uPA Protein Research Articles

Urokinase-type plasminogen activator (uPA) system and invasion of early human extravillous trophoblast is regulated by hypoxia induced factor (HIF) pathway

Objective:Urokinase-type plasminogen activator (uPA) system is a critical protease pathway in human pregnancy. We have been reported the importance of uPA system on decidua and extravillous trophoblast (EVT) in early pregnancy, but the controlling molecule of the system remains unclear. In this study, we investigated the connection of hypoxia-induced factor (HIF) and uPA on primary EVTs to define the intracellular and extracellular protease pathway on early pregnancy. Methods: Human placental villi of 5–10 weeks gestation (obtainedafter informedconsent onartificial abortion) were minced, digested and cytotrophoblast fraction was separated with Percoll-based method. Cells were cultured on Matrigel® for 24h under 20%, 5% and 0.1% (three times of one hour hypoxic stimulation) oxygen condition with siRNA designed for knock-down of HIF. The invasive capacity of EVT (Matrigel-coated invasion assay), localization of HIF (immunofluorescence staining), and productions of HIF, uPA and receptor uPAR protein on EVT (Western Blot and ELISA) were studied. Results: HIF protein was increased under 5% oxygen condition than 20%. Invasive capacity was significantly decreased in the presence of siRNA for HIF. Productions of uPA and uPAR were also decreased in the presence of the siRNA. Conclusions: Hypoxic condition increased HIF expression and invasiveness of human primary EVT like cells, but upon addition of siRNA and knock-down of HIF in the cells, invasive capacity of the cells and expression of uPAR were reduced. It was thus suggested that uPA system in human trophoblast was induced by HIF and enhanced invasive capacity under hypoxic conditions.

Possible induction of murine miscarriages through activation of DEC-205+ dendritic cells by glycolipid antigens

Objective:Urokinase-type plasminogen activator (uPA) system is a critical protease pathway in human pregnancy. We have been reported the importance of uPA system on decidua and extravillous trophoblast (EVT) in early pregnancy, but the controlling molecule of the system remains unclear. In this study, we investigated the connection of hypoxia-induced factor (HIF) and uPA on primary EVTs to define the intracellular and extracellular protease pathway on early pregnancy. Methods: Human placental villi of 5–10 weeks gestation (obtainedafter informedconsent onartificial abortion) were minced, digested and cytotrophoblast fraction was separated with Percoll-based method. Cells were cultured on Matrigel® for 24h under 20%, 5% and 0.1% (three times of one hour hypoxic stimulation) oxygen condition with siRNA designed for knock-down of HIF. The invasive capacity of EVT (Matrigel-coated invasion assay), localization of HIF (immunofluorescence staining), and productions of HIF, uPA and receptor uPAR protein on EVT (Western Blot and ELISA) were studied. Results: HIF protein was increased under 5% oxygen condition than 20%. Invasive capacity was significantly decreased in the presence of siRNA for HIF. Productions of uPA and uPAR were also decreased in the presence of the siRNA. Conclusions: Hypoxic condition increased HIF expression and invasiveness of human primary EVT like cells, but upon addition of siRNA and knock-down of HIF in the cells, invasive capacity of the cells and expression of uPAR were reduced. It was thus suggested that uPA system in human trophoblast was induced by HIF and enhanced invasive capacity under hypoxic conditions.

Effect of adenovirus‑mediated uPA gene transduction on the fibrinolytic activity of human umbilical vein endothelial cells.

