uPA Gene Expression Research Articles

Interleukin-17 modulates uPA and MMP2 expression in human periodontal ligament mesenchymal stem cells: Involvement of the ERK1/2 MAPK pathway

Periodontal disease is a chronic infection of periodontal tissue characterized by extracellular matrix (ECM) degradation due to increased expression of plasminogen activators and matrix metalloproteinases (MMPs) and various proinflammatory cytokines, including interleukin (IL)-17. Successful regeneration of damaged periodontal tissues depends on the proper functionality of periodontal ligament mesenchymal stem cells (PDLMSCs), especially the production of extracellular matrix proteases. We investigated the influence of IL-17 on ECM remodeling through modulation of urokinasetype plasminogen activator (uPA) and MMP2/MMP9 expression in human PDLMSCs at mRNA, protein and activity levels using by RT-PCR, Western blotting and zymography, respectively. Investigation of the involvement of MAPKs in these processes in PDLMSCs was determined by Western blotting, as well as by utilizing specific p38 and MEK1/2 inhibitors. Our results show that IL-17 activates MAPK signaling in PDLMSCs. Moreover, IL-17 had no effect on MMP9 expression, but it stimulated uPA and MMP2 gene and protein expression in PDLMSCs through the activation of the ERK1/2 MAPK signaling pathway. The obtained data suggest that IL-17 contributes to ECM degradation in the periodontal ligament by stimulating uPA and MMP2 expression and activity in PDLMSCs. This information is important for understanding periodontal disease development and defining future directions of its treatment.

Comparison of the effects of olsalazine and decitabine on the expression of CDH1 and uPA genes and cytotoxicity in MDA-MB-231 breast cancer cells.

Background and purpose:Since DNA methyltransferase enzymes play a key role in DNA methylation, they can be used as a target to alter epigenetic changes and treat cancer. Recent studies have shown that olsalazine, through its potent inhibitory effect on the DNA methyltransferase enzyme, can be a good option. The aim of this study was to investigate the effects of olsalazine on cell viability and expression of CDH1 and uPA genes in MDA-MB-231 cells compared with decitabine.Experimental approach:The cytotoxicity of the drugs was determined using a standard MTT assay. MDA-MB-231 cells were treated with olsalazine and decitabine with concentrations less than IC50 to evaluate the effect of drugs on the expression of genes. RNA was extracted from the cells after 24 and 48 h and CDH1and uPA gene expression were evaluated by quantitative real-time polymerase chain reaction method.Findings/Results:The cytotoxicity of the two drugs was comparable. The IC50 values at 24 h were 4000 and 4500 μM for olsalazine and decitabine, respectively. The IC50 values of both drugs were about 300 μM at 48 h. Statistical analyzes showed a significant increase in CDH1 expression after 24-48 h treatment with olsalazine, and 48 h treatment with decitabine, without any significant increase in uPA expression.Conclusion and implications:Our results showed that olsalazine has cellular toxicity comparable to decitabine in MDA-MB-231 cells. Also compared to decitabine, olsalazine causes a greater increase in expression of CDH1 without any significant increase in uPA expression. Therefore, it appears to be a good candidate for cancer treatment.

Staphylococcus aureus on the effect of expression of MMPs/TIMPs and uPA system in bovine mammary fibroblasts.

Bovine mammary fibrosis is characteristic of chronic in injury in response to diverse pathogens. Staphylococcus aureus (S.aureus) is a frequent cause of mastitis in bovine and is prone to persistent infection. Diverse studies have shown MMPs/TIMPs and uPA system as a potent target for the treatment of fibrosis. However, pathogenesis of S. aureus-induced mammary fibrosis has not been completely defined. BMFBs treated with heat-inactivated S. aureus (105, 106, and 108CFU/mL) for 6, 12, 24, and 48h. Total RNA and protein were isolated from the treatments and controls of BMFBs samples. MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, TIMP-1, TIMP-2, COL Ⅰ, uPA, uPAR and PAI-1 gene and protein expression were examined by RT-qPCR and Western blot analysis. Gelatin zymography assay was performed to assess the levels of MMP-2 and MMP-9 enzyme secreted. BMFBs treated with heat-inactivated S. aureus increased mRNA and protein expression levels of MMP-1, MMP-2, MMP-3, MMP-9 and MMP-13, and heat-inactivated S. aureus induced TIMP-1, TIMP-2 and COL Ⅰ expression. There was a clear activation of MMP-2 in the presence of heat-inactivated S. aureus in the conditioned medium from the BMFBs, whereas MMP-9 was no significantly altered. Moreover, uPA system was activated in BMFBs to S. aureus. Activation of the uPA system together with its impact on the MMPs levels could play a significant role in S. aureus-induced BMFBs with mechanism of ECM metabolism, MMPs/TIMPs and uPA system could participate in bovine mammary fibrosis.

