Plasminogen Activator Inhibitor-1: The Expression, Biological Functions, and Effects on Tumorigenesis and Tumor Cell Adhesion and Migration

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1 Plasminogen Activator Inhibitor-1: The Expression, Biological Functions, and Effects on Tumorigenesis and Tumor Cell Adhesion and Migration Chun-Chung Lee and Tze-Sing Huang 1 National Cancer Research Center, National Health Research Institutes, Taipei, Taiwan Review Article Plasminogen activator inhibitor-1 (PAI-1) is a primary regulator of urokinase-type plasminogen activator (upa). It inhibits upa by forming a covalent complex, thus blocking upa s interaction with substrates. The half-life of active PAI-1 is < 1 h and it is easily transformed to a more stable inactive latent form. By binding with vitronectin, PAI-1 can be stabilized and remained its activity. PAI-1 expression is regulated by many intrinsic factors (e.g. cytokines, growth factors, hormones, and lipids) and extrinsic factors (e.g. physical injury and DNA-damaging agents). PAI-1 is an essential regulator in physiological thrombotic/fibrinolytic process in vessel. It is also in the extracellular matrix (ECM) where it controls local proteolysis via inhibiting upa. PAI-1 can regulate cell adhesion via either inhibition of upa or interference with the binding between cellular integrins or upa receptor (upar) and vitronectin. In addition, PAI-1 can regulate cell migration by inducing clearance receptor-mediated cycled attachment-detachmentreattachment of integrins. PAI-1 may have a role in regulating tumor invasion, angiogenesis and metastasis. Elevated levels of both upa and PAI-1 are associated with a poor prognosis in many cancers. However, most in vivo experiments revealed: (1) High level (pharmacological level) of PAI-1 prevented angiogenesis and tumorigenesis; (2) Low level (physiological level) of PAI-1 conversely facilitated tumor growth and angiogenesis, and (3) While in PAI-1-deficient host, tumor growth and angiogenesis could not progress. The conclusions of PAI-1 in cancer development are still controversial. Because PAI-1 may play a role in tumor cell migration and invasion through antiproteinase activity and interference with cell attachment to ECM, some strategies can be considered to apply PAI-1 derivatives to cancer therapy. Keywords: PAI-1 upa upar integrin cell adhesion cell migration Journal of Cancer Molecules 1(1): 25-36, Introduction Plasminogen activator inhibitors (PAIs 2 ) are classified as a subgroup of the serine protease inhibitor (serpin) superfamily with a common characteristic of possessing an arginine in the reactive center [1]. The members, including PAI-1, PAI-2, PAI-3 (protein C inactivator), and protease nexin 1 (PN-1), act as the inhibitors of tissue-type plasminogen activator (tpa), urokinase-type plasminogen activator (upa) and thrombin [1-4]. PAI-1 is a single chain glycoprotein with the Received 8/10/05; Revised 9/14/05; Accepted 9/20/05. 1 Correspondence: Dr. Tze-Sing Huang, National Cancer Research Center, National Health Research Institutes, 7F, No. 161, Min-Chuan East Road Sec. 6, Taipei 114, Taiwan, ROC. Phone: ; Fax: tshuang@nhri.org.tw 2 Abbreviations: PAI-1, plasminogen activator inhibitor-1; serpin, serine protease inhibitor; PN-1, protease nexin 1; tpa, tissue-type plasminogen activator; upa, urokinase-type plasminogen activator; upar, upa receptor; ECM, extracellular matrix; MMP, matrix metalloproteinase; TGF-β1, transforming growth factor-β1; bfgf, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; RCL, reactive center loop; LRP, lipoprotein receptor-related protein; SMB, somatomedin B; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α; PMA, phorbol 12-myristate 13-acetate; HRE, hypoxia-responsive element. molecular weight of 45 kda. It is expressed in many cell types, such as fibroblasts, smooth muscle cells, endothelial cells, hepatocytes, inflammatory cells, let alone the platelets, the main source of the circulating PAI-1 [1]. PAI-2 was originally described in the extracts prepared from placenta and accordingly was called placental PAI [1]. Macrophage lineage cells has also been found to express PAI-2 [1]. PAI-3 was isolated from human urine and this molecule is identical with protein C inactivator found in plasma [4]. PN-1 was isolated from cultured fibroblasts, but is also produced by other cultured cell types [1-2]. Generally, PAIs are specific and fast-acting inactivators with distinct biological characteristics and are present in most body fluids, tissues and cell lines. They can act as a pseudo-substrate to stably couple with target proteases by ester bond formation, and thus convert the target enzymes into an inactive conformation [5]. PAI-1 is the most extensively studied serpin member as we know today. In this article, we will review the biological function of PAI-1 and the factors that regulate PAI-1 expression. Additionally, we will also focus on the effects of PAI-1 on cancer cell growth, adhesion, migration and invasion. The possible role of PAI-1 for cancer therapeutics will be discussed MedUnion Press 25