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

Up-regulation of urinary-type plasminogen activator correlates with high-risk papillary thyroid carcinoma with BRAFV600E mutation and its possible molecular mechanism

The aim of the present study is to investigate the relationship between urinary-type plasminogen activator (uPA) expression and clinicopathological features in papillary thyroid carcinoma (PTC) and to determine the signal transduction of PTC cells in vitro.PTC tissues from 42 patients were analyzed for the expression of uPA and the BRAFV600E mutation. BCPAP, a PTC cell line harboring the BRAFV600E mutation, was used to study MAPK signaling. PCR and direct sequencing were applied to analyze BRAFV600E mutation status. uPA mRNA expression was measured using a quantitative RT-PCR method, and uPA protein was localized using an immunohistochemical method. The ERK protein status was detected by Western blot analysis.uPA gene expression was significantly increased in PTC tissues as compared to the corresponding non-tumor tissues. Furthermore, the up-regulation of uPA mRNAs was correlated with high-risk clinicopathological features, including extrathyroid invasion, loss of cellular polarity/cohesiveness, and the BRAFV600E mutation. Marked dephosphorylation of ERK1/2 and down-regulation of uPA expression were detected when BCPAP was treated with a MEK inhibitor, U0126.MEK inhibitors might be a potential treatment strategy for aggressive PTC with BRAFV600E through inhibition of uPA expression.

Hyperglycemia impairs cytotrophoblast function via stress signaling

Diabetes mellitus is a risk factor for preeclampsia. Cytotrophoblast (CTB) invasion is facilitated from the conversion of plasminogen to plasmin by urokinase plasminogen activator (uPA), regulated by plasminogen activator inhibitor 1 (PAI-1), and may be inhibited in preeclampsia. This study assessed signaling mechanisms of hyperglycemia-induced CTB dysfunction. Human CTBs were treated with 45, 135, 225, 495, or 945 mg/dL glucose for 48 hours. Some cells were pretreated with a p38 inhibitor (SB203580) or a peroxisome proliferator-activated receptor-gamma (PPAR-γ) ligand (rosiglitazone). Expression of uPA, PAI-1, and PPAR-γ levels and p38 mitogen-activated protein kinase phosphorylation were measured by Western blot in cell lysates. Messenger ribonucleic acid of uPA and PAI-1 was measured by quantitative polymerase chain reaction. Levels of interleukin-6, angiogenic (vascular endothelial growth factor [VEGF], placenta growth factor [PlGF]) and antiangiogenic factors (soluble fms-like tyrosine kinase-1 [sFlt-1], soluble endoglin [sEng]) were measured in the media by enzyme-linked immunosorbent assay kits. Statistical comparisons were performed using analysis of variance with a Duncan's post-hoc test. Both uPA and PAI-1 protein and messenger ribonucleic acid were down-regulated (P < .05) in CTBs treated with 135 mg/dL glucose or greater compared with basal (45 mg/dL). The sEng, sFlt-1, and interleukin-6 were up-regulated, whereas the VEGF and PlGF were down-regulated by 135 mg/dL glucose or greater. p38 phosphorylation and PPAR-γ were up-regulated (P < .05) in hyperglycemia-treated CTBs. The SB203580 or rosiglitazone pretreatment showed an attenuation of glucose-induced down-regulation of uPA and PAI-1. Hyperglycemia disrupts the invasive profile of CTB by decreasing uPA and PAI-1 expression; down-regulating VEGF and PlGF; and up-regulating sEng, sFlt-1, and interleukin-6. Attenuation of CTB dysfunction by SB203580 or rosiglitazone pretreatment suggests the involvement of stress signaling.

A3.19 mIR-193B induces UPA in SSC and contributes to the proliferative vasculopathy via uPAR independent pathways

BackgroundWe had reported that miRNA-193b (miR-193b) is significantly downregulated in SSc and that down regulation of miR-193b causes overproduction of urokinase type plasminogen activator (uPA). However, the effects of increased...

