Downregulation of uPAR inhibits migration,invasion, proliferation,FAK/PI3K/Akt signaling and induces senescence in papillary thyroid carcinoma cells

Papillary thyroid carcinoma (PTC) is the most common endocrine and thyroid malignancy. The urokinase plasminogen activator receptor (uPAR) plays an important role in cancer pathogenesis, including breakdown of the extracellular matrix, invasion and metastasis. Additionally, there is increasing evidence that uPAR also promotes tumorigenesis via the modulation of multiple signaling pathways. BRAF^V600E, the most common initial genetic mutation in PTC, leads to ERK1/2 hyperphosphorylation, which has been shown in numerous cancers to induce uPAR. Treatment of the BRAF^V600E-positive PTC cell line, BCPAP, with the MEK/ERK inhibitor U0126 reduced uPAR RNA levels by 90%. siRNA-mediated downregulation of uPAR in BCPAP cells resulted in greatly decreased activity in the focal adhesion kinase (FAK)/phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. This phenomenon was concurrent with drastically reduced proliferation rates and decreased clonigenic survival, as well as demonstrated senescence-associated nuclear morphology and induction of β-galactosidase activity. uPAR-knockdown BCPAP cells also displayed greatly reduced migration and invasion rates, as well as a complete loss of the cells'' ability to augment their invasiveness following plasminogen supplementation. Taken together, these data provide new evidence of a novel role for uPAR induction (as a consequence of constitutive ERK1/2 activation) as a central component in PTC pathogenesis, and highlight the potential of uPAR as a therapeutic target.Key words: urokinase plasminogen activator receptor (uPAR), papillary thyroid carcinoma, invasion, migration, proliferation, senescence, FAK, PI3K, Akt     

Arachidonate 5 lipoxygenase expression in papillary thyroid carcinoma promotes invasion via MMP-9 induction

Arachidonate 5-lipoxygenase (ALOX5) expression and activity has been implicated in tumor pathogenesis, yet its role in papillary thyroid carcinoma (PTC) has not been characterized. ALOX5 protein and mRNA were upregulated in PTC compared to matched, normal thyroid tissue, and ALOX5 expression correlated with invasive tumor histopathology. Evidence suggests that PTC invasion is mediated through the induction of matrix metalloproteinases (MMPs) that can degrade and remodel the extracellular matrix (ECM). A correlation between MMP-9 and ALOX5 protein expression was established by immunohistochemical analysis of PTC and normal thyroid tissues using a tissue array. Transfection of ALOX5 into a PTC cell line (BCPAP) increased MMP-9 secretion and cell invasion across an ECM barrier. The ALOX5 product, 5(S)-hydroxyeicosatetraenoic acid also increased MMP-9 protein expression by BCPAP in a dose-dependent manner. Inhibitors of MMP-9 and ALOX5 reversed ALOX5-enhanced invasion. Here we describe a new role for ALOX5 as a mediator of invasion via MMP-9 induction; this ALOX5/MMP9 pathway represents a new avenue in the search for functional biomarkers and/or potential therapeutic targets for aggressive PTC.     

甲状腺乳头状癌合并桥本氏甲状腺炎的BRAF基因表达及侵袭性

目的:比较甲状腺乳头状癌合并桥本氏甲状腺炎与不合并桥本氏甲状腺炎的BRAFV600E基因表达以及侵袭性的区别。方法:2011年9月到2013年9月四川大学华西医院手术治疗并有BRAFV600E基因测定的甲状腺乳头状癌患者226名,均有病理证实。其中合并桥本氏甲状腺炎者50例为研究组,同期随机抽取50例不合并桥本氏甲状腺炎者作为对照组。比较两组性别、年龄、肿瘤大小、数量、BRAFV600E基因表达以及甲状腺外侵犯和淋巴结转移与侵袭性相关的因素的区别。结果:甲状腺乳头状癌合并桥本氏甲状腺炎在男女性别,发病年龄、肿瘤大小上和对照组相比无差异(P0.05);BRAFV600E突变率、甲状腺外侵犯和淋巴结转移都较对照组更低(P0.05)。BRAF基因突变阳性组甲状腺外侵犯和淋巴结转移率较BRAFV600E基因突变阴性组更高(P0.05)。结论:BRAFV600E基因突变的甲状腺乳头状癌患者有更高的甲状腺腺外侵犯和淋巴结转移。甲状腺乳头状癌合并桥本氏甲状腺炎较不合并桥本氏甲状腺炎有着更低的BRAFV600E突变率,更低的甲状腺外侵犯和淋巴结转移。     

