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Abstracts for the Thirteenth Annual Meeting of the Society for Neuro-Oncology

	CELL BIOLOGY

Top CELL BIOLOGY EPIDEMIOLOGY EXPERIMENTAL THERAPEUTICS GENOMICS/PROTEOMICS IMMUNOBIOLOGY/IMMUNOTHERAPY MEDICAL THERAPIES--ADULT MODELS, PRECLINICAL PATHOLOGY & PROGNOSTIC MARKERS PEDIATRIC BASIC SCIENCE STUDIES QUALITY OF LIFE/SYMPTOM... RADIATION ONCOLOGY STEM CELLS SURGICAL THERAPIES

Bingbing Dai¹, Russell Pieper², Hongbiao Gu¹, Kenneth Aldape¹, Keping Xie¹, Raymond Sawaya¹, and Suyun Huang¹; ¹The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA; ²University of California–San Francisco, San Francisco, CA, USA..

Our recent studies have shown that the FoxM1B transcription factor is overexpressed in human glioma tissues and that the level of its expression correlates directly with the glioma grade. FoxM1B expression in glioblastoma multiforme (GBM) cells contributes to the cells' tumorigenicity and invasiveness. However, whether FoxM1B plays a role in the early development of glioma, i.e., in transformation, is unknown. In this study, we found that in immortalized normal human astrocytes (NHAs), the FoxM1B molecule causes cellular transformation and tumor formation. Moreover, brain tumors that arose from the intracranial injection of FoxM1B-expressing immortalized NHAs displayed GBM phenotypes, suggesting that FoxM1B overexpression in immortalized NHAs not only transforms the cells but also leads to GBM formation. Mechanistically, FoxM1B overexpression induced the hyperactivation of Akt, which led to the phosphorylation and cytoplasmic retention of FoxO3a. The inhibition of FoxM1 in GBM cells suppressed the activation of Akt, which inhibited the phosphorylation and cytoplasmic retention of FoxO3a. Blocking Akt activation inhibited the FoxM1B-induced transformation of immortalized NHAs. Furthermore, the overexpression of FoxM1B in immortalized NHAs increased the expression of survivin, cyclin D1, and cyclin E genes, whereas the inhibition of FoxM1 in GBM cells suppressed the expression of these genes. Our results indicated that FoxM1B promotes astrocyte transformation and GBM formation through multiple mechanisms, including the upregulation of Akt activation and the expression of survivin, cyclin D1, and cyclin E genes.

CB-02. UNDEREXPRESSION OF PROSTAGLANDIN D2 SYNTHASE: A KEY MOLECULAR EVENT IN THE MALIGNANT TRANSITION IN GLIOMA

Cathy Payne¹, Marinella Messina¹, Sanaz Maleki¹, Raymond Cook², Michael Biggs², Nicholas Little², Charles Teo³, Bruce Robinson⁴, and Kerrie McDonald¹; ¹Kolling Institute of Medical Research, St. Leonards, New South Wales, Australia; ²Department of Neurosurgery, Royal North Shore Hospital, New South Wales, Australia; ³Centre for Minimally Invasive Neurosurgery, Prince of Wales Hospital, New South Wales, Australia; ⁴University of Sydney, Sydney, New South Wales, Australia.

BACKGROUND: Early molecular events that initiate the progression of low-grade gliomas to more aggressive and deadly high-grade gliomas have not been well described. Lipocalin-type prostaglandin D2 synthase (PGDS) was identified from a microarray analysis that compared a small cohort of astrocytomas of WHO malignancy grade II and III. Loss of PGDS expression was confirmed in a larger cohort of grade II and III astrocytomas and glioblastomas by quantitative PCR and immunohistochemistry. The underexpression of PGDS was associated with poor overall survival; however, the mechanism for PGDS loss with increased malignancy is unknown. AIM: Loss of gene expression can occur as a result of epigenetic silencing and mutations. We sought to measure the levels of DNA methylation within intron 1 of PGDS and to look for mutations in exons 1–6 of PGDS to formulate a mechanism for the loss of expression. We also sought to determine the functional significance of PGDS in brain tumor malignancy by restoring the levels of PGDS expression in a glioblastoma cell line, which is normally PGDS-deficient, and to monitor the effects of PGDS on cell proliferation and responsiveness to treatment with a COX-2 inhibitor. METHODS: Bisulfite-treated DNA from tumor samples was analyzed for changes in DNA methylation. Functional studies were performed using the A172 and U87MG human glioma cell lines. RESULTS: No mutations were found when examining the PGDS sequence in 18 glioblastomas. However, the PGDS gene was found to be methylated at approximately 20% of CpG's in glioblastoma samples (n=31). The exogenous addition of PGD2, the enzymatic product of PGDS, to cells resulted in an increase in cell death and a significantly increased sensitivity of cells to killing by COX-2 inhibitors. After the PGDS gene was cloned and transfected into the glioblastoma cell line A172 (which did not show any endogenous PGDS expression), the restoration of PGDS expression levels in A172 cells resulted in a 20% reduction in the rate of cell growth measured after 7 days. The overexpression of PGDS in A172 cells also led to an increased sensitivity to COX-2 inhibitors. CONCLUSION: This is the first report to implicate the underexpression of PGDS as a key event in the transition of low-grade astrocytomas to high-grade astrocytoma.

CB-03. LOSS OF AKT CONVERTS GLIOBLASTOMA TO OLIGODENDROGLIOMA IN VIVO

Matthew Vanbrocklin¹, James Robinson¹, Adam Guilbeault¹, and Sheri Holmen¹; ¹Drug Development, Nevada Cancer Institute, Las Vegas, NV, USA.

In human glioblastoma multiforme (GBM), signaling through the phosphatidylinositol 3-kinase/AKT and the RAS/mitogen-activated protein kinase pathways is thought to originate from abnormally activated receptor tyrosine kinases, such as the epidermal growth factor receptor and the platelet-derived growth factor receptor (1, 2). The loss of expression of both phosphatase and tensin homolog deleted on chromosome 10 also results in increased AKT signaling (2). RAS is activated in almost all cases of GBM (1) and AKT is activated in 70% of GBM tumors (2). Once activated, these kinases send signals to several downstream pathways, including multiple survival and cell death signaling cascades. Understanding which of these signaling cascades is required for tumor progression and maintenance has been a major challenge in glioma oncology. We have previously studied the signaling cascades driving GBM maintenance using a mouse model of human GBM based on the RCAS/TVA system that allows the expression of genes to be regulated under the control of tetracycline-responsive elements (TRE) after delivery. Having demonstrated that KRas is required for the maintenance of these tumors in vivo and that the inhibition of KRas expression results in apoptotic tumor regression and significantly increased survival (3), we next sought to determine the reliance of these tumors on Akt expression in vivo. To do so, we induced tumors in the same kind of mice by intracranially injecting them with KRas, Tet-off, and TRE-Akt retroviruses at birth. This combination of genes resulted in tumor formation in 75% of the injected TVA-positive mice. Magnetic resonance imaging was used to confirm the presence of tumors. Doxycycline was fed to one cohort of tumor-bearing mice at weaning (21 days of age) to suppress Akt expression, while a separate cohort received standard feed. Censored survival data were analyzed using a log-rank test of the Kaplan-Meier estimate of survival, and results from untreated mice and mice given doxycycline were compared to determine whether the suppression of Akt expression increased survival. We found that the inhibition of Akt resulted in a modest but highly significant increase in the survival of tumor-bearing mice (P = 0.0001). Histological analysis of brain tissue from these mice revealed Akt-independent KRas expressing tumor cells that were morphologically distinct from tumors in untreated mice. Furthermore, while the tumor cells expressing Akt were negative for the oligodendrocyte marker Olig2, tumor cells lacking Akt were positive for Olig2. These data suggest that Akt is required for glioblastoma maintenance and that loss of Akt expression results in the conversion of glioblastoma to oligodendroglioma in vivo. REFERENCES: (1) Guha, A., Feldkamp, M. M., Lau, N., Boss, G. & Pawson, A. Proliferation of human malignant astrocytomas is dependent on Ras activation. Oncogene 15, 2755–65 (1997). (2) Holland, E. C. et al. Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice. Nat Genet 25, 55–7 (2000). (3) Holmen, S. L. & Williams, B. O. Essential role for Ras signaling in glioblastoma maintenance. Cancer Res 65, 8250–5 (2005).

CB-04. EGFR- AND EGFR VIII–MEDIATED REGULATION OF UROKINASE PROMOTES ASTROCYTIC TUMOR INVASION VIA THE C-SRC/MEK/AP-1 SIGNALING PATHWAYS

Samson Amos¹, Gerard Redpath², Joan Carpenter², Charles Dipierro³, Sarah Parsons¹, Shuang Huang⁴, Amyn Habib⁵, Webster Cavenee⁶, and Isa Hussaini¹; ¹University of Virginia, Charlottesville, VA, USA; ²Department of Pathology, University of Virginia, VA, USA; ³Molecular Physiology and Biological Physics, University of Virginia, VA, USA; ⁴Department of Biochemistry and Molecular Biology, Medical College of Georgia, GA, USA; ⁵University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA; ⁶University of California–San Diego, La Jolla, CA, USA.

One of the major pathophysiological features of malignant astrocytomas is their ability to diffusely infiltrate the surrounding brain tissue. Although it is known that the epidermal growth factor receptor (EGFR) is amplified or overexpressed in primary glioblastomas and that malignant gliomas express higher levels of urokinase-type plasminogen activator (uPA) than normal brain tissue, little is known about the possible interaction between the activation of EGFR or EGFR VIII and uPA and its role in promoting astrocytic tumor invasion. In this study, we characterized the signal transduction pathway by which EGF regulates uPA expression and promotes astrocytoma invasion. Our data showed that the treatment of glioblastoma cell lines with EGF upregulates the expression and activity of uPA in a time-dependent manner. Similarly, the expression of the EGFR VIII mutant receptor also induced high uPA expression levels. The increase in uPA protein by EGF or EGFR VIII was abrogated by the MEK inhibitor UO 126, the tyrosine kinase inhibitor AG 1478, the small interfering RNA (siRNA) targeting c-Src, and the c-Src inhibitor PP2. Also, EGF-increased uPA promoter activity was abrogated by mutations in the AP-1 sites. Furthermore, treatment with UO 126 attenuated the promoter activity, while the phosphatidylinositol 3-kinase inhibitor LY294002 did not affect the EGF-induced increase in promoter activity. Treatment with EGF increased the extent of in vitro invasion as determined by the Boyden chamber assay, and invasion was attenuated by UO 126, siRNA, and short hairpin RNA (shRNA) directed against uPA. In addition, uPA knockdown cells formed fewer colonies on soft agar than wild-type cells and formed smaller, well-circumscribed tumors than parent U1242 cells in a xenograft glioblastoma multiforme mouse model. In summary, we conclude that EGF requires EGFR kinase activity, mitogen-activated protein kinase, and AP-1-dependent pathways to induce uPA expression and to promote glioblastoma invasion.