Peptide P7 inhibits the bFGF-stimulated proliferation and invasion of SKOV3 cells.

Peptide P7 specifically binds with basic fibroblast growth factor (bFGF) to inhibit the proliferation and invasion of numerous types of cancer cell. However, this effect has remained to be demonstrated in ovarian cancer-derived cell lines. In the present study, the protein P7 was used treat bFGF-stimulated SKOV3 epithelial ovarian cancer cells to explore the therapeutic potential of P7. An MTT and a scratch wound assay were used to respectively evaluate the proliferation and migration of bFGF-stimulated SKOV3 cells treated with P7. Reverse transcription-quantitative polymerase chain reaction analysis was used to detect the gene expression of urokinase-type plasminogen activator (uPA), as well as matrix metallopeptidase (MMP)-2 and −9, which have a role in cell migration/invasion. The morphology and proliferation of SKOV3 cells were not significantly affected by different concentrations of P7. However, P7 had an obvious inhibitory effect on the proliferation and migration of bFGF-stimulated SKOV3 cells. Treatment with P7 significantly lowered the gene expression of uPA, MMP-2 and MMP-9 compared with that in the control group. In conclusion, the present results suggested that P7, which, at least in part, acts through inhibition of bFGF, may have a potential therapeutic application in epithelial ovarian cancer.

Anti-metastatic potential of a proton beam is regulated by p38 MAPK/c-Fos signaling pathway in TPA-treated HepG2 human hepatocellular carcinoma

A proton beam therapy is considered the next generation radio-therapeutic tool for liver cancer treatments. However, its effect on metastasis has not been fully elucidated. Herein, we assessed the effects of a proton beam on the metastatic potential in HepG2, Hep3B and SK-Hep1 hepatocellular carcinomas. The result showed that a proton beam suppressed TPA-induced cell migration and invasion in HepG2 cells, but not in Hep3B and SK-Hep1 cells. In addition, matrix metalloproteinase-9 (MMP-9) activity and mRNA expressions were reversed by proton beam irradiation in TPA-treated HepG2 cells only. Furthermore, a proton beam suppressed TPA-induced gene expressions of urokinase plasminogen activator (uPA), uPA receptor (uPAR), Snail-1 and vascular endothelial growth factor (VEGF) in HepG2 cells in a dose-dependent manner. Moreover, we found that proton beam irradiation restrained p38 MAPK phosphorylation and c-Fos expression. Therefore, the result demonstrates that the anti-metastatic effects of a proton beam in TPA-treated HepG2 cells are associated with the inhibition of MMP-9 activity and the down-regulations of MMP-9, uPA, uPAR, Snail-1 and VEGF gene expression via the p38 MAPK/c-Fos signaling pathway. Taken together, this investigation suggests that the establishment of a customized proton beam therapeutic strategy for each liver cancer type is necessary to improve therapeutic efficiency.

Scabraside D Derived from Sea Cucumber Induces Apoptosis and Inhibits Metastasis via iNOS and STAT-3 Expression in Human Cholangiocarcinoma Xenografts.

