uPAR Controls Vasculogenic Mimicry Ability Expressed by Drug-Resistant Melanoma Cells

Malignant melanoma is a highly aggressive skin cancer characterized by an elevated grade of tumor cell plasticity. Such plasticity allows adaptation of melanoma cells to different hostile conditions and guarantees tumor survival and disease progression, including aggressive features such as drug resistance. Indeed, almost 50% of melanoma rapidly develop resistance to the BRAFV600E inhibitor vemurafenib, with fast tumor dissemination, a devastating consequence for patients’ outcomes. Vasculogenic mimicry (VM), the ability of cancer cells to organize themselves in perfused vascular-like channels, might sustain tumor spread by providing vemurafenibresistant cancer cells with supplementary ways to enter into circulation and disseminate. Thus, this research aims to determine if vemurafenib resistance goes with the acquisition of VM ability by aggressive melanoma cells, and identify a driving molecule for both vemurafenib resistance and VM. We used two independent experimental models of drug-resistant melanoma cells, the first one represented by a chronic adaptation of melanoma cells to extracellular acidosis, known to drive a particularly aggressive and vemurafenib-resistant phenotype, the second one generated with chronic vemurafenib exposure. By performing in vitro tube formation assay and evaluating the expression levels of the VM markers EphA2 and VE-cadherin by Western blotting and flow cytometer analyses, we demonstrated that vemurafenib-resistant cells obtained by both models are characterized by an increased ability to perform VM. Moreover, by exploiting the CRISPR-Cas9 technique and using the urokinase plasminogen activator receptor (uPAR) inhibitor M25, we identified uPAR as a driver of VM expressed by vemurafenib-resistant melanoma cells. Thus, uPAR targeting may be successfully leveraged as a new complementary therapy to inhibit VM in drug-resistant melanoma patients, to counteract the rapid progression and dissemination of the disease.     

“莪黄灌肠液”对人大肠癌SW480细胞HIF-1α通路uPA、uPAR基因表达的影响

目的:通过莪黄灌肠液对SW480细胞HIF-1α通路uPA、uPAR基因表达的影响，探讨莪黄灌肠液对SW480细胞侵袭与迁移的影响。方法:根据随机数学分组的原则，将50只健康SD雄性大鼠分成随机5组并逐个编号，体重控制在（200±20）g。通过预实验得出的药物分组量分别灌胃给药:莪黄灌肠液（莪黄汤）每毫升药液中含生药2 g，莪黄灌肠液（莪黄汤）组由低、中、高（6.075 g/kg，12.15 g/kg，24.3 g/kg）剂量组（灌胃剂量2 ml）组成，空白组为生理盐水组(灌胃剂量2 ml，pH 7.2)，阳性对照组由腹腔注射氟尿嘧啶0.025 g/(kg·d)，调整注射剂量1 ml构成。标准化灌胃1周，无菌条件下提取各组含药血清。在体外使用含药血清体培养SW480细胞，划痕实验观察莪黄灌肠液含药血清在体外对SW480细胞迁移能力的影响；Transwell实验检测肿瘤细胞侵袭能力；Western-blot实验检测各组细胞中HIF-1α、uPA、uPAR蛋白表达。结果:预实验提示莪黄灌肠液对大鼠无明显副作用。SW480细胞具有明显的迁移及侵袭能力。各组莪黄灌肠液含药血清能抑制SW480细胞的侵袭与迁移特性，其中莪黄灌肠液各组（24.3 g/kg，12.15 g/kg，6.075 g/kg）和氟尿嘧啶组较空白组均能够有效抑制肿瘤细胞的侵袭与迁移作用，且氟尿嘧啶组作用最强，中药24.3 g/kg组表现出的抑制侵袭作用较12.15 g/kg，6.075 g/kg组均强（P<0.05）。莪黄灌肠液含药血清作用于肿瘤细胞24 h后，其细胞内HIF-1α、uPA、uPAR蛋白含量均有明显的下降，HIF-1α、uPA、uPAR蛋白表现出浓度依赖性（P<0.05)。结论:“莪黄灌肠液”可能通过作用于HIF-1α通路抑制SW480细胞的侵袭与转移。     