CB-05. H-FERRITIN PLAYS A ROLE IN GLIOMA CELL PROGRESSION

Xiaoli Liu¹, A.B. Madhankumar², Nodar Surguladze³, Jonas Sheehan², Becky Webb³, Qing Yang¹, and James Connor⁴; ¹Pennsylvania State University, Hershey, PA, USA; ²NY, USA; ³PA, USA; ⁴Neurosurgery, Pennsylvania State University, Hershey, PA, USA.

The goal of this project was to explore the role of H-ferritin in the biological pathways of glioma cells. Ferritin, an iron storage protein and a regulator of iron homeostasis in the human body, has been discovered in the cytosol, mitochondria, and nucleus. It is known that the properties of nuclear ferritin differ from cytosolic ferritin, in that nuclear ferritin is distributed in the nucleus unevenly, is enriched in an O-glycosylated form, and binds with DNA. However, little is known about the functionality and role of ferritin in the nuclei. Most of the tumor cells we have examined to date express ferritin in their nuclei. In a previous study, we demonstrated that decreasing the level of H-ferritin protein using small interfering RNA (siRNA) significantly increased the efficacy of chemotherapy and radiotherapy against gliomas. We hypothesized that H-ferritin plays a role in the biological pathways that affect the cell cycle, DNA synthesis, or apoptosis. We chose two glioma cell lines, U251 and U87, as our in vitro models. Nuclear ferritin in U251 and U87 cells was identified using immunocytochemistry and Western blotting. SiRNA was delivered by using cationic liposomes to downregulate H-ferritin expression. The glioma cells were exposed to BrdU (5-bromo, 2-deoxyuridine) and radiolabeled 3H-thymidine to study DNA synthesis after decreasing H-ferritin expression by siRNA. Apoptosis was then visualized using immunocytochemistry and flow cytometry. After H-ferritin was downregulated, DNA synthesis was decreased by 5–10% in U251 cells and by 10–20% in U87 cells, as demonstrated by BrdU and 3H-thymidine incorporation with DNA. We are currently studying the apoptotic mechanisms involved after H-ferritin is suppressed by siRNA. However, our present studies suggest a possible mechanism in which H-ferritin expression plays a role in DNA synthesis in glioma cells. This mechanism subsequently leads to the sensitization of gliomas with suppressed H-ferritin to chemotherapy and radiotherapy.

CB-06. HIF-1 AND CXCR4 DOWNREGULATION REDUCE MIGRATION OF GLIOMA CELLS

Olga Mendez Fernandez¹, Elizabeth Newcomb², and David Zagzag¹; ¹Pathology, New York University, New York, NY, USA; ²New York University, New York, NY, USA.

Glioblastoma multiforme is the most common and also the most malignant primary intracranial human neoplasm. Glioblastomas are characterized by the presence of extensive areas of necrosis and hypoxia. Hypoxia and its master regulator, hypoxia-inducible factor 1 (HIF-1), play a key role in glioma invasion. HIF-1 is a heterodimeric transcription factor that consists of a constitutively expressed HIF-1 alpha subunit and a HIF-1 alpha subunit that is stable in hypoxic conditions but is rapidly degraded in normoxia. Upon stabilization, HIF-1 translocates to the nucleus and induces the transcription of its downstream target genes; among these genes are potent activators of angiogenesis, such as vascular endothelial growth factor, and genes involved in invasion, such as the chemokine receptor CXCR4. To understand the role of hypoxia and CXCR4 in glioma cell invasion, we used shRNA to block the expression of either HIF-1 alpha or CXCR4. Our data showed that glioma cells with knocked-down HIF-1 alpha expression migrate less than control cells in both normoxic and hypoxic conditions. Similarly, CXCR4 downregulation reduced the migration of glioma cells compared to control cells, especially in hypoxic conditions. In summary, the ability of tumor cells to migrate was diminished when either CXCR4 or HIF-1 alpha expression was reduced, especially in hypoxic conditions. These data suggest that hypoxia in general, and HIF-1 and its downstream regulators in particular, play an important role in glioma invasion.

CB-07. A HIGHLY SENSITIVE, ONE-STEP QUANTITATIVE RT-PCR METHOD FOR THE DETECTION OF EGFRVIII

Gordon Li¹ and Albert Wong²; ¹Stanford University, Stanford, CA, USA; ²CA, USA.

EGFRvIII is the most common variant of the epidermal growth factor receptor (EGFR). This variant results from deletion of exons 2 to 7, removing 801 bp from the extracellular domain of the receptor. There are conflicting reports on the prevalence of EGFRvIII in various tumors, including glioblastomas (GBMs), which may be due to the fact that GBMs can express vastly different amounts of EGFRvIII, and current methods are not sensitive enough to detect EGFRvIII unless the gene is highly amplified. We examined the difficulties associated with EGFRvIII detection and have devised a one-step, highly sensitive quantitative RT-PCR method for detecting EGFRvIII mRNA from total tumor RNA. Total RNA was extracted from tumor samples and cell lines following a standard protocol (TRIzol, Invitrogen). EGFRvIII and wild-type (wt) EGFR RNA transcripts were made with the Ambion MAXIscript in vitro transcription kit. One-step, quantitative RT-PCR was performed using the Stratagene Brilliant II SYBR Green QRT-PCR Master Mix Kit, 1-Step and the Stratagene Mx3000P Real-Time PCR System. To establish a highly sensitive, one-step quantitative RT-PCR method for EGFRvIII detection, we systematically examined several regions of the EGFR cDNA sequence and empirically identified a primer set that robustly amplifies an 186-bp fragment corresponding to EGFRvIII. To overcome the competitive effect of the wt EGFR sequence, primers with dideoxy-C termini were designed from sequences in exons 2 and 7 to inhibit the amplification of wt EGFR. It was also discovered that slight degradation of the total RNA induced by heating at 80°C for 5 minutes further increased amplification efficiency. Using these parameters, we were able to specifically detect as few as 10³ transcripts of EGFRvIII RNA when the transcripts were added to total tumor RNA. Elucidating the difficulties associated with the detection of EGFRvIII and developing a highly sensitive, one-step quantitative method for EGFRvIII detection will help resolve the discrepancies in findings related to the presence of EGFRvIII in GBMs and other solid tumors.

CB-08. DEVELOPMENT OF A NOVEL IN SITU PROXIMITY LIGATION ASSAY FOR DIMERIZATION ANALYSIS OF EPIDERMAL GROWTH FACTOR RECEPTORS PREVALENT IN GLIOBLASTOMA MULTIFORME

Aaron Gajadhar¹ and Abhijit Guha²; ¹University of Toronto, Toronto, Ontario, Canada; ²Division of Neurosurgery, Western Hospital, Toronto, Ontario, Canada.

In 50% of glioblastomas (GBMs) epidermal growth factor receptor (EGFR) is amplified, mutated, or activated and is overexpressed. These aberrations are not found in normal glial cells or lower grade gliomas. Several growth-promoting mutant forms of EGFR have been identified in GBMs, the most common of which are EGFRvIII and EGFRc958. Dimerization and interaction between wild-type (wt) EGFR monomers is crucial to their activation; however, determining the dimerization status of mutant EGFRs using standard cross-linking and co-immunoprecipitation strategies remains controversial. We developed a novel approach to study the dimerization status of wt EGFR and mutant EGFRs in GBMs, with a proximity ligation assay (PLA) based on dimerization. This system allows individual EGFR dimers in intact cells and tissue samples to be detected and resolved. Antibody-based proximity probes, conjugated to oligonucleotide extensions, bind to monomers of EGFR and are brought into the requisite hybridization proximity upon receptor dimerization. Proximity probes guide the formation of circular DNA strands that are subsequently amplified to create detection sites for fluorescent probes. To create unique PLA proximity probe binding sites, each EGFR subtype has been fused in frame, C-terminally, to FLAG and MYC epitope tags and cloned into constitutive expression vectors. The cotransfection of the panel of constructs into a CHO-K1 cell line, which has no endogenous EGFR expression, and the measurement of the PLA signal permit the investigation of dimerization combinations and the quantitative comparison of the dimerization abilities of the EGFR mutants. Based on the known dimerization of wt EGFR upon EGF stimulation, and as proof-of-principle for the PLA dimerization assay, we observed a PLA signal from wt EGFR dimers in U87 and U343 cell lines. Significantly, dimer formation is strictly observed in an EGF ligand-inducible fashion, as has previously been shown by traditional biochemical experiments. Additionally, experiments have been carried out to ensure the correct functionality of the fusions of wt EGFR, EGFRvIII, and EGFR c958 and FLAG and MYC tags. Fluorescent confocal imaging experiments have verified the membrane localization of the constructs, and phosphotyrosine analyses have confirmed that receptor activation occurred in a manner similar to that observed in untagged receptors. Preliminary PLA experiments in our CHO expression model have demonstrated wt EGFR:MYC and wt EGFR:FLAG dimer signal in an EGF-dependent manner, further highlighting the proximity requirement for dimer formation and PLA signal generation. Forthcoming results should validate previously determined dimerization findings and illuminate new dimerization combinations. Lastly, EGFR mutant dimerization will be assessed in GBM tissue samples to confirm our findings in a clinical context. The aforementioned studies will be instructive for understanding the dimerization behavior of oncogenic EGFRs in GBMs and will use a methodology in intact cells in which relevant physiological and compartmentalization aspects are preserved. EGFR and ErbB dimerization is a sought-after target for molecular therapy, and our novel platform for EGFR dimerization analysis may ultimately be coupled to small molecule or monoclonal screening libraries to identify compounds of therapeutic relevance.

CB-09. FIBULIN-3 IS UNIQUELY UPREGULATED IN THE EXTRACELLULAR MATRIX OF MALIGNANT GLIOMAS AND PROMOTES TUMOR DISPERSION

Bin Hu¹, Keerthi Thirtamara-Rajamani¹, Hosung Sim¹, and Mariano Viapiano²; ¹Center for Molecular Neurobiology, Ohio State University, Columbus, OH, USA; ²Neurological Surgery, Ohio State University, Columbus, OH, USA.