62: Hyperglycemia impairs cytotrophoblast function via stress signaling

Diabetes mellitus is a risk factor for preeclampsia (preE). The normal extent of cytotrophoblast (CTB) invasion of the decidua, which is facilitated by plasmin, may be inhibited in preE. Inactive plasminogen is converted to plasmin by urokinase plasminogen activator (uPA), and uPA is regulated by plasminogen activator inhibitor 1 (PAI-1). Peroxisome proliferator-activated receptor gamma (PPARγ) also is implicated in the dysfunction of CTBs in hyperglycemic conditions. This study assesses the signaling mechanisms of hyperglycemia-induced CTB dysfunction. Human CTBs (Sw. 71) were treated with 45, 135, 225, 495 or 945 mg/dL glucose for 48h. Some cells were pretreated with a p38 inhibitor (SB203580) or a PPARγ ligand (rosiglitazone). Thereafter, cell lysates were utilized to measure uPA, PAI-1 and PPARγ expression and p38 Mitogen Activated Protein Kinase (MAPK) phosphorylation by western blot. The mRNA expression of uPA and PAI-1 in CTB lysates was measured by qPCR. Levels of angiogenic (soluble fms-like tyrosine kinase-1 (sFLT-1), soluble endoglin (sENG)) and anti-angiogenic factors (VEGF, PlGF) and IL-6 were measured in the culture media by ELISA. Both uPA and PAI-1 protein and mRNA expression were downregulated (p<0.05) in CTBs treated with >135 mg/dL glucose compared to basal (45 mg/dL). Anti-angiogenic factors (sENG, sFLT-1) and IL-6 were up-regulated, while the angiogenic factors (VEGF, PlGF) were down-regulated in the presence of >135 mg/dL glucose. The p38 MAPK phosphorylation and PPARγ expression were up-regulated (p<0.05) in hyperglycemia-exposed CTBs. SB203580 and rosiglitazone pretreatment attenuated glucose-induced down-regulation of uPA and up-regulation of p38 MAPK. Exposure of CTBs to excess glucose inhibits the invasive profile of CTBs by decreasing the expression of uPA and PAI-1; by downregulating of VEGF and PlGF; and upregulating of sENG, sFLT-1, and IL-6. Attenuation of CTB dysfunction by SB203580 or rosiglitazone pretreatment suggests the involvement of stress signaling.

Oncogenic K-Ras and Loss of Smad4 Mediate Invasion by Activating an EGFR/NF-κB Axis That Induces Expression of MMP9 and uPA in Human Pancreas Progenitor Cells

Activating K-Ras mutations and inactivating mutations of Smad4 are two common genetic alterations that occur in the development and progression of pancreatic ductal adenocarcinomas (PDAC). To further study the individual and combinatorial roles of these two mutations in the pathogenesis of PDAC, immortalized human pancreas nestin postive cells (HPNE) were genetically modified by either expressing oncogenic K-Ras (HPNE/K-Ras), by shRNA knock down of Smad4 (HPNE/ShSmad4) or by creating both alterations in the same cell line (HPNE/K-Ras/ShSmad4). We previously found that expression of oncogenic K-Ras caused an increase in expression of EGFR and loss of Smad4 further enhanced the up regulation in expression of EGFR and that this increase in EGFR was sufficient to induce invasion. Here we further investigated the mechanism that links mutational alterations and EGFR expression with invasion. The increase in EGFR signaling was associated with up regulation of MMP9 and uPA protein and activity. Moreover, the increase in EGFR signaling promoted a nuclear translocation and binding of RelA (p65), a subunit of NF-κB, to the promoters of both MMP-9 and uPA. Treatment of HPNE/K-Ras/ShSmad4 cells with an inhibitor of EGFR reduced EGF-mediated NF-κB nuclear translocation and inhibitors of either EGFR or NF-κB reduced the increase in MMP-9 or uPA expression. In conclusion, this study provides the mechanism of how a combination of oncogenic K-Ras and loss of Smad4 causes invasion and provides the basis for new strategies to inhibit metastases.

Snail mediates invasion through uPA/uPAR and the MAPK signaling pathway in prostate cancer cells

