Oncogene (2008) 27, 6834–6844 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc ORIGINAL ARTICLE Glioblastoma-secreted factors induce IGFBP7 and angiogenesis bymodulating Smad-2-dependent TGF- b signaling A Pen1,2, MJ Moreno1, Y Durocher3, P Deb-Rinker4 and DB Stanimirovic1,2 1Cerebrovascular Research Group, Neurobiology Program, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada; 2Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada; 3Animal Cell Technology Group, Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada and 4Neurogenesis and Brain Repair Group, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada Insulin-like growth factor-binding protein 7 (IGFBP7) is a Introduction selective biomarker of glioblastoma (GBM) vessels, stronglyexpressed in tumor endothelial cells and vascular Gliomas are the most common tumors of the central basement membrane. IGFBP7 gene regulation and its nervous system (CNS) derived from glial cells and potential role in tumor angiogenesis remain unclear. classified into four clinical grades (World Health Mechanisms of IGFBP7 induction and its angiogenic Organization (WHO) grades I–IV; Kleihues et al., capacitywere examined in human brain endothelial cells 1995; Dai and Holland, 2001). Glioblastoma multiforme (HBECs) exposed to tumor-like conditions. HBEC (GBM; WHO grade IV) is the most malignant type with treated with GBM cell (U87MG)-conditioned media a mean survival time of approximately 9–12 months (-CM) exhibited fourfold upregulation of IGFBP7 mRNA (Vajkoczy and Menger, 2000). Common features and protein compared to control cells. IGFBP7 gene of GBMs include fast cell proliferation, invasion regulation in HBEC was methylation independent. into normal brain parenchyma, intratumoral necrosis, U87MG-CM analysed by enzyme-linked immunosorbent hypoxia and high angiogenic activity (Vajkoczy et al., assaycontained B5pM transforming growth factor 1999; Louis, 2006). (TGF)-b1, a concentration sufficient to stimulate IGFBP7 The microenvironment of the CNS is maintained by in HBEC to similar levels as U87MG-CM. Both pan- the blood–brain barrier (BBB) formed by specialized TGF-b-neutralizing antibody(1D11) and the TGF- b1 endothelial cells, which are distinguished from those in receptor (activin receptor-like kinase 5, ALK5) antago- the periphery by high mitochondrial content (Oldendorf nist, SB431542, blocked U87MG-CM-induced IGFBP7 et al., 1977), lack of fenestrations, minimal pinocytic expression in HBEC, indicating that TGF-b1isan activity and the presence of tight junctions that restrict important tumor-secreted effector capable of IGFBP7 paracellular permeability of hydrophilic molecules induction in endothelial cells. HBEC exposed to either (Kniesel and Wolburg, 2000). GBM vessels lose many U87MG-CM or IGFBP7 protein exhibited increased BBB properties, leading to clinical signs of brain edema capillary-like tube (CLT) formation in Matrigel. Both due to increased vessel permeability (Long, 1970). TGF-b1- and U87MG-CM-induced Smad-2 phosphoryla- Morphologically, GBM vessels are characterized by tion and U87MG-CM-induced CLT formation in HBEC fenestrations, increased number of caveolae, wide were inhibited bythe ALK5 antagonist, SB431542. These intercellular junctions, abnormal pericytes and a dis- data suggest that proangiogenic IGFBP7 maybe induced continuous basement membrane (Hirano and Matsui, in brain endothelial cells byTGF- bs secreted byGBM, 1975; Dinda et al., 1993). GBM vessels express unique most likelythrough TGF- b1/ALK5/Smad-2 pathway. gene and protein biomarkers (Pen et al., 2007), often Oncogene (2008) 27, 6834–6844; doi:10.1038/onc.2008.287; associated with angiogenesis and/or increased perme- published online 18 August 2008 ability, including aquaporin 4 (Davies, 2002), plasma- lemmal vesicle associated protein-1 (Madden et al., Keywords: glioblastoma; angiogenesis; IGFBP7; 2004) and TEM7 (PLXDC1; Beaty et al., 2007). Recent TGF-b; Smad transcriptomic analysis of laser-captured microdissected vessels from GBM tumors identified insulin-like growth factor-binding protein 7 (IGFBP7) as a highly selective biomarker of tumor vessels (Pen et al., 2007). IGFBP7 was expressed by GBM endothelial cells and extensively Correspondence: Dr DB Stanimirovic, Institute for Biological deposited into the vascular basal lamina (Pen et al., Sciences, National Research Council of Canada, 1200 Montreal Road, 2007). Building M-54, Ottawa, Ontario, Canada K1A 0R6. IGFBP7 is a related member of the IGFBP family E-mail: [email protected] Received 25 February 2008; revised 5 June 2008; accepted 1 July 2008; (Hwa et al., 1999), cloned by several groups (Murphy