Small Molecule Therapeutics Targeting Plasminogen Activator Inhibitor-1 Inhibits Angiogenesis and Tumor Growth in a Human Cancer Xenograft Model

Cancers of the urinary bladder result in aggressive and highly angiogenic tumors for which standard treatments have only limited success. Patients with advanced disease have a 5-year survival rate of less than 20%, and no new anticancer agent has been successfully introduced into the clinic armamentarium for the treatment of bladder cancer in more than 20 years. Investigations have identified plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, as being highly expressed in several malignancies, including bladder cancer, inwhich high expression is associatedwith a poor prognosis. In this study,we evaluated PAI-1 as a potential therapeutic target for bladder cancer. PAI-1 expression was manipulated in a panel of cell lines and functional inhibition was achieved using the small molecule tiplaxtinin. Reduction or inhibition of PAI-1 resulted in the reduction of cellular proliferation, cell adhesion, and colony formation, and the induction of apoptosis and anoikis in vitro. Treatment of T24 xenografts with tiplaxtinin resulted in inhibition of angiogenesis and induction of apoptosis, leading to a significant reduction in tumor growth. Similar results were obtained through evaluation of the human cervical cancerHeLa cell line, showing that PAI-1–mediated effects are not restricted to tumor cells of bladder origin. Collectively, these data show that targeting PAI-1 may be beneficial and support the notion that novel drugs such as tiplaxtinin could be investigated as anticancer agents. Mol Cancer Ther; 12(12); 2697–708. 2013 AACR. Introduction Cancer of the urinary bladder can be aggressive and develop resistance to currently available cancer therapies. An estimated 70,980 newly diagnosed cases of bladder cancer and 14,330 deaths from bladder cancer occurred in 2012 (1). When diagnosed as Ta/T1 lesion or even T2 lesion, cure with surgical resection is possible in a high percentage of cases, with a 5-year survival rate of more than 94% and 50%, respectively (2, 3). However, once metastatic disease develops, it is almost always fatal, with an estimated median survival of 12 to 14 months and a 5-year survival of more than 20% (4). The gold standard in the treatment of metastatic bladder cancer is the chemotherapeutic regimen of cisplatin and gemcitabine or methotrexate, vinblastine, adriamycin, and cisplatin (5, 6). Despite administration of these aggressive oncolytic cocktails, survival rates remain dismal (7), and no new anticancer agent has been successfully introduced into the clinic for the treatment of bladder cancer in more than 20 years. Thus, the identification of novel therapeutics against this disease is urgently required. In bladder cancer biomarker discovery studies, we and others have previously identified plasminogen activator inhibitor-1 (PAI-1), also known as SERPINE1, as a molecule that is consistently overexpressed in bladder tumor cells (8–11). PAI-1 is an endogenous inhibitor of urokinase-type plasminogen activator (uPA), and its expression is regulated by a number of intrinsic factors (e.g., cytokines and growth factors) and extrinsic factors (e.g., cellular stress; ref. 12). Perturbation of PAI-1 and the uPA system has been shown to have both proand antitumoral effects in a number of cancer models, primarily by regulating migration, invasion, apoptosis, and angiogenesis (13–17). Thus, PAI-1 seems to play a pivotal role in tumor growth and may represent a potential therapeutic target for bladder cancer. In this study,wemanipulated the expression of PAI-1 in a panel of cell lines, and used tiplaxtinin (PAI-039), a small-molecule inhibitor of PAI-1 (18–20), to investigate the potential of PAI-1 as a therapeutic target. In vitro, silencing of PAI-1 was associated with an impedance of cellular and colony growth and cellular adhesion, and an induction of apoptosis. In vivo, reduced tumor cell PAI1 was associated with dramatically reduced xenograft growth accompanied by an increase in apoptosis and a reduction in angiogenesis. Authors' Affiliations: Cancer Research Institute, MD Anderson Cancer Center; and Nonagen Bioscience Corp, Orlando, Florida Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Corresponding Author:Charles J. Rosser, Cancer Research Institute, MD Anderson Cancer Center, 6900 Lake Nona Boulevard, Orlando, FL 32827. Phone: 407-266-7401; Fax: 407-266-7402; E-mail: deacdoc@aol.com doi: 10.1158/1535-7163.MCT-13-050