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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 brain tissue. Regardless of recent advances in the understanding of brain tumor progression, the molecular mechanisms underlying glioma growth remain elusive. The present study focuses on uncovering the role of the neurofibromatosis type 2 (NF2)/merlin gene product and ezrin in brain tumorigenesis. Although both proteins share a similar molecular structure, NF2/merlin is a known tumor suppressor inactivated in schwanomas, ependymomas, and meningiomas, whereas ezrin has been associated with an increased malignancy of astrocytic tumors. Nevertheless, it is still not known why these two similar proteins have opposite effects in cancer regulation. Here, we show that GBM cell lines present a NF2/merlin-ezrin signature, in which a subset of GBM cells do not express NF2/merlin but do express high levels of ezrin. Because NF2/merlin and ezrin are able to bind to each other, we hypothesized that they function in an interdependent way to control cell growth and metastasis. To test this hypothesis, we performed gain of function experiments and found that the overexpression of NF2/merlin greatly reduced GBM cell growth and downregulated the expression of ezrin. In addition, LN18 and U251 GBM cells presented the lowest cell growth inhibition rate due to higher levels of the phosphorylated inactive form of NF2/merlin, suggesting a mechanism for NF2/merlin inactivation in these cell lines. On the other hand, the overexpression of ezrin increased cell proliferation in LN18 and LN229 GBM cells by displacing NF2/merlin from the membrane to the cytoplasm. Our results strongly suggest that NF2/merlin and ezrin function together to control GBM cell growth.

CB-37. PROTEIN KINASE C (PKC)-ALPHA–MEDIATED REGULATION OF MMP-9 INCREASES GLIOBLASTOMA INVASION

Zachary Kohutek¹ and Isa Hussaini²; ¹Pathology, University of Virginia, Charlottesville, VA, USA; ²University of Virginia, Charlottesville, VA, USA.

Glioblastomas (GBMs) are highly aggressive astrocytic tumors that invade extensively throughout the brain, because in large part of their secretion of extracellular proteases, such as matrix metalloproteinase (MMP)-9. In this study, we have characterized the regulation of MMP-9 by protein kinase C (PKC) in GBM cells and investigated the role of MMP-9, and the PKC isozymes by which it is regulated, in promoting GBM invasion. First, we found that the treatment of U-1242 or U-251 GBM cells with endogenous activators of PKCs, such as epidermal growth factor (EGF) and diacylglycerol (DAG), increased MMP-9 mRNA expression by three- to 20-fold, while the potent PKC activator phorbol 12-myristate 13-acetate (PMA) induced a 100- to 500-fold increase in MMP-9 mRNA, as well as increases in MMP-9 protein expression and gelatinolytic activity. To determine which PKC isozymes mediated the increase in MMP-9, we utilized pharmacological inhibitors and short hairpin RNA (shRNA) knockdown strategies to inhibit individual PKCs. PMA-induced increases in MMP-9 expression and activity were abolished by pretreatment with the pan-PKC inhibitor BIM, while the partial inhibition of MMP-9 was achieved using the PKC-alpha and -beta inhibitor Gö6976 but not the PKC-beta inhibitor hispidin. In addition, stable cell lines expressing shRNA directed against PKC-alpha demonstrated a reduction in the PMA-induced increase in MMP-9 expression and activity, compared with cells containing non-target shRNA. Having identified PKC-alpha as an important regulator of MMP-9, we next determined which signaling pathways and transcription factors were required for PMA-induced increases in MMP-9 expression and activity in GBM cells. A mutation of the nuclear factor-kappa B (NF-kB) binding site in the MMP-9 promoter or a depletion of NF-kB activity via a constitutively active IkB mutant completely abrogated both basal and PKC-mediated increases in MMP-9 promoter activity, thus indicating that NF-kB is required for MMP-9 expression. In contrast, a mutation of the proximal AP-1 site decreased basal but not PKC-mediated increases in MMP-9 promoter function. Pharmacological inhibitors of both MEK and phosphatidylinositol-3 kinase (PI3K) also reduced PKC-mediated MMP-9 expression and activity in GBM cells. Furthermore, the pharmacological inhibition or shRNA silencing of PKC-alpha reduced both NF-kB p65 and Akt phosphorylation, thus indicating that PKC-alpha may regulate MMP-9 via the NF-kB and/or PI3K-Akt pathways in GBM cells. Lastly, to determine the direct effects of PKC-alpha-mediated regulation of MMP-9 on tumor invasion, U-1242 GBM cells expressing shRNA directed against PKC-alpha or MMP-9 were implanted in the brains of nude mice. Both PKC-alpha and MMP-9 knockdown cells were significantly less invasive and formed smaller tumors than GBM cells containing non-target control shRNA in a xenograft GBM mouse model. Together, these results indicate that PKC-alpha increases MMP-9 expression and activity in an NF-kB and Akt-dependent manner, which is important for mediating GBM invasion in vivo.

