Plasminogen Activator Inhibitor-1 in Cancer: Rationale and Insight for Future Therapeutic Testing Veronica R

Published OnlineFirst July 15, 2015; DOI: 10.1158/0008-5472.CAN-15-0876

Cancer Review Research

Plasminogen Activator Inhibitor-1 in Cancer: Rationale and Insight for Future Therapeutic Testing Veronica R. Placencio1,2 and Yves A. DeClerck1,2,3

Abstract

Despite its function as an inhibitor of urokinase and tissue-type led to the testing of small-molecule PAI-1 inhibitors, initially plasminogen activator (PA), PA inhibitor-1 (PAI-1) has a paradox- developed as antithrombotic agents, in animal models of cancer. ical protumorigenic role in cancer, promoting angiogenesis and The review discusses the challenges and the opportunities that lay tumor cell survival. In this review, we summarize preclinical evi- aheadtothedevelopmentofefﬁcacious and nontoxic PAI-1 inhi- dence in support of the protumorigenic function of PAI-1 that has bitors as anticancer agents. Cancer Res; 75(15); 2969–74. 2015 AACR.

Introduction Adding to the complexity of the function of PAI-1 in cancer is the fact that it can have paracrine and autocrine effects as it is Plasminogen activator inhibitor-1 (PAI-1) is a serine prote- produced by tumor cells and nonmalignant cells, including ase inhibitor (serpin) and the main regulator of the plasmin- endothelial cells (EC), macrophage cells, or adipocytes in the ogen activation system. It acts by inhibiting tissue-type plas- tumor microenvironment. minogen activator (tPA) and urokinase-type plasminogen On the basis of the protumorigenic role of uPA in angio- activator (uPA; ref. 1). PAI-1 has a pleiotropic biologic func- genesis, tumor invasion, and metastasis, it was anticipated that tion that stems from its complex structure. Three specific PAI-1 would have an antitumorigenic function. Surprisingly, protein-binding domains in the molecule have been well several studies revealed a paradoxical association between characterized (Fig. 1; ref. 1): a domain in the reactive center elevated levels of PAI-1 in blood and tissue samples of cancer loop (RCL) that binds to PA, a domain in the flexible joint patients and an unfavorable clinical outcome and poor region that binds to vitronectin composed of helix D (hD), response to therapy (1, 5). The observation that the production helix E (hE), and helix F (hF), and a domain within hD and hE of PAI-1 by EC, fibroblasts, adipocytes, smooth muscle cells, that binds to low-density lipoprotein receptor-related protein andmacrophagecellsinthetumormicroenvironmentwas (LRP1). Upon binding to the RCL, PA cleaves PAI-1 at P1-P1' stimulated by protumorigenic factors such as TGFb,IL6,and inducing a dynamic conformational change that inserts the TNFa added further evidence supporting a protumorigenic role RCL as a b-strand (4A) into the core of the protein. The binding (6, 7). In this article, we review the mechanisms and evidence in of PAI-1 to PA forms a catalytically inactive complex, which is mouse models supporting a protumorigenic function for PAI-1. internalized when combined with the receptor for uPA (uPAR) We then present recent pharmacologic approaches and preclin- and LRP1 (2). Premature insertion of the RCL into the b-sheet ical studies aimed at targeting PAI-1 in cancer and discuss the is the basis for the conversion of active PAI-1 into its latent lessons learned from these studies. form. Binding of PAI-1 to LRP1 also promotes cell migration and signaling (3). Binding of the flexible joint region of PAI-1 PAI-1 Is Proangiogenic to vitronectin has multiple consequences: it masks the adjacent fi RGD-binding site for av integrins and inhibits cell attachment A rst clue explaining the paradoxical association of PAI-1 to vitronectin (2) and stabilizes PAI-1 inhibiting its conversion with more aggressive forms of cancer came from the obser- to latency and increasing its anti-uPA ability by 200-fold (4). vation by several laboratories, including our own, that PAI-1 has a proangiogenic activity through its antiprotease and vitronectin-binding functions. The activity of PAI-1 on angiogenesis is, however, dose dependent with a promoting activity 1Division of Hematology, Oncology and Blood and Bone Marrow at physiologic concentrations (8, 9) and an inhibitory activity Transplantation, Department of Pediatrics, University of Southern California, Los Angeles, California. 2The Saban Research Institute of at pharmacologic concentrations (10). This is explained by Children's Hospital Los Angeles, Los Angeles, California. 3Department the fact that at physiologic concentrations, PAI-1 inhibition of of Biochemistry and Molecular Biology, University of Southern Cali- uPA limits the cleavage by pericellular plasmin of membrane- fornia, Los Angeles, California. associated Fas-L at R144-K145 and the release of a soluble Corresponding Author: Yves A. DeClerck, Children's Hospital Los Angeles, 4650 Fas-L fragment that induces Fas-mediated apoptosis in EC Sunset Boulevard, MS#54, Los Angeles, CA 90027. Phone: 323-361-2150; Fax: (11). PAI-1 also stimulates angiogenesis through its vitronec- 323-361-4902; E-mail: [email protected] tin-binding function promoting the detachment of EC doi: 10.1158/0008-5472.CAN-15-0876 from vitronectin and their migration toward fibronectin rich 2015 American Association for Cancer Research. tissues (9).

