTA-AE105 positron emission tomography (PET)

Background and aimsUrokinase-type plasminogen activator receptor (uPAR) is associated with extracellular matrix (ECM) degradation and cancer aggressiveness. Its role in arterial atherogenesis as a molecular imaging target is not well-established. The aim of this study was to non-invasively visualize uPAR expression in atherosclerosis by a novel uPAR-targeting positron emission tomography (PET) tracer [64Cu]Cu-DOTA-AE105. MethodsWe used molecular biology to investigate uPAR expression by analyzing human atherosclerotic plaques and cultured cells. A retrospective analysis was performed on patients, who underwent combined PET/CT (n = 10) to measure [64Cu]Cu-DOTA-AE105 uptake in five large arteries, divided into a high and low-risk group based on coronary artery calcium score (CAC score). ResultsThe in vitro assay for THP-1 monocytes displayed a significantly upregulated uPAR expression upon stimulation (5.2-fold upregulation, p < 0.0001 by a one-way ANOVA followed by Tukey's test) by single-cell flowcytometric analysis. Freshly excised human atherosclerotic plaques underwent flow cytometric and microarray analyses manifesting 73.9 ± 2.9% of mononuclear phagocyte system (MPS) cells expressing uPAR and had a greater than 7-fold higher gene expression of plasminogen activator urokinase receptor (PLAUR, p = 0.002), integrin subunit alpha X (ITGAX, p = 0.0008), and cluster of differentiation 163 (CD163, p < 0.0001). The tissue-to-background ratios (TBRmax) in five large arteries showed a higher [64Cu]Cu-DOTA-AE105 uptake in the group with high CAC score compared to the group with low CAC score (2.4 ± 0.1 vs 1.7 ± 0.1, p = 0.057), significantly higher in the ascending aorta (2.7 ± 0.1 vs 2.0 ± 0.1, p = 0.038) and the abdominal aorta (3.2 ± 0.2 vs 2.0 ± 0.2, p = 0.038) by a non-parametric Mann-Whitney test. ConclusionsuPAR is abundantly expressed by MPS cells in atherosclerotic plaques and can be visualized by the novel PET tracer [64Cu]Cu-DOTA-AE105 that may non-invasively detect extracellular matrix remodeling during atherogenesis.

Abstract PO-127: A uPA/uPAR axis in both the tumor cell and stromal compartment drives PDAC disease progression

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal solid tumor malignancy with a 5-year survival rate of 9%. In both patients and animal models of disease, PDAC is associated with robust coagulation system activity. Intriguingly, in addition to being a rich source of procoagulant factors, PDAC tumors highly express fibrinolytic system components. Supporting this concept, urokinase plasminogen activator (uPA) and uPA receptor (uPAR) expression positively correlates with reduced overall patient survival. Here, we tested the hypothesis that the expression and activity of plasminogen activation (PA) system components are functionally linked to PDAC tumor growth and disease progression. We generated C57Bl/6-derived KPC (i.e., KRasG12D, TRP53R172H) PDAC cell lines in which uPA and uPAR were knocked out using CRISPR-Cas9. We then analyzed orthotopic tumor growth and experimental metastasis in mice carrying null or functional mutations in uPA, uPAR, or plasminogen to evaluate the interplay of PA components derived from tumor cells and/or stromal cells in mediating PDAC progression. Although both KPC cell CRISPR variants retained procoagulant function, elimination of tumor cell uPA or uPAR yielded significantly smaller tumors when compared to Cas9 control tumor cells in wildtype mice. Similarly, the growth of WT KPC tumor cells in C57Bl/6 background uPA-KO or uPAR-KO mice also resulted in reduced tumor growth. To our surprise, the metastasis potential of WT KPC tumor cells in uPA-KO or uPAR-KO mice did not change when compared to wildtype mice. Regarding to the uPA/uPAR axis downstream effector plasminogen, the growth of WT KPC tumors in plasminogen-KO mice was also significantly reduced, but not to the same extent as when eliminating uPA or uPAR. In addition, eliminating plasminogen drastically reduced WT KPC tumor cells metastasis potential. In conclusion, our data suggest a mechanism whereby uPA functions through uPAR in both the tumor cell and stromal cell compartments to promote PDAC progression through plasminogen-dependent and -independent mechanisms. Citation Format: Yi Yang, Sara R. Abrahams, Aditi Kothari, Harshi Matada, Keely Davey, Alisa S. Wolberg, Matthew J. Flick. A uPA/uPAR axis in both the tumor cell and stromal compartment drives PDAC disease progression [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-127.

Plasminogen Activator Urokinase Receptor Implies Immunosuppressive Features and Acts as an Unfavorable Prognostic Biomarker in Glioma.