RNA interference-directed knockdown of urokinase plasminogen activator and urokinase plasminogen activator receptor inhibits prostate cancer cell invasion, survival, and tumorigenicity in vivo

The invasive ability of tumor cells plays a key role in prostate cancer metastasis and is a major cause of treatment failure. Urokinase plasminogen activator-(uPA) and its receptor (uPAR)-mediated signaling have been implicated in tumor cell invasion, survival, and metastasis in a variety of cancers. This study was undertaken to investigate the biological roles of uPA and uPAR in prostate cancer cell invasion and survival, and the potential of uPA and uPAR as targets for prostate cancer therapy. uPA and uPAR expression correlates with the metastatic potential of prostate cancer cells. Thus, therapies designed to inhibit uPA and uPAR expression would be beneficial. LNCaP, DU145, and PC3 are prostate cancer cell lines with low, moderate, and high metastatic potential, respectively, as demonstrated by their capacity to invade the extracellular matrix. In this study we utilized small hairpin RNAs (shRNAs), also referred to as small interfering RNAs, to target human uPA and uPAR. These small interfering RNA constructs significantly inhibited uPA and uPAR expression at both the mRNA and protein levels in the highly metastatic prostate cancer cell line PC3. Our data demonstrated that uPA-uPAR knockdown in PC3 cells resulted in a dramatic reduction of tumor cell invasion as indicated by a Matrigel invasion assay. Furthermore, simultaneous silencing of the genes for uPA and uPAR using a single plasmid construct expressing shRNAs for both uPA and uPAR significantly reduced cell viability and ultimately resulted in the induction of apoptotic cell death. RNA interference for uPA and uPAR also abrogated uPA-uPAR signaling to downstream target molecules such as ERK1/2 and Stat 3. In addition, our results demonstrated that intratumoral injection with the plasmid construct expressing shRNAs for uPA and uPAR almost completely inhibited established tumor growth and survival in an orthotopic mouse prostate cancer model. These findings uncovered evidence of a complex signaling network operating downstream of uPA-uPAR that actively advances tumor cell invasion, proliferation, and survival of prostate cancer cells. Thus, RNA interference-directed targeting of uPA and uPAR is a convenient and novel tool for studying the biological role of the uPA-uPAR system and raises the potential of its application for prostate cancer therapy.     

Soluble urokinase-type plasminogen activator receptor inhibits cancer cell growth and invasion by direct urokinase-independent effects on cell signaling

The urokinase-type plasminogen activator receptor (uPAR) is released from human cancers and is readily detected in blood. In animal models, soluble uPAR (SuPAR) antagonizes cancer progression; however, the mechanism by which SuPAR functions in vivo remains unclear. It is generally thought that SuPAR scavenges uPA and prevents its interaction with membrane-anchored uPAR. In this study, we demonstrate a novel molecular mechanism by which SuPAR may inhibit cancer progression. We show that SuPAR has the potential to directly and in a uPA-independent manner block the signaling activity of membrane-anchored uPAR. Whether SuPAR inhibits signaling is cell type-specific, depending on the state of the endogenous uPA-uPAR signaling system. In uPAR-deficient cells that lack endogenous uPAR signaling, including uPAR-/-murine embryonic fibroblasts and human embryonal kidney 293 cells, SuPAR functions as a partial signaling agonist that activates ERK/mitogen-activated protein kinase. By contrast, in cells with potent autocrine uPA-uPAR signaling systems, including MDA-MB 231 breast cancer cells and low density lipoprotein receptor-related protein-1-deficient murine embryonic fibroblasts, SuPAR substantially decreases ERK activation. The mechanism probably involves competitive displacement of membrane-anchored uPAR-uPA complex from signaling adaptor proteins. As a result of its effects on cell signaling, SuPAR blocks cell growth and inhibits cellular invasion of Matrigel. Cleavage of SuPAR by proteinases increases its signaling agonist activity and reverses its inhibitory effects on growth and invasion. Thus, proteolytic cleavage represents a molecular switch that neutralizes the anticancer activity of SuPAR.     