2 Lee et al. J. Cancer Mol. 1(1): 25-36, 2005 Figure1: Plasminogen activator inhibitor- 1 is a regulator of plasminogen activators. There are two types of plasminogen activator, i.e. tissue-type plasminogen activator (tpa) and urokinase-type plasminogen activator (upa). In the vessel, tpa binds to fibrin and converts plasminogen into plasmin for proteolytic degradation of the clot. upa is the major plasminogen activator for migrating cells, and it is activated by binding with upa receptor (upar) and subsequently initiates a proteinase cascade. Plasmin is a proteinase with a wide range of substrates. It is able to degrade fibrin or other extracellular matrix (ECM) components, to cleave and activate other proteinases sush as matrix metalloproteinases, and to activate latent transforming growth factor-β1 (TGFβ1), basic fibroblast growth factor (bfgf) and vascular endothelial growth factor (VEGF). Plasminogen activator inhibitor-1 (PAI-1) is a primary inhibitor to inhibit the activation of both tpa and upa. PAI-1 is a regulator of PA system PAI-1 is an important regulatory protein involved in the proteolytic [6,7] and fibrinolytic [8,9] pathways of plasminogen activator. It is the major inhibitor of two types of plasminogen activator, i.e. tpa and upa. tpa binds to fibrin and convert plasminogen to plasmin in the clot and is known as the primary fibrinolytic activator [10](Figure 1). Distinctly, upa has very low affinity for fibrin, but it is the major PA form expressed by the migrating cells and its activity is mediated by upa receptor (upar). upa binds to upar on cell surface to initiate a proteinase activity, which in turn leads to the activation of plasmin [11-13]. Plasmin is a proteinase exhibiting a wide range of substrate spectrum. It is able to degrade many glycoproteins and proteoglycans of extracellular matrix (ECM) such as laminin, fibronectin, vitronectin, fibrin, etc. [14]. It also can activate other proteinases such as matrix metalloproteinase (MMP)-1, -3 and -9 [15], and activate or release growth factors from ECM including latent transforming growth factor (TGF-β) [16], basic fibroblast growth factor (bfgf) [14], and vascular endothelial growth factor (VEGF) [17] (Figure 1). Cellular releasing of plasmin can therefore promote ECM degradation and cell migration/invasion [18]. By preventing the activation of plasmin, the biological function of PAI-1 is not restricted in the vascular fibrinolytic system, PAI-1 can play an important role in many other physiological and pathological processes of tissue remodeling including wound healing, embryogenesis, and tumor cell migration and invasion [19]. Molecular structure of PAI-1 and its interaction with PA PAI-1 is a single-chain glycoprotein belonging to the serpin family. All members of this family consist of about 400 amino acid residues and have apparent molecular weights from 38 to 70 kda, depending on their degree of glycosylation [20]. From the nucleotide sequence analysis, PAI-1 cdna encodes a protein containing 402 amino acids with a predicted nonglycosylated molecular mass of 45 kda [21]. The mature secreted form of PAI-1 consists of 379 amino acids and contains ~13% carbohydrate, which increases the molecular weight to ~50 kda. The tertiary structure of the serpin family protein exhibits three β-sheets (A, B and C), nine α-helices, and an exposed reactive center loop (RCL) that contains the reactive site Arg 346 -Met 347 for the target serine protease [22]. The reactive residues Arg 346 -Met 347 are designated as P1-P1 residues of the RCL (Figure 2). The amino acids located N-terminal from this site are referred to as P-residues P16-P1 and the C-terminal residues are P - residues P1 -P13 (Figure 2A). The strength of binding between the P1 residue and target protease determines the inhibitory activity of the serpin family protein [23]. PAI-1 inhibits PA by the formation of a covalent complex, thus blocking PA for further interaction with its substrate [24]. The inhibitory process involves the formation of a noncovalent reversible Michaelis-like complex, followed by an acyl intermediate and finally the formation of an ester bond between the carboxyl group of the P1 residue and the hydroxyl group of the serine residue of protease [25](Figure 2A upper panel). Once the initial complex is formed, the P1-P1 bond in the RCL is cleaved, and immediately followed by an insertion of the P1-PA complex from the initial interaction site into the opposite side of the PAI-1. This causes a distortion of the protease and inhibits its catalytic activity (Figure 2B). In this covalent complex, the PAI-1 is cleaved at the P1-P1 site implicating that the inhibitory activity of PAI-1 is limited to a single encounter with its target protease [26-28]. Besides the active and cleaved forms, there exists a latent form of PAI-1 that can be transited from both active and cleaved forms. The active form spontaneously converts to the latent form with a half-life of < 1 h [29]. In this structure, the entire amino terminal side of the RCL is inserted as the central strand of β- sheet A (Figure 2C). This increases the stability of PAI-1 but lacking of its inhibitory activity. The latent form can be reactivated partially by denaturing agents [30]. Interaction of PAI-1 with proteins other than PAs In addition to the ability to bind and inactivate PAs, PAI-1 also has other ligands that might mediate functions of PAI-1. These ligands include glucosaminoglycans (eg. heparin)[31], ECM components (eg. vitronectin)[32], and scavenger receptors such as members of the low-density lipoprotein receptor (LDLR) superfamily, specifically the lipoprotein receptorrelated protein (LRP)[33-37]. Vitronectin Vitronectin is a 459-aa glycoprotein. It is divided into a number of specific domains, including the 44-residue somatomedin B (SMB) domain at the amino terminus, the connecting region that is immediately adjacent to the SMB domain and contains a single RGD sequence for binding with integrin, and two hemopexin-like repeats. Vitronectin is rela- 26 Print ISSN