Scabraside D, a sulfated triterpene glycoside, was extracted from the sea cucumber Holothuria scabra. It shows anti-proliferation in many of cancer cell lines, but the function and mechanisms of action of scabraside D in human cholangiocarcinoma (HuCCA) have not previously determined. In this study, we investigated the activity of scabraside D on HuCCA cell apoptosis, lymphangiogenesis and metastasis in a nude mouse model. Scabraside D induced signs of apoptosis, such as cell shrinkage, nuclear condensation, nuclear fragmentation and DNA fragmentation on TUNEL assays, while effectively decreasing expression of BCl-2 but increasing caspase-3 gene level expression. Immunohistochemistry revealed that scabraside D significantly reduced lymphatic vessel density (LVD). Moreover, scabraside D treatment significantly decreased VEGF-C, MMP-9 and uPA gene expression, which play important roles in the lymphangiogenesis and invasion of cancer cells in metastasis processes. Quantitative real-time PCR showed that scabraside D significantly decreased iNOS and STAT-3 gene expression. This study demonstrated that scabraside D plays a role in activation of HuCCA tumor apoptosis and inhibition of lymphangiogenesis, invasion and metastasis through decreasing BCl-2, MMP-9, uPA and VEGF-C and increasing caspase-3 expression by suppression of iNOS and STAT-3 expression. Therefore, scabraside D could be a promising candidate for cholangiocarcinoma treatment.

Abstract A71: TMPRSS4 proteolytically activates pro-uPA to induce cancer cell invasion

Abstract The invasive nature of tumor cells is critical for cancer metastasis. Urokinase-type plasminogen activator (uPA), which is usually derived from stromal cells, is a well-known serine protease involved in invasion and metastasis. uPA is produced as an inactive single-chain protein (known as pro-uPA or sc-uPA) that is processed into the active disulfide-linked two-chain form of uPA by a proteolytic event. TMPRSS4 is a novel type II transmembrane serine protease that is highly expressed on the cell surface in pancreatic, thyroid, colon, and other cancer tissues. Previously, we demonstrated that TMPRSS4 mediates colorectal tumor cell invasion, migration, and metastasis and increased TMPRSS4 expression is associated with colorectal cancer progression. We also demonstrated that TMPRSS4 upregulates uPA gene expression through c-Jun N-terminal kinase signaling activation to induce cancer cell invasion. However, it remains unknown how proteolytic activity of TMPRSS4 contributes to invasion. Here, we report that TMPRSS4 directly converted inactive pro-uPA into the active form through its proteolytic activity. Analysis of conditioned medium from cells overexpressing TMPRSS4 demonstrated that the active TMPRSS4 protease domain is released from the cells and is associated with the plasma membrane. Furthermore, TMPRSS4 could increase pro-uPA-mediated invasion in a serine proteolytic activity-dependent manner. These observations suggest that TMPRSS4 is an upstream regulator of invasion through the regulation of uPA. This study provides valuable insights into the proteolytic function of TMPRSS4 as well as mechanisms for the control of invasion. Citation Format: Hye-Jin Min, Semi Kim. TMPRSS4 proteolytically activates pro-uPA to induce cancer cell invasion. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A71. doi:10.1158/1538-7445.CHTME14-A71

Expression of urokinase type plasminogen activator receptor (uPAR) in the bovine oviduct: Relationship with uPA effect on oviductal epithelial cells

Urokinase type plasminogen activator (uPA) is an oviductal fluid component whose activity is regulated by binding to urokinase type plasminogen activator receptor (uPAR). In this study uPAR and uPA gene expression in bovine oviduct were evaluated and similar expression patterns for both uPAR and uPA mRNAs were observed during the estrous cycle. Immunolocalization of uPAR at the apical zone of epithelial cells suggests that uPA action would be focalized in the oviductal lumen, triggering intracellular signaling pathways. As uPAR expression was also observed in in vitro cultures of oviductal epithelial cells, the effect of uPA was explored using this culture model. Real-time RT-PCR demonstrated that c-fos expression in oviductal cell cultures increases under uPA stimulation. These results suggest that uPA/uPAR binding would be involved in signaling pathways that activate transcription factors and would regulate the synthesis of molecules concerned with the arrangement of a particular oviductal microenvironment.