Malignant gliomas have an almost invariably rapid and lethal outcome. Current treatments for gliomas fail to remove the invasive cells that remain diffusely embedded within normal tissue even after aggressive surgery and chemotherapy. The dispersion of glioma cells is the major cause of disease progression after initial treatment and, therefore, of therapeutic failure. Glioma cells produce a distinctive type of extracellular matrix (ECM) that differs from that of normal neural cells and of other tumors that metastasize to the central nervous system. This unique microenvironment facilitates glioma cell dispersion through motility-enhancing signals; strategies to target these signals could thus hinder the invasive ability of gliomas and provide an important advance in the clinical management of these tumors. We have found that the protein fibulin-3, a recently identified member of the fibulin family, is a novel component of the glioma ECM that is highly upregulated in motile glioma cells. This is a protein of poorly known function, usually associated with the basal lamina in peripheral tissues and generally downregulated in non-neural tumors. Here, we investigated the expression and functions of fibulin-3 in gliomas. A meta-analysis of microarray data from 75 independent studies deposited in the ONCOMINE database showed that fibulin-3 was downregulated in most cancers when compared to the normal tissue of origin but was instead highly upregulated in gliomas compared to normal brain tissue. Additional analysis of microarray data from the National Cancer Institute Repository of Molecular Brain Neoplasia Data showed that fibulin-3 was the most upregulated member of the fibulin family in high-grade gliomas and that this upregulation was well correlated to poor patient survival. Western blotting results confirmed that all isoforms of fibulin-3 were virtually absent from normal brain tissue or cultured astrocytes but were expressed in surgical samples of gliomas, as well as in glioma cell lines and primary cultures of glioma cells. The overexpression of fibulin-3 in cultured glioma cells did not affect their morphology or proliferation rate, but it did enhance cell adhesion, motility, and dispersion on organotypic brain slice cultures. Moreover, the orthotopic implantation of fibulin-3-overexpressing glioma cells in a syngeneic rat model resulted in much larger tumors, with increased antero-posterior dispersion, than were found in controls. Taken together, our results suggest that fibulin-3 has a unique role in promoting glioma dispersion and is a potential target in reducing tumor progression. Targeting strategies against this previously unexplored component of the glioma matrix may contribute to a disruption of the invasive mechanisms in the glioma microenvironment and improve the long-term treatment of these tumors.

CB-10. MOLECULAR MECHANISMS OF BREVICAN, A NEURAL-SPECIFIC PROTEOGLYCAN THAT PROMOTES GLIOMA INVASION

Bin Hu¹ and Mariano Viapiano¹; ¹Center for Molecular Neurobiology, Ohio State University, Columbus, OH, USA.

A fundamental challenge in the treatment of malignant gliomas is their striking ability to infiltrate normal neural tissue, which makes them difficult or impossible to completely remove using conventional therapies. Understanding the mechanisms and molecules involved in glioma invasion is essential to designing effective treatments against the growth and dispersion of these tumors. Invasive glioma cells are uniquely able to disrupt the extracellular matrix (ECM) of the central nervous system (CNS), which is a major barrier to cell movement in the neural microenvironment. Brevican is a predominant chondroitin sulfate proteoglycan of the neural ECM and inhibits cell migration and axonal extension in the adult CNS. However, this proteoglycan is highly upregulated in malignant gliomas and promotes glioma invasion, in stark contrast with its inhibitory role in the normal CNS. The pro-invasive effect of brevican has been described in detail in vivo, but the precise mechanisms underlying this effect remain unclear. Here, we investigated the mechanisms by which brevican produced by glioma cells regulates the cells' motility. Our results indicated that brevican enhances substrate-dependent cell adhesion and motility, leading to increased glioma dispersion in vitro and in vivo. This effect was mimicked by an N-terminal fragment of brevican, produced by ADAMTS-mediated proteolytic cleavage, but was not observed with a C-terminal fragment or with a mutated version of brevican resistant to ADAMTS cleavage. At the molecular level, brevican increased EGFR and Erk1/2 phosphorylation, upregulated fibronectin synthesis and accumulation on the cell surface, and increased the levels of phosphorylated beta3 integrin. In addition, the N-terminal fragment of brevican, but not the full-length protein, associated with fibronectin in cultured cells and in surgical samples of glioma. Furthermore, the inhibition of EGFR signaling reduced the motogenic effects of brevican while fibronectin knockdown completely abolished them. Taken together, our results a) suggest that the EGFR-dependent upregulation of mesenchymal ECM proteins, such as fibronectin, underlies the pro-invasive role of brevican in gliomas, and b) highlight the importance of regulated proteolysis as a trigger of brevican's signaling cascade. These findings also emphasize the relevance of the ECM-processing metalloproteases of the ADAMTS family as potential targets against glioma invasion.

CB-11. DOWNREGULATION OF THE HYALURONAN- AND PROTEOGLYCAN-BINDING LINK PROTEINS IN MALIGNANT GLIOMAS

Hosung Sim¹ and Mariano Viapiano¹; ¹Center for Molecular Neurobiology, Ohio State University, Columbus, OH, USA.

Malignant gliomas are the most common and deadly primary brain tumors because of their striking ability to infiltrate into the normal neural tissue, which makes these tumors virtually impossible to eliminate using conventional therapies. Invasive glioma cells are uniquely able to disrupt the extracellular matrix (ECM) of the central nervous system (CNS), which forms a major barrier to motility in the neural microenvironment. This matrix is formed by a hyaluronic acid scaffold, with associated glycoproteins and chondroitin sulfate proteoglycans (CSPG) that inhibit cell motility and axonal extension in the adult CNS. However, we and other have demonstrated that two CSPGs (brevican and versican) are highly upregulated in glioma cells and surprisingly act as motogenic signals, promoting glioma invasion. In the normal CNS, these CSPGs are thought to associate to the insoluble ECM scaffold through interaction with small glycoproteins of the link protein (LP) family. Therefore, we investigated the expression of LPs in gliomas and the effect of a brain-specific LP on glioma cell motility. A meta-analysis of curated microarray data from the National Cancer Institute Repository of Molecular Brain Neoplasia Data and western blotting analyses from tissue samples indicated that the brain-specific members of the LP family, HAPLN2 and HAPLN4, were strongly downregulated in gliomas. RT-PCR and western blotting analysis of cultured cells failed to detect these LPs in glioma cell lines and primary cultures of glioma cells, although they could be detected in cultured astrocytes and in a subset of glioma stem cells. We hypothesized that the downregulation of LPs is a necessary factor for the pro-invasive gain of function that upregulated CSPGs have in gliomas, and accordingly, we reintroduced HAPLN4 into glioma cells expressing the CSPG brevican. However, HAPLN4 alone failed to reduce the motogenic effect of brevican and, surprisingly, exerted a pro-adhesive and pro-motility effect on glioma cells. Importantly, the expression of HAPLN4 in glioma cells resulted in its being predominantly secreted to the culture medium, whereas native HAPLN4 is exclusively membrane-associated and poorly solubilized in normal neural tissue. This suggests that other ECM components are required to associate this LP and that brevican could also be absent in glioma cells. Experiments involving the coprecipitation and expression of additional ECM components are underway to test this hypothesis. The current results, however, are the first characterization of LP family members in malignant brain tumors and suggest that these proteins can modulate the invasive behavior of glioma cells.

CB-12. MEDIATORS OF GLIOBLASTOMA INVASION DURING ANTI-VEGF TREATMENT

Agda Karina Lucio-Eterovic¹, Yuji Piao¹, Howard Colman¹, and John De Groot¹; ¹Department of Neuro-oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.

The growth of highly aggressive glioblastoma tumors depends on angiogenesis is regulated by a balance of pro- and anti-angiogenic factors. Vascular endothelial growth factor (VEGF) has been identified as the most critical molecule involved in this process. Currently, several different strategies are being employed to target the VEGF and VEGFR signal transduction pathway in these tumors. Although anti-angiogenic therapy appears to be effective in blocking vascular permeability and slowing tumor growth, some studies have demonstrated that one mechanism of tumor escape from this therapy is via vessel co-option, which results in tumor infiltration into the normal brain. In the present study, we evaluated the effect of anti-angiogenic treatment on glioma cell invasion in vitro. Glioma cells that secrete high levels of VEGF and have minimal or no in vitro or in vivo invasion were selected. In the selected cell lines, the influence of increasing doses of the anti-VEGF monoclonal antibody bevacizumab (Avastin) on transwell migration was tested. We also measured the total levels (using western blotting) and activity (using zymography) of invasion-related proteins matrix metalloproteinase (MMP)-2 and MMP-9 using conditioned, serum-free media from control and treated cells. The levels of total secreted proteins were also evaluated using an antibody array. The transwell migration of U87 and the glioma cancer stem cell NSC23 increased after treatment with bevacizumab compared to migration in IgG-treated control cells. In agreement with the increased invasion observed, the activity of MMP-2 (in both cell lines) and MMP-9 (in U87 cells) was higher after treatment in a concentration-dependent manner. A dose-dependent increase in MMP-2 and MMP-9 proteins levels was also observed in U87 cells after treatment. Results from the antibody array demonstrated that both cell lines had increased levels of bFGF, TIMP-1, and TIMP-2 after bevacizumab treatment. However, some proteins were differentially secreted by U87 and NSC23 cells: the levels of angiogenin and IL-1alpha were higher in U87 glioblastoma cells but lower in NSC23 cells. Similarly, the levels of IL-6 and IL-8 were decreased in U87 cells, although this trend was not observed in NSC23 cells. We conclude that anti-angiogenic treatment induces invasion in U87 and NSC23 cells. Although the levels of invasion-related proteins, such as MMP-2, TIMP-1, and TIMP-2, increased after treatment of both cell lines, the invasive process may also be regulated by the secretion of other growth factors and cytokines. These findings suggest that alternate pathways that are induced by anti-angiogenic treatment are involved in tumor invasion in these cell lines. The mechanisms leading to enhanced tumor invasion after anti-angiogenic therapy is under investigation.

CB-13. DIFFERENTIAL EXPRESSION OF STAT-5 AND ITS ROLE IN GLIOBLASTOMA MULTIFORME

Barbara Merk¹ and Isa Hussaini¹; ¹University of Virginia, Charlottesville, VA, USA.

Glioblastomas (GBMs) are the most malignant type of primary central nervous system tumor. They are characterized by rapid growth and diffuse infiltration into surrounding brain tissue, which is further exacerbated by their intrinsic resistance to current radiation and chemotherapy regimens. To facilitate the development of novel therapies, an understanding of the mechanisms mediating the growth, invasion, and apoptotic resistance of GBM tumors is critical. Signal transducers and activators of transcription (STATs) have been shown to affect cellular proliferation and sensitivity to apoptosis in various cellular models of carcinogenesis, and several STATs are upregulated or constitutively active in cancer. While a number of STAT gene targets have been demonstrated, the mechanisms regulating the expression and activation of STATs in GBMs have not been fully characterized, and it is not known exactly how increased STAT levels affect the tumor phenotype. We found that STAT-5 protein levels were increased in GBM cell lines compared to normal astrocytes, and we confirmed these findings in GBM patient specimens. Biologically relevant growth factors, including epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), activated STAT-5 in these cells. Furthermore, the constitutively active mutant EGF receptor vIII, which is expressed in 40–60% of GBMs and correlates with poor prognosis, constitutively activated STAT-5 in our GBM cell lines. Functional studies using siRNA indicate that STAT-5 may regulate GBM cell invasion through a Type IV collagen matrix in vitro. We conclude that STAT-5 is likely active in GBM tumors and may mediate cellular behaviors, such as the ability to degrade a basement membrane and invade remote brain structures.