3 PAI-1 s Expression, Biological Function, and Effects on Cancer Development Figure 2: Molecular structure of PAI-1 and its interaction with upa. (A) PAI-1 contains an exposed reactive center loop (RCL)(red). The active site Arg 346 (red ball)-met 347 (green ball), designated as P1-P1 residues, is responsible for contacting with its target protease (blue ball). The inhibitory process involves the formation of a reversible acyl intermediate, cleavage at the P1-P1 bond, and the final formation of an irreversible ester bond between the carboxyl group of the P1 residue and the hydroxyl group of the serine residue of the protease. (B) Once the initial acyl intermediate is formed, the P1-P1 bond in the RCL is cleaved, and immediately followed by a translocation of the P1-PA complex from the initial interaction site to the opposite side of the PAI-1. This causes a distortion of the PA and thus inhibition of the proteinase activity. (C) Besides the active and cleaved forms, there exists a latent form of PAI-1. In this structure, the entire amino-terminal part of RCL (i.e. P1- P16) is inserted as the central strand of β- sheet A. This increases PAI-1 s stability but decreases its inhibitory activity. tively unique among adhesive proteins not only because PAI-1 binds to it with high affinity but also cells can attach to it through integrins, upar, or both [38,39](Figure 3A). Vitronectin-bound PAI-1 inhibits the proteolytic degradation of ECM by inhibiting upa/tpa, which in turn inhibits cell migration and invasion [40]. The binding site of vitronectin for PAI-1 is localized to the SMB domain [41]. All of the active PAI-1 in plasma circulates in complex with vitronectin [32], and the binding with vitronectin increases the stability of PAI-1 [42]. In tissue, vitronectin also binds to upar [43] and the binding site for upar is located in the SMB domain as well [44]. The binding sites for PAI-1 and upar are partially overlapping but not identical. The affinity of PAI-1 for the SMB domain is much higher than that of upar [45] (Figure 3B). Thus, PAI-1 can competitively inhibit the upardependent attachment of cells to vitronectin [38,39,45](Figure 3B). Binding of PAI-1 to SMB also inhibits integrin-mediated cell adhesion by sterically blocking the adjacent RGD site [36] (Figure 3B). The stability of PAI-1 in plasma is increased by 2-fold when binding with vitronectin [46]. The crystal structure of the PAI-1 SMB complex suggests a simple mechanism for the stabilization of PAI-1 activity: SMB slows the transition of PAI-1 to the latent form by blocking the associated sliding movement of strands 1A and 2A of the main β-sheet into gap between helices E and F [47] (Figure 2B). By binding to vitronectin, the target specificity of PAI-1 is altered, because interaction with vitronectin enables PAI-1 to inhibit another serine protease, thrombin [48]. Lipoprotein receptor-related protein (LRP) Once PAI-1 forms the complex with upa that is specifically bound to upar, it loses its high affinity for vitronectin but instead increases its affinity toward the clearance receptors such as lipoprotein receptor-related protein (LRP)[37]. After PAI-1 is complexed with proteases, the heparin-binding domain of PAI-1, which contains a cryptic receptor-binding site (Lys 69 ) with the high affinity for LRP, becomes exposed and accessible for LRP binding. The complex is endocytosed by binding to clathrin-coated pit-localized endocytosis receptors (Figure 3C and Figure 4). The upa and PAI-1 then undergo lysosomal degradation, whereas upar is recycled and reappears on the cell surface [52] (Figure 4). However, another report demonstrate that GPI-anchored upar is also endocytosed by piggybacking on LRP and that direct binding of occupied upar to LRP is essential for internalization of occupied upar [53]. Especially, direct binding of domain 3 (D3) of upar to LRP is required for clearance of upa-pai- 1-occupied upar [53]. PAI-1 also contains a cryptic receptor-binding site that is exposed upon complex formation with tpa, and plays a role in the clearance of tpa [54]. These studies suggest that PAI-1, by interacting with upa/upar and LRP, can be a potent chemoattractant molecule, which induces cell migration via regulating morphological attachment/dettachment changes (Figure 4). Recently, PAI-1 was reported to induce cell migration with corresponding cytoskeleton reorganization and phosphotyrosine redistribution [55]. Using several PAI-1 mutants and specific inhibitors, the activity of PAI-1 was shown not to depend on its interactions with upa, tpa or vitronectin, but rather with LRP. This interaction leads to the activation of the Jak/Stat signaling pathway and the induction of cell migration [55]. Heparin It has been demonstrated that the PAI-1-binding site on vitronectin is adjacent to a heparin-binding site [49]. In addition, a heparin binding site can be predicted from the amino acid sequence of PAI-1, which is conserved in antithrombin III and heparin cofactor II. Consequently, It have been investigated that PAI-1 indeed interacts with heparin. In the presence of heparin, the reactivity of PAI-1 toward thrombin is substantially increased. In contrast to vitronectin, heparin does not stabilize the active conformation of PAI-1 [50,51]. Biochemical function of glycosylation of PAI-1 PAI-1 has 3 potential sites for N-linked glycosylation. A heterogeneous glycosylation pattern was noted to occur at the Asn 209 and Asn 265 sites of PAI-1, while the Asn 329 site is not utilized for glycosylation yet in human cell lines [56]. The latent transition of non-glycosylated PAI-1 was more easily enhanced by a non-ionic detergent if comparison with glycosylated PAI-1. The glycosylation at Asn 265 seems to determine the conformation of PAI-1. Also, the PAI-1 binding protein vitronectin may reverse the conformational changes induced by the lack of glycosylation at Asn 265 [56] MedUnion Press 27