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A s4A uPA/ tPA binding site (RCL insertion) RCL

sB/sC pocket

Figure 1. Structure of PAI-1 and inhibitor Active Latent targeting sites. A, structure of PAI-1 and targeted binding sites. The hD reactive center loop (RCL) is shown in Vitronectin LRP1 binding red, which inserts itself to become s4A binding site site in the latent form of PAI-1. The sB/sC (hD, hE, and hF) hE pocket is indicated by the arrow (purple). The vitronectin (hD, hE, and hF hF) and LRP1 (hD and hE) binding regions are shown in purple and blue, respectively. Molecular modeling was B carried out using Pymol software Apoptosis, Migration, Angiogenesis, 123version 1.2r1 and the PDB ﬁles 1B3K for angiogenesis signaling migration active PAI-1 (57) and 1DVN for latent PAI-1 (58). B, roles of PAI-1 in tumorigenesis: (1) the inhibitory role Plasminogen of PAI-1 on uPA action to prevent the cleavage of plasminogen into plasmin uPA Vitronectin and subsequent substrates on the cell surface, leading to angiogenesis or intracellular apoptotic signaling; (2) the ability of PAI-1 to bind to LRP1, Plasmin directly leading to intracellular signaling and cell migration; and (3) the ability of PAI-1 to bind to uPAR LRP1 Integrin vitronectin in the extracellular matrix to promote endothelial cell migration and angiogenesis.

PAI-1 Inhibits Spontaneous Apoptosis in maximum antitumor effect was shown upon PAI-1 suppression in Cancer Cells both host and tumor cells pointing to a combined role for extracellular PAI-1. Similar observations were made when PAI- PAI-1 protects tumor cells from apoptosis through multiple 1–deficient human tumor cells were xenotransplanted in immu- mechanisms. Similar to EC, it inhibits Fas-mediated apoptosis in nodeficient PAI-1 null mice. These studies typically demonstrated several human cancer cells, including brain metastasis through its that suppression of PAI-1 expression in human tumor cells and in control over pericellular plasmin activity (12, 13). PAI-1 also mouse host cells resulted in poor tumor take and slower tumor affects intrinsic apoptosis, as the absence of extracellular PAI-1 in growth (11, 12, 22, 23). However in some situations, such as in tumor cells results in higher levels of activated caspase-9 (14). skin carcinoma, the effects observed depended on the tumor grade Intracellular PAI-1 also promotes cell survival through its ability with minimal effects observed in high-grade tumors (23). to inhibit caspase-3 protecting tumor cells from chemotherapy- In contrast with the above data, experiments using genetically induced apoptosis (15). engineered mice (GEM) prone to develop cancer crossed with PAI- 1 null mice failed to demonstrate any effect of PAI-1 suppression What Have We Learned from Mouse Tumor on tumor initiation, growth, or metastasis. For example, when FVB- PymT mice prone to develop mammary tumors were crossed with Models? PAI-1 null mice, there was no effect on the development of Observations in PAI-1–deficient mice have provided additional mammary tumors, metastasis, or survival (24). Similarly, genetic and important clues on the function of PAI-1 in tumorigenesis ablation of PAI-1 had no effect on tumor development in Apc/ – – and angiogenesis. The vast majority of experiments using PAI-1– Apc1638N/PAI-1 / mice prone to develop colon cancer (25), in deficient murine tumor cells syngeneically implanted into WT or TRP-1SV40 Tag/PAI-1 / mice prone to develop ocular tumors PAI-1–deficient mice consistently demonstrated the contributory (26), and in K14-HPV16/PAI-1 / mice prone to develop skin role of host and tumor-derived PAI-1 to tumor growth, angio- cancer (27). An explanation for this difference of effect genesis, and metastasis (Table 1; refs. 10, 16–21). Consistently, between transplanted and GEM mice is the possible presence of