TMPRSS4 induces cancer cell invasion through pro-uPA processing

TMPRSS4 is a novel type II transmembrane serine protease that is highly expressed on the cell surface in pancreatic, thyroid, colon, and other cancer tissues. Previously, we demonstrated that TMPRSS4 mediates cancer cell invasion, epithelial-mesenchymal transition, and metastasis and that increased TMPRSS4 expression correlates with colorectal cancer progression. We also demonstrated that TMPRSS4 upregulates urokinase-type plasminogen activator (uPA) gene expression to induce cancer cell invasion. However, it remains unknown how proteolytic activity of TMPRSS4 contributes to invasion. In this study, we report that TMPRSS4 directly converted inactive pro-uPA into the active form through its proteolytic activity. Analysis of conditioned medium from cells overexpressing TMPRSS4 demonstrated that the active TMPRSS4 protease domain is released from the cells and is associated with the plasma membrane. Furthermore, TMPRSS4 could increase pro-uPA-mediated invasion in a serine proteolytic activity-dependent manner. These observations suggest that TMPRSS4 is an upstream regulator of pro-uPA activation. This study provides valuable insights into the proteolytic function of TMPRSS4 as well as mechanisms for the control of invasion.

Controllable Urokinase Gene Expression in Trabecular Meshwork Cells

Purpose: Glaucoma is a type of progressive optic neuropathy that finally leads to blindness related to elevated intraocular pressure (IOP). Accumulation of extracellular matrix in trabecular meshwork (TM) and juxta-canalicular connective tissues, which form an aqueous outflow pathway, may be a major cause of increased IOP, thus the fibrinolytic system may be associated with regulation of IOP. We examined the possibility of controllable urokinase plasminogen activator (uPA) gene transfer into TM cells.Methods: TM cells were freshly isolated from porcine eyes and human TM cells obtained during trabeculectomy procedures and cultured. Total RNA was extracted from human TM cells and reverse transcribed into cDNA. A reverse-transcribed polymerase chain reaction (RT-PCR) method was then performed to detect the gene expression of uPA. The cDNA of human uPA was sub-cloned into an expression vector (pEYFP-N1 vector) and controllable expression vector (TRE-Tight vector), then each vector was independently transfected into cultured porcine TM cells. Doxycycline was added to the culture medium to activate the pTet-ON and TRE-Tight vectors. Finally, the expression of uPA was examined using an enzyme linked immunosorbent assay (ELISA).Results: ELISA findings revealed the expression of uPA (3.5 ng/ml) in medium from cultured porcine TM cells that had been transfected with the human uPA gene using the pEYFP-N1 vector. Doxycycline induced human uPA in the pTet-On and TRE-Tight vectors with human uPA gene co-transfected TM cells in a dose-dependent manner.Conclusions: Controllable gene transfer of uPA, which may degrade the extracellular matrix in juxta-canalicular connective tissue, into TM cells was achieved using a Tet-On system. The present method may be useful as a novel therapy for glaucoma.

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

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

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

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

DEXAMETHASONE DOWNREGULATES UROKINASE PLASMINOGEN ACTIVATOR (uPA) AND INHIBITS TUMOUR PROGRESSION AND METASTASIS IN MICE WITH EHRLICH MAMMARY SOLID CARCINOMA

In many types of cancer, increased levels of the tumour-associated serine proteases uPA (urokinase-type plasminogen activator) are linked to tumour progression and metastasis. Dexamethasone (DEX) is a glugocorticoid used in the treatment of cancer and found to have inhibitory effect on cancer progression and metastasis. In the present study, we used BALB/c mice with spontaneous Ehrlich murine mammary adenocarcinoma to assess the effect of DEX on uPA gene expression and metastasis. Mice bearing Ehrlich solid carcinoma (ESC) were treated with 10 mg/kg DEX for 3, 6 and 9 days. Animals were sacrificed and ESC cells were subjected to RT-PCR and Western blot analyses to assess the uPA gene expression in term of mRNA and protein levels, respectively. To investigate metastasis occurrence in liver as secondary tumour site, liver paraffin sections were subjected to hematoxylin and eosin (H & E) staining for carcinoma cells detection. Our results revealed that: 1) uPA was overexpressed in ESC cells compared to control skeletal muscles; 2) treatment with DEX down regulated uPA expression in ESC. 3) Metastasis was observed in liver cells of mice with ESC and disappeared after treatment with DEX. We concluded that DEX treatment inhibited tumour progression and metastasis through its down regulatory effect on uPA expression.