4 Lee et al. J. Cancer Mol. 1(1): 25-36, 2005 Figure 3: PAI-1 involved in cell adhesion. (A) Vitronectin contains the somatomedin B (SMB) domain at its amino terminus, and immediately adjacent to the SMB domain is the single RGD sequence that is a binding site for integrins. Vitronectin is relatively unique among adhesive proteins not only because PAI-1 binds to it but also because cells can attach to it through integrins, upar, or both. The binding sites of vitronectin for upar and PAI-1 are both located in the SMB domain. (B) The binding sites of vitronectin for PAI-1 and upar are partially overlapping but not identical. The affinity of PAI-1 for the SMB domain is much higher than the affinity of upar for this domain. Thus, PAI-1 can competitively inhibit the upar-dependent attachment of cells to vitronectin. Binding of PAI-1 to SMB also inhibits integrin-mediated cell adhesion via a steric hindrance to the adjacent RGD site. (C) PAI-1 can bind with upa/upar complex. This association renders the cryptic receptor-binding site (CRB) of PAI-1 exposed out and increases PAI-1 s affinity for the clearance receptor lipoprotein receptor-related protein (LRP). By binding to clathrin-coated pit-localized endocytosis receptors, the complex of PAI-1/uPA/uPAR can be endocytosed. The PAI-1 and upa then undergoes lysosomal degradation. Many factors regulate PAI-1 expression in vitro and in vivo PAI-1 promoter The human PAI-1 gene is located on the long arm of chromosome 7. The genomic DNA of human PAI-1 is ~12.2 kb long, comprising nine exons and eight introns [21,57]. There are two distinct transcripts (3 and 2 kb, respectively) of PAI-1 mrna with different lengths of polyadenylation in the 3 untranslated region (3 UTR)[21,58]. The sequence of 5'-flanking DNA contains the essential cis-acting elements CCAAT and TATAA for RNA polymerase binding [59]. Several known consensus sequences of regulatory elements are located in the region of ~800 bp in front of the PAI-1 transcription initiation site. These cis-acting elements and the trans-acting regulatory factors involved in the regulation of PAI-1gene expression include: SP-1 (-76 to -71 and -44 to -39) [60,61], CTF/NF-1 (-119 to -105, -477 to -445 and -542 to -519), AP-1 (-65 to -50), AP-2 and SP-1-like (-82 to -65)[62], and two repressor elements (-764 to -628 and -266 to -188)[57]. Interestingly, wild-type p53 acts as a trans-activator and mutant p53 (His 273 ) acts as a repressor to the binding region at -160 to -139 of PAI-1 promoter [63]. There are three CAGA conserved elements (-280, -580 and -70) that are specific for TGF-β1-activated Smad 3 and Smad 4 binding in the PAI-1 promoter [64]. One hypoxia-responsive element at the human PAI-I promoter -194 to -187 is identified, and is necessary and sufficient for hypoxia-mediated response [65](Figure 5). PAI-1 mrna stability The level of PAI-1 gene expression is determined not only by the rate of gene transcription but also by the rate at which the mrna is degraded. The AU-rich sequence in the 3 UTR of PAI-1 transcript determines its mrna stability [66,67]. Moreover, PAI-1 mrna is especially degraded easily when cells have been treated with 8-bromo-cAMP [68]. Using serial deletion and insertion of PAI-1 3 UTR sequence, the region from 2926 to 3054 of PAI-1 mrna is defined as PAI-1 camp-responsive element (PAI-1 CRE) [69]. Recently, a PAI- 1 RNA binding protein 1 (PAI-RBP1) that specifically interacts with the PAI-1 CRE has been identified to involve in camp-mediated PAI-1 mrna degradation [70]. Factors regulate PAI-1 expression Expression of PAI-1 can be regulated at the transcriptional level by many factors including: growth factors and cytokines (e.g. TGF-β1, interleukin-1, FGF, and VEGF), hormones (e.g. glucocorticoids and insulin), inflammatory factors (e.g. tumor necrosis factor-α and lipopolysaccharide), glucose or lipid metabolites (e.g. glucose, free fatty acid, triglycerol, and very-low-density lipoprotein), vascular tone regulating factors (e.g. angiogentensin II); chemicals (e.g. phorbol ester), and other environmental or physical factors (e.g. reactive oxygen species, hypoxia, stress, wound, adhesion to matrix). Some examples are described as below: TGF-β1: TGF-β1 is one of the earliest cytokines that was described to regulate PAI-1 expression at the transcriptional level. TGF-β1 is abundant in the α-granules of platelets. After vascular injury, it can be released from platelets and stimulate local endothelial PAI-1 expression. This will slow down the lytic process of thrombin, and result in increase of fibrin deposit and progression of atherosclerosis. The TGFβ1-responsive CAGA boxes in the PAI-1 gene promoter are the binding sites for SMA/Mothers against decapentaplegic (Smad) proteins, which play the key role in TGF-β1-mediated activation of PAI-1 transcription [64]. Inflammatory factors: Lipopolysaccharide (LPS) induces PAI-1 levels in the plasma of patients with Gram-negative septicemia and in the cultured human and bovine endothelial cells [71]. LPS stimulates PAI-1 synthesis but decreases tpa production in endothelial cells, resulting in an antifibrinolytic outcome. The stimulatory effect of LPS on PAI-1 expression is mediated in part by tumor necrosis factor-α (TNF-α). The 5' distal TNF-α-responsive element of PAI-1 gene located 15 kb upstream of the transcription start site that is a conserved NF-κB-binding site and mediates PAI-1 s response to TNF-α [72]. Lipids: Free fatty acids induces PAI-1 expression by activating a transcription factor to bind with the sequence 5 - TG(G/C) 1-2 CTG-3 that is repeated four times in the PAI-1 promoter (-528 and 599)[73]. A very-low-density lipoproteinresponsive element that locates at the PAI-1 promoter region from -672 to -657 is responsible for the effect of plasma triglycerides on PAI-1 expression [74]. Notably, the 4G/5G polymorphism site locates at Higher level of PAI-1 synthesis is associated with the 4G/4G genotype. The 4G/4G allele is preferentially bound with an enhancer protein, whereas the 5G/5G allele is bound with an enhancer plus a 28 Print ISSN