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Table 1. Outcome of the genetic ablation of PAI-1 in the host and/or implanted tumor cells in murine models of cancer Model PAI-1 host mouse Implanted cells Tumor growth Angiogenesis Metastasis Reference Syngeneic implantation þ/þ B16 melanoma No difference n/e No difference 21 / B16 melanoma No difference n/e No difference 21 / Malignant keratinocytes Decreased Decreased n/e 17 / T241 fibrosarcoma Decreased Decreased Decreased 16 þ/þ Malignant keratinocytes Decreased Decreased n/e 10 / Malignant keratinocytes Decreased Decreased n/e 10 / Spontaneously transformed Decreased n/e n/e 18 primary lung fibroblasts / Spontaneously transformed Decreased n/e n/e 19 primary fibrosarcoma / Spontaneously transformed Decreased n/e n/e 20 primary fibrosarcoma Xenograft / Human neuroblastoma Decreased Decreased n/e 11 þ/þ Human M21 melanoma Increased Increased n/e 22 / Human M21 melanoma Decreased Decreased n/e 22 / Human HaCaTII-4 and HaCaT Decreased Decreased n/e 23 A5-RT3 / Human PAI-1 KD HT1080 Decreased Decreased n/e 12 fibrosarcoma, colon cancer HCT116, breast cancer MDA- MB-231, lung cancer A549 GEMM MMTV-PymT/PAI-1/ No difference No difference No difference 24 Apc/Apc1638N/PAI-1/ No difference n/e n/e 25 TRP-1/SV40 Tag/PAI-1/ No difference No difference Decreased 26 K14-HPV16/PAI-1/ No difference No difference n/e 27 Abbreviations: GEMM, genetically engineered mouse model; þ/þ, transgenic overexpressing PAI-1 mice; /, PAI-1 deficient mice; n/e, the experimental parameter was not evaluated.

