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Biomedicine & Pharmacotherapy 67 (2013) 179–182 Available online at www.sciencedirect.com Review The urokinase plasminogen activator system in breast cancer invasion and metastasis Linlin Tang a, Xiuzhen Han a,*,b a b Department of Pharmacology, School of Pharmaceutical Sciences, Shandong University, 44, West Wenhua Road, Jinan, Shandong Province 250012, China Key Laboratory of Chemical Biology of Natural Products, Ministry of Education, Shandong University, 44, West Wenhua Road, Jinan 250012, China A R T I C L E I N F O A B S T R A C T Article history: Received 1 September 2012 Accepted 22 October 2012 The urokinase plasminogen activator system, which is a serine protease family include urokinase-type plasminogen activator (uPA), the uPA receptor and plasminogen activator inhibitors (PAIs). uPA catalyzes the transformation of plasminogen to its active form plasmin, which is able to degrade the extracellular matrix (ECM) and basement membranes, directly or indirectly through activating promatrix metalloproteinases (pro-MMPs), promoting cancer cell metastasis and invasion. Both uPA and PAI-1 are poor prognosis markers in primary breast cancer. Evidence has been presented that the uPA system facilitates breast cancer metastasis by several different mechanisms, such as the Ras-ERK pathway and p38 MAPK pathway. This review focuses on uPA system, summarizes their biological effects, highlights the molecular mechanism and pathway, and discusses the role of uPA system in the prevention and treatment of human breast cancers. ß 2012 Elsevier Masson SAS. All rights reserved. Keywords: uPA system Breast cancer Metastasis Invasion Ras-ERK pathway 1. Introduction 2. The urokinase plasminogen activator system Breast cancer is one of the major malignant tumors to threaten women well being. About 25 to 40% of breast cancer patients develop distant metastases [1]. Various proteolytic enzymes play an important action in tumor invasion and metastasis process. These proteases involve collagenases, cathepsins, plasmin, or plasminogen activators [2]. The urokinase plasminogen activator (uPA) system, which is a serine protease family, plays a crucial role in tumor invasion and metastasis. The system includes urokinasetype plasminogen activator (uPA), the glycolipid-anchored cell membrane receptor for the uPA (uPAR), plasminogen activator inhibitors (PAIs). The biological system is implicated in multiple physiological and pathologic processes including cell migration, angiogenesis, inflammation, embryogenesis, tumor growth, and metastasis [3]. uPA and uPAR were over-expressed in diverse human malignant tumors in contrast to the corresponding normal tissue. The uPA system catalyzes the inactive plasminogen to the active plasmin, which lead to the degradation and regeneration of the basement membrane and extracellular matrix (ECM) that result in metastasis. It is beyond reasonable doubt that this enzyme system plays a central role in tumor biology and represents a high potential target for therapeutic intervention of tumor growth and metastasis. 2.1. uPA uPA protein is 411 amino acid residues long, consists of two a helices and two anti-parallel b strands, and is secreted as a 53 KD zymogen (pro-urokinase) [4]. The pro-uPA is an one-chain zymogen, with an activity that is at least several hundred-fold lower than that of two-chain uPA. The former consists of two disulfide bridge–linked polypeptide chains; a carboxyl terminal serine proteinase domain and an amino terminal contained a kringle and a growth factor domain (GFD). The GFD contains all the determinants required for binding to uPAR. Conversion of pro-uPA to uPA occurs by cleavage of the peptide bond Lys158–Ile159. This conversion can be catalyzed by plasmin. In turn, uPA can catalyze the inactive plasminogen to the active plasmin though splitting the single peptide bond Arg561Val562. Thus, uPA and plasmin generate a positive feedback loop to active each other [5]. The serine proteinase plasmin consists of two disulfide bridge–linked polypeptide chains. The C-terminal contains a typical serine proteinase domain which is responsible for catalytic activity and binding to inhibitors. The N-terminal contains five socalled kringle domains. Plasmin is able to be inhibited by a2-antiplasmin (a2AP) [6]. 2.2. uPAR * Corresponding author. Tel.: +86 531 88382490; fax: +86 531 88382490. E-mail addresses: [email protected], [email protected] (X. Han). 0753-3322/$ – see front matter ß 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biopha.2012.10.003 uPAR is over-expressed in various tumour cells, including colon, liver, breast, lung, stomach, ovary cancer et al.; additionally in 180 L. Tang, X. Han / Biomedicine & Pharmacotherapy 67 (2013) 179–182 several tumour assisting cells, such as endothelial cells, macrophages and fibroblasts [7]. uPAR is a 50–60 kDa extracellular glycoprotein with riched cysteine and congregated in lipid rafts [8]. uPAR is composed of three homologous domains (D1, D2 and D3) belonging to the Ly-6/uPAR/alpha-neurotoxin protein domain family [9], the last of which attached to the cell membrane by a glycosyl phosphatidyl inositol (GPI) anchor. This GPI-anchor is hypothesized to have high intramembrane mobility. Furthermore, the uPAR is glycosylated at N-residues of glucosamine and sialic acid within the binding site, thereby regulating its affinity (KD of 0.1–1.0 nM) for uPA [10]. uPAR binds with high affinity to uPA, prouPA and the ECM protein vitronectin. The interaction between uPA and uPAR require the intact three-domain (D1, D2 and D3) structure of uPAR, among which D1 is most important during the interaction. The crystal structure of a soluble form of human uPAR reveals that the receptor-binding module of uPA engages the uPAR central cavity, thus leaving the external receptor surface accessible for vitronectin (VN) and integrins [11]. uPAR shed from the membrane by phospholipase or proteolytic cleavage the GPIanchor. The soluble uPAR can scavenge the uPA and interfere in the functions of uPAR [12]. The release of DI fragment from the rest receptor (DIIDIII) induces uPAR cleavage, which inactivate the binding to most ligands [13]. 