Epithelial-mesenchymal transition (EMT) is a process by which cancer cells acquire mesenchymal properties, such as induction of vimentin, while epithelial-associated genes like E-cadherin are lost. This enables cells to be more metastatic. Factors that are able to induce EMT include growth factors such as transforming growth factor-β (TGF-β) and epidermal growth factor, and transcription factors such as Snail. Snail-induced EMT promotes migration and invasion and we hypothesized that this may be mediated by the activity of urokinase-type plasminogen activator (uPA) and its receptor (uPAR). LNCaP, 22Rv1 and ARCaP human prostate cancer (CaP) cells stably transfected with empty vector control (Neo) or constitutively active Snail exhibited increased cell invasion. Superarray analysis revealed an upregulation in uPA and uPAR RNA expression in Snail-transfected ARCaP cells compared with that of a Neo control. In addition, the protein expression levels of Snail, uPA and uPAR were measured by western blot analysis which showed that overexpression of Snail increased uPA and uPAR protein levels. The activity of uPA in conditioned media was measured using an ELISA which revealed that uPA activity was elevated in LNCaP, 22Rv1 and ARCaP cells overexpressing Snail. Additionally, transient silencing of uPAR in ARCaP cells overexpressing Snail using short interfering RNA resulted in abrogation of Snail-mediated invasion. Snail overexpression was associated with increased extracellular-signal-regulated kinase activity, and antagonism of this activity with mitogen-activated protein (MAPK) inhibitor, UO126, inhibited cell invasion and decreased uPA activity. Therefore, Snail-mediated cell invasion in human CaP cells may occur via the regulation of uPA/uPAR and the MAPK signaling pathway.

Abstract 547: Excess Glucose Dowregulates Factors for Invasion and Induces an Anti-angiogenic Milieu in First Trimester Cytotrophoblast Cell

Objective: Diabetes mellitus is a known risk factor for the development of preeclampsia (preE). First trimester cytotrophoblastic (CTB) cells invade the decidua using the serine protease plasmin. The enzymatic conversion from the inactive plasminogen to the active form plasmin, is performed by urokinase plasminogen activator (uPA), which is regulated by plasminogen activator inhibitor 1 (PAI-1) in CTBs. Disruption of placental development, setting the stage for preE development is believed, in part, to be caused by a dysfunction of the invasive nature of CTBs. The aim of this study is to determine whether excess glucose induces anti-invasive and anti-angiogenic effects in CTBs in vitro . Methods: Human CTB cells (Sw. 71) were derived from first trimester placenta. The CTB cells were treated with 45, 135, 225, 495 or 945 mg/dL glucose for 72h. Thereafter, cell lysates were utilized to measure uPA and PAI-1 protein expression and phosphorylation of p38 MAPK by western blot. The mRNA expression of uPA and PAI-1 in glucose-induced CTB cell lysates was measure by qPCR. Levels of angiogenic and anti-angiogenic factors (sFLT-1, sENG, VEGF165, PlGF) and the level of inflammatory cytokine IL-6 were measured in the culture media by the commercially available kits. Statistical comparisons were performed using analysis of variance with Dunnett's post hoc tests. Results: Both uPA and PAI-1 protein and mRNA expression were downregulated (p&lt;0.05) in CTB cells treated with &gt;135 mg/dL glucose as compared to those in basal (45 mg/dL). The anti-angiogenic factors (sENG and sFLT-1) were upregulated, while the angiogenic factors (VEGF165 and PlGF) were downregulated in the presence of &gt;135 mg/dL glucose. The level of IL-6 was higher (p&lt;0.05) in the presence of glucose &gt;135 mg/dL compared to basal (45 mg/dL) conditions. The p38 MAPK phosphorylation was upregulated (p&lt;0.05) in excess glucose-treated CTBs. Conclusions: Exposure of CTB cells to glucose at levels associated with diabetes causes a disruption in the invasive profile of CTB cells by decreasing the synthesis of uPA and PAI-1 (both protein and mRNA expression); by downregulating expression of VEGF165 and PlGF; and by upregulating expression of sENG, sFLT-1, and IL-6. Moreover, glucose induced a stress signaling in CTBs.

OP0229 MIR-193B Induces uPA in SSC and Contributes to the Proliferative Vasculopathy Via uPAR Independent Pathways

BackgroundWe had reported that miRNA-193b (miR-193b) is significantly downregulated in SSc and that down regulation of miR-193b causes overproduction of urokinase type plasminogen activator (uPA). However, the effects of increased...