Clinical outcomes of patients with glioma are still poor, even after standard treatments, including surgery combined with radiotherapy and chemotherapy. New therapeutic strategies and targets for glioma are urgently needed. Plasminogen activator urokinase receptor (PLAUR), a highly glycosylated integral membrane protein, is reported to modulate plasminogen activation and extracellular matrix degradation in many malignant cancers, but its role in gliomas remains unclear. Glioma samples with mRNA sequencing data and clinical information from the Chinese Glioma Genome Atlas (n = 310) data set and The Cancer Genome Atlas (n = 611) data set were collected for this study. Analyses using Kaplan-Meier plots, time-dependent receiver operating characteristic curves, Cox regression, and nomograms were conducted to evaluate the prognostic performance of PLAUR expression. Analyses using Metascape, ESTIMATE, EPIC, and immunohistochemical staining were performed to reveal the potential biological mechanism. The statistical analysis and graphical work were completed using SPSS, R language, and GraphPad Prism. PLAUR was highly expressed in phenotypes associated with glioma malignancy and could serve as an independent prognostic indicator. Functional analysis revealed the correlation between PLAUR and immune response. Further studies found that samples with higher PLAUR expression were infiltrated with fewer CD8 T cells and many more M2 macrophages. Strong positive correlation was demonstrated between PLAUR expression and some immunosuppressive markers, including immune checkpoints and cytokines. These findings were also confirmed in patient samples. Our results elucidated the clinical significance and immunosuppressive effect of PLAUR in gliomas, which might provide some clues in glioma immunotherapy. Although the efficacy of immunotherapy has been verified in other tumors, its application in glioma is impeded because of the unique microenvironment. Tumor-associated macrophages, which are particularly abundant in a glioma mass, contribute much to the immunosuppressive microenvironment and offer new opportunities in glioma immunotherapy. The results of this study identified plasminogen activator urokinase receptor (PLAUR) expression as a potential marker to predict the infiltration of macrophages and the status of immune microenvironment in patients with glioma, suggesting that treatment decisions could be based on PLAUR level when administering immunotherapeutics. The soluble PLAUR in blood and other body fluids would make this approach easy to implement in the clinic.

Cytokine-Specific Regulation of Urokinase Receptor (CD87) Expression by U937 Mononuclear Phagocytes

Mononuclear phagocytes concentrate urokinase-type plasminogen activator (uPA) at the cell surface by expressing membrane uPA receptors (uPAR). This study examines the ability of exogenous cytokines to alter expression of membrane-associated uPA and uPAR in U937 mononuclear phagocytes. Cells were stimulated with recombinant interferon gamma (IFN gamma) or tumor necrosis factor alpha (TNF alpha), followed by immunolabeling for uPA or uPAR and flow cytometry. IFN gamma increased surface uPA 2.2-fold relative to unstimulated controls (P < .001), whereas TNF alpha had no significant effect. Likewise, maximal uPA binding capacity was increased 2.8-fold by IFN gamma (P < .02), but was not affected by TNF alpha. In unstimulated cells, 50% of receptors were occupied by endogenously generated uPA, and this proportion was not affected by either cytokine. IFN gamma upregulated uPAR 2.1-fold relative to unstimulated controls (P < .001), whereas TNF alpha had no effect. In contrast to effects on surface protein, TNF alpha induced a substantial increase in uPAR mRNA, equaling the effect of IFN gamma. In addition, both cytokines doubled the intracellular uPAR pool (P < .01). By contrast, TNF alpha induced a 2.5-fold increase in the level of uPAR protein released into conditioned medium (compared with unstimulated cells), whereas IFN gamma had no effect. These results indicate that uPAR expression is regulated in a cytokine-specific fashion. Some stimuli, such as TNF alpha, may increase uPAR synthetic activity without a corresponding change in membrane expression, because of enhanced release of uPAR from the cell. Cytokine-specific modulation of uPAR may be important in regulating the function of mononuclear phagocytes in inflammation and tissue repair.

Urokinase receptor and tissue plasminogen activator as immediate-early genes in pentylenetetrazole-induced seizures in the mouse brain.