Abstract 3948: An inhibitor of uPA reduces osteosarcoma metastasis by blocking signaling in tumour cells and the bone marrow microenvironment .

Abstract Osteosarcoma (OS) is the most common primary malignant bone tumour in children and adolescents. Pulmonary metastasis is the major complication of OS and results in just 10-20% long-term survival. We have shown that metastatic potential is an inherent characteristic of OS, and that the bone microenvironment contributes to OS metastasis (Endo-Munoz et al. Cancer Res 70:7063-72, 2010). Specifically, loss of osteoclasts increases the metastatic potential of OS, and bone marrow cells (BMC) increase the migration of OS cells. However, the factors regulating inherent and BMC-mediated metastasis have not been elucidated. We have used a multi-omics approach to identify genes and proteins that may mediate OS metastasis. We found very high levels of mRNA and protein expression and secretion of the urokinase plasminogen activator (uPA) and its receptor (uPAR) exclusively in metastatic OS. Addition of uPA-rich OS conditioned medium to metastatic OS cells significantly (P < 0.007) increased their basal in vitro migration to levels similar to those observed with recombinant human uPA. On the other hand, overall migration was significantly (P < 0.005) inhibited by addition of a neutralizing mAb against uPAR or an uPA inhibitor. Silencing of uPAR also abrogated the response to uPA and decreased metastasis in an orthotopic model, indicating that uPA/uPAR signaling acts in a positive feedback loop in the regulation of inherent OS metastasis. To investigate whether BMC could also be signaling through uPA/uPAR, we performed a comparative transcriptomic analysis of metastatic and non-metastatic OS cell lines before and after treatment with BMC conditioned medium, and found that uPA was the most significantly (FC = 7.97, B = 18.82) upregulated gene after BMC treatment. Moreover, immunohistochemistry on femurs of mice bearing OS tumours showed high expression of uPA in the bone marrow as well as in the leading edges of the tumour. In addition, BMC conditioned medium containing high levels of secreted uPA also significantly increased metastatic OS cell migration. Our data indicate that increased uPA gene expression induced by BMC could contribute to enhanced secretion of uPA by OS cells, which in turn could augment signaling through uPAR to increase migration. In addition, BMC secrete uPA into the microenvironment to further increase migration of OS tumour cells in close proximity. Finally, we used a therapeutic inhibitor of uPA in an orthotopic mouse model of OS. The uPA inhibitor reduced metastasis significantly (P = 0.0004) in the treated group. This opens the possibility that uPA inhibitors, which are already in clinical trials for a number of cancers, may also be a useful therapeutic in the treatment for OS metastasis. Citation Format: Liliana B. Endo-Munoz, Na Cai, Andrew Cumming, Dorothy Loo, Michelle Hill, Pamela Mukhopadhyay, Nicholas A. Saunders. An inhibitor of uPA reduces osteosarcoma metastasis by blocking signaling in tumour cells and the bone marrow microenvironment . [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 3948. doi:10.1158/1538-7445.AM2013-3948

Shear stress modulates macrophage-induced urokinase plasminogen activator expression in human chondrocytes

IntroductionSynovial macrophages, which can release proinflammatory factors, are responsible for the upregulation of cartilage-breakdown proteases and play critical roles in cartilage degradation during the progression of osteoarthritis (OA). In addition, shear stress exerts multifunctional effects on chondrocytes by inducing the synthesis of catabolic or anabolic genes. However, the interplay of macrophages, chondrocytes, and shear stress during the regulation of cartilage function remains poorly understood. We investigated the mechanisms underlying the modulation of human chondrocyte urokinase plasminogen activator (uPA) expression by macrophages and shear stress.MethodsHuman chondrocytes were stimulated by peripheral blood-macrophage- conditioned medium (PB-MCM), or exposure of chondrocytes cultured in PB-MCM to different levels of shear stress (2 to 20 dyn/cm2). Real-time polymerase chain reaction was used to analyze uPA gene expression. Inhibitors and small interfering RNA were used to investigate the mechanism for the effects of PB-MCM and shear stress in chondrocytes.ResultsStimulation of human chondrocytes with PB-MCM was found to induce uPA expression. We demonstrated that activation of the JNK and Akt pathways and NF-κB are critical for PB-MCM-induced uPA expression. Blocking assays by using IL-1ra further demonstrated that IL-1β in PB-MCM is the major mediator of uPA expression in chondrocytes. PB-MCM-treated chondrocytes subjected to a lower level of shear stress showed inhibition of MCM-induced JNK and Akt phosphorylation, NF-κB activation, and uPA expression. The PB-MCM-induced uPA expression was suppressed by AMP-activated protein kinase (AMPK) agonist. The inhibitor or siRNA for AMPK abolished the shear-mediated inhibition of uPA expression.ConclusionsThese data support the hypothesis that uPA upregulation stimulated by macrophages may play an active role in the onset of OA and in the shear-stress protection against this induction.