published online 18 August 2008 et al., 1993; Akaogi et al., 1994; Yamauchi et al., 1994), Tumor cells induce IGFBP7 in endothelium APenet al 6835 which, unlike the other family members (IGFBP1–6), exposed to U87MG-CM responded with a time-depen- exhibits a low affinity for IGF but a high and specific dent upregulation of IGFBP7 mRNA (Figure 1a, upper affinity for insulin (Oh et al., 1996). IGFBP7 is a cell panel) and IGFBP7 protein (Figure 1a, lower panel), adhesive glycoprotein of about 30kDa (Akaogi et al., reaching maximal levels after 3–6 days of incubation in 1994), which is regulated by proteolytic cleavage into a U87MG-CM. The pronounced upregulation of IGFBP7 two-chain form by a membrane-bound serine proteinase protein at 6 days of exposure to U87MG-CM was matriptase (Ahmed et al., 2006). Varied IGFBP7 detected by western blot in both HBEC lysates expression patterns have been reported in different (Figure 1c) and in the secreted fractions (Figure 1a, tumor types (Ruan et al., 2007). IGFBP7 transcription lower panel, d). Conditioned media (CM) from another in tumor cells is modulated by DNA methylation human GBM cell line, T98G, induced IGFBP7 mRNA (Komatsu et al., 2000), retinoic acid and transforming and protein in HBEC in a similar fashion as that growth factor (TGF)-b1 (Hwa et al., 1999). IGFBP7 has observed with U87MG-CM (Supplementary Figure S1). been implicated in tumor growth suppression (Burger IGFBP7 protein was not detected in either U87MG-CM et al., 1998; Sprenger et al., 1999) through induction of (Figure 1d) or T98G-CM (Supplementary Figure S1, apoptosis or cellular senescence (Wilson et al., 2002; lower panel). Mutaguchi et al., 2003). Recently, Wajapeyee et al. Heat-inactivated U87MG-CM lost the ability to (2008) identified a mechanism by which BRAF proto- induce IGFBP7 mRNA (Figure 1b) and protein oncogene triggers cellular senescence in melanocytes (Figures 1c and d) expression in HBEC, suggesting that through induction of IGFBP7. heat-liable mediator(s), most likely proteins, produced In contrast to the tumor suppressor role of IGFBP7 in by GBM cells were responsible for IGFBP7 induction. cancer cells, IGFBP7 is highly upregulated in vessels of several tumors, including GBM (Akaogi et al., 1996; Pen IGFBP7 gene methylation in HBEC is not affected et al., 2007), and interacts in vitro with various by glioblastoma-conditioned media extracellular matrix (ECM) proteins to stimulate adhe- To investigate whether IGFBP7 induction in HBEC sion and migration/invasion of vascular endothelial cells treated with U87MG-CM was the result of DNA on plastic substrates (Sato et al., 1999; Kishibe et al., demethylation, the methylation status of IGFBP7 gene 2000); hence, the original name ‘angiomodulin’ (Akaogi in HBEC treated with either Dulbecco’s modified Eagle’s et al., 1996). Although literature evidence suggests that medium (DMEM; control) or U87MG-CM was ana- IGFBP7 might exhibit angiogenesis-modulating proper- lysed (as described in Supplementary Materials and ties (Akaogi et al., 1996; van Beijnum et al., 2006), the Methods; Miller et al., 1998; Deb-Rinker et al., 2005) role of IGFBP7 in tumor angiogenesis remains poorly using primer sets (Supplementary Table 1) designed to understood. amplify cytosine-phosphate-guanine (CpG) sites present As IGFBP7 is selectively expressed in GBM vessels, in the promoter region (primer sets A and B) or exon 1 the goal of this study was to identify the components of and exon 1/intron 1 (primer sets C and D, respectively) the glial tumor microenvironment that induce IGFBP7 of IGFBP7 gene (Chen et al., 2007). The methylation expression in brain endothelial cells and to determine status of DMEM- and U87MG-CM-treated HBEC in the potential role of IGFBP7 in angiogenesis. IGFBP7 the exon 1 region ( þ 73 to þ 472; primer pair IGFBP7- expression in brain endothelial cells was found to be C) of IGFBP7 spanning 45 CpG sites could not be unresponsive to hypoxia, but upregulated by secreted determined, possibly due to the high density of CpG factors from GBM cells through TGF-b1/ALK5/Smad- islands present in this region resulting in unspecific PCR 2 signaling pathway. products. The PCR-amplified fragment spanning 19 CpG sites ( þ 464 to þ 649; primer pair IGFBP7-D) showed a higher (but not statistically significant) number of methylation sites in control HBEC DNA compared to Results U87MG-CM-treated HBEC DNA (Supplementary Fig- ure S2, graph). No differences in methylation state were Glioblastoma-conditioned media induces IGFBP7 detected within the 5 CpG (À941 to À766; primer pair in human brain endothelial cells IGFBP7-A) and 6 CpG (À789 to À577; primer pair To