2.3. Serine proteinase inhibitor The activation of uPA and tissue plasminogen activator (tPA) can be inhibited by the endogenous serine proteinase inhibitor PAI-1 and PAI-2 among which the fast acting PAI-1plays the predominant role [10]. PAI-1 reacts quickly with both uPA and tPA [6], modulating the fibrinolytic activity in the vasculature. PAI-1 is mainly produced by endothelial cells, megakaryocytes as well as smooth muscle cells and stored in platelets [14]. After release into the bloodstream, the majority of PAI-1 is active and circulates in complex with VN [15]. PAI-1 binds active uPA forming an uPAR-uPA-PAI-1 covalent complex and brings about the internalization of the whole complex. This internalization is mediated by a member of the low density lipoprotein receptor-related protein (LRP) family. This process involves the formation of clathrin-coated vesicles; uPA-PAI-1 complex is degraded in lysosomes and uPAR is recycled back to the cell surface, which is necessary to sustain plasminogen activation on the cell surface [4,16,17]. PAIs react with active uPA, but not with pro-uPA [18]. PAI-1 and u-PAR can interact with the ECM protein VN and its integrin [6]. VN contains one somatomedin B (SMB) domain, one Arg-Gly-Asp (RGD) sequence, one collagenbinding region and two hemopexin-like domains [19]. The SMB domain binds PAI-1 and u-PAR and the RGD sequence binds the integrins. PAI-1 and u-PAR can compete for binding to VN. And PAI-1 may impair the binding of integrins to VN. 3. Biological functions uPA catalyzes the transformation of plasminogen to its active form plasmin, which is able to degrade many ECM proteins, such as fibronectin (FN), VN and fibrin. It can also catalyze activation of the zymogen forms of several metalloproteinases [6]. In this regard, uPA may have a central role in initiating the proteolytic cascade that facilitates the invasion of blood vessels by tumor cells, their dissemination through the circulation, and their final deposition and growth at distant sites [20]. uPA triggers the series cascade reactions relying on combination with uPAR. The effect of uPAR on cancer cell migration may be divided into proteolytic as well as non-proteolytic functions. The proteolytic function catalyze by uPAR-bound uPA. The non-proteolytic function will rely on the interaction with VN, integrin family and G protein-coupled receptors [21]. The total functions of uPAR contain cellular movement by proteolytic extracellular matrix degradation for tumor cell invasion, chemotaxis, and cellular adhesion; activation diacylglycerol accumulation, modulation of cAMP levels, angiogenesis, and alterations in inositol phosphate, interactions with integrins, tyrosine kinases and serine/threonine kinases [8,22]. The uPAR also have influence the development of inflammatory and immune responses [17]. Cells are resistant to traditional chemotherapies, and could serve as critical targets for more effective therapeutic interventions [23]. The data of Hollas et al. suggest that invasion is a function of the amount of cell surface receptor bound urokinase for cultured colon cancer [24]. uPAR activates various intracellular signaling molecules such as the tyrosine kinase Src, the serine kinase Raf, focal adhesion kinase (FAK), p130Cas and extracellular-signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK). Activation of these proteins leads to deep changes in cell proliferation, adhesion and metastasis [12]. PAI-1’s role in cancers is dual character that it can affect cell surface expression and internalization of uPA-uPAR leading to inhibition of invasion and metastasis and also it has been reported to facilitate tumor growth and dissemination [4]. PAI-1 is a poor prognostic marker in various tumors. PAI-1 promotes cancer invasion and metastasis through preventing excess degradation of the ECM, modulating cell adhesion, playing a role in angiogenesis, and stimulating cell proliferation [25]. 4. uPA system and breast cancer uPA is involved in regulating breast cancer invasion and metastasis, explained by its ability to facilitate ECM degradation, cell proliferation, angiogenesis, migration and adhesion[26,27]. The traditional prognostic factors for breast cancer focus on age, tumor size, tumor grade, lymph node status, steroid receptor status, menopausal status and histologic type [25,28]. uPA and PAI-1 are the first novel tumor biological prognostic factors confirmed in the highest level of evidence regarding their clinical utility in breast cancer[29]. As a marker for breast cancer, uPA may be an important independent variable to identify the recurrence rate [2] and is stronger than most of the traditional prognostic factors [25], especially in the node-negative subtype. uPA and PAI1 can classify about half of node-negative breast cancer