Specific interference of urokinase-type plasminogen activator receptor and matrix metalloproteinase-9 gene expression induced by double-stranded RNA results in decreased invasion, tumor growth, and angiogenesis in gliomas

We have previously demonstrated the effectiveness of adenovirus-mediated expression of antisense urokinase-type plasminogen activator receptor (uPAR) and matrix metalloproteinase-9 (MMP-9) in inhibiting tumor invasion in vitro and ex vivo. However, the therapeutic effect of the adenovirus-mediated antisense approach was shown to be transient and required potentially toxic, high viral doses. In contrast, RNA interference (RNAi)-mediated gene targeting may be superior to the traditional antisense approach, because the target mRNA is completely degraded and the molar ratio of siRNA required to degrade the target mRNA is very low. Here, we have examined the siRNA-mediated target RNA degradation of uPAR and MMP-9 in human glioma cell lines. Using RNAi directed toward uPAR and MMP-9, we achieved specific inhibition of uPAR and MMP-9. This bicistronic construct (pUM) inhibited the formation of capillary-like structures in both in vitro and in vivo models of angiogenesis. We demonstrated that blocking the expression of these genes results in significant inhibition of glioma tumor invasion in Matrigel and spheroid invasion assay models. RNAi for uPAR and MMP-9 inhibited cell proliferation, and significantly reduced the levels of phosphorylated forms of MAPK, ERK, and AKT signaling pathway molecules when compared with parental and empty vector/scrambled vector-transfected SNB19 cells. Furthermore, using RNAi to simultaneously target two proteases resulted in total regression of pre-established intracerebral tumor growth. Our results provide evidence that the use of hairpin siRNA expression vectors for uPAR and MMP-9 may provide an effective tool for cancer therapy.     

Urokinase-type plasminogen activator receptor crosslinking in an NK cell line increases integrin surface expression by the MAP kinase/ERK 1/2 signaling pathway

Urokinase-type plasminogen activator receptor (uPAR) is attached to cell membranes by a glycosylphosphatidylinositol (GPI) anchor, and as such is devoid of an intracellular domain, but is nevertheless able to initiate signal transduction. Herein, we report a relationship between integrins and uPAR on the surface of the human NK cell line, YT. Our data reveals that crosslinking uPAR, which mimics uPAR clustering at focal adhesion sites, causes increases in expression of the alpha(M), alpha(V), and beta(2) integrins on the surface of YT cells. Activation of the MEK/ERK signaling cascade occurs following uPAR crosslinking, as phosphorylation of both MEK 1/2 and ERK 1/2 results from receptor clustering. The MEK-specific inhibitors PD98059 and U0126 blocked MAP kinase phosphorylation; furthermore, PD98059 inhibited the increase in integrin expression induced by uPAR clustering. This study suggests that uPAR is a signaling receptor and regulator of integrins in NK cells and may impact NK cell function, including the potential for their accumulation within tumor metastases following adoptive transfer.     