CB-38. GATA4, A NOVEL TUMOUR SUPPRESSOR GENE IN GLIOBLASTOMA MULTIFORME, MEDIATES ITS EFFECT THROUGH P15INK4B AND P21CIP INDUCTION

Sameer Agnihotri¹, Cynthia Hawkins², and A. Guha¹; ¹University of Toronto, Toronto, Ontario, Canada; ²Toronto, Ontario, Canada.

INTRODUCTION: Several gain- and loss-of-function genetic alterations have been implicated in gliomagenesis leading to glioblastomas (GBMs); however, it is likely that many remain to be elucidated for this highly heterogenous tumor. Recently, using gene-trap strategies in our spontaneous transgenic mouse glioma models, we identified GATA6, a member of the GATA family of transcription factors, as a novel tumor suppressor gene (TSG) involved in the progression of human GBMs. We now report, that GATA4, a close family member of GATA6, also functions as a TSG in gliomagenesis. We have previously demonstrated that GATA4 is widely expressed in various cell types of the normal murine and human central nervous system at different developmental time-points. Specifically, in normal astrocytes, GATA4 expression has a pro-apoptotic and an antiproliferative effect, suggesting its role as a negative regulator of the cell cycle. At the molecular level, GATA4-induced anti-proliferation was mediated through p15Ink4b induction and the attenuation of cyclinD1. RESULTS: In transformed astrocytes, GATA4 loss was found in a majority of human GBM cell lines and 87 of 149 operative specimens at the RNA and protein level, similar to our previous findings with GATA6. Overall, patients with GATA4 loss demonstrated decreased survival, with current studies stratifying these results to other major prognostic factors in GBMs. GATA4 silencing in GBMs was primarily due to epigenetic silencing by the hyper-methylation of the GATA4 promoter. In addition to promoter methylation, GATA4 somatic mutations in the DNA binding domain and nuclear localization sequence (nls) were also observed. The loss of GATA4 by shRNA in non-transformed astrocytes harboring a loss of p53 or oncogenic V12Ha-Ras increased the cells' resistance to apoptosis and, synergistically, their ability to proliferate. The stable knockdown of GATA4 induced the in vitro transformation of these astrocytes, with current studies focusing on in vivo xenograft models. The reintroduction of GATA4 in several human GBM cell lines increased sensitivity to apoptosis when subjected to radiation and chemotherapy (camptothecin and temozolomide). In addition, GATA4 expression in these GBM cells significantly reduced cell proliferation and cell viability. Mechanistically, the reintroduction of GATA4 induced its anti-proliferation effects through the induction of the TSGs p15Ink4b and p21Cip. Chromatin immunoprecipitation (ChIP) assays revealed that GATA4 binds to GATA elements in the p15 promoter. In addition, GATA4 was able to transactivate the p15 and p21 promoters, highly suggesting that they are transcriptional targets of GATA4. TGFB, a key inducer of anti-proliferation in non-transformed astrocytes, mediates its anti-proliferation effects through the induction of p21 and p15. We demonstrated that TGFB positively induces GATA4 and that TGFB stimulation of astrocytes treated with GATA4 siRNA had a significantly reduced anti-proliferation effect. Several glioma cell lines are refractory to the growth suppression of TGFB signaling, and current studies are being undertaken to investigate whether the restoration of GATA4 in gliomas can restore the growth suppressive effects of TGFB through p15 and p21. CONCLUSIONS: Collectively, these findings support GATA4 as a novel TSG in GBMs and suggest that the identification of GATA4 targets may be of genetic, prognostic, and therapeutic relevance.

CB-39. FUNCTIONAL ROLE OF AKT ISOFORMS IN HUMAN MALIGNANT GLIOMAS

Hideo Mure¹, Kazuhito Matsuzaki², Keiko Kitazato³, Yoshifumi Mizobuchi⁴, Kazuyuki Kuwayama³, Teruyoshi Kageji³, and Shinji Nagahiro³; ¹The Department of Neurosurgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan; ²Tokushima, Japan; ³Asia, Japan; ⁴University of Tokushima, Tokushima, Japan.

BACKGROUND: The Akt proto-oncogene, which is the major downstream effector of phosphatidylinositol 3-kinase (PI3K), has been shown to be hyperexpressed and activated in a variety of cancers, including glioblastomas. There are three Akt isoforms: Akt1/PKB-alpha, Akt2/PKB-beta, and Akt3/PKB-gamma, which share a high degree of structural similarity. It has been shown that each Akt isoform possesses distinct roles including cell-cycle progression, migration, invasion, survival, and angiogenesis. However, the expression profiles of Akt isoforms and their functional roles in malignant gliomas have not been well clarified. PURPOSES: To clarify the role of each Akt isoform, we examined the protein and mRNA expression pattern of Akt isoforms in tissue extracted from human astrocytomas, glioblastomas, and non-neoplastic regions. We also explored the biological roles of each Akt isofom in malignant glioma cells using RNA interference-mediated knockdown of each isoform. RESULTS: In glioma tissues, Akt1 protein and mRNA expression was similar to that in the normal control tissues. Increases in both protein and mRNA levels of Akt2 were dependent on the pathological grade of malignancy. The expression of Akt3 in mRNA and protein level decreased according to the pathological grade. However, mRNA expression of Akt3 was remarkably higher than that of other isoforms in each tissue sample. In U87MG and T98MG cells, the downregulation of Akt2 by RNA interference induced caspase-dependent apoptosis. The downregulation of Akt3 also induced apoptosis. The overexpression of Akt3 with plasmid vector facilitated cell growth and concomitantly increased the expression of phosphorylated Akt. Akt1 knockdown did not affect cell growth. CONCLUSION: Akt2 increases and Akt3 decreases depend on the grade of malignancy, while Akt1 expression is not altered. Akt2 and Akt3 play important roles in the cell viability of human malignant gliomas. It is suggested that alteration of the expression profiles of Akt isoforms may lead to tumorigenesis or the progression of malignant gliomas. Thus, therapy targeting Akt2 and Akt3 might hold promise as a potent approach.