5 PAI-1 s Expression, Biological Function, and Effects on Cancer Development Figure 4: PAI-1 involved in cell migration. PAI-1 binds upa/upar complex. This leads to a large shift in PAI-1 s relative affinity from vitronectin to the clearance receptor LRP. Subsequently, PAI-1, LRP, upa/upar, and associated integrins will be endocytosed. Cell will detach not only from the vitronectin but also the general matrixes. In the endosome, PAI-1/uPA complex separates from the receptors and is targeted to the lysosome for degradation. The LRP, upar and integrins are then recycled to another edge of the cell surface for the next attachment to ECM. This model provides for the PAI-1-mediated cycled attachmentdetachment-reattachment of integrins that is necessary for cell migration. suppressor, resulting in lower levels of PAI-1 transcription. This polymorphism site responds to triglyceride. Increased levels of PAI-1 will occur in the individuals with the 4G/4G genotype and elevated triglyceride levels in plasma [75,76]. Despite the plasma levels of PAI-1 seem to be the result of both environmental and genetic influences, many studies have reported that higher PAI-1 plasma levels occur in individuals with 4G/4G genotype [77-80]. However, the data on the clinical relevance of these polymorphisms are controversial [81]. The transcription factor PPARγ may also participate in PAI-1 regulation, and PPARγ might involve in vascular diseases [82]. Glucose and Insulin: In pig aortic endothelial cells, the increase of PAI-1 secretion is significantly associated with extracellular glucose concentration [83]. It has also been found that insulin stimulates synthesis of PAI-1 in human hepatic cells [84]. Glucose-induced PAI-1 expression could interpret the frequent occurrence of attenuated fibrinolytic activity of plasma and premature cardiovascular disease in the patients with type II diabetes mellitus. Glucose regulates PAI-1 gene expression through two adjacent SP-1 sites located between -85 and -42 of the PAI-1 5'-flanking region [61]. Release of a transcriptional repressor from the SP-1 complexes may explain the induction of PAI-1 gene under high glucose conditions in vascular smooth muscle cells [61]. Hyperglycemia stimulates Rho-kinase activity via PKC and oxidative stress-dependent pathways, leading to increased PAI-1 gene transcription [85]. p53 and the cell cycle: A p53 binding site in the region to -139 of the human PAI-1 gene promoter has been identified that is responsible for stimulation of the PAI-1 transcription by p53 [63]. Conversely, a p53 mutant (His 273 ) inhibits PAI-I promoter activity. This result suggests that an oncogenic p53 form may reduce PAI-1 expression and thus alter the plasminogen/plasmin system during tumor progression. In addition, PAI-1 is expressed by a cell cycledependent manner with increasing expression during growth activation (G 0 -to-g 1 transition), and PAI-1 could therefore be an indirect cell proliferation indicator [86,87]. Phorbol Ester: Phorbol 12-myristate 13-acetate (PMA) induces the PAI-1 levels in human rhabdomyosarcoma and hepatocellular carcinoma cell lines [88,89]. There is also an increase in the PAI-1 mrna and gene transcription levels in PMA-treated cells. The PMA response elements (i.e. AP-1 site) in the PAI-1 gene promoter are also identified [62]. Hypoxia: Oxygen deprivation, as occurred during tissue ischemia, usually results in coagulation. When mice are placed in a hypoxic environment, the plasma levels of PAI-1 increase in a time-dependent manner accompanied by an increase in plasma PAI-1 activity [90]. Northern blot analysis of the hypoxic murine lung tissues have demonstrated an increase in PAI-1 mrna compared with normoxic controls [90]. In contrast, the levels of transcripts for both tpa and upa are decreased under hypoxic conditions. Several putative hypoxia-responsive elements (HRE-1, at -158 to 151; HRE-2, at -194 to -187; HRE-3, at -453 to -446; HRE-4, at -566 to -559; HRE-5, at -681 to -674) were identified in the human PAI-I gene promoter [65]. Reporter gene assays show that the HRE-2 site is necessary and sufficient for the hypoxiainduced PAI-1 expression [65]. Cell adhesion: In anchorage-dependent cells, the level of PAI-1 mrna expression is increased when cells begins to attach onto the substrate and subsequently turns to be down regulated when cells have attached already [91]. The PAI-1 gene expression is induced only in adherent but not in nonadhered cells. Regulation of PAI-1 gene expression by cell adhesion can be initiated through the interaction between vitronectin, fibronectin or collagen and integrins, and is mediated by PI-3 kinase/akt and MEK/ERK pathways [92-94]. The role of PAI-1 in regulation of cell adhesion, migration and invasion PAI-1 in cell adhesion PAI-1 has dual roles in regulation of cell adhesion. upa functions as an anti-adhesion molecule by proteolytic degradation of ECM components. As an inhibitor of upa, PAI-1 would be expected to promote adhesion. Indeed, PAI-1 has the ability to selectively protect vitronectin from proteolysis by inhibiting local plasminogen activator and thus stabilizes the vitronectin-dependent cell adhesion [95](Figure 3A). However, PAI-1 also exhibits an anti-adhesive function. Its association with vitronectin blocks the binding of both upar and integrin to vitronectin [39,96-99](Figure 3B). In addition, an anti-adhesive effect of PAI-1 can be through the down MedUnion Press 29

6 Lee et al. J. Cancer Mol. 1(1): 25-36, 2005 Figure 5: The transcription regulatory sites in the PAI-1 promoter. PAI-1 gene expression is regulated by many intrinsic factors (e.g. cytokines, growth factors, hormones, and lipids) and extrinsic factors (e.g. physical injury and DNA-damaging agents). The sequence of 5'-flanking DNA contains the essential cis-acting elements CCAAT and TATAA for RNA polymerase binding. Several known consensus sequences of regulatory elements are located in the region of ~800 bp in front of the PAI- 1 transcription initiation site. These cisacting elements and the trans-acting regulatory factors include: SP-1 (-76 to -71 and -44 to -39), CTF/NF-1 (-119 to -105, -477 to -445 and -542 to -519), AP-1 (-65 to -50), AP-2 and SP-1-like (-82 to -65), and two repressor elements (-764 to -628 and -266 to -188). Interestingly, wild-type p53 acts as a trans-activator and mutant p53 (His 273 ) acts as a repressor to the binding region at -160 to There are three CAGA conserved elements (-280, -580 and -70) that are specific for TGF-β1-activated Smad 3 and Smad 4. One hypoxia-responsive element at the human PAI-I promoter -194 to -187 is identified, and is necessary and sufficient for hypoxia-mediated response. regulation