Integrin αvβ3, metalloproteinases, and sphingomyelinase-2 mediate urokinase mitogenic effect

Plasminogen activators are implicated in the pathogenesis of several diseases such as inflammatory diseases and cancer. Beside their serine-protease activity, these agents trigger signaling pathways involved in cell migration, adhesion and proliferation. We previously reported a role for the sphingolipid pathway in the mitogenic effect of plasminogen activators, but the signaling mechanisms involved in neutral sphingomyelinase-2 (NSMase-2) activation (the first step of the sphingolipid pathway) are poorly known. This study was carried out to investigate how urokinase plasminogen activator (uPA) activates NSMase-2. We report that uPA, as well as its catalytically inactive N-amino fragment ATF, triggers the sequential activation of MMP-2, NSMase-2 and ERK1/2 in ECV304 cells that are required for uPA-induced ECV304 proliferation, as assessed by the inhibitory effect of Marimastat (a MMP inhibitor), MMP-2-specific siRNA, MMP-2 defect, and NSMase-specific siRNA. Moreover, upon uPA stimulation, uPAR, MT1-MMP, MMP-2 and NSMase-2 interacted with integrin α_vβ₃, evidenced by co-immunoprecipitation and immunocytochemistry experiments. Moreover, the α_vβ₃ blocking antibody inhibited the uPA-triggered MMPs/uPAR/integrin α_vβ₃ interaction, NSMase-2 activation, Ki67 expression and DNA synthesis in ECV304. In conclusion, uPA triggers interaction between integrin α_vβ₃, uPAR and MMPs that leads to NSMase-2 and ERK1/2 activation and cell proliferation. These findings highlight a new signaling mechanism for uPA, and suggest that, upon uPA stimulation, uPAR, MMPs, integrin α_vβ₃ and NSMase-2 form a signaling complex that take part in mitogenic signaling in ECV304 cells.     

Suppression of urokinase plasminogen activator receptor inhibits proliferation and migration of pancreatic adenocarcinoma cells via regulation of ERK/p38 signaling

Pancreatic ductal adenocarcinoma (PDAC) expresses high levels of urokinase-type plasminogen activator (uPA), its receptor (uPAR) and plasminogen activator inhibitor (PAI)-2, which may play an important role in PDAC progression. The overexpression of uPAR predicted short survival in PDAC patients. In this study, two different PDAC cell lines were used to examine the effect of small interfering (si) RNAs to uPAR, uPA and PAI-2 on proliferation, apoptosis, migration and MAP kinase activation. In both PDAC cell lines, siRNA to uPAR significantly inhibited cell proliferation and migration and stimulated apoptosis, to a greater extent than uPA siRNA. When either PDAC cell line was treated with uPAR siRNA, the level of phosphorylated ERK (p-ERK) decreased substantially, whereas phosphorylated p38 (p-p38) increased when compared to non-silencing control, uPA siRNA or PAI-2 siRNA treatment. This resulted in enhancement of the p-p38/p-ERK ratio which favors cancer cell arrest. Interestingly, uPAR protein expression was suppressed by p-ERK inhibition and stimulated with p-p38 inhibition, suggesting the presence of a positive feedback loop between uPAR and ERK. In summary, our data indicate that, of the uPA system, uPAR exerts the strongest effects on PDAC cells, by acting through the ERK signaling pathway via a positive feedback loop. Disruption of this loop with uPAR siRNA or inhibitor of p-ERK, inhibits PDAC proliferation and migration and promotes apoptosis. These findings suggest that uPAR strongly contributes to PDAC progression and may be considered as a potential anti-pancreatic cancer target.     

Physical association of uPAR with the alphaV integrin on the surface of human NK cells

The urokinase-type plasminogen activator receptor (uPAR) serves as a receptor for urokinase plasminogen activator (uPA) and plays a role in invasion and migration of certain immune cells, including NK cells. Although uPAR is anchored to the plasma membrane via a glycosylphosphatidylinositol lipid moiety, we have previously shown that uPAR crosslinking results in MAP kinase signaling and increased integrin expression on the surface of the human NK cell line, YT. We report, herein, that the binding of uPA to uPAR also activates the MAP kinase signaling cascade. Furthermore, we show the physical association between uPAR and integrins on YT cells using cocapping and fluorescence microscopy. These results suggest that signaling initiated by either uPAR binding to uPA or by uPAR clustering may depend on the physical association of uPAR with integrins, a process that may be a prerequisite for NK cell accumulation within established tumor metastases during adoptive therapy.     