CB-40. EVALUATION OF ELK-1 ACTIVATION BY PHOSPHATIDYLINOSITOL 3-KINASE/AKT PATHWAY IN GLIOBLASTOMA

Melike Mut¹, Ozlem Demir², Isil Kurnaz², and Nejat Akalan³; ¹Department of Neurosurgery, Hacettepe University, Ankara, Turkey; ²Istanbul, Turkey; ³Ankara, Turkey.

Glioblastoma multiforme (GBM) is the most malignant tumor among the glial tumors. GBM pathogenesis is closely linked to the epidermal growth factor receptor (EGFR) and the downstream signal transduction pathways, mainly the mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 kinase (PI3K), and protein kinase C (PKC) pathways. A survival transcription factor, Elk-1, has been shown to be downstream of the MAPK and PKC pathways, and these pathways exert some of their proliferative effects via Elk-1 activation in GBM. However, a possible interaction with Elk-1 and another major survival pathway, PI3K, has not been studied in GBM. In this study, Elk-1 transcriptional activation was demonstrated after 12-O-tetradecanoylphorbol-13-acetate (TPA) or epidermal growth factor (EGF) was used to stimulate the MAPK, PI3K, and PKC pathways in rat C6 glioma cells, our in vitro GBM model. Elk-1 transcriptional activity paralleled the phosphorylation of its serine 383 residue in response to EGF or TPA stimulation. This activation was blocked mainly by UO 126, an MEK inhibitor. Inhibiting the PI3K pathway with LY 294002 did not affect Elk-1 transcriptional activation, but the phosphorylation of Elk-1 was blocked by LY 294002. EGF preferentially stimulated the PI3K pathway, while TPA did so for the MAPK pathway in C6 rat glioma cells. Interestingly, PI3K phosphorylated Erk 1/2 in response to EGF, but not in response to TPA. PI3K had no direct effect on the activation of Elk-1; rather, it interacted with MAPK to phosphorylate Elk-1. Elk-1 activation did not have a physiological co-response in regard to the viability, proliferation, and apoptosis observed in C6 rat glioma cells. Taken together, our findings suggest that Elk-1 is activated after phosphorylation at the serine 383 residue mainly by the MAPK pathway, and the PI3K pathway has an indirect role in Elk-1 activation via the MAPK pathway. Elk-1 may not be the major survival factor in C6 rat glioma cells. The physiological significance of Elk-1 activation and of the blocking of both the MAPK and PI3K pathways have yet to be identified in other human GBM-derived cells.

CB-41. ISOLATION AND CHARACTERIZATION OF AN N-LINKED OLIGOSACCHARIDE THAT IS INCREASED IN GLIOBLASTOMA TISSUE AND CELL LINE

Ryuya Yamanaka¹, Naoto Tsutchiya², and Kazuhiro Ikenaka³; ¹Research Center for Innovative Cancer Therapy, Kurume University, Kurume, Fukuoka, Japan; ²Neurosurgery, Niigata University, Japan; ³Division of Neurobiology and Bioinformatics, National Institutes of Natural Sciences, National Institute for Physiological Sciences, Japan.

We have isolated and characterized N-linked oligosaccharides that are significantly increased in glioblastoma tissue and cell lines. The structures of N-linked oligosaccharides in three human normal brain tissue samples, 15 patients with glioblastoma, and three glioma cell lines were analyzed using a partially automated technique for the isolation and fluorescent labeling of N-linked sugar chains from glycoproteins. The characterization of the sugar chains was achieved with the use of a combination of high performance liquid chromatography columns and a highly sensitive fluorescence detector at femtomole levels. By collecting peaks that accounted for 0.1% or more, 16 different oligosaccharide structures were characterized from glioblastoma tissue and cell lines, which accounted for 48.9% of the total N-linked oligosaccharides present in the glioblastoma tissue. The major components of total oligosaccharides were similar to those of normal brain tissue. The amount of a biantennary bigalactosylated structure with one core fucosylation (A2G2F) was present in increased levels in glioblastoma tissue (mean= 2.90%) and glioma cell lines (mean= 5.60%), while being less than 0.1% in normal brain tissues. The expression of the highly branched tetra-antennary N-glycans that are usually detected in the lungs or in hepatocellular cancer was not observed. Tissue glioma cells and cultured cells also displayed strong leukocyte common antigen (LCA)-lectin binding, which bound to sugar chains with core fucose (including A2G2F), while normal brain tissue did not. Moreover, LCA-lectin inhibited the proliferation of glioma cells, and this inhibition resulted from the induction of apoptosis. A2G2F in glioma specimens may provide a novel marker and target for the diagnosis and treatment of glioblastoma.