of the integrin levels on cell surface by the mechanism that PAI-1 binds and induces LRP-mediated endocytosis of PAI-1/uPA/uPAR/integrin complex [100](Figure 3C). PAI-1 in cell migration Cell migration is the locomotion of a cell over an ECM substratum. upa has been recognized as to stimulate cell migration by catalyzing plasminogen activation for proteolysis of substratum and thus releasing cells from the substratum [101]. PAI-1 would be expected to inhibit plasminogen activation-dependent cell migration. However, it has been shown that PAI-1 can directly block cell attachment and migration by a mechanism independent of its anti-proteolytic activity [102,103]. PAI-1 can play a role in cell migration owing to its dual roles in regulating the cell adhesion. It can be either pro-migratory or anti-migratory, depending on whether PAI-1 locates at the leading or trailing edge of the cell and on the PAI-1 concentration [97, ]. Once PAI-1 is binding with the upa/upar complex, PAI-1 decreases its affinity for vitronectin but increases its affinity for the clearance receptors LRP and very-low-density lipoprotein receptor [37,51]. Subsequently, cell will detach not only from the vitronectin but also the general matrixes by the clearance receptor-mediated endocytosis mechanism [98], because the endocytosis will recruit the PAI-1/uPA/uPAR complex and associated integrins [ ]. In the endosome, PAI-1/uPA complex separates from the receptors and is targeted to the lysosome for degradation. The clearance receptors, upar and integrins are then recycled to another edge of the cell surface. Thus this model provides for the cycled attachment-detachment-reattachment of integrins that is necessary for cell migration [109]. Hence, the cell migration is not only as simple as through the proteolysis of ECM or blocking the binding between vitronectin and integrin, but also through the induction of endocytosis by PAI-1-conjugated upa/upar complex (Figure 4). PAI-1 in cell invasion When cells progress from migration to invasion, not only locomotion but also penetration through ECM of the cells can be observed. In the cell invasion models, the proteolysis of ECM by upa is well associated with the cellular penetration in ECM. However, the observatory results of PAI-1 action influencing cell invasion are not consistent. Some reports suggest that PAI-1 inhibits cell invasion through its ability to inhibit the proteinase activity ( ), but coexpression of upa, upar and PAI-1 is required for optimal invasiveness of human lung cancer cells [113]. In addition, transfection of PAI-1 cdna into PC-3 prostate carcinoma cells has been reported not to alter cell invasiveness [114]. PAI-1 in tumor growth, angiogenesis and metastasis Association of upa with cancer development has been noticed for decades. A simple conception is that ECM and basement membrane are degraded through the proteinases that are activated by upa during cancer cell invasion. High levels of upa in extracts of primary breast carcinoma can be used to predict an early relapse [115,116]. Several studies with specific disruptions of the genes for plasminogen and upa also support the hypothesis that upa activation is a rate-limiting step for tumor growth, local invasion and distant metastasis [ ]. However, as an inhibitor of upa and originally regarded as an anti-cancer molecule, PAI-1 is surprised to be a poor prognostic marker in breast and other types of cancer [101, ]. What on earth is the role of PAI-1 during tumor cell progress toward malignancy? It is an important stimulant for cancer scientists to investigate the biological function(s) of PAI-1 in tumor. PAI-1 has been extensively investigated in tumor growth, invasion, and metastasis by experimental animal models (some important examples summarized in Table 1). The results obtained are quite controversial as different experimental systems are used. For example, high levels of PAI-1 expression in human or murine cancer cells were associated with the retardation of tumor growth, invasion, and metastasis in immunodeficient mice [112,114,127]. Application of high level of PAI-1 protein to the immunodeficient mice bearing transplanted human tumors caused tumor growth inhibition [128,129], but low level of PAI-1 enhanced tumor growth [129]. Deficiency in PAI-1 or upa expression in host mice prevented murine T241 fibrosarcoma growth compared with control wild-type mice [130]. Also, PAI-1 deficiency in the host mice prevented local invasion and tumor vascularization of transplanted malignant keratinocytes [131]. While this prevention in PAI-1-deficient mice was aborted by the exogenous adenoviral vector expressing PAI-1, the tumor invasion and angiogenesis were restored [131]. This PAI-1-recovered tumor vascularization is through its proteinase inhibition activity but not through its interference with vitronectin-integrin 30 Print ISSN

7 PAI-1 s Expression, Biological Function, and Effects on Cancer Development Table 1: Some important examples studying the roles of PAI-1 in cancer development by experimental animal models Model System PAI-1 Effect Reference Overexpression of PAI-1 in PC-3 human prostate carcinoma in athymic mice Overexpression of PAI-1 reduced the aggressive phenotype of PC-3 human prostate carcinoma. Soff et al [114] B16 melanomas implanted into PAI-1-overexpression or PAI-1 deficient mice Intraocular melanomas by adenovirus-mediated gene transfer of PAI-1 in an athymic mouse model LNCaP prostate carcinoma in SCID mice treated with mutated form of PAI-1 to confirm a correlation between the inactivation of upa and tumor size Malignant keratinocytes implanted into PAI-1-deficient mice No difference in tumor growth, metastasis, and survival was observed between control and PAI-1-deficient or - overexpressed mice. Metastatic tumor burden was reduced and host survival was significantly prolonged. PAI-1 inhibited tumor growth through its inhibition of PA activity. Deficient PAI-1 expression in host