CB-14. HEXOKINASE 2 IS AN IMPORTANT MEDIATOR OF THE WARBURG EFFECT IN GLIOBLASTOMAS

Amparo Wolf¹, Joydeep Mukherjee¹, Cynthia Hawkins¹, and Abhijit Guha²; ¹Brain Tumor Research Centre, University of Toronto, Toronto, Ontario, Canada; ²Neurosurgery and Cell Biology, Toronto Western Hospital & the Hospital for Sick Children Research Institute, Toronto, Ontario, Canada.

INTRODUCTION: Tumor cells undergo a shift in glucose metabolism from oxidative phosphorylation to glycolysis and lactate formation even in the presence of oxygen, a phenomenon referred to as the Warburg effect. This glycolytic switch is believed to confer a selective survival advantage to tumor cells. The molecular basis of the Warburg effect remains elusive, although it likely involves the interplay of oncogenic signaling, the aberrant expression of metabolic enzymes, and the tumor microenvironment. We provide evidence that Hexokinase 2 (HK2), the first rate-limiting enzyme of glycolysis that converts glucose to glucose-6-phosphate, is an important mediator of the Warburg effect in glioblastomas (GBMs) and that HK2 promotes in vivo tumorigenesis. RESULTS: Human GBM tissue and GBM cells subjected to hypoxia in culture demonstrated a marked increase in the HK2 isozyme, relative to levels of the normal brain isozyme HK1. The expression of HK2 was found to be a marker of poor outcome in 56 GBM specimens. The inhibition of HK2 in GBM cell lines by siRNA led to a decrease in tumor cell viability by favoring the release of cytochrome c and the activation of caspase-mediated apoptosis, especially under hypoxia. Inhibiting HK2 increased the susceptibility of GBM cells to apoptosis induced by radiation therapy and chemotherapy, including temozolomide. The stable knockdown of HK2 with shRNA in U87 cells promoted mitochondrial membrane permeability, reduced lactate formation, and increased the expression of proteins required for oxidative phosphorylation and the genes implicated in mitochondrial biogenesis. Increased total hexokinase activity by the transient expression of HK1 cDNA in U87 HK2 shRNA did not lead to a return in aerobic glycolysis. Lastly, the stable knockdown of HK2 led to a reduced ability to grow tumors in an in vivo xenograft model. CONCLUSIONS: These results demonstrate that HK2, rather than the more ubiquitously expressed HK1, plays an important role in the Warburg effect in GBMs and confers an anti-apoptotic growth advantage. Targeting this enzyme may be of therapeutic benefit.

CB-15. JNK2ALPHA2 IS CRITICAL FOR GLIOMA TUMORIGENESIS AND ITS DIMERIZATION IS IMPORTANT FOR ITS CONSTITUTIVE ACTIVITY

Ryan Nitta¹ and Albert Wong¹; ¹Stanford University, Stanford, CA, USA.

c-Jun N-terminal kinases (JNKs) are important for regulating cell growth, proliferation, and apoptosis. The activation of the JNK pathway has been implicated in several human tumors, including glioblastoma multiforme (GBM). Evidence has shown that a specific JNK isoform, JNK2alpha2, has increased expression and activity in 86% of primary GBMs. Interestingly, this JNK isoform is constitutively active, since it possesses the ability to autophosphorylate. The overexpression of JNK2alpha2 in GBM cells upregulates eIF4E and AKT activity and enhances several tumorigenic phenotypes, including those linked to cell growth and tumor formation in mice. Since the increased expression of JNK2alpha2 induces glial tumor formation, we studied the effects of reducing JNK2alpha2 activity in GBM cells. Using short interfering RNAs, we showed that decreasing JNK2 alpha2 expression in GBM cells ameliorates many tumorigenic phenotypes. The siRNA we used reduced JNK2alpha2 expression nine-fold, compared to expression in scrambled controls, resulting in a four-fold reduction in cell growth and a three-fold reduction in colony formation in soft agar. Our findings suggest that decreasing JNK2alpha2 expression or JNK2 alpha2 activity may prevent glial tumor growth. One method of reducing JNK2alpha2 activity is to inhibit its autophosphorylation activity. To this end, we studied the mechanism of JNK2alpha2 autophosphorylation and autoactivation. Here, we used size exclusion chromatography to demonstrate that JNK2alpha2 exists as both a constitutive dimer and a monomer. Using chimeras between JNK2alpha2 and JNK1alpha2, which is not constitutively active, we previously demonstrated that a nine-aminoacid region from JNK2alpha2, known as the alpha-region, is necessary and sufficient for activation. Also, JNK2alpha2 chimeras that possess the alpha region can coimmunoprecipitate with wild-type JNK2alpha2 in GBM cells. Alanine-scanning mutagenesis of the alpha region revealed that five specific mutants (L218A, K220A, G221A, I224A, and F225A) prevented JNK2alpha2 dimerization. This finding indicates that these amino acids are either directly involved in the JNK2alpha2 dimer interface or are necessary for the proper protein confirmation that enables dimerization. Lastly, we showed that JNK2alpha2 autophosphorylation is dependent on dimerization. In vitro kinase assays of the five specific alpha-region mutants showed a correlation between loss of dimerization and loss of autophosphorylation. In addition, immunoprecipitation and kinase assays showed that a kinase inactive JNK2alpha2 mutant can interact with and inhibit wild-type JNK2alpha2 autophosphorylation. Together, our results illustrate that JNK2alpha2 is important in glial tumorigenesis and that JNK2alpha2 dimerization may be a novel therapeutic target for treating patients with GBM.

CB-16. PLASMINOGEN KRINGLE 5 INDUCES APOPTOSIS OF BRAIN MICROVESSEL ENDOTHELIAL CELLS: SENSITIZATION BY RADIATION AND REQUIREMENT FOR GRP78 AND LRP1

Braden Mcfarland¹, Jerry Stewart², Amal Hamza², Robert Nordal³, Don Davidson⁴, Jack Henkin⁴, and Candece Gladson¹; ¹University of Alabama at Birmingham, Birmingham, AL, USA; ²Pathology, University of Alabama at Birmingham, Birmingham, AL, USA; ³Radiation Oncology, University of Alabama at Birmingham, Birmingham, AL, USA; ⁴Abbott Laboratories, Abbott Park, IL, USA.

Recombinant plasminogen kringle 5 (rK5) has been shown to induce apoptosis in dermal microvessel endothelial cells (MvECs) (Davidson et al., 2005). As we are interested in antiangiogenic therapy for glioblastoma tumors, and the effectiveness of antiangiogenic therapy can be enhanced when combined with radiation therapy, we investigated the pro-apoptotic effect of rK5 on brain MvECs with and without prior irradiation. We found that rK5 treatment induced apoptosis, measured as the cleavage of caspase-7 or -3 and terminal transferase dUTP nick end labeling (TUNEL) positivity, in brain MvECs in a dose- and time-dependent manner. Prior irradiation significantly sensitized (500-fold) the cells to the pro-apoptotic effect of rK5. The pro-apoptotic effect of rK5 required the expression of glucose-regulated protein 78 (GRP78), based on blocking studies with an antibody directed toward GRP78 and the downregulation of GRP78 with siRNA. In addition to requiring GRP78, the pro-apoptotic effect of rK5 post-irradiation required the expression of low density lipoprotein receptor-related protein 1 (LRP1), a scavenger receptor. The necessity of LRP1 was demonstrated by blocking the pro-apoptotic effect of rK5 with recombinant receptor-associated protein, a competitive inhibitor of ligand binding to LRP1, and by the downregulation of LRP1 with siRNA. Our findings have potential applications as new therapy for glioblastoma tumors, given that we also demonstrate that the expression of the GRP78 protein is upregulated in brain MvECs in glioblastoma tumor samples compared to samples from the normal brain. Immunoblotting experiments confirmed the upregulation of GRP78 in the tumor samples. Overall, these data suggest that irradiation sensitizes brain MvECs to the pro-apoptotic effect of rK5 and that this effect requires the LRP1 internalization of GRP78.

CB-17. PLATELET-DERIVED GROWTH FACTOR STIMULATES CHORDOMA GROWTH AND INVASION BY ACTIVATING THE PI3K/AKT/MTOR SIGNALING PATHWAY

Myung-Jin Park¹, Amin Kassam¹, Paul Gardner¹, Daniel M. Prevedello¹, Akio Soeda¹, Frank Lieberman¹, Andrew Rosenberg², and Deric M. Park¹; ¹University of Pittsburgh, Pittsburgh, PA, USA; ²Pathology, Massachusetts General Hospital, Boston, MA, USA.

Chordoma is a rare bone tumor of the axial skeleton believed to originate from the remnants of the embryonic notochord. The tumor cells are characterized by a physaliferous morphology and the expression of brachyury, a transcription factor critical for mesoderm specification. Although most chordomas are histologically low-grade, treatment remains challenging because of uniformly poor response to conventional chemotherapy and the tumor cells' insidious, invasive growth into surrounding tissues. Therefore, a better understanding of the pathways regulating the growth and invasion of chordoma cells may lead to the formulation of more effective treatment strategies. Here, we show the feasibility of establishing brachyury-positive primary cultures of chordomas from acutely resected surgical specimens and that platelet-derived growth factor (PDGF) is a mitogen for chordoma cells and promotes invasion in Matrigel transwell assays. Under a serum-free cell culture condition that allowed for the serial assessment of the role of various recombinant growth factors, PDGF stimulated the phosphorylation of PDGF receptors A and B, ERK1/2, Akt, and mTOR. The PDGF-induced cell division and invasion of chordoma cells were abrogated by treatment with inhibitors of the PDGF receptor (PDGFR) (3-fluoro-N-(6,7-dimethoxy-2,4-dihydroindeno[1,2-c]pyrazol-3-yl) phenylamine) and phosphatidylinositol-3 kinase (PI3K) (LY294002) but not by an ERK1/2 inhibitor (PD98059). Further confirming the involvement of the Akt pathway, we found that overexpression of the constitutively active form of Akt (myr-Akt) led to the growth and invasion of chordoma cells. In contrast, the introduction of the dominant-negative form of Akt strongly suppressed PDGF-induced growth and invasion. These results suggest that the stimulation of the PDGFR with the subsequent activation of the PI3K-Akt signaling pathway contributes to the growth and invasive character of chordoma cells. The PDGFR signaling pathway may represent one of several important targets to consider in the multimodal treatment of chordomas.