CB-42. AUTOCRINE REGULATION OF GLIOBLASTOMA PROLIFERATION AND SURVIVAL THROUGH THE VEGF-VEGFR2 INTERPLAY

Petra Knizetova¹, Jiri Ehrmann², Alice Hlobilkova², Zdenek Kolar², Ondrej Kalita², and Jiri Bartek¹; ¹Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark; ²Laboratory of Molecular Biology, Faculty of Medicine, Olomouc, Czech Republic.

Given the key role of the tumor microenvironment in general and the role of vascular endothelial growth factor (VEGF) signaling in glioblastoma biology in particular, we have explored the potential autocrine control of human astrocytoma behavior by VEGF. Using a range of cell and molecular biology approaches to study a panel of astroglial tumor (grade III/IV)-derived cell lines and a series of clinical specimens from low- and high-grade astrocytomas, we showed the following: (i) VEGF and VEGF receptors (VEGFRs) were co-expressed in astroglioma cells in vitro; (ii) VEGF was secreted and biological effects (modulation of proliferation and enhanced viability of astroglioma cells) operated in an autocrine manner; (iii) autocrine VEGF signaling was mediated via VEGFR2 and involved the co-activation of the Ras/c-Raf/MAPK (mitogen-activated protein kinase), phosphatidylinositol-3 kinase (PI3K)/Akt, and phosphoinositide phospholipase C/protein kinase C (PLC/PKC) signaling pathways; (iv) the selective inhibitor (SU1498) of VEGFR2 Tyr kinase activity blocked the biological effects mentioned above and potentiated ionizing radiation–induced tumor inhibitory effect; (v) data obtained from the examination of clinical specimens indicated a correlation between increased VEGF expression and astrocytoma progression, thereby supporting the importance of functional VEGF-VEGFR signaling in vivo. Overall, our results are consistent with the VEGF-VEGFR2 interplay having a potential autocrine role in promoting malignant astrocytoma growth and support the emerging value of this signaling cascade as a therapeutic target, possibly in combination with radiotherapy. This work was supported by grants MSM6198959216 and IGLFUP91110131/39.

CB-43. REIC/DKK-3 INDUCES CELL DEATH IN HUMAN MALIGNANT GLIOMA

Yoshifumi Mizobuchi¹, Kazuhito Matsuzaki², Hideo Mure³, Keiko Kitazato⁴, Kazuyuki Kuwayama⁴, Teruyoshi Kageji⁴, and Shinji Nagahiro⁴; ¹University of Tokushima, Tokushima, Japan; ²Tokushima, Japan; ³Japan; ⁴Asia, Japan.

The progression of glioma to more malignant phenotypes results from the stepwise accumulation of genetic alterations and the consequent disruption of the apoptotic pathway and the augmentation of survival signaling. REIC/Dkk-3, a member of the human Dickkopf (Dkk) family, plays a role as a suppressor of the growth of several human cancers; however, to date, it has not been identified in brain tumors. We compared the gene and protein expression of REIC/Dkk-3 in human malignant glioma and normal brain tissues using quantitative real-time PCR, Western blotting, and immunohistochemistry. We also performed siREIC/Dkk-3 knock-down and REIC/Dkk-3 overexpression experiments to examine the role of REIC/Dkk-3 in human malignant glioma cells in vitro. In brain tissue from patients with malignant glioma, the gene and protein expression of REIC/Dkk-3 was lower than in normal brain tissue and was related to the malignancy grade. In the primary glioblastoma cell line, REIC/Dkk-3 transfection led to apoptosis, owing to the activation of phosphorylated JUN, caspase-9, and caspase-3 and the reduction of beta-catenin; in REIC/Dkk-3 knockdown experiments, cell growth was augmented. Our results suggest that REIC/Dkk-3 regulates the growth and survival of these cells in a caspase-dependent and -independent way via the modification of the Wnt signaling pathway. Our work is the first documentation that the gene and protein expression of REIC/Dkk-3 is downregulated in human malignant glioma. Our demonstration of the mechanisms underlying REIC/Dkk-3-induced cell death indicates that REIC/Dkk-3 plays a pivotal role in the biology of human malignant glioma and suggests that REIC/Dkk-3 is a promising candidate for molecular target therapy.

CB-44. MICRORNA-7 TARGETS NOTCH AND OTHER KEY PATHWAYS IN GLIOMAS

Benjamin Kefas¹, Jakub Godlewski², Laurey Comeau¹, Roger Abounader³, Antonio Chiocca⁴, Sean Lawler⁵, and Benjamin Purow¹; ¹University of Virginia, Charlottesville, VA, USA; ²OH, USA; ³Charlottesville, VA, USA; ⁴OR, USA; ⁵Ohio State University, Columbus, OH, USA.

