Cell Cycle and Senescence PAI-1 Leads to G 1-Phase Cell-Cycle Progression through Cyclin D 3 / cdk 4 / 6 Upregulation

The canonical function of plasminogen activator inhibitor-1 (PAI-1/SERPINE1) is as an inhibitor of urokinasetype plasminogen activator for blood clot maintenance, but it is now also considered a pleiotropic factor that can exert diverse cellular and tumorigenic effects. However, the mechanism controlling its pleiotropic effects is far from being understood. To elucidate the tumorigenic role of PAI-1, we tested the effects of PAI-1 after manipulation of its expression or through the use of a small-molecule inhibitor, tiplaxtinin. Downregulation of PAI-1 significantly reduced cellular proliferation through an inability to progress from the G0–G1 phase of the cell cycle. Accordingly, overexpression of PAI-1 augmented proliferation by encouraging S-phase entry. Biochemically, cell-cycle arrest was associated with the depletion of theG1-phase transition complexes, cyclinD3/cdk4/6 and cyclin E/cdk2, in parallel with the upregulation of the cell-cycle inhibitors p53, p21Cip1/Waf1, and p27Kip1. PAI-1 depletion significantly decreased the tumor size of urothelial T24 and UM-UC-14 xenografts, and overexpression of PAI-1 substantially increased the tumor size of HeLa xenografts. Finally, immunohistochemical analysis of human bladder and cervical tumor tissue microarrays revealed increased expression of PAI-1 in cancerous tissue, specifically in aggressive tumors, supporting the relevance of this molecule in human tumor biology. Implications: Targeting PAI-1 has beneficial antitumoral effects and should be further investigated clinically.Mol Cancer Res; 12(3); 322–34. 2014 AACR. Introduction PAI-1 is an endogenous inhibitor of urokinase-type plasminogen activator (uPA). Specifically, PAI-1 normally functions as part of the plasminogen activation system, which includes the serine protease uPA, its receptor uPA-R, tissuetype plasminogen activator (tPA), and inhibitors PAI-1 and PAI-2 (1–3). PAI-1 expression is regulated bymany intrinsic factors (e.g., cytokines and growth factors) and extrinsic factors (e.g., cellular stress; ref. 4). Although the canonical function of PAI-1 has been known as an inhibitor of uPA to maintain clot formation, it is now regarded as a pleiotropic factor exerting diverse cellular effects, many potentially related to tumorigenesis, including cell migration, invasion, adhesion, and angiogenesis. Some reports suggest that PAI-1 influences tumor progression indirectly, through effects on the extracellular matrix that perturb migration, invasion, and angiogenesis (5, 6), but other reports claim that PAI-1 itself promotes tumor growth directly (7). For example, high levels of PAI-1 in human and murine cancer cells were associated with the retardation of tumor growth and invasion in a rodent model (8, 9). Thus, the role of PAI-1 in tumorigenesis and growth is complicated, varying with experimental design and its cellular origin, so more preclinical and mechanistic studies are required to elucidate the role of PAI-1 in human cancers. Dysregulation of cellular proliferation is a hallmark of cancer (10–13). The cell cycle is a highly controlled process that involves tight regulation of keymolecules that allow cells to progress between phases of the cell cycle. In an attempt to investigate the effect of PAI-1 on cell proliferation and cellcycle progression, we created stable knockdown clones of PAI-1 in the urothelial T24 and UM-UC-14 cells and overexpressing PAI-1 clones in the cervical tumor cell HeLa. Next, we also inhibited PAI-1 activity with the smallmolecule antagonist of PAI-1, tiplaxtinin (PAI-039; refs. 14, 15). Silencing of PAI-1 in T24 and UM-UC-14 cells via short hairpin RNA (shRNA) or tiplaxtinin treatment was associated with a marked inhibition of cellular proliferation causing a cell-cycle arrest in G1–S phase. The silencing of PAI-1 by either genetic or pharmacologic techniques conferred depletion of theG1–S transitionmolecules, cyclinD3/ cdk4/6 and cyclin E/cdk2, with a concomitant increase in the cell-cycle inhibitors p21 and p27. In addition, in vitro results were corroborated in a xenograft model Authors' Affiliations: Cancer Research Institute, MD Anderson Cancer Center Orlando; Nonagen Bioscience Corp; Department of Pathology, Orlando Health, Orlando, Florida; and Department of Health Sciences Research, Mayo Clinic, Jacksonville, Florida Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). Correspondence Author: Charles J. Rosser, Clinical and Translational Research, University of Hawaii Cancer Center, University of Hawaii School of Medicine, 701 Ilalo Street, Room 327, Honolulu, HI 96813. Phone: 808564-3812; Fax: 808-586-2984; E-mail: crosser@cc.hawaii. edu doi: 10.1158/1541-7786.MCR-13-0543 2014 American Association for Cancer Research. Molecular Cancer Research Mol Cancer Res; 12(3) March 2014 322 on June 18, 2017. © 2014 American Association for Cancer Research. mcr.aacrjournals. org Downloaded from Published OnlineFirst January 24, 2014; DOI: 10.1158/1541-7786.MCR-13-0543