Peritoneal Fluid Reduces Angiogenesis-Related MicroRNA Expression in Cell Cultures of Endometrial and Endometriotic Tissues from Women with Endometriosis

Endometriosis, defined as the presence of endometrium outside the uterus, is one of the most frequent gynecological diseases. It has been suggested that modifications of both endometrial and peritoneal factors could be implicated in this disease. Endometriosis is a multifactorial disease in which angiogenesis and proteolysis are dysregulated. MicroRNAs (miRNAs) are small non-coding RNAs that regulate the protein expression and may be the main regulators of angiogenesis. Our hypothesis is that peritoneal fluid from women with endometriosis could modify the expression of several miRNAs that regulate angiogenesis and proteolysis in the endometriosis development. The objective of this study has been to evaluate the influence of endometriotic peritoneal fluid on the expression of six miRNAs related to angiogenesis, as well as several angiogenic and proteolytic factors in endometrial and endometriotic cell cultures from women with endometriosis compared with women without endometriosis.MethodsEndometrial and endometriotic cells were cultured and treated with endometriotic and control peritoneal fluid pools. We have studied the expression of six miRNAs (miR-16, -17-5p, -20a, -125a, -221, and -222) by RT-PCR and protein and mRNA levels of vascular endothelial growth factor-A, thrombospondin-1, urokinase plasminogen activator and plasminogen activator inhibitor-1 by ELISA and qRT-PCR respectively.ResultsControl and endometriotic peritoneal fluid pools induced a significant reduction of all miRNAs levels in endometrial and endometriotic cell cultures. Moreover, both peritoneal fluids induced a significant increase in VEGF-A, uPA and PAI-1 protein levels in all cell cultures without significant increase in mRNA levels. Endometrial cell cultures from patients treated with endometriotic peritoneal fluid showed lower expression of miRNAs and higher expression of VEGF-A protein levels than cultures from controls. In conclusion, this “in vitro” study indicates that peritoneal fluid from women with endometriosis modulates the expression of miRNAs that could contribute to the angiogenic and proteolytic disequilibrium observed in this disease.

Abstract 2623: Snail 1 regulates invasion through uPA-uPAR signaling using the MAPK pathway in prostate cancer cells.

Abstract Epithelial-mesenchymal transition (EMT) is a process by which cancer cells acquire mesenchymal properties, such as induction of N-cadherin, while epithelial-associated genes like E-cadherin are lost. This enables the cells to be more invasive, migratory and metastatic. Factors that can induce EMT include growth factors like transforming growth factor -β (TGF-β) and epidermal growth factor (EGF), and transcription factors like Snail1. Snail1-induced EMT promotes migration and invasion and we hypothesized that this may be mediated by urokinase (uPA) and its receptor (uPAR) activities. uPA, a serine protease, may be activated by integrin engagement to bind to its receptor, uPAR, and initiate a signaling cascade that regulates a variety of biological pathways including invasion. Androgen-dependent LNCaP and 22Rv1 and androgen-independent ARCaP human prostate cancer (PC) cells were stably transfected with empty vector control (Neo) or constitutively active Snail1 which led to increased cell migration and invasion. Superarray analysis revealed an upregulation in uPAR expression in Snail-transfected cells as compared to Neo control. Next, the protein expression levels of Snail1, uPA, and uPAR were measured by Western Blot analysis. uPA activity in conditioned media was measured using the Elisa uPA activity assay kit. Also, uPAR was transiently silenced in ARCaP Snail-overexpressing cells using uPAR siRNA. Additionally, cells were treated with MAPK inhibitor, UO126, at set time points. The data suggests that overexpression of Snail1 increased uPA and uPAR protein levels, and uPA activity was elevated in conditioned media from LNCaP, 22Rv1 and ARCaP Snail-overexpressing cells, as compared to Neo controls. Additionally, the silencing of uPAR decreased cell invasion in ARCaP Snail-overexpressing cells. Finally, MAPK inhibitor, UO126, inhibited cell invasion and decreased uPA activity. Therefore, Snail-mediated cell invasion in human prostate cancer cells may occur via the regulation of the MAPK and uPA/ uPAR signaling pathway. Acknowledgements: These studies were supported by the: NIH/NCRR/RCMI grant G12RR03062 and NIH P20 grant 1P20MD002285. Citation Format: Diandra D. Randle, Valerie Marah. Snail 1 regulates invasion through uPA-uPAR signaling using the MAPK pathway in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2623. doi:10.1158/1538-7445.AM2013-2623