MicroRNAs are small, non-coding RNAs that downregulate a large subset of human genes, with some microRNAs possessing powerful oncogenic or tumor suppressor functions. MicroRNAs have been shown to play major roles in numerous cancers, including brain tumors. We have previously reported that microRNA-7 inhibits both the epidermal growth factor receptor (EGFR) and the Akt pathway, is downregulated in glioblastomas, and impairs glioma cell viability and invasion. Subsequent work has now indicated that microRNA-7 also suppresses the Notch pathway, which we and others have shown plays an oncogenic role in gliomas. Results from bioinformatic analyses suggested several potential targets of microRNA-7 in the Notch pathway. To validate one of these targets, we demonstrated that transfection with microRNA-7 decreased the protein expression of Notch-2 and the activity of a Notch-2 3'-UTR reporter plasmid. Importantly, microRNA-7 significantly reduced the overall Notch activity indicated by a well-established reporter. This work represents the first demonstration of a microRNA inhibiting the Notch pathway. It suggests a major new target for a putative tumor suppressor microRNA, with potential biological and therapeutic implications.

CB-45. GRP78, A NOVEL INTERACTOR OF PKCDELTA IN THE ENDOPLASMIC RETICULUM, REGULATES THE RESPONSE OF GLIOMA CELLS TO TRAIL AND GAMMA-RADIATION

Hae Kyung Lee¹, Susan Finniss², Cunli Xiang², Simona Cazacu², and Chaya Brodie¹; ¹Henry Ford Hospital, Detroit, MI, USA; ²MI, USA.

Protein kinase C delta (PKCdelta) plays a major role in the regulation of cell apoptosis and survival. Most studies indicate that PKCdelta is a proapoptotic kinase that mediates the apoptosis of various cells in response to different stimuli. Conversely, it was recently demonstrated that this isoform can also protect cells from apoptosis; however, the mechanisms underlying this effect have not yet been characterized. The anti-apoptotic effect of PKCdelta has been associated with its localization in the endoplasmic reticulum (ER) in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-treated and gamma-irradiated cells. We further characterized the anti-apoptotic effects of PKCdelta in the ER using a PKCdelta plasmid targeted to this subcellular site (PKCdelta-ER). The overexpression of PKCdelta-ER decreased the cell apoptosis induced by etoposide and TRAIL and the activation of caspase-3 and caspase-7. Since GRP78 exerts similar effects in etoposide- and TRAIL-treated cells, we examined the interaction of PKCdelta and GRP78. We found that GRP78 and PKCdelta co-localized in the ER as determined by immunofluorescence staining. In addition, we used co-immunoprecipitation to demonstrate that these two proteins were associated. Stimulation of the cells with TRAIL and gamma-radiation, which induce the translocation of PKCdelta to the ER, increased the co-localization and association of GFRP78 with PKCdelta, as determined by co-immunoprecipitation and fluorescence resonance energy transfer analysis. Using limited tryptic digestion studies of isolated microsomes from PKCdelta-ER overexpressing cells, we demonstrated that subpopulations of both PKCdelta and GRP78 existed as ER transmembrane proteins. To examine the role of GRP78 on the anti-apoptotic effect of PKCdelta-ER, we silenced the expression of GRP78 and found that silencing this protein inhibited the protective effect of PKCdelta-ER against TRAIL and etoposide-induced apoptosis and increased the activation of caspase-7. In addition, we found that the overexpression of PKCdelta-ER increased the association of GFRP78 and pro-caspase-7 and decreased the activation and generation of cleaved caspase-7, suggesting that the interaction between PKCdelta and GRP78 is important for the protective effect of the two proteins. In addition, we found that the association of PKCdelta and GRP78 mediated the resistance of glioma cells to radiation in a mechanism that was not related to caspase-7 activation. Recent studies in our lab have demonstrated that GRP78 is upregulated in gliomas, compared to normal brain specimens Collectively, our results indicate that GRP78 is a novel interactor of PKCdelta in the ER and that the association of GRP78 and PKCdelta is essential for the protective effect of this kinase and for the resistance of glioma cells to chemotherapeutic drugs and gamma radiation.

CB-46. P14ARF INHIBITS GLIOMA-INITIATED PLASMA COAGULATION BY ACTIVATING TISSUE FACTOR PATHWAY INHIBITOR-2

Abdessamad Zerrouqi¹ and Erwin G. Van Meir¹; ¹Emory University, Atlanta, GA, USA.