A transformation in the mechanism by which the urokinase receptor signals provides a selection advantage for estrogen receptor-expressing breast cancer cells in the absence of estrogen

Binding of urokinase-type plasminogen activator (uPA) to its receptor, uPAR, in estrogen receptor-α (ERα) expressing breast cancer cells, transiently activates ERK downstream of FAK, Src family kinases, and H-Ras. Herein, we show that when uPAR is over-expressed, in two separate ERα-positive breast cancer cell lines, ERK activation occurs autonomously of uPA and is sustained. Autonomous ERK activation by uPAR requires H-Ras and Rac1. A mutated form of uPAR, which does not bind vitronectin (uPAR-W32A), failed to induce autonomous ERK activation. Expression of human uPAR or mouse uPAR but not uPAR-W32A in MCF-7 cells provided a selection advantage when these cells were deprived of estrogen in cell culture for two weeks. Similarly, MCF-7 cells that express mouse uPAR formed xenografts in SCID mice that survived and increased in volume in the absence of estrogen supplementation, probably reflecting the pro-survival activity of phospho-ERK. Autonomous uPAR signaling to ERK was sensitive to the EGFR tyrosine kinase inhibitors, Erlotinib and Gefitinib. The transition in uPAR signaling from uPA-dependent and transient to autonomous and sustained is reminiscent of the transformation in ErbB2/HER2 signaling observed when this gene is amplified in breast cancer. uPAR over-expression may provide a pathway for escape of breast cancer cells from ERα-targeting therapeutics.     

The urokinase receptor: Focused cell surface proteolysis, cell adhesion and signaling

Plasma membrane urokinase-type plasminogen activator (uPA)-receptor (uPAR) is a GPI-anchored protein that binds with high-affinity and activates the serine protease uPA, thus regulating proteolytic activity at the cell surface. In addition, uPAR is a signaling receptor that often does not require its protease ligand or its proteolytic function.uPAR is highly expressed during tissue reorganization, inflammation, and in virtually all human cancers. Since its discovery, in vitro and in vivo models, as well as retrospective clinical studies have shown that over-expression of components of the uPA/uPAR-system correlates with increased proliferation, migration, and invasion affecting the malignant phenotype of cancer. uPAR regulates the cells-extracellular matrix interactions promoting its degradation and turnover through the plasminogen activation cascade.     

Multiple signaling conduits regulate global differentiation-specific gene expression in PC12 cells

PC12 cells serve as a model for exploring nerve growth factor (NGF)-stimulated signal pathways that mediate neural differentiation. We previously demonstrated that neurofilament light chain (NFLC) gene induction by NGF requires collaborative extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling. Herein, we investigate the broader requirement for integrated ERK and JNK signaling in NGF-stimulated gene expression. NGF stimulates differentiation as well as maintenance of cell viability while insulin-like growth factor-1 (IGF-1) stimulates only trophic actions in PC12 cells. Affymetrix Genechips were used to identify genes whose expression specifically increased in response to NGF, but not IGF-1. From the set of NGF-specific genes, the induction by NGF of ten genes with diverse predicted cellular functions was tested for ERK and JNK pathway requirements using the protein kinase inhibitors, PD98059 and SP600125, respectively. Like NFLC, induction of urokinase plasminogen activator (uPAR), transin/matrix metalloproteinase 3 (MMP3), Fra-1 and transforming growth factor beta 1 (TGF beta 1) required collaborative ERK and JNK signaling while the increased expression of cortexin, rat collapsin response mediator protein 4 (rCRMP4), rat growth and transformation-dependent protein (RGT), and synapsin II required neither mitogen-activated protein kinase (MAPK) pathway. NGF-induction of the bradykinin B2 receptor and c-Ret mRNAs was partially inhibited by SP600125, but not PD98059. Reporter constructs containing the promoters for ERK/JNK-dependent genes (NFLC, transin, uPAR) as well as an ERK/JNK-independent gene (synapsin II) revealed that both sets of genes required functional Ras signaling for activation by NGF. Integrated signaling through the ERK and JNK MAPKs, therefore, represents a general conduit for NGF-dependent gene expression, but additional Ras-dependent signaling pathways distinct from the ERKs and JNKs must contribute as well. Thus, multiple signaling conduits control global differentiation-specific gene expression in PC12 cells.     