CD44 enhances invasion of basal-like breast cancer cells by upregulating serine protease and collagen-degrading enzymatic expression and activity

IntroductionBasal-like breast cancers (BL-BCa) have the worst prognosis of all subgroups of this disease. Hyaluronan (HA) and the HA receptor CD44 have a long-standing association with cell invasion and metastasis of breast cancer. The purpose of this study was to establish the relation of CD44 to BL-BCa and to characterize how HA/CD44 signaling promotes a protease-dependent invasion of breast cancer (BrCa) cells.MethodsCD44 expression was determined with immunohistochemistry (IHC) analysis of a breast cancer tissue microarray (TMA). In vitro experiments were performed on a panel of invasive BL-BCa cell lines, by using quantitative polymerase chain reaction (PCR), immunoblotting, protease activity assays, and invasion assays to characterize the basis of HA-induced, CD44-mediated invasion.ResultsExpression of the hyaluronan (HA) receptor CD44 associated with the basal-like subgroup in a cohort of 141 breast tumor specimens (P = 0.018). Highly invasive cells of the representative BL-BCa cell line, MDA-MB-231 (MDA-MB-231Hi) exhibited increased invasion through a basement membrane matrix (Matrigel) and collagen. In further experiments, HA-induced promotion of CD44 signaling potentiated expression of urokinase plasminogen activator (uPA) and its receptor uPAR, and underpinned an increased cell-associated activity of this serine protease in MDA-MB-231Hi and a further BL-BCa cell line, Hs578T cells. Knockdown of CD44 attenuated both basal and HA-stimulated uPA and uPAR gene expression and uPA activity. Inhibition of uPA activity by using (a) a gene-targeted RNAi or (b) a small-molecule inhibitor of uPA attenuated HA-induced invasion of MDA-MB-231Hi cells through Matrigel. HA/CD44 signaling also was shown to increase invasion of MDA-MB-231 cells through collagen and to potentiate the collagen-degrading activity of MDA-MB-231Hi cells. CD44 signaling was subsequently shown to upregulate expression of two potent collagen-degrading enzymes, the cysteine protease cathepsin K and the matrix metalloprotease MT1-MMP. RNAi- or shRNA-mediated depletion of CD44 in MDA-MB-231Hi cells decreased basal and HA-induced cathepsin K and MT1-MMP expression, reduced the collagen-degrading activity of the cell, and attenuated cell invasion through collagen. Pharmacologic inhibition of cathepsin K or RNAi-mediated depletion of MT1-MMP also attenuated MDA-MB-231Hi cell invasion through collagen.ConclusionHA-induced CD44 signaling increases a diverse spectrum of protease activity to facilitate the invasion associated with BL-BCa cells, providing new insights into the molecular basis of CD44-promoted invasion.

High level of urokinase plasminogen activator contributes to cholangiocarcinoma invasion and metastasis.

