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Fibulin-3 Promotes Glioma Growth and Resistance Through a Novel Paracrine Regulation of Notch Signaling

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Fibulin-3 Promotes Glioma Growth and Resistance Through a Novel Paracrine Regulation of Notch Signaling Published OnlineFirst June 4, 2012; DOI: 10.1158/0008-5472.CAN-12-1060 Cancer Tumor and Stem Cell Biology Research Fibulin-3 Promotes Glioma Growth and Resistance through a Novel Paracrine Regulation of Notch Signaling Bin Hu1, Mohan S. Nandhu1, Hosung Sim1, Paula A. Agudelo-Garcia1, Joshua C. Saldivar1, Claire E. Dolan1, Maria E. Mora1, Gerard J. Nuovo2, Susan E. Cole3, and Mariano S. Viapiano1 Abstract Malignant gliomas are highly invasive and chemoresistant brain tumors with extremely poor prognosis. Targeting of the soluble factors that trigger invasion and resistance, therefore, could have a significant impact against the infiltrative glioma cells that are a major source of recurrence. Fibulin-3 is a matrix protein that is absent in normal brain but upregulated in gliomas and promotes tumor invasion by unknown mechanisms. Here, we show that fibulin-3 is a novel soluble activator of Notch signaling that antagonizes DLL3, an autocrine inhibitor or Notch, and promotes tumor cell survival and invasion in a Notch-dependent manner. Using a strategy for inducible knockdown, we found that controlled downregulation of fibulin-3 reduced Notch signaling and led to increased apoptosis, reduced self-renewal of glioblastoma-initiating cells, and impaired growth and dispersion of intracranial tumors. In addition, fibulin-3 expression correlated with expression levels of Notch-dependent genes and was a marker of Notch activation in patient-derived glioma samples. These findings underscore a major role for the tumor extracellular matrix in regulating glioma invasion and resistance to apoptosis via activation of the key Notch pathway. More importantly, this work describes a noncanonical, soluble activator of Notch in a cancer model and shows how Notch signaling can be reduced by targeting tumor-specific accessible molecules in the tumor microenvironment. Cancer Res; 72(15); 1–13. Ó2012 AACR. Introduction The discovery and characterization of glioma-initiating Malignant gliomas are the most common primary brain stem-like cells (GIC) that share properties with neural stem tumors and one of the types of cancer with worst prognosis cells (9) has brought attention to a number of signaling path- (1, 2). Despite significant advances in neurosurgery and ways in glioma that are also involved in neural development, chemoradiotherapy, gliomas remain highly resistant to con- such as Hedgehog, Wnt, and Notch (10, 11). These pathways are ventional treatments and improvements in patient outcome necessary for the maintenance of GICs (12, 13) and critical for have been modest (3). A major challenge for glioma therapy glioma initiation, self-renewal, and progression through stages is the typical scattering of invasive tumor cells in the brain, of malignancy (11, 14), all of which makes them appealing escaping resection and leading to recurrence (4). Cumulative pharmacologic targets. While Hedgehog and Wnt are activated evidence suggests that these invasive cells are highly resis- by soluble factors, Notch activation is usually a juxtacrine tant to cytotoxic therapies (5, 6) and that their dispersion signaling mechanism that mediates close cell-to-cell signaling may be triggered, in part, by these treatments (7, 8). (15), which becomes less likely as tumor cells scatter. A few Improved targeting of the mechanisms that promote glioma soluble, noncanonical activators of Notch signaling have been invasion and facilitate chemoresistance is therefore critical described in mammals [e.g., MAGP-1/2, CCN3, and YB-1 to increase the long-term efficacy of current therapeutic (ref. 15)] but their relationship with Notch signaling in cancer approaches. progression has not been elucidated. The fibulins are a family of secreted proteins that associate to the extracellular matrix (ECM) scaffold, forming anchoring structures that can regulate cell proliferation and migration Authors' Affiliations: 1Department of Neurological Surgery, Dardinger Center for Neuro-Oncology and Neurosciences, 2Department of Patholo- (16, 17). Several fibulins have been associated with the devel- gy, The Ohio State University Wexner Medical Center; and 3Department of opment of solid tumors such as ovarian, breast, and colorectal Molecular Genetics, The Ohio State University College of Arts and Sciences, Columbus, Ohio cancers (16, 17), but their role in cancer initiation and pro- gression has been difficult to define (16–18). Fibulin-3, also Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). known as EFEMP1, is a protein of restricted expression in the body, predominantly localized