uPA、uPAR、PAI-1血浆含量与卵巢恶性肿瘤的相关性研究

目的：探讨尿激酶型纤溶酶原激活物（uPA）及其受体（uPAR）和抑制剂（PAI-1）血浆含量与卵巢恶性肿瘤之间的关系。方法：收集52例卵巢恶性肿瘤患者血液标本,以30例健康人作对照,用ELISA法分别检测uPA、uPAR和PAI-1的含量。结果：uPA、uPAR在卵巢恶性肿瘤各期之间均有极显著性差异（P〈0．01）,PAI-1在卵巢恶性肿瘤FIGO Ⅰ～Ⅲ期的含量逐渐升高,但在FIG0Ⅳ期时显著下降（P〈0．05）。患者组uPA、uPAR、PAI-1均较对照组升高,差异极显著（P〈0．01）。结论uPA、uPAR在卵巢恶性肿瘤患者可作为预后的判断指标,PAI-1与卵巢恶性肿瘤的分期有一定的相关性。     

黑素瘤组织中乙醛脱氢酶1、上皮性钙黏附蛋白及尿激酶型纤溶酶原激活物受体的表达及临床意义

目的研究黑素瘤组织中乙醛脱氢酶1(aldehyde dehydrogenase 1,ALDH1)、上皮性钙黏附蛋白(E-cadherin,E-cad)及尿激酶型纤溶酶原激活物受体(urokinase-type plasminogen activator receptor,uPAR)的表达及与临床病理特征的关系。方法选择50例黑素瘤和50例色素痣患者的病变组织作为研究对象,采用免疫组化法检测病变组织中的ALDH1、E-cad及uPAR的表达情况,分析这3种蛋白表达与患者临床组织病理特征、肿瘤分期的关系。结果黑素瘤组织中ALDH1和uPAR表达阳性率分别为82.00%(41/50)和86.00%(43/50),均显著高于色素痣组(P0.05);黑素瘤组织中E-cad阳性表达率为38.00%(19/50),显著低于色素痣组(P0.05)。ALDH1、uPAR表达阳性率与患者年龄、性别、肿瘤厚度、浸润深度、是否淋巴结转移和组织病理分型均无相关性,但肿瘤厚度≥4 mm、浸润深度Ⅳ~Ⅴ、发生淋巴结转移等患者黑素瘤组织中ALDH1、uPAR表达阳性率均90%。浸润深度Ⅳ~Ⅴ的黑素瘤患者E-cad表达阳性率显著低于浸润深度Ⅰ~Ⅲ的患者(P0.05),黑素瘤患者E-cad表达阳性率与其他临床特征无相关性。结论黑素瘤在发生和进展过程中ALDH1、uPAR表达上调,E-cad表达下调,E-cad表达下调与肿瘤程度有关。     

uPA‐uPAR molecular complex is involved in cell signaling during neuronal migration and neuritogenesis

Background: In the development of the central nervous system (CNS), neuronal migration and neuritogenesis are crucial processes for establishing functional neural circuits. This relies on the regulation exerted by several signaling molecules, which play important roles in axonal growth and guidance. The urokinase‐type plasminogen activator (uPA)—in association with its receptor—triggers extracellular matrix proteolysis and other cellular processes through the activation of intracellular signaling pathways. Even though the uPA‐uPAR complex is well characterized in nonneuronal systems, little is known about its signaling role during CNS development. Results : In response to uPA, neuronal migration and neuritogenesis are promoted in a dose‐dependent manner. After stimulation, uPAR interacts with α₅‐ and β₁‐integrin subunits, which may constitute an αβ‐heterodimer that acts as a uPA‐uPAR coreceptor favoring the activation of multiple kinases. This interaction may be responsible for the uPA‐promoted phosphorylation of focal adhesion kinase (FAK) and its relocation toward growth cones, triggering cytoskeletal reorganization which, in turn, induces morphological changes related to neuronal migration and neuritogenesis. Conclusions : uPA has a key role during CNS development. In association with its receptor, it orchestrates both proteolytic and nonproteolytic events that govern the proper formation of neural networks. Developmental Dynamics 243:676–689, 2014. © 2014 Wiley Periodicals, Inc.     

Enhanced levels of urokinase plasminogen activator and its soluble receptor in common variable immunodeficiency

Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by defective immunoglobulin production and high frequency of bacterial infections, autoimmunity and manifestations of chronic inflammation.The urokinase plasminogen activator (uPA), its cell bound and soluble receptor (uPAR, suPAR) have complex biological functions involving innate immune defense mechanisms and regulation of inflammation. Based on this dual role, we hypothesized that the uPA system could be affected in CVID, and examined expression of components of the uPA system in subgroups of CVID. All CVID-patients had increased plasma levels of suPAR with particularly high levels in those with splenomegaly and thrombocytopenia. Plasma uPA levels were also raised in these patients, and both suPAR and uPA levels correlated with the monocyte activation marker neopterin. Monocytes from CVID patients had increased expression of uPAR. We show an increased activation of the uPA system possibly contributing to the inflammatory phenotype seen in subgroups of CVID patients.     