Epileptogenesis progressively leads to the rearrangement of normal neuronal networks into more excitable ones and can be viewed as a form of neuroplasticity, the molecular mechanisms of which still remain obscure. Here, we studied pentylenetetrazole seizure-induced regulation of genes for plasminogen activator system in the mouse brain. We found that expression of tissue plasminogen activator (tPA) and urokinase receptor (uPAR) mRNA was strongly increased in the mouse cerebral cortex, hippocampus, striatum and amygdala as early as 3hr after pentylenetetrazole seizures. Such early activity-induced expression of uPAR in the central nervous system has not been demonstrated before. uPAR mRNA accumulation was followed by elevation of uPAR protein, indicating a complete transcription-translation process. Both tPA gene induction and uPAR gene induction were independent of the protein synthesis, suggesting that they are regulated by neural activity as immediate-early genes. In contrast to tPA and uPAR genes, the expression of which returned to the basal level 6hr following seizures, urokinase and plasminogen activator inhibitor-1 gene expression showed a delayed activation only at 3days after seizures. In conclusion, our results suggest an important sensitivity of the brain plasminogen activator system to seizure activity which raises the question of its role in activity-dependent neural tissue remodeling in pathological and normal conditions.

Fiber-Reinforced Silk Composite for Enhanced Urokinase Production Using High-Density Perfusion Culture and Bioactive Molecule Supplementation.

Urokinase plasminogen activator (uPA) has been extensively used as a thrombolytic drug in cases of myocardial infarction, thromboembolism, and ischemic brain stroke. Media optimization and high-density perfusion culture are the decisive factors that facilitate enhanced urokinase production in a conditioned medium. In this study, we have aimed for a high-density perfusion culture of HT1080, a human fibrosarcoma cell line, by formulating optimal media for enhanced urokinase productivity. Four scaffold variants were fabricated from silk fibroin and microfibers of Bombyx mori (BM) and Antheraea assamensis (AA) and physico-chemically characterized. Field emission scanning electron microscopy studies revealed a heterogeneous distribution of pores with interconnected networks supporting cell infiltration, attachment, and long-term viability. AA-based fiber-reinforced scaffolds (ASAF) demonstrated superior mechanical strength, integral stability, and increased cell proliferation as compared to pure silk scaffolds. Media formulation was accomplished by limiting serum concentration (2% FBS) and supplementing with 20 μg/mL arginine and 20 ng/mL TGF-β1 to retain the stationary phase of cells and augment the urokinase production. A perfusion bioreactor culture of HT1080-laden scaffolds in the presence of formulated media was performed for improving the production of urokinase, with a maximum activity of 432 U/L. Also, gene expression analysis revealed that the individual silk scaffolds have different effects on regulating the expression of plasminogen activator urokinase and plasminogen activator urokinase receptor. In brief, our results suggest that a perfusion bioreactor culture of HT1080-laden ASAF scaffolds in formulated media promotes an increased urokinase production, such that it can be further used as a novel 3D matrix platform for industrial production of the lifesaving uPA drug.

Abstract 3469: CYP1B1 induces cancer progression through regulation of TRAIL pathway and uPA-uPAR system

Abstract Human CYP1B1 is known as a major metabolizer for estrogen and shows tumor-specific hyper-expression. To explore the role of CYP1B1 on progression of human cancer cells, we studied the effects of CYP1B1 in MCF-7, MDA-MB-231, and HeLa cells. CYP1B1 significantly induced invasion of cancer cells while strongly inhibited cancer cell apoptosis. Suppression of CYP1B1 by si/shRNA or treatment with TMS, a specific inhibitor for CYP1B1, markedly increased the protein expression level along with the level of release from cells of TNF-related apoptosis inducing ligand (TRAIL), a death ligand for cancer cells, whereas significantly decreased the expression of urokinase-type plasminogen activator receptor (uPAR) as well as uPA. We also found that CYP1B1 showed to inhibit the expression of key factors in TRAIL-induced apoptotic pathway and promote the inducers in uPA-uPAR metastasis system including integrin β1 and α5. Interestingly, uPAR overexpression only caused a significant increase of integrin β1 and α5 in protein levels, indicating protein degradation may play an important role in regulating integrin protein level. Surprisingly, CYP1B1 down-regulated p53 expression through MDM2 activation and nutlin-3a, an inhibitor for MDM2, blocked the promoting effects of CYP1B1 inducer, DMBA, on uPAR, which explains that CYP1B1 induced uPAR expression through regulation of MDM2-p53 system. Furthermore, we found that CYP1B1 suppresses TRAIL-related apoptosis through not only inhibition of TRAIL expression but also induction of DNA methylation on the promoter region of DR4, a receptor for TRAIL. Taken together, our data suggest that CYP1B1 promotes cancer cell metastasis via activating uPA-uPAR pathway which is one of the targets of p53 signaling and suppresses cancer cell apoptosis through inhibition of TRAIL-induced apoptosis by suppression of TRAIL release as well as post-translational regulation on DR4. Citation Format: Yeo-Jung Kwon, Young-Jin Chun. CYP1B1 induces cancer progression through regulation of TRAIL pathway and uPA-uPAR system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3469.