CB-18. P16INK4A IMPARTS TEMOZOLOMIDE RESISTANCE TO TUMOR CELLS CARRYING AN HFE MUTATION

Sang Lee¹, Ryan Mitchell², Jonas Sheehan², and James Connor²; ¹Neurosurgery, Penn State Hershey College of Medicine, M.S. Hershey Medical Center, Hershey, PA, USA; ²PA, USA.

We developed a line of human neuroblastoma cells to study the effect of variants of the HFE gene on cell phenotype. HFE polymorphisms are the most common genetic variants in Caucasians, and we have previously reported that levels of one of its variants, H63D, is increased in a number of neurodegenerative diseases. In the course of these studies, we observed that the neuroblastoma cells carrying the other common HFE gene variant, C282Y, proliferated at a significantly greater rate than the other neuroblastoma cells that contained the wild-type or H63D variant. Surprisingly, we also observed that the cells carrying the C282Y allelic variant were resistant to the chemotherapeutic agent temozolomide (temodar) and to gamma radiation. The resistance to temozolomide and gamma radiation was found not only in neuroblastoma cell lines but also in glioma cell lines with the C282Y allele. To better understand the temozolomide resistance mechanism in C282Y-expressing cells, we determined the status of O6-methylguanine methyltransferase (MGMT) promoter methylation and MGMT protein expression. C282Y-expressing neuroblastoma cell lines have the methylated MGMT promoter and have low expression levels of the MGMT protein. C282Y-expressing glioma cell lines also have the methylated MGMT promoter and undetectable levels of MGMT protein expression. These data suggest that MGMT is not involved in temozolomide resistance in the C282Y-expressing cells. Therefore, we performed a series of targeted gene array studies for cell cycle, signal transduction, and drug resistance on human neuroblastoma cells. Based on the gene array analysis, we identified the p16ink4a (cyclin dependent kinase inhibitor 2A) gene for further analysis, because the difference in the expression of this gene was greater after quantitative real-time PCR in the C282Y-expressing cells than in the wild-type cell lines. Consistent with the gene expression analysis results, p16INK4A protein expression was elevated in association with the C282Y allele, relative to wild-type HFE. C282Y-expressing glioma cells also expressed high levels of p16INK4A protein. Thus, we determined the role of p16INK4A in temodar resistance using p16ink4a siRNA. Cells treated with p16ink4a small interfering RNA (siRNA) have decreased p16INK4A protein expression after transfection, and the decreased expression of p16INK4A is associated with C282Y-expressing cells having increased sensitivity to temozolomide. This increased sensitivity is evident when we compare these cells to those in other control groups, such as the untreated, mock, and negative siRNA control groups. Taken together, these data suggest a novel function of p16INK4A in tumor resistance and provide a novel anti-cancer target. (The temozolomide used in this study was a gift from Schering Plough.)

CB-19. ABERRANT EGFR SIGNALING IN GLIOMA

Yeohyeon Hwang¹, Khatri Latha¹, Vaibhav Chumbalkar¹, Anupama Gururaj¹, Marta Rojas¹, Rebecca Maywald¹, Webster Cavenee², Frank Furnari³, and Oliver Bogler⁴; ¹Neurosurgery, University of Texas M. D. Anderson Cancer Center, Houston, TX, USA; ²University of California, San Diego, La Jolla, CA, USA; ³LaJolla, CA, USA; ⁴Neurosurgery and Neuro-Oncology, University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.

Aberrant EGFR signaling is a major contributing force to glioma progression and treatment resistance. The most prevalent mutation, EGFRvIII, is an in-frame deletion of the extracellular domain that occurs in about 40% of glioblastomas and promotes the growth and survival of cancer cells. More recently, new point mutations in the extracellular domain of EGFR have been identified by The Cancer Genome Atlas. We are investigating the signaling of these abnormal receptors to identify the basis of their oncogenicity. The signaling of EGFRvIII is ligand-independent, does not involve receptor dimerization, and is of low intensity. This low-intensity signal has made it challenging to uncover whether there are components of EGFRvIII signaling that are distinct from wild-type EGFR signaling. We have created a chimeric EGFRvIII molecule that can be dimerized experimentally, using a variant FKBP12 domain and a cognate small molecule, a process termed chemically-induced dimerization (CID). CID increases the intensity of EGFRvIII signaling several fold, without leading to ubiquitination and degradation, and allows us to investigate the nature of the EGFRvIII signal in greater depth than before. In addition to analyzing known signaling pathways downstream of EGFR, we are using shotgun phosphoproteomics based on the recovery of phosphopeptides and mass spectrometry to study glioma cell lines expressing EGFRvIII, wild-type EGFR, and mutant EGFR and cell lines with different PTEN backgrounds. Another possible mechanism behind EGFRvIII's impact on glioma biology is differential cellular localization, and we are currently investigating whether it partitions to the nucleus with different kinetics than EGFR. Lastly, new EGFR mutants, identified by high-throughput resequencing, are being investigated for their role in glioma biology.

CB-20. ARF6 REGULATES GLIOMA CELL INVASION THROUGH THE IQGAP1-RAC1-MEDIATED PATHWAY

Bo Hu¹, Binhai Shi², Michael Jarzynka³, Jia-Jean Yiin³, Crislyn D'Souza-Schorey⁴, and Shi-Yuan Cheng³; ¹Cancer Institute & Dept. of Medicine, University of Pittsburgh, Pittsburgh, PA, USA; ²Cancer Institute & Dept. of Pathology, University of Pittsburgh, Pittsburgh, PA, USA; ³Cancer Institute & Pathology, University of Pittsburgh, Pittsburgh, PA, USA; ⁴Biology, University of Notre Dame, Notre Dame, IN, USA.

A common pathobiological feature of malignant gliomas is the insidious infiltration of single tumor cells into the brain parenchyma, which renders these deadly tumors virtually incurable using available therapies. Glioma cell invasion is a highly complex process, and mechanisms regulating cell motility may represent key element of the invasive cascade. The ADP-ribosylation factor 6 (Arf6), which belongs to the Arf family of small GTP-binding proteins with multiple roles in fundamental biological processes, has recently been shown to play an important role in tumor cell invasion. In gliomas, the exogenous expression of EFA6A, a guanidine exchange factor (GEF) for Arf6 in glioma cells, enhanced cell motility and invasiveness in vitro. However, whether Arf6 exerts a direct impact on glioma cell invasion is largely unknown. In this study, we report that Arf6, a small GTPase of the Ras superfamily, is expressed at high levels in invasive human glioma cells. In vitro, the inhibition of Arf6 by siRNA impaired HGF- and serum-stimulated glioma cell migration. In vivo and ex vivo, the stable knockdown of Arf6 in invasive glioma cells suppressed glioma cell invasion in the brain. Conversely, the ectopic expression of Arf6 by glioma cells promoted cell migration through activation of Rac1. Upon stimulation, IQGAP1, a key regulator of cell adhesion and migration, was recruited to the membrane of the leading edges of migrating cells together with Arf6, allowing forward protrusion. The depletion of endogenous Arf6 by siRNA abrogated the recruitment of IQGAP1 into the cell membrane and attenuated the formation of the protrusions at the invasion fronts. Finally, using co-immunoprecipitation assays, we found that Arf6 was associated with Rac1 and IQGAP1 in glioma cells upon HGF stimulation. The knockdown of IQGAP1 by siRNA significantly inhibited Arf6-promoted Rac1 activation and cell migration. Taken together, these data suggest that Arf6 signaling is pivotal for glioma cell invasion in the brain, and IQGAP1 is required for Arf6-mediated Rac1 activation and glioma cell invasion.

CB-21. SIMULTANEOUS DOWNREGULATION OF UPAR AND MMP-9 INDUCES APOPTOSIS VIA THE CASPASE 9 APOPTOSOME CASCADE AND INHIBITS THE NUCLEAR LOCALIZATION OF CD44ICD AND NFB IN HUMAN GLIOMA XENOGRAFT CELLS

Christopher Gondi¹, Meena Gujrati², Dzung Dinh³, and Jasti Rao¹; ¹Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL, USA; ²Pathology, University of Illinois College of Medicine at Peoria, Peoria, IL, USA; ³Neurosurgery, University of Illinois College of Medicine at Peoria, Peoria, IL, USA.

Glioblastoma multiforme is a highly malignant, primary central nervous system neoplasm that is extremely refractory to therapy. Glioblastomas are resistant to treatment because of the tendency of the tumor cells to invade normal brain tissue. We have previously demonstrated that the simultaneous downregulation of urokinase-type plasminogen activator (uPAR) and matrix metalloproteinase (MMP)-9 resulted in the regression of pre-established intracranial tumors in nude mice. In this study, we have attempted to further evaluate the molecular mechanisms involved in glioma tumor regression via the simultaneous downregulation of uPAR and MMP-9. To determine the involvement of CD44 in uPAR and MMP-9 downregulated cells, CD44 levels were measured in cytoplasmic and nuclear extracts. These analyses indicated that the levels of CD44ICD in the nucleus were reduced, compared to those in controls. CD44 has been shown to be involved in multiple functions and is also suspected to have transcriptional activity. Our Western blots of nuclear and extracellular fractions of CD44 under various treatment conditions using antibody for total CD44, showed CD44ICD to be localized in the nucleus. We were also able to detect CD44 in the conditioned media; its cleavage was indicated by various proteases, such as MT1-MMP and MMP-9. Total CD44 levels decreased in uPAR- and MMP-9–downregulated cells, and the CD44ICD domain in the nucleus was almost undetectable. We also observed that caspase 9 activation was initiated, and a collapse in the mitochondrial DeltaPsi was also observed, indicating the release of cytochrome c and the activation of the apoptosome complex. The dephosphorylation of ERK1/2 and a decrease in the nuclear and cytoplasmic levels of nuclear factor kappa beta (NF-kappa B) p65 and 50 were also observed. A mobility shift assay with oligos specific for NF-kappa B further confirmed NF-kappa B down-regulation. The real time RT-PCR array results showed that MEK levels did not change, whereas the levels of MAP4K1 levels increased, thereby indicating the activation of the JUN pathway. Previous studies have speculated that MAP4K1 plays a role in response to environmental stress. It is also speculated that the decrease in the kinase activity of these molecules is related to cell surface components associated with the target molecules uPAR and MMP-9. Taken together, it is evident that glioma cells, which overexpress uPAR and MMP-9, have the potential to undergo apoptosis upon the downregulation of uPAR and MMP-9. Hence, the simultaneous targeting of uPAR and MMP-9 holds promise for glioma therapy.