Prognostic significance of urokinase plasminogen activator receptor and its cleaved forms in blood from patients with non‐small cell lung cancer

Urokinase plasminogen activator (uPA) cleaves its three‐domain cell surface receptor, uPAR, liberating domain I [uPAR(I)] and leaving the cleaved uPAR(II‐III) on the cell surface. Both intact and cleaved uPAR can be shed from the cell surface. uPAR(I) was previously shown to be a prognostic factor in lung tumour extracts. Here we analyse uPAR forms in blood from patients with non‐small cell lung cancer (NSCLC). Preoperatively sampled plasma/serum from 32 patients with NSCLC was analysed. Three time‐resolved fluoroimmunoassays (TR‐FIAs) measuring intact uPAR(I‐III) (TR‐FIA 1), uPAR(I‐III) + uPAR(II‐III) (TR‐FIA 2) and uPAR(I) (TR‐FIA 3) were applied. The Spearman rank correlations between plasma and serum levels of uPAR(I‐III), uPAR(I‐III) + uPAR(II‐III), and uPAR(I) were 0.89, 0.94 and 0.68 respectively. Survival analysis demonstrated that high levels of all uPAR forms were associated with shorter survival. Adjusted for histological subtype high plasma uPAR(I‐III) and uPAR(I) levels as well as serum uPAR(I) levels were significantly associated with shorter OS (hazards ratios = 4.3, 2.8 and 3.8 respectively). High blood levels of intact uPAR and its cleaved forms are associated with poor prognosis in NSCLC.     

Urokinase and its receptor in follicular and inflammatory cysts of the jaws

Oral Diseases (2010) 16 , 753–759 Objective: Proteases are considered critical in peri‐cystic tissue degradation required in jaw cyst expansion. We studied the expression of the plasminogen activation system (plasminogen activators; their inhibitor type‐1, PAI‐1; the receptor for the urokinase‐type plasminogen activator, uPAR) in follicular and inflammatory cysts of the jaw, to identify a possible role of this system in jaw cyst enlargement. Materials and Methods: Jaw cysts were collected by therapeutic enucleation. ELISA and casein zymography were used to measure and characterize plasminogen activators in cyst fluid. By immunohistochemistry we examined the presence of uPAR in cyst walls and inflammatory cells, and by Western blotting the molecular forms of uPAR within the cyst fluid. Results: Inflammatory cysts fluid contained higher amounts of plasminogen activators of the urinary‐type (uPA), and lower amounts of PAI‐1, when compared to follicular cysts fluid. Epithelial layers of both types of cysts and inflammatory cells expressed uPAR. Native 3‐domain uPAR was scarcely detectable within cysts, where its cleavage was accounted for by uPA. Conclusion: These data suggest a plasminogen activation‐dependent mechanism of cyst enlargement, where only the outward uPAR expressed on epithelial cells and on inflammatory cells direct the peri‐cystic protease cascade, in a way similar to tumor enlargement within tissues.     

Plasminogen activator inhibitor-1 is an aggregate response factor with pleiotropic effects on cell signaling in vascular disease and the tumor microenvironment

In hemostasis, the serine protease inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1) functions to stabilize clots via inhibition of tissue plasminogen activator (tPA) with subsequent inhibition of fibrinolysis. In tissues, PAI-1 functions to inhibit extracellular matrix degradation via inhibition of urokinase plasminogen activator (uPA). Elevated levels of PAI-1 in the vasculature and in tissues have long been known to be associated with thrombosis and fibrosis, respectively. However, there is emerging evidence that PAI-1 may participate in the pathophysiology of a number of diseases such as atherosclerosis, restenosis, and cancer. In many of these disease states, the canonical view of PAI-1 as an inhibitor of tPA and uPA cannot fully account for a mechanism whereby PAI-1 contributes to the disease. In these cases, one must consider recent data, which indicates PAI-1 can directly promote pro-proliferative and anti-apoptotic signaling in a variety of cell types. Given the wide variety of inflammatory, hormonal, and metabolic signals that increase PAI-1 expression, it is important to consider mechanisms by which PAI-1 can directly participate in disease etiology.