The malignant progression of many tumors, including malignant gliomas, involves the loss of the p14ARF tumor suppressor gene. This genetic alteration occurs with the transition to high-grade glioma and precedes the associated pathological features of intravascular thrombosis, the formation of hypoxic regions, and dramatically increased neovascularization. The mechanisms underlying and possibly connecting these biological features are poorly understood. The tissue factor pathway inhibitor-2, called also placental protein 5 (PP5) is a kunitz-type serine protease inhibitor associated with extracellular matrices. PP5 inhibits the initial blood coagulation reactions involving the tissue factor, which consists of the factor VIIa complex and factor Xa. PP5 has been found to be down-regulated or lost during the progression of astrocytoma toward glioma. Given the high frequency of ARF gene deletions in high-grade glioma and the decreased expression of PP5, we hypothesized that ARF loss is one of the molecular events that dysregulates PP5 expression and promotes the thrombosis within glioma. To determine the biological functions of ARF, we generated Tet-inducible glioma cells to conditionally express the ARF gene in ARF-deficient cells and determined the expression of PP5 mRNA by Northern blot. The specific effects of PP5 on plasma coagulation were analyzed by gene silencing using RNA interference technology. These effects were studied by timing the plasma clotting. Pharmacological inhibitors, RNA interference technology, and chromatin immunoprecipitation assays were used to examine the effects of ARF on PP5 transcription. Our findings showed that ARF upregulates PP5 at the transcriptional level. Furthermore, the ability of cancer cells to promote plasma clotting was significantly reduced, as demonstrated by the increase in coagulation time of plasma in contact with ARF-induced versus uninduced glioma cells. ARF increases c-jun phosphorylation as an immediate response to JNK activation and, therefore, increases the binding of the AP-1 transcription factor to AP-1 binding sites present on the PP5 gene promoter, resulting in PP5 gene upregulation. In summary, these data present the first evidence that the p14ARF tumor suppressor can control plasma coagulation through the AP1-mediated activation of PP5 expression. These findings suggest that therapies directed toward restoring ARF activity or toward inducing PP5 expression or its activity could reverse the intra-tumoral thrombotic cascade that may initiate hypoxia-driven malignant progression.

CB-47. PROMYELOCYTIC LEUKEMIA PROTEIN INDUCES APOPTOSIS BY THE REPRESSION OF NFKAPPAB ACTIVATION IN GLIOBLASTOMA CELLS

Kazuhito Matsuzaki¹, Kazuyuki Kuwayama², Yoshifumi Mizobuchi², Hideo Mure², Keiko Kitazato², Teruyoshi Kageji², and Shinji Nagahiro²; ¹The University of Tokushima Graduate School, Tokushima, Asia, Japan; ²Asia.

PURPOSE: The promyelocytic leukemia (PML) protein plays an essential role in the induction of apoptosis; its expression is reduced in various cancers. As the functional roles of PML in glioblastoma multiforme (GBM) have not been clarified, we assessed the expression of the PML protein in GBM tissues and explored the mechanisms of PML-regulated cell-death in GBM cells. EXPERIMENTAL DESIGN: We examined the PML mRNA level and the expression of PML protein in surgical GBM specimens. PML-regulated apoptotic mechanisms in GBM cells transfected with plasmids expressing the PML gene were examined. RESULTS: The protein expression of PML was significantly lower in GBM than in non-neoplastic tissues; approximately 10% of GBM tissues were PML-null. The PML mRNA levels were similar in both tissue types. The overexpression of PML activated caspase-8 and induced apoptosis in GBM cells. In these cells, PML decreased the expression of transactivated forms of nuclear factor-kappa B (NFkB)/p65, and c-FLIP gene expression was suppressed. Therefore, PML-induced apoptosis resulted from the suppression of the transcriptional activity of NFkB/p65. PML overexpression decreased phosphorylated IkappaB-alpha (IkB-alpha) and nuclear NFkB/p65 and increased the expression of the suppressor of cytokine signaling (SOCS)-1. A proteasome inhibitor blocked the reduction of activated p65 by PML. CONCLUSIONS: The reduction of PML is associated with the pathogenesis of GBM. PML induces caspase-8–dependent apoptosis via the repression of NFkB activation, by which PML facilitates the proteasomal degradation of activated p65 and the sequestration of p65 with IkB-alpha in the cytoplasm. This novel mechanism of PML-regulated apoptosis may represent a therapeutic target for GBM.

CB-48. DETERMINATION OF MOLECULAR REQUIREMENTS FOR GBM SENSITIVITY TO EGFR INHIBITON BY THE ANTI-EGFR MONOCLONAL ANTIBODY ERBITUX

Hetal Bhanushali¹ and Gregory Canute²; ¹SUNY - Upstate Medical University, Syracuse, NY, USA; ²SUNY - Upstate Medical University, NY, USA.