A novel oncogenic role for urokinase receptor in leukemia cells: molecular sponge for oncosuppressor microRNAs.

Urokinase receptor (uPAR) expression is up-regulated and represents a negative prognostic marker in most cancers. We previously reported that uPAR and CXCR4 can be regulated by common microRNAs in leukemia cells. Transcripts containing response elements for shared microRNAs in their 3’UTR may regulate their availability.We investigated uPAR 3’UTR capability to recruit microRNAs, thus regulating the expression of their targets. uPAR 3’UTR transfection in KG1 leukemia cells up-regulates the expression of endogenous uPAR. Transfection of uPAR 3’UTR, inserted downstream a reporter gene, increases uPAR expression and simultaneously down-regulates the reporter gene expression. Transfection of uPAR 3’UTR also increases CXCR4 expression; accordingly, uPAR silencing induces down-regulation of CXCR4 expression, through a mechanism involving Dicer, the endoribonuclease required for microRNA maturation.Transfection of uPAR 3’UTR also increases the expression of pro-tumoral factors and modulates cell adhesion and migration, consistently with the capability of uPAR3’UTR-recruited microRNAs to target several and different transcripts and, thus, functions.Finally, we found 3’UTR-containing variants of uPAR transcript in U937 leukemia cells, which show higher levels of uPAR expression as compared to KG1 cells, in which these variants are not detected.These results suggest that uPAR mRNA may recruit oncosuppressor microRNAs, allowing the expression of their targets.

Effects of Dahuang zhechong pill on doxorubicin-resistant SMMC-7721 xenografts in mice

Ethnopharmacological relevanceDahuang zhechong pill (DHZCP) is a famous traditional Chinese medicinal prescription from the “Synopsis of Prescriptions of the Golden Chamber (Jin Kui Yao Lue)”,Lue)”, an ancient Chinese medical classic. DHZCP is commonly used for clinical treatment of liver cancer by promoting blood circulation to dissolve blood stasis and by removing pathogenic vegetations.vegetations. DHZCP-based treatment has been derived from Traditional Chinese Medicine (TCM) and is officially recorded in the Chinese Pharmacopoeia. Aim of the studyThe aim of this study was to investigate the ability of DHZCP to reverse doxorubicin (DOX) resistance of SMMC-7721 cells in a xenograft mouse model, and to explore the underlying mechanisms. Materials and methodsLiquid chromatography–mass spectrometry was used to verify the composition of DHZCP. H&E staining was used to observe the pathological changes in hepatocellular carcinoma samples. Intracellular DOX accumulation was observed as intrinsic fluorescence by microscopy. Cell apoptosis was detected by the TUNEL assay. Human antibody arrays were used to analyze the expression of apoptotic- and angiogenic-related proteins. ATP levels were assessed and western blots were used to detect the protein expression of key enzymes of energy metabolism. ResultsDHZCP significantly reduced the tumor volume and weight of subcutaneous xenografts of drug-resistant hepatoma cells, and combining DHZCP with lower doses of DOX significantly increased the content of DOX in tumor tissue, increased the apoptosis of hepatoma cells, and reversed Dox resistance. With respect to 43 apoptosis-associated proteins, DHZCP regulated the expression of 5 of them. When combined with low-dose DOX, the expression of 40 apoptosis-related proteins was significantly altered. With respect to 23 angiogenesis-associated proteins, DHZCP upregulated the expression of endostatin and inhibited the expression of matrix metallopeptidase 9. When combined with low-dose DOX, DHZCP significantly downregulated protein expression of urokinase receptor, as well as vascular endothelial growth factor receptors 2 and 3. Especially, DHZCP significantly inhibited the expression of key enzymes of the tricarboxylic acid cycle and of oxidative phosphorylation, reducing the level of ATP in tumor tissue. ConclusionsDHZCP inhibited the growth of DOX-resistant hepatocellular carcinoma subcutaneous xenografts in nude mice and promoted increased apoptosis caused by DOX, thus reversing DOX resistance. This was associated with a decline in energy metabolism and regulated expression of pro-apoptotic proteins.