Characterization of Spontaneous and TGF-β-Induced Cell Motility of Primary Human Normal and Neoplastic Mammary Cells In Vitro Using Novel Real-Time Technology

The clinical complications derived from metastatic disease are responsible for the majority of all breast cancer related deaths. Since cell migration and invasion are a prerequisite for metastasis their assessment in patient cancer cells in vitro may have prognostic value for the tumor's metastatic capacity. We employed real-time cell analysis (RTCA) on the xCELLigence DP system to determine in vitro motility of patient-derived primary human breast cancer epithelial cells (HBCEC). Initially, the RTCA assay was validated using established human breast cancer cell lines with either an invasive (MDA-MB-231, MDA-MB-435s) or a non-invasive phenotype (MCF-7, MDA-MB-468), and primary NSCLC cells (Tu459). Previous standard assays of cell migration/invasion revealed that only MDA-MB-231, −435s, and Tu459 cells exhibited spontaneous and TGF-β1-stimulated migration and invasion through a Matrigel barrier. In the present study, the TGF-β1-stimulated activities could be blocked by SB431542, a potent kinase inhibitor of the TGF-β type I receptor ALK5. Application of the RTCA assay to patient-derived tumor cells showed that 4/4 primary HBCEC and primary NSCLC cells, but not normal human mammary epithelial cells (HMEC), displayed high spontaneous migratory and invasive activity which correlated with higher MMP-2 expression and uPA protein levels in HBCEC compared to HMEC. Upon treatment with TGF-β1, HBCEC exhibited morphologic and gene regulatory alterations indicative of epithelial-to-mesenchymal transition. However, exclusively the invasive but not the migratory activity of HBCEC was further enhanced by TGF-β1. This indicates the requirement for molecular, e.g. integrin interactions with Matrigel components in HBCEC in order to become responsive to pro-invasive TGF-β effects. Together, these results show for the first time that tumorigenic HBCEC but not normal HMEC possess a strong basal migratory as well as a basal and TGF-β1-inducible invasive potential. These findings qualify the RTCA assay as an in vitro migration/invasion testing system for patient-specific primary breast cancer cells.

TGF-β1 alters microRNA profile in human gastric cancer cells.

MicroRNAs (miRNAs) are important regulators that play a key role in tumorigenesis and tumor progression. Transforming growth factor-β1 (TGF-β1) is involved in invasion and metastasis in many tumors. In this study, we investigated the microRNAs (miRNA) profiles altered by TGF-β1 in gastric cancer (GC) cells. We detected the expression profiles of miRNA by miRNA microarray and quantitative real-time polymerase chain reaction. Migration and invasion, wound-healing assay, prediction of miRNA targets, Western blot and qRT-PCR analysis were carried out to determine the role of one selected miRNA, namely miR-193b, in affecting the biological behaviors of GC BGC823 cells. Among 847 human miRNAs in the microarray, three miRNAs (miR-27a, miR-29b-1 and miR-194) were up-regulated and three (miR-574-3p, miR-193b and miR-130b) were down-regulated in BGC823 cells treated with TGF-β1 compared with control. miR-193b suppressed the invasion and metastasis of GC cells in vivo and in vitro, and down-regulated urokinase-type plasminogen activator (uPA) protein in GC cells. TGF-β1 altered miRNA expression profile in BGC823 cells. Among the altered miRNAs, TGF-β1 induced the down-regulation of miR-193b, which inhibited cell invasion and metastasis in vivo and in vitro, and down-regulated uPA protein in GC cells.