Cetuximab (Erbitux), a monoclonal antibody that competitively inhibits the epidermal growth factor receptor (EGFR), produced dose-dependent cytotoxicity (in vitro) in two EGFR-overexpressing glioblastoma multiforme (GBM) cell lines, as opposed to its producing very little or no cytotoxicity in two low-EGFR-expressing GBM cell lines. The purpose of this study was to look at the expression profiles of key molecules of the survival pathways, such as the phosphatidyl-inositol 3-kinase (PI3K/Akt) and mitogen-activated protein kinase (MAPK/ERK) pathways, which are commonly activated by EGFR stimulation, to determine expression requirements for GBM sensitivity to EGFR inhibition. Previous work has shown that forced EGFR expression in EGFR-transfected U87 cells does not sensitize that cell line to EGFR inhibition with cetuximab. We subcloned a cetuximab-resistant GBM cell line that exhibits EGFR amplification but expresses low levels of EGFR from a cetuximab -sensitive EGFR-amplified and -overexpressing GBM cell line. When the subclone was injected into mice, the tumors that formed re-expressed high levels of EGFR and were resensitized to cetuximab treatment. Cetuximab-sensitive, EGFR-overexpressing GBM cell lines expressed lower levels of activated pAKT than cetuximab-resistant, low-EGFR expressing U87 cels. These cetuximab-sensitive GBM cells contained wild-type PTEN, while U87 cells contained mutated PTEN. Total ERK, but no active ERK, was detected in all in vivo tumors of cetuximab-sensitive, EGFR-overexpressing GBM cells. Cetuximab treatment decreases the total and active EGFR in cetuximab-sensitive, EGFR-overexpressing GBM tumors in vitro and in vivo. The findings from this study suggest that EGFR amplification or EGFR overexpression alone are not sufficient for sensitizing GBM to EGFR inhibition with cetuximab. Our cetuximab-sensitive GBM cells all exhibited EGFR amplification, EGFR overexpression, low pAKT expression, and wild-type PTEN expression. In contrast, cetuximab-resistant U87 cells lacked EGFR amplification and had low EGFR, high pAKT, and mutant PTEN expression. We are currently attempting to determine whether wild-type PTEN and/or low pAKT levels are required for GBM sensitivity to EGFR inhibition by cetuximab.

CB-49. CHLOROQUINE INDUCES DEATH RESPONSE IN GLIOMA CELLS BY P53-DEPENDENT AND P53-INDEPENDENT MECHANISMS

Ella Kim¹, Gabi Warneke², Christoph Schmitz-Salue¹, Wolfgang Deppert², Veit Rohde¹, and Alf Giese¹; ¹Neurosurgery, Georg-August University of Goettingen, Goettingen, Germany; ²Heinrich-Pette-Institute, Hamburg, Germany.

Chloroquine (ClQ) is a lysosomotropic weak base capable of inducing a death response by autophagy and apoptosis. It has recently emerged that ClQ can elicit cytotoxic effects in different types of cancer cells(1–3). The mechanisms underlying ClQ-mediated cytotoxicity appear versatile and may be utilized differently in different types of cells. Ataxia telangiectasia mutated (ATM), p53, and nuclear factor-kappaB have been implicated as molecular targets of ClQ. Although the effects of ClQ on glioma cells has not been investigated, empirical clinical evidence suggests that ClQ retards glioma progression (4). We examined the impact of ClQ on glioma proliferation and survival in vitro and in vivo. Our results revealed that ClQ potently induces a death response in glioma cells in vitro and induces glioma progression in an orthotopic glioma mouse model. The molecular mechanism underlying the death-inducing effects of ClQ in glioma cells involves the activation of the p53 transcriptional response and p53-independent apoptosis. Interestingly, while inducing the autophosphorylation of ATM, ClQ does not induce the phosphorylation of p53 at Ser15, a major site targeted by the ATM kinase in response to DNA damage. Thus, the mechanism of p53 activation by ClQ appears to be distinct from that underlying p53 activation by DNA damage. The versatile modes of cytotoxicity mediated by ClQ in glioma cells and the long history of its relatively safe clinical usage warrant further explorations of the potential benefits of ClQ as a candidate for the treatment of glioma. Further underscoring this notion, our data show that ClQ treatment sensitizes glioma cells to BCNU, temozolomide, and irradiation. REFERENCES: 1. Boya et al. (2005) Mol.Cell. Biol., 25:1025–1040. 2. Gurova et al. (2005) Proc.Natl Acad. Sci. USA, 102: 17448–17453. 3. Maclean et al. (2008) J. Clin.Invest. 118: 79–88. 4. Briceno et al. (2007) Surgical Neurology, 676: 388–391.

CB-50. THE APOPTOTIC FUNCTION OF P53 IS REGULATED BY THE ALTERNATIVELY SPLICED ISOFORM P53BETA

Ella Kim¹, Sven Hanson¹, Eva-Maria Buecker¹, Christoph Schmitz-Salue¹, Veit Rohde¹, and Alf Giese¹; ¹Neurosurgery, Georg-August University of Goettingen, Goettingen, Germany.