Abstract P1-01-08: The evolution of breast cancer circulating tumor cells mediating brain metastasis

Abstract Multiple studies concur that CTCs - the “seeds” of fatal metastasis - intravasate into the bloodstream throughout the early stages of cancer promoting generation of micro-metastatic reservoirs, some of which ultimately evolve to metastatic tumors. However, while it may take decades for CTCs to progress from cells in circulation to clinically detectable metastases, once detected, the tumor usually grows at an exponential rate. This difference in growth dynamics at two ends of the metastatic spectrum should also be reflected in the behavior of CTCs shed from these organ sites. Having a focus towards breast cancer brain-colonizing CTCs and their properties, we hypothesized that a gradual evolution of dormant CTCs into mitotically active CTCs with brain-metastatic potency is central to the progression of primary breast cancer to brain metastasis. We isolated CD44+/CD24- (stem-like) and Pan-Cytokeratin+ (epithelial) breast cancer CTCs from peripheral blood of two groups of breast cancer patients – five with brain metastases (BCBM) (MRI-detectable) and five without brain metastases (No BCBM). CTCs were isolated by multi-parametric flow cytometry and the expression of CTC-specific antigen was confirmed by immuno-cytochemistry and DEPArrayTM platform. Intra-cellular flow cytometry showed that No BCBM patients had 3-fold higher mitotically dormant (Ki67-) CTCs compared to BCBM patients. Whole genome expression arrays demonstrated low generalized transcriptional activity in CTCs suggestive of mitotic/metabolic dormancy. Comparison of CTC transcriptomes with ER+/PR+, HER2+ and triple negative breast cancers identified a unique CTC gene signature valid not only across the three molecular subtypes but also independent of the molecular subtype patients were initially diagnosed as. Pathway analyses predicted increased activation of pluripotency-related pathways along with decreased protein translational machinery and proliferative pathways in CTCs. Further, to dissect mechanisms of metastatic reactivation in the brain microenvironment, we compared the CTC transcriptome in BCBM vs No BCBM patients and identified a 126 gene signature potentiated in BCBM CTCs. Subsequent pathway analyses revealed upregulation of known CTC pathways like Notch along with novel hematopoietic and immune evasion networks. Cellular and functional annotations of cell migration and chemotaxis were significantly activated in BCBM along with pro-inflammatory (TNF, IL1β, NF-kB), immunomodulatory chemokines (CXCL8, CXCR4, CD86) and mitogenic growth factors (PDGF-BB). Lastly, lack of cell surface expression of Urokinase Plasminogen Activator Receptor (uPAR) and integrin beta-1 (int-β1) were used to identify CTCs in a state of metastatic dormancy. Whole genome sequencing showed significantly higher incidence of genomic mutations in proliferating CTCs compared to their dormant counterparts. We provide first time evidence of signaling pathways keenly implicated in breast cancer CTC biology based on comprehensive analyses of CTC transcriptomes. We discovered the existence of CTCs in a state of metabolic/mitotic dormancy and identified genomic instability as a switch for CTCs to revert to their proliferative phenotype. Citation Format: Boral D, Vishnoi M, Liu HN, Yin W, Hong DS, Scamardo AT, Marchetti D. The evolution of breast cancer circulating tumor cells mediating brain metastasis [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-08.

Effect of mycophenolic acid in experimental, nontransplant glomerular diseases: new mechanisms beyond immune cells

Mycophenolic acid (MPA) was introduced into clinical practice as immunosuppressive drug therapy to prevent allograft rejection. Since then, its clinical application has widened. Our goal was to review the lessons learned from experimental nontransplant glomerular disease models on the mechanisms of MPA therapy. T and B lymphocytes are preferentially dependent on de novo purine synthesis. By inhibiting the rate-limiting enzyme of de novo purine synthesis, MPA depletes the pool of deoxyguanosine triphosphate (dGTP) and inhibits proliferation of these immune cells. Furthermore, MPA can also induce apoptosis of immune cells and is known to inhibit synthesis of fucose- and mannose-containing membrane glycoproteins altering the surface expression and binding ability of adhesion molecules. However, MPA exerts a direct effect also on nonimmune cells. Mesangial cells are partially dependent on de novo purine biosynthesis and are thus susceptible to MPA treatment. Additionally, MPA can inhibit apoptosis in podocytes and seems to be beneficial in preserving the expression of nephrin and podocin, and by attenuation of urokinase receptor expression leads to decreased foot-process effacement. In summary, our manuscript sheds light on the molecular mechanisms underlying the antiproteinuric effect of MPA. Overall, MPA is an excellent treatment option in many immunologic glomerulopathies because it possesses immunosuppressive properties, has a remarkable effect on nonimmune cells and counteracts the proliferation of mesangial cells, expansion of mesangial matrix, and foot-process effacement of podocytes combined with a low systemic toxicity.

Clinical Significance of CD87 Expression in Acute Myeloblastic Leukemia.