mice prevented invasion and tumor vascularization of transplanted malignant keratinocytes. But it was restored by intravenous injection of a adenoviral vector expressing human PAI-1. Eitzman et al [137] Ma et al [127] Jankun et al [128] Bajou et al [131] T241 fibrosarcomas implanted into upa or PAI-1 deficient mice upa or PAI-1 deficiency disturbed tumor growth. Only PAI- 1 deficiency prevented angiogenesis. Gutierrez et al [130] Chicken chorioallantoic membrane treated with pharmacological levels of PAI-1 mutants Malignant keratinocytes implanted into PAI-1 deficient mice and treated with adenoviral PAI-1 mutants M21 human melanomas in nude mice treated with different concentrations of PAI-1: matrigel implants in wild type mice treated with increasing concentrations of PAI-1 PAI-1 inhibited angiogenesis either through inhibiting proteinase activity or blocking cellular access to vitronectin. PAI-1 restored tumor vascularization through inhibition of proteinase activity but not by interacting with vitronectin. PAI-1 stimulated tumor growth and angiogenesis at low amounts while it inhibited tumor growth at higher amounts. Stefansson et al [133] Bajou et al [132] McMahon et al [129] Overexpression of PAI-1, PAI-1 (VN-), PAI-1 (INH-) PAI- 1 (VN-, INH-) and tumor cell migration in vitro and metastasis in mice in vivo Aortic ring explant in PAI-1 deficient mice treated with increasing concentration of PAI-1 or adenovirus expression of PAI-1 mutants. Only overexpression of wild-type PAI-1 reduced the burden of metastasis by 68% compared with the control. PAI-1 stimulated angiogenesis at low doses while it inhibited angiogenesis at higher doses. Praus et al [112] Devy et al [134] T241 fibrosarcomas implanted into PAI-1 deficient mice MMTVPym-T-induced breast tumor that mimic human mammary adenocarcinoma in PAI-1 deficient or normal mice PAI-1 deficiency had no effect on tumor growth and angiogenesis. No difference of breast tumor development and vascular density was observed between PAI-1 deficient and normal mice. Curino et al [138] Almholt et al [140] Doses of recombinant PAI-1 protein intraperitonealy injected in PAI-1 deficient animals to see the angiogenic potential with a model of laser-induced choroidal neovascularization. (CNV) PAI-1-overexpressed PVDA keratinocytes transplanted into PAI-1-overexpressed, -deficient or wild type mice PAI-1 exhibited both pro- and anti-angiogenic effects depending on the dose and merely through its anti-proteolytic activity. Overexpression of PAI-1 in host or in tumor cells reduced tumor development in vivo. High level of PAI-1 in cancer cells did not overcome the PAI-1 deficiency in the host for tumor growth. Lambert et al [135] Bajou et al [136] Low grade (HaCaT II-4) and high grade (HaCaT A5-RT3) of malignant skin keratinocytes subcutaneously injected in PAI-1-deficient and wild type mice Tumor incidence was reduced for HaCaT II-4 (low grade) cells in PAI-1 deficient mice, but not reduced for HaCaT A5-RT3 (high grade) cells. Maillard et al [141] binding [132]. Many studies have suggested that PAI-1 at high level prevents tumor growth through inhibiting angiogenesis, while enhances tumor growth by promoting angiogenesis at low level [129,133,134]. Recently, many investigations more support that null expression of PAI-1 in the host and supraphysiologic level of PAI-1 expression either by host or tumor cells exhibit preventive effect on tumor growth and angiogenesis, but the physiological concentration of PAI-1 promotes in vivo tumor invasion and angiogenesis [135,136]. Interstingly, when the PAI-1-overexpressed tumor cells were transplanted into the PAI-1-deficient mice, the PAI-1-overexpressed cells did not compensate the PAI-1 deficiency in the host to recover the angiogenesis and tumorigenesis [136]. However, some reports exhibit no significant difference in the tumor growth and angiogenesis between PAI-1-deficient and wild-type mice. For example, when intravenous inoculation of murine melanoma cells into PAI-1-overexpressed or PAI-1-deficient mice in comparison with wild-type control, no differences were observed in the extent of pulmonary metastasis, tumor size and overall sur MedUnion Press 31

8 Lee et al. J. Cancer Mol. 1(1): 25-36, 2005 vival in these mice [137]. PAI-1 deficiency has also no effect on the tumor growth and angiogenesis of the transplantation of T241 fibrosarcoma cells [138]. One report illustrates that using MMTVPym-T-induced breast cancer to mimic spontaneously human mammary adenocarcinoma [139], there are no differences in the primary tumor growth and vascular density between PAI-1-deficient and wild-type mice [140]. In addition, PAI-1 deficiency is not sufficient to prevent neoplastic growth of the aggressive tumors in human skin, it still exerts its tumor-promoting effect in a tumor stage dependent manner [141]. The proangiogenic effect of PAI-1 at suitable concentrations may be through the inhibitory activity of PAI-1 to limit upa-dependent plasminogen activation, and thus stabilize the basement membranes around newly formed vessels and also stabilize the matrix scaffold required for endothelial cell migration and the assembly of endothelial cells into capillaries. A critical balance between proteases and their inhibitors is thought to be essential for optimal tumor cell invasion and angiogenesis [113,142]. Elevated expression of PAI-1 in tumor cells tends to inhibit tumor growth, invasion, and metastasis. This result is opposite to the clinical observation that PAI-1 overexpression correlates with the malignancy in breast or colon cancer. By immunohistochemistry assay, the PAI-1 is expressed in the leading edge of myofibroblasts in breast cancer tissues, which is responsible for the correlation between the PAI-1 level in breast tumor extracts and poor prognosis [143]. Similar observation is also obtained in colon cancer [144]. These results are consistent with other results that PAI-1 is mostly expressed by stromal cells but not