Small-molecule inhibition of the uPAR·uPA interaction: Synthesis, biochemical, cellular, in vivo pharmacokinetics and efficacy studies in breast cancer metastasis

The uPAR·uPA protein–protein interaction (PPI) is involved in signaling and proteolytic events that promote tumor invasion and metastasis. A previous study had identified 4 (IPR-803) from computational screening of a commercial chemical library and shown that the compound inhibited uPAR·uPA PPI in competition biochemical assays and invasion cellular studies. Here, we synthesize 4 to evaluate in vivo pharmacokinetic (PK) and efficacy studies in a murine breast cancer metastasis model. First, we show, using fluorescence polarization and saturation transfer difference (STD) NMR, that 4 binds directly to uPAR with sub-micromolar affinity of 0.2μM. We show that 4 blocks invasion of breast MDA-MB-231, and inhibits matrix metalloproteinase (MMP) breakdown of the extracellular matrix (ECM). Derivatives of 4 also inhibited MMP activity and blocked invasion in a concentration-dependent manner. Compound 4 also impaired MDA-MB-231 cell adhesion and migration. Extensive in vivo PK studies in NOD-SCID mice revealed a half-life of nearly 5h and peak concentration of 5μM. Similar levels of the inhibitor were detected in tumor tissue up to 10h. Female NSG mice inoculated with highly malignant TMD-MDA-MB-231 in their mammary fat pads showed that 4 impaired metastasis to the lungs with only four of the treated mice showing severe or marked metastasis compared to ten for the untreated mice. Compound 4 is a promising template for the development of compounds with enhanced PK parameters and greater efficacy.

Expression of Tax-interacting protein 1 (TIP-1) facilitates angiogenesis and tumor formation of human glioblastoma cells in nude mice

Glioblastoma is the most common and fatal type of primary brain tumors featured with hyperplastic blood vessels. Here, we performed meta-analyses of published data and established a correlation between high TIP-1 expression levels and the poor prognosis of glioblastoma patients. Next, we explored the biological relevance of TIP-1 expression in the pathogenesis of glioblastoma. By using orthotopic and heterotopic mouse models of human glioblastomas, this study has characterized TIP-1 as one contributing factor to the tumor-driven angiogenesis. In vitro and in vivo functional assays, along with biochemical analyses with microarrays and antibody arrays, have demonstrated that TIP-1 utilizes multiple pathways including modulating fibronectin gene expression and uPA protein secretion, to establish or maintain a pro-angiogenic microenvironment within human glioblastoma. In conclusion, this work supports one hypothesis that TIP-1 represents a novel prognostic biomarker and a therapeutic target of human glioblastoma.

Relationships of uPA and VEGF Expression in Esophageal Cancer and Microvascular Density with Tumorous Invasion and Metastasis

To investigate uPA and VEGF expression in esophageal cancer and relations with tumorous invasion and metastasis. Immunohistochemistry was used to detect uPA and VEGF expression in the normal epithelial tissue of esophageal mucosa and cancer tissue and detect CD34 labeled micrangium and analyze the relationships with clinical pathological features and tumor angiogenesis. Positive rates for uPA and VEGF protein expression were significantly greater in esophageal cancer than normal epithelial tissue (P < 0.05), the two being linked (P <0.05). In addition, uPA and VEGF protein expression of the high microvessel density (MVD) group was significantly lower than in the low MVD group (P < 0.05), with relation to clinical pathological staging, differentiation and lymph node metastasis (P < 0.05). In esophageal cancer tissue, uPA and VEGF proteins are overexpressed and promote tumor angiogenesis, indicative of a poor prognosis.