CB-22. A MECHANISM UNDERLYING FAVORABLE PROGNOSIS BY HIGH PAX6 AND PTEN IN MALIGNANT ASTROCYTIC GLIOMAS: CO-REGULATION EXPRESSION OF GENES ENCODING VEGF AND EFEMP1

Yi-Hong Zhou¹, Yuanjie Hu², Debra Mayes³, Eric Siegel⁴, Marlon Mathews⁵, and Mark Linskey⁵; ¹Neurological Surgery and Biological Chemistry, University of California, Irvine, Irvine, CA, USA; ²Neurological Surgery, University of California, Irvine, Irvine, CA, USA; ³Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; ⁴Department of Biostatistics, University of Arkansas for Medical Sciences, Little Rock, AR, USA; ⁵Neurological Surgery, University of California, Irvine, Orange, CA, USA.

INTRODUCTION: Two pathways, activated by PAX6 and PTEN, have been reported to greatly suppress the tumorigenicity of glioma cell lines, and a higher expression of both genes is correlated with a better prognosis for patients with malignant astrocytic gliomas. We investigated the mechanisms underlying glioma progression by examining the role of both PAX6 and PTEN in angiogenesis, a hallmark of the highest grade of glioma, glioblastoma multiforme (GBM), through the regulation of two related genes, VEGFA and EFEMP1, that respectively encode proangiogenic and antiangiogenic factors. METHODS AND RESULTS: The stable overexpression of PAX6 in glioma cell lines and the quantitative analysis of VEGFA and EFEMP1 expression showed that PAX6 suppressed VEGFA while enhancing EFEMP1 expression in vitro under normoxic condition and in vivo in subcutaneous xenografts. The regulation of EFEMP1 by PAX6 is dose-dependent and requires its DNA binding activity. The stable overexpression of EFEMP1 in glioma cells suppressed tumor growth and VEGFA expression, which is in accordance with blood-vessel-density decrease in xenografts. Decreasing EFEMP1 in PAX6-overexpressing cells partially restored the PAX6 suppression effect on tumor growth and VEGF, confirming the role of EFEMP1 in the suppression of GBM tumorigenicity via the suppression of angiogenesis. Interestingly, PTEN was also shown to enhance EFEMP1 expression. PTEN is known to suppress VEGFA expression by blocking the phosphatidylinositol-3 kinase (PI3K)/AKT pathway and subsequently suppressing HIF1-inhibitor activity. The overexpression of PAX6, however, did not alter AKT and GSK3 phosphorylation in glioma cells, suggesting that PAX6 does not block the PI3K/AKT pathway. In agreement with findings that the independent regulation of EFEMP1 and VEGF expression occurs via PAX6 and PTEN, an enhancing regulatory effect on VEGFA and EFEMP1 was observed to result from the cointroduction of PAX6 and PTEN or the PI3K inhibitor LY294002, when compared to the introduction of the three factors alone. CONCLUSION: Overall data revealed that EFEMP1 is a critical downstream target of PAX6, which affects its tumor suppression function in glioma cells. PAX6 and PTEN work through parallel and complementary gene regulatory pathways to suppress angiogenesis-driven glioma progression.

CB-23. IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF THE HUMAN GSTP1 GENE AS A NOVEL TRANSCRIPTIONAL TARGET OF THE P53 TUMOR SUPPRESSOR GENE

Hui-Wen Lo¹, Lisa Stephenson², Xinyu Cao³, Raphael Pollock², Mira Milas⁴, and Francis Ali-Osman¹; ¹Surgery, Duke University, Durham, NC, USA; ²TX, USA; ³Department of Surgery, Duke University, Durham, NC, USA; ⁴OH, USA.

The glutathione S-transferase P1 (GSTP1) is involved in multiple cellular functions, including phase II metabolism, stress response, signaling, and apoptosis. The mechanisms underlying the significantly high GSTP1 expression in many human tumors are, however, currently not well understood. We report here that the GSTP1 gene is a heretofore, unrecognized downstream transcriptional target of the tumor suppressor p53. We identified a p53-binding motif comprising two consecutive half-sites located in intron 4 of the GSTP1 gene that is highly homologous to consensus p53-binding motifs in other p53-responsive genes. Using a combination of electrophoretic mobility shift assay and DNase I footprinting analyses, we showed that wild-type p53 protein binds to the GSTP1 p53 motif; luciferase reporter assays showed the motif to be transcriptionally functional in human tumor cells. In temperature-sensitive p53 mutant cells, levels of both p21/WAF1 and GSTP1 gene transcripts increased time-dependently when cells were switched from the inactive mutant state to the wild-type p53 state. The siRNA-mediated reduction of p53 expression resulted in a specific decrease in GSTP1 expression and in tumor cells with mutated p53; the adenovirally mediated expression of wild-type p53 increased GSTP1 expression significantly. In a panel of early passage brain tumor cultures from patients, high levels of GSTP1 transcripts and protein were associated with wild-type p53 and, conversely, low GSTP1 levels were associated with mutant p53. The knockdown of p53 expression by siRNA increased cisplatin sensitivity. The ability of wild-type p53 to transcriptionally activate the human GSTP1 gene defines a novel mechanism of protecting the genome and, potentially, of tumor drug resistance.

CB-24. CONSTITUTIVE STAT3 ACTIVATION FREQUENTLY COEXISTS WITH EGFR EXPRESSION IN HIGH-GRADE GLIOMAS AND TARGETING STAT3 SENSITIZES THEM TO ANTI-EGFR AND ALKYLATING AGENTS

Hui-Wen Lo¹, Xinyu Cao², Hu Zhu², and Francis Ali-Osman¹; ¹Surgery, Duke University, Durham, NC, USA; ²Department of Surgery, Duke University, Durham, NC, USA.

Malignant gliomas and medulloblastomas are the most frequent brain malignancies in adults and children, respectively. The relationship between the oncogenic transcription factor STAT3 and glioma grade remains unknown. Also uninvestigated is whether high STAT3 activity is a mechanism underlying the resistance of malignant gliomas and medulloblastomas to chemotherapy. Here, we found STAT3 to be constitutively activated in 60% of primary high-grade and malignant gliomas and the extent of activation to be positively correlated with glioma grade. High levels of activated or phosphorylated STAT3 were also present in cultured human malignant glioma and medulloblastoma cells. Three STAT3-activating kinases, JAK2, epidermal growth factor receptor (EGFR), and EGFRvIII, contributed to STAT3 activation. An inhibitor to JAK2/STAT3, JSI-124, significantly reduced the expression of STAT3 target genes, cyclin D1, and vascular endothelial growth factor; suppressed cell growth; and induced apoptosis in malignant glioma and medulloblastoma cells. Furthermore, we found that STAT3 constitutive activation coexisted with EGFR expression in 27.2% of primary high-grade gliomas and that such coexpression correlated positively with glioma grade. The combination of an EGFR agent, gefitinib (Iressa), and a JAK2/STAT3 inhibitor synergistically suppressed STAT3 activation and potently killed two human glioblastoma cell lines that expressed wild-type EGFR or EGFRvIII. JSI-124 also sensitized malignant glioma and medulloblastoma cells to temozolomide, BCNU, and cisplatin; a synergism was observed between JSI-124 and cisplatin. Together, our findings suggest that STAT3 constitutive activation alone and with EGFR expression plays an important role in de novo primary high-grade gliomas and/or secondary tumors, as a result of malignant progression from low-grade gliomas, and that targeting STAT3 sensitizes these tumors to anti-EGFR and alkylating agents.

CB-25. NESTIN IS REQUIRED FOR GLIOMA CELL MIGRATION

Eli Bar¹, Alex Lin², David Berman¹, and Charles Eberhart¹; ¹Johns Hopkins University, Baltimore, MD, USA; ²Pathology, Johns Hopkins University, Baltimore, MD, USA.

Expression of the intermediate filament protein nestin is commonly used as a marker of stem and progenitor cells in neural tissues, but its functional role is poorly understood. Recently, Kleeberger et al. have shown that nestin may promote the migration and metastasis of prostate cancer cells. We therefore investigated the role of nestin in glioblastoma. Nestin expression was reduced using short hairpin RNA (shRNA) in several glioblastoma neurosphere lines. An reduction of more than 90% in Nestin mRNA expression was confirmed using quantitative PCR. A similar level of nestin protein decrease was documented using immunofluorescence microscopy. These profound reductions of nestin level had no effect on glioblastoma proliferation in culture or on tumor neurosphere formation. Expression of the stem cell marker CD133 was similarly unaffected by nestin level. However, the reduction of Nestin using shRNA inhibited the spread of some glioblastoma neurosphere lines over Matrigel-coated plates by more than 80%, suggesting that nestin may be required for tumor invasion. We are currently examining whether nestin depletion inhibits the migration of these cell lines following orthotopic xenografting in nude mice. Further dissection of the functional role played by nestin may lead to new strategies to prevent glioblastoma invasion.

CB-26. BCL2L12-MEDIATED INHIBITION OF EFFECTOR CASPASES 3 AND 7 VIA DISTINCT MECHANISMS IN GLIOBLASTOMA

Alexander Stegh¹, Santosh Kesari², John Mahoney², Harry Jenq², Kristin Forloney², Alexei Protopopov², David Louis³, Lynda Chin², and Ronald Depinho²; ¹Dana-Farber Cancer Institute, Boston, MA, USA; ²Boston, MA, USA; ³Pathology and Cancer Center, MGH, Charlestown, MA, USA.

Glioblastoma multiforme (GBM) is a highly aggressive brain cancer that is characterized by the paradoxical features of intense apoptosis resistance and a marked propensity to undergo necrosis. Bcl2L12 (Bcl2-Like12) is a nuclear and cytoplasmic oncoprotein that is universally overexpressed in primary GBM and functions to block post-mitochondrial apoptosis signaling by neutralizing effector caspase-3 and -7 maturation. This post-mitochondrial block in apoptosis engenders the alternate cell fate of cellular necrosis, thus providing a molecular explanation for GBM's classical features. While the Bcl2L12-mediated neutralization of caspase-7 maturation involves physical interaction, the mechanism governing the Bcl2L12-mediated inhibition of caspase-3 activity is not known. The nuclear localization of Bcl2L12 prompted expression profile studies of primary astrocytes engineered to overexpress Bcl2L12. The Bcl2L12 transcriptome revealed a striking induction of the small heat shock protein alpha-basic-crystallin (alphaB-crystallin/HspB5) – a link reinforced by robust alphaB-crystallin expression in Bcl2L12-expressing orthotopic glioma cells and by strong co-expression of alphaB-crystallin and Bcl2L12 proteins in human primary GBMs. On the functional level, enforced alphaB-crystallin or Bcl2L12 expression enhances orthotopic tumor growth. Conversely, the RNAi-mediated knockdown of alphaB-crystallin in Bcl2L12-expressing astrocytes and in glioma cell lines with high endogenous alphaB-crystallin enhanced apoptosis, yet decreased necrotic cell death with an associated increase in caspase-3, but not in caspase-7 activation. Mirroring this specific effect on effector caspase-3 activation, alphaB-crystallin selectively binds pro-caspases-3 and its cleavage intermediates in vitro and in vivo. Thus, alphaB-crystallin is a Bcl2L12-induced oncoprotein that enables Bcl2L12 to block the activation of both effector caspases via distinct mechanisms, thereby contributing to GBM pathogenesis and its hallmark biological properties.