by tumor cells [131,140]. Moreover, whatever the amounts of PAI-1 are produced by tumor cells, blood vessels are limited under the collagen gel and tumor cells fail to invade in the PAI-1-deficient host tissue [136]. These findings suggest that the site of PAI-1 production (stromal cells rather than tumor cells) is a critical factor in tumor angiogenesis rather than the total amount of PAI-1 in tumor. However, some reports exhibit no differences in the tumor growth and angiogenesis between PAI-1-overexpressed or - deficient and wild type mice. These controversial results may implicate that the influence of diverse factors exist on animal tumor models, such as cell numbers implant, cell types (stages of malignancy), expression of some other types of PAI (PAI-2 or PAI-3), and the activation of PA system. Nevertheless, in despite of many reasons for the incompatible results, most of these studies result from the transplantation of tumor models that may not be reflected onto the real pathogenesis of cancer. Till now, only one report illustrates the spontaneous development of primary breast tumors, but shows no significant difference in the rates of tumor formation, growth, angiogenesis, and metastasis. From these results, how does PAI-1 involve in the real process of tumorigenesis is still not certified. It still needs to accumulate more investigations using such spontaneous carcinogenesis models. It has been suggested recently that the prognostic impact of PAI-1 is not based on its involvement in angiogenesis alone [145]. Another important efferent is that high levels of PAI-1 contribute to tumor growth by inhibiting apoptosis of tumor cells. Addition of PAI-1 to culture medium can inhibit tumor cell apoptosis [146]. The spontaneous fibrosarcoma with wild-type PAI-1 shows less sensitivity to chemotherapyelicited apoptosis [147]. This phenomenon was not noted in PAI-1-deficient fibrosarcoma. However either in wild type or PAI-1 deficiency, the mice display similar sensitivity to the treatment of etoposide [147]. Hence, PAI-1 might regulate apoptosis of transformed cells but not normal cells, suggesting a differential effect of PAI-1 between cancer cells and normal cells. Is PAI-1 a target or drug for cancer therapy Since the early observations of elevated PAI-1 expression in several tumor types, PAI-1 had been expected to be a potential target for cancer therapy. But actually, it still remains a controversy of the role of PAI-1 in tumorgenesis. From many experimental results, low level (physiological level) of PAI-1 in the host may facilitate angiogenesis and tumor growth [129,131, ]. In PAI-1-deficient background, angiogenesis and tumor will not progress [130,131]. Conversely, many other studies indicate that administration of high level (pharmacological level) of PAI-1 seems to prevent the angiogenesis, tumor growth and metastasis [112,114, ]. It is notable that all these results are derived from the model of tumor transplanted, and many experiments with different cell types or different protocols exhibit the different conclusions. The only one study that used spontaneously induced tumors in the PAI-1-deficient host also shows no correlation of PAI-1 with tumorigenesis [140]. However, administration of pharmacological levels of PAI- 1 to inhibit tumor growth and angiogenesis has been demonstrated by many reports [112,114, ,133,134,136]. Because PAI-1 is multifunctional and exhibits short half-life in vivo, more studies needed to be performed to render PAI-1 really usable in the treatment of cancer. It should be concerned that if treatment of high level of the PAI-1, the inhibitory activity of PAI-1 may increase the rate of the process of atherosclerosis and thrombosis. The strategy of using the specific mutant is intriguing to scientists. For example, the mutant of PAI-1 that interferes with the binding of integrin with vitronectin but without inhibiting PA activity is found to inhibit angiogenesis in vivo, but not found to promote thrombosis because it does not inhibit fibrinonlysis [133]. Up to date, more than 600 PAI-1 mutants have been prepared and all these mutants have been utilized for studying the structure-and-function of PAI-1 [148]. The availability of these mutants and the information of the PAI-1 s function may provide us some directions to develop useful agents to inhibit tumor angiogenesis and growth. As demonstrated by many studies, PAI-1 has been administrated in its protein form. Delivery of PAI-1 gene into the target tissue can be another considerable way. Adenovirus-mediated PAI-1 gene expression is found to reduce the tumor growth, migration and metastasis in vivo [127,149,150]. Some factors, such as the efficiency of the gene delivery system (adenovirus, retrovirus or liposome), the design for PAI-1 expression construction (driven by which promoter and what the mutant design), and the possibility of accompanying side effects during the delivery process, are all needed to concern. Another intriguing strategy is to develop antibodies that partially modulate PAI-1 s function. It has been demonstrated that antibodies to PAI-1 can suppress the metastasis of human tumor cells in mouse xenograft models [ ]. In addition, the scientists have screened some small molecules bearing the specific inhibitory activity on PAI-1 [ ], although the detailed mechanisms of these small molecules are unclear yet. Conclusion Further studies should be done to fully realize PAI-1 s biological functions in vivo. For understanding of PAI-1 s role in cancer development, accumulation of more data from consistent animal models and protocols is necessary. Moreover, spontaneous tumorigenesis models should be adopted to investigate the actual role of PAI-1 in tumorigenesis. The developments of several PAI-1 mutants, gene delivery systems, and some antibodies or small molecules for antagonizing the partial or complete PAI-1 s function may 32 Print ISSN

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