Abstract 2407: Snail 1 regulates invasion through uPA-uPAR and MMP-9 signaling in prostate cancer cells

Abstract Epithelial-mesenchymal transition (EMT) is a process by which cancer cells acquire mesenchymal properties, such as induction of N-cadherin, while epithelial-associated genes like E-cadherin are lost. This enables the cells to be more invasive, migratory and metastatic. Factors that can induce EMT include growth factors like transforming growth factor -α (TGF-β) and epidermal growth factor (EGF), and transcription factors like Snail1. Snail1-induced EMT promotes migration and invasion and we hypothesized that this may be mediated by urokinase (uPA) and its receptor (uPAR) activities. uPA, a serine protease, may be activated by integrin engagement to bind to its receptor, uPAR, and initiate a signaling cascade that regulates a variety of biological pathways including invasion. Since Snail1 can induce matrix metalloproteinases (MMP 2 and 9) which are also involved in cell invasion, we also examined MMP activity. Androgen-dependent LNCaP, and 22Rv1 and androgen-independent ARCaP human prostate cancer (PC) cells were stably transfected with empty vector control (Neo) or constitutively active Snail1 which led to increased cell migration and invasion. The levels of Snail1, uPA, and uPAR were measured by Western Blot analysis. uPA activity in conditioned media was measured using the Elisa uPA activity assay kit. Additionally, cells were treated with MAPK inhibitor, UO126, at set time points. Gelatin zymography analysis was utilized to detect MMP-9 activity in the conditioned media. Currently, ARCaP-Snail cells are being transiently transfected with uPAR siRNA to assay for the effects of uPAR knockdown on uPA and MMP activity. The data suggests that overexpression of Snail1 increased uPA and uPAR protein levels, while uPA activity was elevated in conditioned media from LNCaP, 22Rv1 and ARCaP cells. Also, MAPK inhibited MAPK expression in the cell and decreased uPA activity. In addition gelatin zymography showed higher MMP 9 activity in ARCaP-Snail overexpressing cells that was inhibited by MAPK inhibitor. Therefore, Snail-mediated cell migration and invasion in human prostate cancer cells may occur via the regulation of uPA and uPAR activities in addition to MMPs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2407. doi:1538-7445.AM2012-2407

Abstract 565: Benzyl isothiocyanate-mediated inhibition of epithelial-mesenchymal transition in human breast cancer cells is associated with downregulation of urokinase-type plasminogen activator and its receptor

Abstract Novel approaches for chemoprevention of breast cancer are clinically attractive not only because many risk factors associated with this disease are not easily modifiable (e.g., genetic predisposition) but also due to the fact that the currently available chemopreventive options targeted against breast cancer (e.g., estrogen receptor antagonists) are sub-optimal and plagued with adverse side effects. Needless to emphasize that breast cancer continues to be a leading cause of cancer-related death in American women despite significant advances towards targeted therapies. Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables (e.g., garden cress), seems promising for chemoprevention of breast cancer based on our own preclinical work. For example, we have shown previously that BITC administration not only inhibits growth of human breast cancer xenografts (MDA-MB-231) in athymic mice but also imparts significant protection against mammary cancer development in a transgenic mouse model (MMTV-neu). More recent studies from our laboratory have revealed that BITC is a potent inhibitor of epithelial-mesenchymal transition (EMT) in human breast cancer cells, but the mechanism underlying this effect is not known. In the present study, we addressed this question using MDA-MB-231, MDA-MB-468, and SUM159 human breast cancer cells as a model. Overexpression of superoxide dismutase, which confers significant protection against proapoptotic and proautophagic cell death responses to BITC, did not have any meaningful impact on BITC-mediated inhibition of EMT. Likewise, ectopic expression of constitutively active STAT3, a transcription factor implicated in EMT regulation, had no effect on EMT inhibition by BITC. Instead, the BITC-mediated inhibition of EMT was associated with suppression of protein and mRNA levels of urokinase-type plasminogen activator (uPA) and its receptor (uPAR). Secretion of uPA in culture media of breast cancer cells was also decreased significantly upon BITC treatment. BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was accompanied by downregulation of uPA and uPAR protein expression in the tumor and suppression of uPA secretion in plasma. Ectopic expression of uPAR in MDA-MB-468 and MCF-7 cells conferred partial but significant protection against BITC-mediated inhibition of EMT, cell migration, and/or cell invasion. Collectively, these results indicate that BITC-mediated inhibition of EMT is caused, at least in part, by suppression of the uPA/uPAR system. This investigation was supported by the USPHS grant RO1 CA129347-05, awarded by the National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 565. doi:1538-7445.AM2012-565