CB-27. SIGNALING CASCADE INVOLVED IN TENASCIN-C STIMULATED GLIOMA INVASION IN A THREE-DIMENSIONAL COLLAGEN MATRIX

Susobhan Sarkar¹ and Voon Wee Yong²; ¹University of Calgary, Calgary, Alberta, Canada; ²Departments of Oncology and Clinical Neurosciences, University of Calgary, Calgary, Alberta, Canada.

Glioma invasiveness within the central nervous system is a major cause of the high morbidity of this disease. The invasiveness of glioma cells involves the attachment of invading tumor cells to the extracellular matrix (ECM), the disruption of ECM components, and the subsequent penetration of cells into adjacent brain structures. These processes are accomplished in part by tumor-secreted matrix metalloproteinases (MMPs). That tenascin-C (TN-C), an ECM protein widely expressed in glioma specimens, has a role in glioma invasiveness has been suggested by several groups. We have previously shown that in a three-dimensional matrix of type 1 collagen (3D CL-I) gel, TN-C increased the invasiveness of glioma cells and that the downstream production of MMP-12 is an associated mechanism (Sarkar et al. Cancer Res 66:11771, 2006). The present study investigated the signaling mechanisms involved in the TN-C–stimulated glioma invasiveness in a 3D CL-I matrix and the correspondent production of MMP-12 in glioma cells. By using a panel of pharmacological inhibitors with relative selectivity for particular signaling pathways, we found that the pan protein kinase C (PKC) inhibitor, bisindolylmaleimide I, decreased TN-C–mediated glioma invasion in 3D CL-I. In addition, calphostin C, an inhibitor of conventional and novel PKC as well a relatively selective PKC inhibitor (rottlerin), decreased TN-C–stimulated glioma invasion in a concentration- and time-dependent manner. In concordance with this finding, we determined that PKC is activated after exposure to TN-C, as ascertained by immunoblotting and the translocation of PKC from the cytosolic to the membrane fraction using the subcellular fractionation of cells grown in 3D CL-I. An increase in PKC enzyme activity confirmed the activation of PKC with TN-C, as determined by immunofluorescence microscopy with immunopurified PKC from cells and small interfering RNA to PKC, which attenuated the TN-C–stimulated glioma invasion. Finally, the blocking of PKC in glioma cells in 3D CL-I with rottlerin downregulated TN-C–induced MMP-12 expression. Our results implicate PKC as a therapeutic target in TN-C–stimulated MMP-12 expression and glioma invasion.

CB-28. THE DEATH DOMAIN KINASE RIP1 INHIBITS P53 INDUCTION AND CONFERS A WORSE PROGNOSIS IN GBM

Seongmi Park¹, Kimmo Hatanpaa², Yang Xie³, Bruce Mickey⁴, Deepti Ramnarain¹, Christopher Madden⁴, and Amyn Habib⁵; ¹Neurology, University of Texas Southwestern Medical Center at Dallas, TX, USA; ²Pathology, University of Texas Southwestern Medical Center at Dallas, TX, USA; ³University of Texas Southwestern Medical Center, TX, USA; ⁴Neurosurgery, University of Texas Southwestern Medical Center at Dallas, TX, USA; ⁵University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA.

Inflammation appears to play a causal role in certain types of human cancer. However, while signs of inflammation are common in glioblastoma multiforme (GBM), the significance of inflammation is unknown. We have found that the receptor interacting protein (RIP, RIP1), a key component of inflammation and stress-induced nuclear factor (NF)-kappa B activation, is expressed at high levels in about 30% of GBM but not in grade II-III glioma (p=0.03) and that the protein confers a worse prognosis in GBM. RIP1 levels were assessed by Western blotting in 70 GBM and 22 grade II-III glioma specimens and quantitated by densitometry. GBMs were dichotomized into high and low RIP1 groups based on RIP1 levels in the normal brain. A multivariate proportional hazards model was fitted to adjust for the effects of age and for Karnofsky performance score; a high RIP1 was significantly associated with a short survival time (hazard ratio = 4.1 and p=0.003) and disease-free survival time (hazard ratio=2.5 and p=0.016). We further show that the increased expression of RIP1 inhibits p53 induction in response to ionizing radiation in U87MG glioma cells. An ablation of p53 in glioma cells, in turn, alters the biological response of glioma cells to RIP1 expression. While a high level of RIP1 induces apoptosis in U87MG cells, p53 ablation renders these cells resistant to apoptosis. Thus, in cells lacking functional p53, RIP1 seems to activate prosurvival pathways, such as the NF-ÛB and phosphatidylinositol-3 kinase (PI3K)-Akt pathways, without activating cell death pathways. Finally, we show that the combination of increased RIP1 and p53 mutation confers the worst prognosis in GBM. Comparison of the Kaplan-Meier survival curves of overall survival in high-RIP1 patients divided on the basis of p53 status showed a significant difference (p=0.022). Among high-RIP1 GBM patients, the median survival times were 22.3 months for patients with wild-type p53 and 7.1 months for those in the p53 mutation group, despite treatment with surgery, radiation, and temozolomide. Thus, inflammatory signals may contribute to gliomagenesis by inhibiting classical tumor suppressor signaling pathways.

CB-29. MIR-185 IS LOST IN GLIOBLASTOMA MULTIFORME (GBM) AND INHIBITS PROLIFERATION IN GLIOMA CELL LINES

Colin Morrow¹, Ivan Smirnov², Alex Adai², Ru-Fang Yeh³, Anjan Misra⁴, and Burt Feuerstein¹; ¹Barrow Neurological Institute, Phoenix, AZ, USA; ²CA, USA; ³University of California, San Francisco, San Francisco, CA, USA; ⁴Phoenix, AZ, USA.

MicroRNAs (miRNAs) are small non-coding RNAs composed of 17 to 25 nucleotides. MiRNAs pair with the 3' untranslated regions of mRNA transcripts via eight nucleotide seed sequences on their 5' ends. This pairing regulates both the translation of proteins by ribosomal inhibition and the stability of transcription by poly A tail degradation. We have identified miR-185 as being expressed in the normal brain but frequently lost in GBM. We first inferred the loss of miR-185 from an analysis of gene expression and comparative genomic hybridization array data from GBM tissue. We later observed its loss in miRNA microarrays. The loss was validated by real-time PCR. We procured a list of miR-185 targets to infer the functional significance of miR-185 loss in GBM. An analysis of miR-185 targets suggested that miR-185 regulates cell proliferation and migration. The proliferation-associated targets include p85alpha, mitogen-activated protein kinase (MAPK)14, and vascular endothelial growth factor, and migration targets include two major cytoskeletal regulators, RhoA and CDC42, and the ephrin ligand and receptors. To assess the effects of miR-185 in vitro, the glioma cell lines U87 and U251 were transfected with either a non-targeting negative control miRNA or miR-185. MiR-185-transfected cells exhibited growth inhibition. We have also observed qualitative migration differences in miR-transfected cells in this model. Supported by the Barrow Neurological Foundation, Dianne & Bruce Halle, and CA97874.

CB-30. ENZASTAURIN INDUCES H2AX PHOSPHORYLATION AND CHK2 ACTIVATION IN GLIOMA CELLS

Esther Jane¹ and Ian Pollack²; ¹University of Pittsburgh, Pittsburgh, PA, USA; ²PA, USA.

Enzastaurin (LY317615), an acyclic bisindolylmaleimide, is an oral inhibitor of PKCbeta as well as other PKC isoforms. We determined the cytotoxicity of enzastaurin in a panel of malignant glioma cell lines with diverse genomic alterations. Cell proliferation was determined from dose-response curves. The activation of checkpoint kinase 2 (Chk2) and an increase in the phosphorylation of histone H2AX in response to enzastaurin occurred in a dose-dependent fashion. H2AX is a marker that correlates with DNA damage and the induction of apoptosis. Although enzastaurin independently produced a dose-dependent inhibition of cellular proliferation, decreased cell viability, and partially downregulated Akt and GSK3beta phosphorylation, the median effective concentrations were at the upper limits of or above the clinically achievable range in all the glioma cell lines tested. Therefore, we questioned whether the inhibition of mitogen-activated protein kinase (MAPK) signaling using the MAPK inhibitor U0126 might potentiate therapeutic efficacy. Enzastaurin and U0126 reduced proliferation in all cell lines when used as single agents, and the combination produced a marked potentiation of growth inhibition. Cells stained with Annexin V-propidium iodide and an immunocytochemical assessment of cytochrome c release demonstrated that the addition of U0126 resulted in significantly higher levels of apoptosis than did enzastaurin alone. Our results clearly indicate that the inhibition of MAPK signaling enhances the antiproliferative effect of enzastaurin in malignant human glioma cell lines and support the examination of combinations of signaling inhibitors in these tumors.

CB-31. P53 MUTATIONS AFFECT GLIOMA CELL GROWTH AND CHEMOSENSITIVITY

Christine Billecke¹, Oliver Bogler², Nicholas Farrell³, and Tom Mikkelsen¹; ¹Neurosurgery, Henry Ford Hospital, Detroit, MI, USA; ²Neurosurgery, The University of Texas M. D. Anderson, Houston, TX, USA; ³Chemistry, Virginia Commonwealth University, Richmond, VA, USA.

Treatment options for primary brain tumors, also termed glioblastomas (GBMs), are limited, with few effective modalities available. The response to cytotoxic agents is often influenced by cellular p53 status, with p53 mutations common in treatment-resistant tumors. These mutations are often missense mutations that allow for the potential residual activity of p53. We have focused on the p53 mutations most commonly found in gliomas: V143A, R175H, R248W, and R273H. We hypothesized that specific p53 mutations gain functionality that affect cellular response to chemotherapy agents and influence tumor progression. We have created p53-null astrocytes that express these p53 mutants, thus allowing us to explore the potential gain-of-function aspects of these mutant proteins. We have begun to examine how these specific p53 mutations affect cell growth and proliferation and the cellular response of glioma cells to chemotherapy agents. Here, we show that the presence of mutant p53 influenced the growth rate of these cells, indicating that these mutants are capable of exerting effects on cells in vitro. In addition, the expression of mutant p53 altered the cell cycle distribution of these astrocytes and influenced the expression of p53-responsive genes. Because we saw changes in cell signaling in the presence of mutant p53 alone, we next looked to see if these p53 mutants influenced response to chemotherapy agents. Using a clonogenic assay, we found that p53 status influenced sensitivity to chemotherapy agents, depending on the specific mutation present. In addition, we found that the expression of mutant p53 influenced the expression of bcl-2, caspase-3, and poly ADP-ribose polymerase after exposure to cisplatin. In summary, we show that mutant forms of p53 could influence cell growth rate and cell signaling patterns in the presence and absence of drug exposure. Furthermore, cellular response to various chemotherapy agents depended on the p53 protein expressed, suggesting that p53 mutants can modulate chemosensitivity. Thus, knowledge of a tumor's precise p53 mutation may aid in treatment design and in predicting disease response to therapy.