To investigate the role of urokinase plasminogen activator (uPA) in cholangiocarcinoma (CCA) invasion and its correlation with clinicopathological parameters. uPA expression in CCA tissue was determined by immunohistochemistry. The level of uPA from two CCA cell lines (HuCCA-1 and KKU-M213) and a non-cancer immortalized cholangiocyte cell line (H69) was monitored by plasminogen-gelatin zymography and western blotting, whereas that of plasminogen activator inhibitor type 1 (PAI-1) protein and uPA receptor (uPAR) mRNA was monitored by western blotting and quantitative real-time reverse transcriptase polymerase chain reaction, respectively. Two independent methods were employed to suppress uPA function: a synthetic uPA inhibitor (B428) and silencing of uPA gene expression using siRNA. In vitro invasion of the uPA-disrupted cells was assessed by Matrigel-coated Transwell assay. The immunohistochemical study showed that 75.3% (131/174) of CCA tissues expressed uPA. High uPA expression was correlated with lymphatic invasion and metastasis of CCA patients. Plasminogen-gelatin zymography of the conditioned media and cell-surface eluates showed that both CCA cell lines, but not H69, expressed both secreted and membrane-bound forms of uPA. Although the two CCA cell lines, HuCCA-1 and KKU-M213, expressed a relatively high level of uPA and uPAR, the latter exhibited a much lower degree of in vitro invasiveness, correlating with a high expression of PAI-1 in the latter, but not in the former. Suppressing uPA function with a specific uPA inhibitor, B428, or with siRNA against uPA reduced in vitro invasiveness of KKU-M213 cells, demonstrating the requirement for uPA in the invasiveness of CCA cells. Therefore, our in vivo and in vitro studies suggest that uPA is an important requirement for the invasion process of CCA. uPA expression correlates with lymphatic invasion and metastasis in vivo and is required for CCA cell invasion in vitro, suggesting its potential as a therapeutic target.

Microarray-based gene expression profiles to predict ELISA-derived uPA and PAI-1 levels in breast cancer biopsies: A comparison between fresh frozen (FF) and formalin-fixed, paraffin-embedded (FFPE) samples.

194 Background: The urokinase plasminogen-activator (uPA) and its inhibitor (PAI-1) predict the benefit of node-negative breast cancer patients from chemotherapy. The determination of uPA and PAI-1 need(s) large amounts of tumor tissue. In contrast, microarray-based gene expression profiling (MGEP) requires only small quantities of FF tissue and is applicable to FFPE archival tissue. Identification of MPEG-derived surrogate markers for ELISA-based uPA/PAI-1 may facilitate their analysis together with MPEG-based predictive genes and metagenes. Methods: Three groups of breast cancer biopsies were analysed. Group A: 136 FF tissues from 2008-2009, group B: 85 FF tissues from 2010, group C: 20 independent FFPE tissues. Gene expression was assessed by Agilent 4x44K microarrays. Results: For group A a significant correlation between protein expression of uPA and uPA gene (PLAU) expression (r = 0.627 p < 0.001) and PAI-1 and PAI-1 genes (SERPINE1)(r = 0.281 p = 0.001) was found. Top sets of genes correlated with uPA/PAI-1 protein expression were extracted. For group B additional sets of top correlated genes were identified. The correlation of individual genes with uPA/PAI-1 protein expression was highest for their genes themselves (PLAU: r = 0.694 p < 0.001; SERPINE1 (r = 0.469 p < 0.001). Subsequently consensus gene sets were identified with a good performance for both biopsy sets. Only genes which were significantly correlated with their counterparts in group C were selected, resulting in a final 11-gene model (uPA metagene) and a 6-gene model (PAI-1 metagene). They significantly correlated with uPA/PAI-1 protein expression in all groups of biopsies (uPA: group A: r = 0.621, p < 0.001; group B: r = 0.771, p < 0.001; group C: r = 0.665, p = 0.005; for PAI-1: group A: r = 0.569, p < 0.001; group B: r = 0.687, p < 0.001; group C: r = 0.683, p = 0.008). Conclusions: Reliable MPEG-derived metagenes as surrogate markers for ELISA-based uPA/PAI-1 expression were identified.

Microarray-based gene expression profiles reliably predict ELISA-derived uPA and PAI-1 levels in breast cancer biopsies, a comparison between fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) samples.