CD87 or uPAR (urokinase type plasminogen activator receptor) expression leads to conversion of plasminogen to plasmin to mediate extravasation by proteolytic breakdown of adhesion molecules responsible from cell contact. uPA (urokinase type plasminogen activator) system consists of a proteinase, its receptor uPAR, and two inhibitors (plasminogen activator inhibitor [PAI] 1 and 2). CD87 expression may have implications for diagnosing acute myeloblastic leukemia (AML) and determining its prognosis, as well as for follow-up of minimal residual disease (MRD). In this study we determined CD87 levels in bone marrow or peripheral blood immunophenotypically and investigated its relationship with some prognostic factors in 35 newly diagnosed and untreated AML patients. While there was no significant correlation between CD87 expression and age, sex, admission white cell count, and CD34 expression, a significant relationship was found between CD87 expression and organomegaly. This relationship was prominent in cases with AML showing monocytoid differentiation. No correlation was found between CD87 expression and treatment response. In AML patients hepatosplenomegaly and mucocutaneous infiltrations are associated with increased CD87 expression. Extramedullary extension is common in leukemias with monocytoid differentiation. uPA, by binding to CD87, initiates conversion of plasminogen into plasmin to mediate extravasation of cells through endothelium by proteolytic breakdown of endothelium-associated adhesion molecules. It plays role in adhesion, migration and metastasis of leukemia cells. This condition may also explain the relationship between CD87 and the rate of organomegaly. It may prove a useful marker for follow-up of MRD when detected at the time of diagnosis.

UPA receptor and its interaction partners: Impact as potential therapeutic targets in triple-negative breast cancer.

150 Background: Triple-negative breast cancers (TNBC), which lack expression of estrogen (ER), progesterone (PR), and HER2 receptors are characterized by tumor tissue expression of the urokinase-type plasminogen activator receptor (uPAR) system. Methods: We evaluated uPAR and its interaction partners (uPAR-interactome) by immunohistochemistry (IHC) in tissue microarrays (TMA) prepared from ~300 TNBC tumor tissue specimens and in the TNBC model cell lines BT549 and MDA-MB231. Protein expression of 25 different factors constituting the uPAR-interactome was evaluated by IHC; several of the uPAR-protein complexes were identified employing the proximity ligation assay (PLA). Results were correlated with histomorphological parameters and survival of the patients. RNA interference was employed in the TNBC cell lines for several of the parameters assessed. To analyze effects of uPAR and/or uPA depletion on malignant cell behavior, proliferation, migration, and invasion assays as well as Western blot, PLA, immunoprecipitation, mass spectrometry, and immunohistochemistry were conducted. Results: After downregulation of uPAR and uPA in TNBC cell lines, significantly reduced cell proliferation, invasion, and migration were observed. Furthermore, interaction between uPAR and IGF1R (insulin-like growth factor1 receptor) was shown, and several novel interaction proteins of uPAR could be identified, such as Cyr61 (cysteine-rich angiogenic inducer61). In TNBC tumor specimens, association between uPAR expression and histological grade plus expression of cathepsin B, IGF1R, IR (insulin receptor), and plasminogen was shown. Expression of KLK4 (stromal) and Ki67 was correlated with disease-free survival of the patients. Presence of uPAR/IGF1R protein complexes was associated with histological grade, with the expression of PAI-1, Ki-67, p27, and cathepsin B. Conclusions: Our findings demonstrate that uPAR and its interacting partners, expressed in TNBC tumor tissues, may be considered as potential novel therapeutic targets for this fatal disease.

Abstract 363: Dissecting CTC phenotypes: insights into mechanisms of breast cancer dormancy