patients as low risk, of which low levels have a very good prognosis, and half as high risk, because of high levels of them are correlated with shortened disease-free interval and poor overall survival [30,31]. uPA and PAI-1 play a key role in selecting appropriate therapies for patients with breast cancer[25]. High concentrations of uPA and PAI-1 in node-negative breast cancer women could benefit from adjuvant chemotherapy, whereas those with low concentrations of both proteins could be spared the side effects and costs of this treatment [25]. Node-negative patients with high uPA/PAI-1 are more than double risk of disease recurrence compared to that of patients with three or more tumor cell positive axillary lymph nodes [31]. Some study showed the bad prognostic impact of high uPA and PAI-1 levels was higher in patients treated with adjuvant chemotherapy than patients treated with adjuvant hormone therapy [32], while other results suggested that patients with increased concentrations of either uPA or PAI-1 fail to respond to hormone therapy in advanced disease [25]. In lymph nodepositive patients, PAI-1 protein displayed stronger prognostic impact than uPA [33]. uPA and PAI-1 median levels are higher in ductal than in lobular tumors[28]. In conclusion, uPA and PAI-1 levels in primary tumor tissue provide clinically relevant information on relapse risk and treatment response that will help to tailor adjuvant therapy concepts in breast cancer, accounting for individual biological tumor characteristics [34]. L. Tang, X. Han / Biomedicine & Pharmacotherapy 67 (2013) 179–182 181 Fig. 1. Schematic representation of components and biological functions about uPA system. 5. Mechanisms of uPA system in cancer invasion and metastasis The uPA system promotes tumor metastasis by several different mechanisms (Fig. 1), and not just only by breaking down the ECM [35]. uPA and uPAR initiate the activation of MMPs as well as the conversion of plasminogen to plasmin [36], then degradate the ECM and reduce the interaction between cell and cell, cell and ECM. Expression of uPA and uPAR can be up-regulated by mitogen, growth factors, the oncogenes v-Src and v-Ras, cytokines, protein kinase C and ligation of integrin with extracellular matrix protein [37,38]. Binding of uPA to uPAR can activate Ras-Raf-MEK-ERK pathway [39]. Because uPAR has no transmembrane structure, many studies propose integrins act as an associated protein to transduce proliferative or migratory signals [40,41]. In the transduction events, some intracelluar enzymes and adaptor proteins should play key roles in the signal transduction processes. The FAK has been implicated to mediate signal transduction events initiated by integrins through recruitment c-Src or other Src family tyrosine kinases [42]. Src phosphorylates Tyr-925 in FAK and creates a binding site for Grb2/Sos complex to activate Ras. c-Src and FAK may also phosphorylate Shc serving as an adaptor protein to recruit Grb2. FAK, c-Src, and Shc affiliates the uPAR-initiated pathway and integrin-mediated ERK activation [43]. uPA-induced Ras-ERK signaling pathway is dependent on the downstream effectors Raf and MEK [44]. uPA-initiated cell migration requires the integration of diverse cell signaling pathways. Jo et al. support Rho-Rho kinase pathway cooperates to promote Ras-ERK-stimulated cell migration [44]. The p38 MAPK pathway also participates in uPA secretion and inhibits the MEK/ERK signaling pathway [45]. Myosin light chain kinase (MLCK) acts as downstream of Ras/ERK and involves in uPA-promoted cell migration. Activated MLCK can induce serine phosphorylation of the myosin II regulatory light chain and thereby promotes contraction of the actomyosin cytoskeleton [44]. There are other signal pathways involved in uPA-initiated cell migration to be reported. Present studies suggest that the p38 MAPK pathway participates in invasive breast cell migration by regulating uPA expression. p38a, rather than p38b, MAPK activity is essential for uPA expression [36]. The Rac1-MKK3-p38MAPKAPK2 pathway was found to mediate Vn/av integrinmediated uPA up-regulation [46]. Bagheri-Yarmand et al. showed that upregulation of the uPA system contributed to the invasive function of LIMK1 in MDA-MB-435 breast cancer cells and suggested a signaling pathway connecting LIMK1 and the uPA system to actin reorganization and increased cell invasiveness [47]. Another reporter showed that uPA transcription was regulated through canonical JAG- Notch signaling, facilitating the invasion and metastasis in breast cancer [3]. In MDA-MB-231 cells, active PI3K, via transactivation of NF-kB, induces expression and secretion of uPA. PI3K is constitutively active in highly invasive breast cancer cell, MDA-MB-231[48]. And PKC through the active transcription factors AP-1 and NF-kB regulates secretion of uPA in breast cancer cells [38]. The correlation between PAI-1 and integrin aV and HER3 also were found [49,50]. 6. Conclusion uPA system plays an important role in breast cancer growth, invasion, and metastasis. As a candidate target, uPA is crucial in the choice of adjuvant therapy scheme in node-negative breast cancer because it provides relevant information on relapse risk and treatment response. uPA system may be worked through Ras-ERK or p38-MAPK pathway to facilitate tumor cell metastasis and invasion, but it need to be further investigated. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. 182 L. Tang, X. 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