compensatory serpins, including PAI-2, protein C inhibitor, pro- rombotic activity in preclinical rat and canine models of acute tease nexin-1, or maspin (28–31), whose overexpression in trans- arterial thrombosis (38, 39). Other inhibitors derived from the formed cells may have compensated for a lack of PAI-1 (27–31). structure of this inhibitor (PAI-749 and PAZ-417) then underwent human phase I clinical trials in healthy volunteers and in patients with Alzheimer's disease (40–42). To date, the results of these Pharmacologic Inhibition of PAI-1 studies have not been reported. Over the last two decades, several laboratories and pharmaceu- As an alternative approach, using computer simulation models tical companies have developed a variety of small-molecule PAI-1 mimicking the RCL insertion within the strands of b-sheet A, inhibitors using high-throughput screening (32). The vast major- conformation disrupting agents were designed. Such compounds ity of these inhibitors are molecules interfering with the molecular include a family of dimeric 2-acylamino-3-thiophenecarboxylic interactions of PAI-1 to inhibit tPA and uPA by binding to the acid derivatives (TM5001 and TM5007) developed with the reactive center loop and by inducing a conformational change that anticipation that by binding to the s4A position of the A b-sheet, promotes an irreversible conversion of PAI-1 into its latent form. they would induce the conversion of PAI-1 into its latent and To our knowledge, no inhibitors specifically interacting with the inactive form (43). These inhibitors were found active against vitronectin-binding domain of PAI-1 have been reported yet. The vascular thrombosis in a rat model and against lung fibrosis in main focus of investigations on the activity of these inhibitors in murine models. Their pharmacokinetic properties, however, were biologic processes has been in cardiovascular diseases (thrombus suboptimal due to their relatively high lipophilicity [calculated repermeabilization), lung fibrosis, Alzheimer's disease and to a octanol/water partition coefficient (ClogP) value of 5.79, above lesser degree in cancer. A first family of PAI-1 inhibitors developed the ideal 2–3 value]. A second-generation molecule (TM5275) consisted of diketopiperazine inhibitors that interfere with uPA with better oral pharmacokinetic properties and lower ClogP of binding to the protease-binding domain of the RCL. These inhi- 3.37 was then developed (44). TM5275 showed activity in rat and bitors (XR334, XR1853, XR5082, and XR5118) block tPA inhibi- mouse models of acute arterial thrombosis and lung fibrosis. tion by PAI-1 in vitro (33, 34). However, their in vivo efficacy is Importantly, this inhibitor had no systemic toxicity as it did not limited by their low solubility, poor oral availability, or high IC50 prolong bleeding time in nonhuman primates (45). values (in the 5 to >1,000 mmol/L range) that are unachievable in A limiting element in the activity of these inhibitors so far, has the blood upon administration in animals. Other menthol-based, been their lack of activity against the stable form of PAI-1 bound to benzothiophene, and butadiene small molecules with lower IC50 vitronectin (46). Thus, the recent focus has been on the devel- values (0.64 to 44 mmol/L) in vitro were developed (35–37); opment of inhibitors active against vitronectin-bound PAI-1. however, despite their good anti-PAI-1 activity in vitro, they were These efforts, however, have been limited by a lack of crystal either not tested or not further pursued in animal experiments for structure information on vitronectin-bound PAI-1 (47). Polyphe- reasons not reported. nolic inhibitors of PAI-1 (CDE-066 and 096) synthesized on the One indole oxoacetic acid PAI-1 inhibitor, PAI-039 (tiplaxti- basis of the structure of small molecules identified by a high- nin; ref. 38), was extensively and successfully tested for its antith- throughput screen were found to bind to the sB/sC pocket of PAI-1

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Table 2. Outcome of pharmacologic inhibition of PAI-1 in preclinical murine models of cancer Efﬁcacy in cancer models

Compound Inhibitor class IC50 (mmol/L) Half-life (h) Cancer model In vitro Tumor growth Angiogenesis Metastasis PAI-039 Indole oxoacetic acid 2.7 2.95–3.73 Bladder and cervical cancer Yes Yes Yes n/e TM5275 N-acylanthranilic acid 6.9 2.5 Ovarian cancer Yes n/e n/e n/e SK-116, SK-216 Not stated 35, 44 n/e ApcMin/þ GEMM Yes Yes n/e n/e SK-216 Not stated 44 n/e Lung cancer and melanoma Yes Yes Yes Yes Abbreviation: n/e, the experimental parameter was not evaluated.