CB-32. ROLE OF RAGE IN MICROGLIA INACTIVATION IN GLIOMAS

Leying Zhang¹, Darya Alizadeh², and Behnam Badie²; ¹City of Hope, Duarte, CA, USA; ²CA, USA.

To better understand the mechanisms responsible for glioma immune suppression, we evaluated the role of the receptor for advanced glycation end products (RAGE) on macrophage and microglia activation in vitro. A multi-ligand member of the immunoglobulin superfamily of cell surface molecules, RAGE interacts with distinct molecules implicated in homeostasis, development, inflammation, and certain diseases, such as diabetes and Alzheimer's disease. Engagement of RAGE by a ligand triggers the activation of key cell signaling pathways, such as p21ras, mitogen-activated protein (MAP) kinases, nuclear factor (NF)-B and cdc42/rac. Recently, we demonstrated that conditioned medium (CM) from gliomas can activate STAT3 and enhance the expression of anti-inflammatory cytokines, such IL-10, in microglia. Because, under oxidative stress, gliomas can release a number of RAGE ligands (such as AGEs, S100b, and HMGB1), we hypothesized that STAT3 activation in microglia may be mediated through the RAGE pathway. Exposure of N9 microglia to GL261 glioma CM increased STAT3 binding (by electrophoretic mobility shift assay) but inhibited NF-B expression (by western blotting). RAGE inhibition with blocking antibodies completely inhibited STAT3 activity, reversed NF-B suppression, and enhanced TNF-alpha expression in N9 cells exposed to glioma CM. These findings suggest that RAGE may play very important role in macrophage and microglia inactivation in gliomas.

CB-33. MICRORNA-BASED REGULATION OF GLIOMA CELL GROWTH—THE ROLE OF MIR-128

Jakub Godlewski¹, Agnieszka Bronisz², Michal Nowicki¹, Shanté Williams¹, Herbert Newton³, E. Antonio Chiocca⁴, and Sean Lawler⁵; ¹Department of Neurological Surgery, Ohio State University Medical Center, Columbus, OH, USA; ²Department of Molecular and Cellular Biochemistry, Ohio State University Medical Center, Columbus, OH, USA; ³Ohio State University, Columbus, OH, USA; ⁴Department of Neurological Surgery, Ohio State University Medical Center, OH, USA; ⁵Department of Neurological Surgery, Ohio State University, Columbus, OH, USA.

Glioblastoma multiforme (GBM, glioma) represents the most common and the most aggressive primary tumor of the brain. Since cancer formation is closely connected with altered expression of oncogenes and/or tumor suppressor genes, great efforts have been made to identify such changes during tumor formation and progression. Recent evidence indicates that small non-protein-coding RNA molecules, called microRNAs (miRs), might also function as expression regulators of oncogenes and tumor suppressor genes. MiRs have been shown to control cell growth, differentiation, and apoptosis; consequently, impaired miR expression has been implicated in tumorigenesis. Here, we show that the expression of miR-128, one of the miRs normally abundant in the brain, is significantly impaired in tumor samples (18.75-fold reduction) compared to levels in adjacent, non-pathological tissue. The ectopic expression of miR-128 using an oligonucleotide precursor or a lentiviral vector reduced the growth of glioma cells considerably – both in vitro (in U87, U251cells) and in vivo (in flank cells). Direct effects on several target genes were functionally linked to the regulation of oncogenic growth-related processes, as well as the impact exerted on stem cells – like self-renewal are also discussed. Our research provides the basis for future studies in which miRs may be used as diagnostic or therapeutic agents and also a novel approach to tumorigenesis that may yield significant mechanistic insights.

CB-34. ROLE OF THE PROTEIN KINASE MRK IN GLIOBLASTOMA CELL INVASION

Rosamaria Ruggieri¹, Zhiwan Dong², Issai Vanan², and Marc Symons²; ¹Oncology and Cell Biology, Feinstein Institute for Medical Research, Manhasset, NY, USA; ²Feinstein Institute for Medical Research, Manhasset, NY, USA.

Glioblastomas (GBMs) are characterized by a high tendency to infiltrate into adjacent brain tissue, which impedes complete surgical excision. In addition, the invasive cells are resistant to radio- and chemotherapy and are responsible for tumor recurrence. We have identified the protein kinase MRK, a member of the mitogen-activated protein 3 kinase (MAP3K) family, as a signaling molecule that mediates these two important aspects of GBM biology. MRK is activated by lysophosphatidic acid (LPA), an extracellular lipid mediator that elicits mitogenic and motogenic signals in tumor cells. Here, we explore the signaling pathway through which MRK controls cell invasion downstream of LPA. We have used gene silencing to downregulate the endogenous levels of various signaling molecules to address their relationship in the signaling pathway stimulated by LPA and mediated by MRK. We have also used Matrigel matrix-coated chambers to study the cell invasion of glioblastoma cells. MRK downregulation impairs the LPA-stimulated invasion of glioblastoma cell lines. MRK depletion also reduces the LPA-mediated stimulation of the ERK and p38 MAP kinase signaling pathways, and it affects the organization of the actin cytoskeleton. MRK-depleted cells have increased levels of phosphorylated myosin light chain, which correlates with sustained myosin activity. Data from published studies on the Oncomine site indicate that the mRNA levels of several members of the LPA-stimulated pathway, including MRK, RhoC, MKK3, and p38, increase with the severity of brain tumors. Thus, this pathway may play an important role in tumor cell invasion. In addition to this function in LPA-stimulated tumor cell invasion, MRK is activated by ionizing radiation (IR) and is necessary for checkpoint regulation triggered by DNA damage. MRK downregulation causes failure to arrest in the cell cycle after radiation and, as a consequence, it sensitizes glioblastoma cells to killing by radiation. Recent in vitro studies have shown that IR can increase the invasive potential of glioblastoma cells, potentially limiting the benefits of radiotherapy. We have discovered that MRK depletion severely impairs the invasion of GBM cells stimulated by sublethal doses of IR. This dual function of MRK in GBM makes it an attractive drug target to improve the response to radiotherapy, while potentially reducing the potential side-effect of radiation on GBM invasion.

CB-35. ELUCIDATING INHIBITORS OF APOPTOSIS PROTEIN (IAPS)-MEDIATED SURVIVAL AND PROLIFERATIVE SIGNALING IN HUMAN GLIOBLASTOMA MULTIFORME (GBM)

Joydeep Mukherjee¹, Amparo Wolf², Cynthia Hawkins³, and A. Guha¹; ¹University of Toronto, Toronto, Ontario, Canada; ²University of Toronto, Ontario, Canada; ³Ontario, Canada.

Transformation in glial cells resulting in the formation of human glioblastomas (GBMs) requires not only aberrant proliferation but also aberrant inhibition of regulators of apoptosis. Regarding the latter, little is known about the expression and function of a family of proteins known as inhibitors of apoptosis proteins (IAPs), which includes cIAP1, cIAP2, XIAP, and survivin, in GBMs. To date, variable expression of c-IAP1, c-IAP2, and XIAP has been demonstrated in glioma cell lines and increased expression of survivin in GBMs. Previously, we examined in detail the expression levels of cIAP1, cIAP2, XIAP, and survivin in human GBM cells and operative samples, including micro-isolated regional differences between the "center" and "periphery" of human GBMs. Real-time quantitative PCR and immunohistochemistry demonstrated no significant expression of any IAPs in normal white matter or immortalized normal human astrocytes. The only IAP with significantly elevated expression in low-grade astrocytoma specimens was cIAP1. All four IAPs were highly expressed in GBM cells and operative specimens, with increased XIAP and survivin expression in the "center" vs. "periphery" of GBMs. This study explores the mechanisms of XIAP and survivin in GBM cell survival and proliferation. Two established human GBM cell lines, U87-p53:wild-type (wt) and U373-p53:mutation (mut), with elevated levels of XIAP and survivin had knockdown of these two IAPs by siRNA and shRNA. Knock-down of both IAPs resulted in increased apoptosis, especially in response to apoptotic inducers such as chemotherapy, as measured by the activation of caspase-3 and caspase-8. JNK activation, downstream of nuclear factor-kappa B, which transcriptionally regulates IAPs, plays a critical role in cell death and survival signals. We found that the knockdown of XIAP resulted in increased JNK activation, while the knockdown of survivin did not. In addition to apoptosis, IAPs may also promote proliferation. Decreased XIAP resulted in a moderate inhibition of proliferation, with decreased cyclin-D, while the inhibition of survivin resulted in a much more profound anti-proliferative effect, with a marked decrease in both cyclin-D and cyclin-E. The upstream signals resulting in the overexpression of IAPs and how they may be linked to known aberrant signaling pathways in GBMs are also of interest. As previously demonstrated by our group, human GBM cells and specimens have elevated levels of activated Ras compared to normal brain specimens, which correlates to an increased expression of IAPs in GBMs. To further explore this link, we utilized our previously described GFAP:12V-HaRas (RasB8) transgenic mouse glioma model. Elevated Ras activity in mouse and human astrocytes induced expression of both XIAP and survivin. Additional experiments to determine the linkage of aberrant signaling pathways (growth factor and receptor, phosphatidylinositol-3 kinase) to IAP expression in GBMs are underway. Collectively, our data suggest that aberrant signaling pathways in GBMs result in the increased expression of IAPs, which in turn contributes to resistance to endogenous and therapeutic apoptotic inducers. Therefore, modulating these IAPs may be another strategy to sensitize GBMs to apoptotic inducers such as chemotherapy and radiation.

CB-36. INTERDEPENDENT MOLECULAR FUNCTION OF NF2/MERLIN AND EZRIN IN GLIOBLASTOMA

Fabiana Morales¹, Jennifer Molina¹, and Maria-Magdalena Georgescu¹; ¹Neuro-Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.

Malignant gliomas are the most common primary brain tumor in adults. Glioblastoma multiforme (GBM) is the most aggressive type of glioma, and one of the main challenges in treating patients with glioma is the tumor's high propensity to invade distant ^</