in the ECM of elastic tissues Corresponding Author: Mariano S. Viapiano, Department of Neurological Surgery, The Ohio State University Wexner Medical Center, 226B Rightmire (19). This protein is absent from normal brain (19, 20) and is Hall, 1060 Carmack Rd., Columbus, OH 43210. Phone: 614-292-4362; Fax: downregulated in several types of solid tumors (21, 22). Sur- 614-292-5379; E-mail: [email protected] prisingly, fibulin-3 is highly upregulated in gliomas, where it doi: 10.1158/0008-5472.CAN-12-1060 promotes tumor growth and invasion (22). Recent results Ó2012 American Association for Cancer Research. suggest that fibulin-3 is also upregulated in some highly www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst June 4, 2012; DOI: 10.1158/0008-5472.CAN-12-1060 Hu et al. metastatic tumors, where it correlates with the progression of 20 mg/mL doxycycline for 48 hours to induce shRNA and eGFP these tumors toward the invasive phenotype (23, 24). The expression. Inducible fibulin-3 knockdown was confirmed in 3 molecular mechanisms of this protein in cancer are still independent glioma cell lines (Supplementary Fig. S3). essentially unknown. Here, we report that fibulin-3 is a novel soluble activator of Immunohistochemistry Notch signaling, acting through an unconventional mecha- Human tissue sections (5 mm) were deparaffinized and nism that reduces cis-inhibition of Notch. Our results show processed for immunohistochemistry with antibodies against that fibulin-3 regulates the Notch pathway in glioma and fibulin-3 and Hes1 (listed in Supplementary Table S1). Scoring promotes resistance to apoptosis as well as tumor growth and was conducted blindly by a pathologist (G.J. Nuovo), using a invasion in a Notch-dependent manner. This is the first dem- scale from 0 (absent staining) to 100 (whole visual field stained; onstration of a secreted matrix molecule that regulates the ref. 29). Frozen mouse brains were coronally sectioned at 20 Notch pathway in cancer to promote tumor progression. mm and every fourth section stained with 0.1% w/v cresyl violet (22). Imaging software (ImageJ v.1.45) was used to measure maximum coronal area and distance from the geometric Materials and Methods center of the tumor to the most distant border. Sections Cells and tissue specimens selected for immunohistochemistry were probed with an The human glioblastoma cell lines U87 and U251 and the rat antibody against the proliferation marker Ki67 (22) or pro- glioblastoma cell line CNS1 were cultured as previously cessed for terminal deoxynucleotidyl transferase–mediated described (22) and authenticated using the Cell Check service dUTP nick end labeling (TUNEL) staining (Apoptag Red Kit, provided by the Research Animal Diagnostic Laboratory Millipore). (RADIL, Columbia, MO). Primary glioblastoma-derived initi- ating cells (GICs G2, G8, G34, and G146) were kindly provided Biochemical assays by Drs. E. Chiocca and I. Nakano of the Department of Cells were recovered from culture, lysed, and processed for Neurological Surgery, The Ohio State University (OSU; Colum- Western blotting using standard protocols (ref. 22; antibodies bus, OH), and cultured as floating neurospheres in DMEM: are listed in Supplementary Table S1). To analyze protein Ham's F-12 supplemented with 2 mmol/L glutamine, 20 ng/mL phosphorylation, cells were incubated in serum-free media EGF, 20 ng/mL basic fibroblast growth factor (bFGF), 2 mg/mL overnight before treatments. In vitro effects of fibulin-3 were grade-1A heparin, and B27 supplement (Invitrogen). These analyzed by incubating glioma cells with purified fibulin-3 (100 cells were validated for self-renewal, ability to form tumors ng/mL) for 2 hours. Short-time incubations (15 minutes) were in low numbers, and multilineage differentiation. Tissue also carried out to compare activity of fibulin-3 against a microarrays containing multiple replicate cores of formalin- canonical EGF receptor (EGFR) ligand (EGF, 5 ng/mL). For fixed, paraffin-embedded glioma specimens were purchased semiquantitative real-time PCR (RT-PCR), cells or tissue sam- from US Biomax, yielding 65 independent samples. ples were processed using TRIzol reagent (Invitrogen) and total RNA was purified by ethanol precipitation. For Notch DNA constructs, siRNAs, and lentivirus reporter assays, cells were transfected with the Notch reporter A clone containing the full-length coding sequence of fibu- construct and Renilla luciferase as loading control. Reporter lin-3 was previously described (22). From this clone, the cells were exposed to purified fibulin-3 for 8 hours or cotrans- variants fib-3Dn and fib-3Dc were generated by deleting the fected with different constructs and processed after 24 hours to sequences Glu19-Ser106 and Cys379-Phe493, respectively. Full- quantify luciferase activity. length DLL3 cDNA (25) was
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