Abstract Uncovering phenotypes of patient-derived Circulating Tumor Cells (CTCs) offers the promise to dissect CTC heterogeneity in relation to metastatic competence, and to determine biomarkers of therapeutic utility for improved treatment. However, it is still unknown whether and how CTCs differ in their capacity to circulate while maintaining metastatic potential. Rates of CTC survival are highly variable, lasting less than few hours in some patients but in the order of decades in others. This can lead to many questions for yet unexplored mechanisms of CTCs responsible for dormancy, along with their properties and biomarker functionalities. We hypothesized that breast cancer CTC subsets possessing markers of pluripotency avoid organ arrest with extreme efficiency by the concomitant presence of quiescence and stem cell properties; and that expression of urokinase plasminogen activator receptor (uPAR) and beta-1 integrin (β1int), two biomarkers known to be directly implicated in tumor cell dormancy, are relevant in controlling the recurrence of breast cancer brain metastasis (BCBM). First, we isolated CTC subsets not expressing the epithelial cell adhesion molecule (EpCAM-negative CTCs), and characterized these subsets using DEPArrayTM, a new CTC platform able to dissect CTC heterogeneity at a single-cell level, thus interrogating the smallest functional unit of cancer. We captured EpCAM-negative/CD45-/CD44+/CD24- breast cancer CTC subsets that possessed combinatorial uPAR and β1int expression using multiparametric flow cytometry. Second, CTC subsets grew in vitro and were further characterized by DEPArrayTM. Markers expression was confirmed by confocal microscopy with subsets possessing a specific breast cancer gene profiling. Third, EpCAM-negative CTC subsets (uPAR+/β1int+ and uPAR-/β1int-) were interrogated for human embryonic stem cell markers by RT2 PCR arrays. Gene expression profiling was consistently distinct among uPAR+/β1int+ vs. uPAR-/β1int- CTC subsets and dependent upon patients’ BCBM status: expression of genes implicated in cell cycle progression (e.g., CDK42, CDK1), angiogenesis (e.g., FGF-2), and pluripotency (e.g., KLF4) was &amp;gt;30-fold higher than controls. Third, CTC subsets gene patterns isolated from patients with BCBM possessed RT2 profiles that were strikingly distinct from ones derived from patients with no BCBM. Of note, gene expression for RIF-1, a protein that counteracts actions of the breast cancer suppressor BRCA1, was highest (&amp;gt;50-fold) with distinct RIF-1 nuclear patterns in BCBM CTC subsets. In summary, we have linked EpCAM-negative uPAR/β1int CTC subsets and their properties to clinical BCBM; and will assess the therapeutic inhibition of uPAR/β1int CTC biomarkers on BCBM development and its timing. Deciphering the relevance of uPAR/β1int as key CTC biomarkers of dormancy vs. metastatic competence will elucidate CTC mechanisms responsible for BCBM onset. Citation Format: Sirisha Peddibhotla, Monika Vishnoi, Wei Yin, Yizhen Chen, Antonio Scamardo, David Hong, Dario Marchetti. Dissecting CTC phenotypes: insights into mechanisms of breast cancer dormancy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 363. doi:10.1158/1538-7445.AM2015-363

Effects of uPA on mesangial matrix changes in the kidney of diabetic rats

Objective: To investigate the effect of urokinase-type plasminogen activator (uPA) on mesangial matrix in the kidney of diabetic rats and its related mechanisms. Methods: Diabetic Sprague-Dawley (SD) rats induced by intraperitoneal injection of streptozotocin (STZ) were randomly and evenly divided into two groups: DM + vehicle, and DM + uPA (2500 U kg−1 uPA via tail vein once a day for four weeks). The normal SD rats without diabetes were considered as control group. Rats in the three groups were executed and the heart blood was sampled for determination of blood glucose and serum creatinine. Meanwhile, kidney tissues of rats were also harvest for measurement of glomerular area, volume, and mesangial area by periodic acid silver methenamine (PASA) staining. The expression of urokinase-type plasminogen activator receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1), and collagen IV in renal tissues was tested with immunohistochemistry. Results: Compared with control, the DM rats had obvious albuminuria, significantly (p < 0.01) increased glomerular volume and mesangial matrix area, and significantly (p < 0.05) higher expression of uPAR, PAI-1 and collagen IV in mesangial matrix, significantly up-regulated (p < 0.05) glomerular uPAR, PAI-1, and collagen IV expression. After treated with uPA, the diabetic rats had significantly (p < 0.05) reduced albuminuria, significantly (p < 0.01) improved glomerular volume and mesangial matrix, significantly (p < 0.05) down-regulated PAI-1 and collagen IV expression in mesangial matrix. However, the uPAR expression in renal tissues were unchangeable (p > 0.05) and PAI-1 and collagen IV expression were significantly (p < 0.05) reduced when diabetic rats were treated with uPA. Conclusion: uPA can down-regulate glomerular PAI-1 expression in the DM rats but not significantly influence uPAR expression, suggesting that uPA might regulate the mesangial cell (MC) and its matrix expression and improve diseased diabetic mesangial matrix via its combination with uPAR to uptake PAI-1 and accelerate its degradation.

Urokinase, urokinase receptor, and plasminogen activator inhibitor-1 expression on podocytes in immunoglobulin A glomerulonephritis

Background/AimsThe purpose of this study was to investigate the expression of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), and plasminogen activator inhibitor (PAI)-1 on podocytes in immunoglobulin A (IgA) glomerulonephritis (GN).MethodsRenal biopsy specimens from 52 IgA GN patients were deparaffinized and subjected to immunohistochemical staining for uPA, PAI-1, and uPAR. The biopsies were classified into three groups according to the expression of uPA and uPAR on podocytes: uPA, uPAR, and a negative group. The prevalences of the variables of the Oxford classification for IgA GN were compared among the groups.ResultsOn podocytes, uPA was positive in 11 cases and uPAR was positive in 38 cases; by contrast, PAI-1 was negative in all cases. Expression of both uPA and uPAR on podocytes was less frequently accompanied by tubulointerstitial fibrosis.ConclusionsOur results suggest a possible protective effect of podocyte uPA/uPAR expression against interstitial fibrosis.