with an IC50 of 32 mmol/L and 25 nmol/L, respectively (Fig. 1; cancer progression, its potential as a target for therapeutic inter- refs. 48, 49). CDE-096 induces allosteric conformational changes vention in cancer has only been recently considered and explored. affecting the flexibility of PAI-1 and preventing it from binding to These studies provide important insight on the need to develop PA and decreasing vitronectin binding (49). CDE-096 was still better inhibitors for cancer. The need for chronic administration able to interact with vitronectin-bound PAI-1, although the mag- and thus a pharmacologic profile with a prolonged plasma half- nitude of polarization was decreased 7.3-fold compared with life of orally available inhibitors is an important consideration. binding-free PAI-1. The in vivo pharmacokinetic profile of these Although much work has been done on the development of orally inhibitors is unknown. available compounds, the half-life of these inhibitors in vivo is 2 to 3 hours and therefore would be impractical in long-term cancer Pharmacologic Inhibition of PAI-1 in Cancer therapy. The fact that most inhibitors developed until now are Therapy active in vitro at concentrations in the mmol/L range represents another limitation. Such concentrations are typically difficult to Pharmacologic inhibition of PAI-1 in cardiovascular diseases reach in blood and more importantly in tumor tissues for an has been the primary goal with the objective to prevent inhibition extensive period of time. The lack of activity of most inhibitors fi of intravascular brinolysis and subsequently promote thrombus against the stable vitronectin-bound form of PAI-1 is a third repermeabilization in an acute setting. In contrast, preclinical limitation, since this is the predominant form of PAI-1 in vitro- fi evidence supporting the therapeutic ef cacy of small PAI-1 inhi- nectin-rich tumor tissues (55). A fourth limitation is the potential bitors in cancer has been limited (Table 2). In cancer, the objective systemic effect that the chronic administration of PAI-1 inhibitors is to prevent inhibition of pericellular activation of plasminogen may have on hemostasis. By suppressing the control that PAI-1 and interfere with PAI-1-vitronectin interactions. Although in exerts on plasmin-mediated fibrinolysis, small-molecule PAI-1 cardiovascular and thrombotic disease, the desired inhibition is inhibitors may impair hemostasis and cause excessive spontane- short-term; in cancer (or other chronic conditions), it is long-term ous bleeding (56). Although studies in nonhuman primates have fi requiring a pharmacologic pro le suitable for chronic adminis- shown an absence of side effects on systemic hemostasis follow- tration (32). ing the chronic administration of some PAI-1 inhibitors (44, 45), Insofar, four small-molecule PAI-1 inhibitors have been tested this aspect deserves more extensive investigation. in preclinical models of cancer. Although limited, these studies In summary, targeting PAI-1 in cancer therapy remains an have shown antitumor activity. SK-116 and SK-216 inhibitors attractive but also challenging approach that will require the administered orally for 9 weeks to Apc/Apc1638N mice that design and development of inhibitors that address some of the spontaneously developed intestinal polyps caused an almost 2- current limitations outlined above. As new inhibitors with better fold reduction in the number of small intestinal polyps (50). pharmacologic profiles are developed, they should continue to be When administered to PAI-1 producing Lewis lung carcinoma and tested in preclinical models of cancer. PAI-1–nonproducing B16 melanoma tumor-bearing mice, SK- 216 caused a 2-fold reduction in subcutaneous primary tumor fl size and inhibited angiogenesis and metastases (51). The oral Disclosure of Potential Con icts of Interest fl administration of PAI-039 to mice xenotransplanted with human No potential con icts of interest were disclosed. T24 bladder and HeLa cervical cancer cells resulted in a 2-fold reduction of tumor volume after 14 days associated with a Acknowledgments decrease in tumor cell proliferation and vascularization and an The authors thank Dr. Marta Kubala for reading through the article and increase in apoptosis (52). The TM5275 inhibitor was recently showing them how to generate the crystal structure to highlight the inhibitor- shown to increase apoptosis in vitro in ovarian cancer cell lines binding sites. (53). The in vivo activity of these inhibitors in cancer models have not been reported with the exception of TM5275 and TM5441 in a Grant Support most recent study (54). This work was supported by the U.S. Department of Health and Human Services/National Institutes of Health with a grant (Y.A. DeClerck; grant 5R01 Challenges and Opportunities CA 129377).

Despite the fact that much is known on the structure of PAI-1, Received March 31, 2015; revised April 27, 2015; accepted April 28, 2015; its complex biologic function, and its protumorigenic role in published OnlineFirst July 15, 2015.

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2974 Cancer Res; 75(15) August 1, 2015 Cancer Research

Plasminogen Activator Inhibitor-1 in Cancer: Rationale and Insight for Future Therapeutic Testing

Veronica R. Placencio and Yves A. DeClerck

Cancer Res 2015;75:2969-2974. Published OnlineFirst July 15, 2015.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-15-0876

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