PTEN and Hypoxia Regulate Tissue Factor Expression and Plasma Coagulation by Glioblastoma Yuan Rong, 1 Dawn E. Post, 4 Russell O. Pieper, 5 Donald L. Durden, 2,3 Erwin G. Van Meir, 3 and Daniel J. Brat 1 Departments of 1 Pathology and Laboratory Medicine, 2 Pediatrics, 3 Hematology/Oncology, and 4 Neurosurgery, Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia; and 5 Department of Neurological Surgery and the Brain Tumor Research Center, University of California-San Francisco, San Francisco, California Abstract We have previously proposed that intravascular thrombosis and subsequent vasoocclusion contribute to the development of pseudopalisading necrosis, a pathologic hallmark that distin- guishes glioblastoma (WHO grade 4) from lower grade astrocy- tomas. To better understand the potential prothrombotic mechanisms underlying the formation of these structures that drive tumor angiogenesis, we investigated tissue factor (TF), a potent procoagulant protein known to be overexpressed in astrocytomas. We hypothesized that PTEN loss and tumor hypoxia, which characterize glioblastoma but not lower grade astrocytomas, could up-regulate TF expression and cause intravascular thrombotic occlusion. We examined the effect of PTEN restoration and hypoxia on TF expression and plasma coagulation using a human glioma cell line containing an inducible wt-PTEN cDNA. Cell exposure to hypoxia (1% O 2 ) markedly increased TF expression, whereas restoration of wt- PTEN caused decreased cellular TF. The latter effect was at least partially dependent on PTEN’s protein phosphatase activity. Hypoxic cells accelerated plasma clotting in tilt tube assays and this effect was prevented by both inhibitory antibodies to TF and plasma lacking factor VII, implicating TF-dependent mecha- nisms. To further examine the genetic events leading to TF up- regulation during progression of astrocytomas, we investigated its expression in a series of human astrocytes sequentially infected with E6/E7/human telomerase, Ras, and Akt. Cells transformed with Akt showed the greatest incremental increase in hypoxia-induced TF expression and secretion. Together, our results show that PTEN loss and hypoxia up-regulate TF expression and promote plasma clotting by glioma cells, suggesting that these mechanisms may underlie intravascular thrombosis and pseudopalisading necrosis in glioblastoma. (Cancer Res 2005; 65(4): 1406-13) Introduction Glioblastoma (WHO grade 4) is the most common and the highest grade astrocytoma (1). Currently incurable, it has a mean survival of only 50 weeks following standard surgical and adjuvant therapies (2). More telling of its explosive natural behavior, survival after surgical resection alone averages 14 weeks (3). Compared to lower grade astrocytomas (grades 2 and 3), glioblastomas have radial growth rates nearly 10 times as rapid (4). One pathologic feature that distinguishes glioblastoma from lower grade astrocytomas and may explain the abrupt change in biological behavior is pseudopalisading necrosis—a dense collection of neoplastic cells that surround a central necrotic focus. The origins and potential function of pseudopalisades in tumorigenesis have remained obscure since their recognition in the early 1900s (5). The first indication of their central role in glioblastoma biology was the discovery that they express high levels of hypoxia-inducible regulators of angiogenesis including vascular endothelial growth factor and IL-8, which promote a new vasculature and drive rapid tumor growth (6, 7). More recent studies have shed light on events that may initiate pseudopalisade formation, hypoxia, and necrosis in glioblastoma. Based on morphologic and functional studies of human gliomas, we proposed that pseudopalisades represent a wave of tumor cells actively migrating away from a central hypoxic zone that is created following vascular compromise and associated with intravascular thrombosis (8). The latter is a frequent finding in glioblastoma tissue, present in >90% of well-sampled cases. Deep venous thrombosis of the lower extremities occurs in 20% to 30% of glioblastoma patients, indicating a profound systemic disturbance in coagulation. In contrast, lower grade astrocytomas are not associated with the same degree of coagulopathy. Tissue factor (TF), one of the body’s most potent procoagulants, is up-regulated in a variety of neoplasms including astrocytomas, and its levels correlate with tumor grade (9–11). TF is usually a 47 kDa transmembrane receptor expressed by perivascular stromal cells but also exists as a soluble form. Upon disruption of vascular integrity, TF comes in contact with its activating ligand from the plasma, factor VII/VIIa, which in turn causes thrombin activation, platelet aggregation, fibrin deposition, and local hemostasis. TF is expressed in >90% of malignant astrocytomas, but only in 10% of grades 1 and 2 astrocytomas. Its expression has been localized to neoplastic cells by in situ hybridization and levels correlate with the extent of necrosis (12). This led us to hypothesize that thrombosis in glioblastomas could be promoted by increased TF expression. Precise mechanisms that up-regulate TF in astrocytomas are not known, but triggering events would be expected to be specific to glioblastoma, where intravascular thrombosis and necrosis are apparent, and not present in lower grade astrocytomas. PTEN is a tumor suppressor with dual lipid and protein phosphatase activity (13). The lipid phosphatase activity of PTEN antagonizes phosphoinositide 3 (PI 3)-kinase) by its conversion of phosphatidylinositol 3,4,5-trisphosphate (PIP3) to phosphatidyli- nositol 3,4-bisphosphate (PIP2). Loss of PTEN leads to Akt activation, a frequent finding in glioblastomas (14–16). PTEN lipid phosphatase activity also normally suppresses the Ras/MEK/ERK signaling cascade that is up-regulated in the majority of glioblastomas (17). PTEN protein phosphatase activity is less well characterized, but likely has critical tumor suppressive functions of its own (18). PTEN is inactivated by mutation in 20% to 40% of Requests for reprints: Daniel J. Brat, Department of Pathology and Laboratory Medicine, Emory University Hospital, H-176, 1364 Clifton Road Northeast, Atlanta, GA 30322. Phone: 404-712-1266; Fax: 404-727-3133; E-mail: dbrat@emory.edu. I2005 American Association for Cancer Research. Cancer Res 2005; 65: (4). February 15, 2005 1406 www.aacrjournals.org Research Article Research. on July 20, 2015. © 2005 American Association for Cancer cancerres.aacrjournals.org Downloaded from