Urokinase-type plasminogen activator receptor in IgA nephropathy

Urokinase-type plasminogen activator receptor in IgA nephropathy

The critical role of dysregulated FOXM1–PLAUR signaling in human colon cancer progression and metastasis.

405 Background: The mammalian Forkhead Box (Fox) transcription factor FOXM1 is implicated in tumorigenesis including mouse intestinal cancer. However, the clinical significance of FOXM1 signaling in human colorectal cancer (CRC) pathogenesis remains unknown. Methods: We investigated FOXM1 expression in 203 cases of primary colon cancer and matched normal colon tissue specimens and explored the underlying mechanisms of altered FOXM1 expression and the impact of this altered expression on colon cancer growth and metastasis using in vitro and animal models of colon cancer. Results: We found weak expression of FOXM1 protein in the colon mucosa, whereas we observed strong FOXM1 expression in tumor-cell nuclei of colon cancer and lymph node metastases. A Cox proportional hazards model revealed that FOXM1 expression was an independent prognostic factor in multivariate analysis. Experimentally, overexpression of FOXM1 by gene transfer significantly promoted the growth and metastasis of colon cancer cells in orthotopic mouse models, whereas knockdown of FOXM1 expression by small interfering RNA did the opposite. Promotion of colon tumorigenesis by FOXM1 directly and significantly correlated with activation of urokinase plasminogen activator receptor (PLAUR) expression and elevation of invasion and metastasis. Conclusions: Given the importance of FOXM1 in regulation of the expression of genes key to cancer biology, dysregulated expression and activation of FOXM1 may play important roles in colon cancer progression and metastasis.

Urokinase Receptor Mediates Doxorubicin-Induced Vascular Smooth Muscle Cell Senescence via Proteasomal Degradation of TRF2

The anthracycline doxorubicin is a widely used effective anti-cancer drug. However, its application and dosage are severely limited due to its cardiotoxicity. The exact mechanisms of doxorubicin-induced cardiotoxic side effects remain poorly understood. Even less is known about the impact of doxorubicin treatment on vascular damage. We found that low doses of doxorubicin induced a senescent response in human primary vascular smooth muscle cells (VSMC). We observed that expression of urokinase receptor (uPAR) was upregulated in response to doxorubicin. Furthermore, the level of uPAR expression played a decisive role in developing doxorubicin-induced senescence. uPAR silencing in human VSMC by means of RNA interference as well as uPAR knockout in mouse VSMC resulted in abrogation of doxorubicin-induced cellular senescence. On the contrary, uPAR overexpression promoted VSMC senescence. We further found that proteasomal degradation of telomeric repeat binding factor 2 (TRF2) mediates doxorubicin-induced VSMC senescence. Our results demonstrate that uPAR controls the ubiquitin-proteasome system in VSMC and regulates doxorubicin-induced TRF2 ubiquitination and proteasomal degradation via this mechanism. Therefore, VSMC senescence induced by low doses of doxorubicin may contribute to vascular damage upon doxorubicin treatment. uPAR-mediated TRF2 ubiquitination and proteasomal degradation are further identified as a molecular mechanism underlying this process.

Invasion-associated genes identified by gene expression profiling in extranodal natural killer/T-cell lymphoma, nasal type

To identify invasion-associated genes in extranodal natural killer (NK)/T-cell lymphoma, we performed microarray analysis on seven tumor samples and two control pools (composed of normal NK cells and T cells, tonsil and spleen) using Affymetrix GeneChip. Compared with all control pools, 59 uniquely expressed genes were discovered in the tumor samples. Overexpressed genes related to proteolysis, cell motility and chemotaxis, including CTSL, uPAR, TIMP-1, CXCL9, CXCL11 and DEFB1, were identified. Comparing the gene expression profiles of five upper aerodigestive tract (UAT) cases with two non-UAT cases, we found some overexpressed genes in non-UAT cases related to proteolysis and cell adhesion function, including matrix metalloproteinase 9 (MMP-9). Immunohistochemistry detection was performed on 34 paraffin sections to evaluate the expression of selected genes. A correlation of urokinase-type plasminogen activator receptor (uPAR) expression with MMP-9 expression was revealed. Analysis of prognosis demonstrated that expressions of MMP-2 and MMP-9 were closely correlated with a poor prognosis. These invasion-associated genes may become targets for diagnostic and therapeutic procedures.