Group A streptococcal surface GAPDH, SDH, recognizes uPAR/CD87 as its receptor on the human pharyngeal cell and mediates bacterial adherence to host cells

Streptococcal surface dehydrogenase (SDH) is a multifunctional, anchorless protein present on the surface of group A Streptococcus (GAS). It plays a regulatory role in GAS-mediated intracellular signaling events in human pharyngeal cells. Using ligand-binding assays, we have identified an approximately 55 kDa protein as an SDH-specific receptor protein on the surface of Detroit human pharyngeal cells. LC-MS/MS analyses identified this SDH-binding pharyngeal cell-surface-exposed membrane-bound protein as uPAR (urokinase plasminogen activator receptor)/CD87. Ligand-binding assays also revealed that only the N-terminal domain (D1) of uPAR bound to SDH. uPAR-D1 more specifically bound to the C-terminal alpha-helix and two immediate flanking regions of the S-loop of the SDH molecule. Site-directed mutagenesis in GAS resulting in SDH with altered C-terminal ends, and the removal of uPAR from pharyngeal cells by phosphatidylinositol-phopsholipase C treatment decreased GAS ability to adhere to pharyngeal cells. When compared to uninfected Detroit pharyngeal cells, GAS-infected pharyngeal cells showed a transient but a significant increase in the expression of uPAR-specific mRNA, and a prolonged recycling process of uPAR on the cell surface. Together, these results indicate that the specific streptococcal surface protein-pharyngeal cell receptor interaction mediated by SDH and uPAR is modulated during GAS infection of human pharyngeal cells. This interaction significantly contributes to bacterial adherence and thus may play a significant role in GAS pathogenesis by regulating intracellular signaling events in pharyngeal cells.     

Mannose 6-Phosphate/Insulin-like Growth Factor 2 Receptor Limits Cell Invasion by Controlling αVβ3 Integrin Expression and Proteolytic Processing of Urokinase-type Plasminogen Activator Receptor

The multifunctional mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) is considered a tumor suppressor. We report here that RNA interference with M6P/IGF2R expression in urokinase-type plasminogen activator (uPA)/urokinase-type plasminogen activator receptor (uPAR) expressing human cancer and endothelial cells resulted in increased pericellular plasminogen activation, cell adhesion, and higher invasive potential through matrigel. M6P/IGF2R silencing led also to the cell surface accumulation of urokinase and plasminogen and enhanced expression of αV integrins. Genetic rescue experiments and inhibitor studies revealed that the enhanced plasminogen activation was due to a direct effect of M6P/IGF2R on uPAR, whereas increased cell adhesion to vitronectin was dependent on αV integrin expression and not uPAR. Increased cell invasion of M6P/IGF2R knockdown cells was rescued by cosilencing both uPAR and αV integrin. Furthermore, we found that M6P/IGF2R expression accelerates the cleavage of uPAR. M6P/IGF2R silencing resulted in an increased ratio of full-length uPAR to the truncated D2D3 fragment, incapable of binding most uPAR ligands. We conclude that M6P/IGF2R controls cell invasion by regulating αV integrin expression and by accelerating uPAR cleavage, leading to the loss of the urokinase/vitronectin/integrin-binding site on uPAR.