10555 Background: The urokinase plasminogen-activator (uPA) and its inhibitor (PAI-1) predict the benefit of node-negative breast cancer patients from chemotherapy. The determination of uPA and PAI-1 need(s) large amounts of tumor tissue. In contrast, microarray-based gene expression profiling (MGEP) requires only small quantities of FF tissue and is applicable to FFPE archival tissue. Identification of MPEG-derived surrogate markers for ELISA-based uPA/PAI-1 may facilitate their analysis together with MPEG-based predictive genes and metagenes. Methods: Three groups of breast cancer biopsies were analysed. Group A: 136 FF tissues from 2008-2009, Group B: 85 FF tissues from 2010, Group C: 20 independent FFPE tissues. Gene expression was assessed by Agilent 4x44K microarrays. Results: For group A a significant correlation between protein expression of uPA and uPA gene (PLAU) expression (r=0.627 p<0.001) and PAI-1 and PAI-1 genes (SERPINE1; r=0.281 p=0.001) was found. Top sets of genes correlated with uPA/PAI-1 protein expression were extracted. For group B additional sets of top correlated genes were identified. The correlation of individual genes with uPA/PAI-1 protein expression was highest for their genes themselves (PLAU: r=0.694 p<0.001; SERPINE1 (r=0.469 p<0.001). Subsequently consensus gene sets were identified with a good performance for both biopsy sets. Only genes which were significantly correlated with their counterparts in group C were selected, resulting in a final 11-gene model (uPA metagene) and a 6-gene model (PAI-1 metagene).They significantly correlated with uPA/PAI-1 protein expression in all groups of biopsies ( uPA: group A: r=0.621, p<0.001; group B: r=0.771, p<0.001; group C: r=0.665, p=0.005; for PAI-1: group A: r=0.569, p<0.001; group B: r=0.687, p<0.001; Group C: r=0.683, p=0.008). Conclusions: Reliable MPEG-derived metagenes as surrogate markers for ELISA-based uPA/PAI-1 expression were identified.

Abstract LB-298: The bone marrow microenvironment increases osteosarcoma tumour cell migration by signaling through uPA/uPAR

Abstract Osteosarcoma (OS) is the most common primary malignant bone tumour in children and adolescents. Pulmonary metastasis is the major complication of OS and can occur in up to 50% of cases, resulting in just 10–20% long-term survival for these patients. We have shown that the bone microenvironment contributes to the metastatic potential of osteosarcoma (Endo-Munoz et al. Cancer Res 70:7063–72, 2010). Specifically, loss of osteoclasts increases the metastatic potential of OS in vitro and in vivo, and bone marrow cells (BMC) increase the migration of OS cells in vitro. However, the factors mediating this increase in migration has not been elucidated. Using a combined transcriptomic and proteomic (SILAC) approach, we found very high levels of mRNA and protein expression of the urokinase plasminogen activator (uPA) and its receptor (uPAR) in metastatic, but not in non-metastatic OS. Furthermore, examination of the cytokines secreted in the conditioned medium of metastatic and non-metastatic OS cells in an antibody array showed high and selective secretion of uPA by metastatic OS cells only. Addition of this conditioned medium to metastatic OS cells significantly (P &amp;lt; 0.007) increased their in vitro migration to levels similar to those observed with recombinant human uPA. No effect was observed for non-metastatic cells. Overall migration was significantly (P &amp;lt; 0.005) inhibited by addition of a neutralizing monoclonal antibody against uPAR, indicating that uPA/uPAR signaling acts in a positive feedback loop in the regulation of inherent OS cell migration. To investigate whether the uPA/uPAR system could also be involved in signaling by BMC, we performed a comparative transcriptomic analysis of metastatic and non-metastatic OS cell lines before and after treatment with BMC conditioned medium. We found uPA to be the most significantly upregulated gene after BMC treatment (FC = 7.97, B = 18.82), suggesting that increased uPA gene expression induced by BMC could contribute to enhanced secretion of uPA by OS cells, which in turn could augment signaling through uPAR to increase migration. Finally, we explored the possibility that BMC may also secrete uPA into the microenvironment to further increase migration of OS tumours in close proximity. Immunohistochemistry on femurs of mice bearing OS tumours showed high expression of uPA in the bone marrow as well as in the leading edges of the tumour. These data indicate that BMC increases OS cell migration not only by increasing uPA gene expression in the tumour, but also by secreting uPA which serves to amplify the migration-inducing signaling of tumour-secreted uPA. This opens the possibility that uPA inhibitors, which are already in clinical trials for a number of cancers, may also be a useful therapeutic in the treatment for OS metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-298. doi:10.1158/1538-7445.AM2011-LB-298