INTRODUCTION: The functional disruption of the p53 pathway plays an important role in tumor genesis and progression. In primary glioblastoma multiforme (GBM), p53 is often non-mutated but is functionally impaired by mechanisms involving the inactivation of p53's cooperating factors or the aberrant hyperactivation of p53-inhibiting factors. The identification of cellular factors interfering with the tumor suppressing activities of wild-type p53 is of crucial importance for understanding the mechanisms of p53 inactivation in tumor cells lacking TP53 mutations. The roles of alternatively spliced isoforms of p53 in the modulation of p53 activity have recently emerged. Of the particular interest is the C-terminally truncated isoform p53beta implicated in the augmentation of the apoptotic activity of p53 (1). The aim of our study was to elucidate the roles of p53beta in apoptosis. RESULTS: As a first report, we demonstrated that p53beta possesses an intrinsic anti-apoptotic activity that is independent from and antagonistic to the apoptosis-inducing activity of p53. The overexpression of p53beta increased the resistance of glioma cells to apoptosis. We showed that p53beta, although itself transcriptionally inactive, interferes with the potential of p53 to activate the transcription of the apoptotic genes. In contrast to mutant p53 proteins, whose apoptosis-inducing activity is abolished by hot-spot TP53 mutations, in the corresponding forms of mutant p53beta, anti-apoptotic activity is preserved. Our biochemical assessments revealed that p53beta is targeted by post-translational modifications relevant to p53 protein stabilization, is not susceptible to MDM2-mediated degradation, and is capable of localizing to the mitochondria under cytotoxic stress. CONCLUSION: Our findings reveal for the first time that p53beta acts as a functional antagonist of p53 in apoptosis. REFERENCES: 1. Bourdon JC et al. Genes Dev. 2005;19(18):2122–37.

CB-51. PP6R1 REGULATES THE RADIATION SENSITIVITY OF GLIAL CELL LINES

James Larner¹, David Brautigan², and Jun Mi¹; ¹University of Virginia, Charlottesville, VA, USA; ²Department of Microbiology, University of Virginia, Charlottesville, VA, USA.

Ionizing radiation in combination with temozolomide is the current standard of care for patients with high-grade gliomas. Double-strand breaks (DSBs) are thought to be the critical lesions produced by ionizing radiation (IR). The DNA-dependent protein kinase (DNA-PK) plays a central role in regulating non-homologous end joining (NHEJ), which is the primary pathway for the repair of DSBs. Serine/threonine (Ser/Thr) phosphatase 6 (PP6) is a novel protein Ser/Thr phosphatase classified as a type 2A phosphatase family member based on its sequence homology to the catalytic subunit of PP2A. PP6 is a heterotrimeric enzyme that consists of a catalytic subunit, one of three SAPS regulatory subunits, and an ankyrin repeat subunit (ARS). PP6R1 is one of the SAPS regulatory subunits. Our data indicate that DNA-PKcs (DNA-PK catalytic subunit) and PP6R1 co-immunoprecipitate in several glial cell lines (M059J, M059K, U138, and U118). In addition, we observed that 1) IR-induced nuclear trafficking of PP6R1 is DNA-PK–dependent, since it was noted in DNA-proficient but not DNA-PK–deficient cells; 2) siRNA knockdown of PP6R1 significantly decreases the IR-induced increase in DNA-PK activity; and 3) siRNA knockdown of PP6R1 sensitizes glioblastoma cells to IR, as measured by clonogenic survival. We conclude that PP6R1 may be a molecular target for radiation sensitization.

CB-52. A20 INHIBITS CASPASE-8 CLEAVAGE AND TRAIL-INDUCED APOPTOSIS IN GLIOBLASTOMAS

Anita Bellail¹, Ling Qi¹, C. David James², and Chunhai (Charlie) Hao¹; ¹Pathology & Laboratory Medicine, Emory University, Atlanta, GA, USA; ²University of California, San Francisco, San Francisco, CA, USA.

BACKGROUND: TRAIL (tumor necrosis factor–related apoptosis-inducing ligand) is currently under clinical development as a cancer therapeutic agent; however, phase I-II trials have shown that the majority of human cancers are resistant to TRAIL. Here, we report that A20, an ubiquitin-modifying enzyme, mediates the ubiquitination of RIP (receptor-interacting protein) that in turn inhibits caspase-8 dimerization and cleavage, and thereby TRAIL-induced apoptosis in glioblastomas. METHODS AND RESULTS: Our earlier studies have shown that TRAIL-induced apoptosis occurs through its binding on the death receptor 5 (DR5) on the glioblastoma cell surface. DR5 in turn recruits intracellular FADD (Fas-associated death domain) and caspase-8 for the assembly of the DISC (death-inducing signaling complex). In the DISC, caspase-8 zymogen monomers form dimers, become cleaved, and thus initiate an apoptotic cascade in TRAIL-sensitive glioblastoma cells. In contrast, however, the dimerization and cleavage of caspase-8 are inhibited in the DISC in TRAIL-resistant glioblastoma cells. In search of the molecular basis for the caspase-8 inhibition, we identified A20 and RIP in the DISC in TRAIL-resistant cells. TRAIL treatment led to the ubiquitination of RIP and the inhibition of caspase-8 dimerization and cleavage in the DISC. The ectopic expression of A20 at physical levels inhibited caspase-8 cleavage and thereby TRAIL-induced apoptosis in sensitive cells. The knockdown of A20 by RNAi (RNA interfering) eliminated the RIP ubiquitination, the caspase-8 inhibition, and the TRAIL resistance. The examination of genomic DNA, RNA transcripts, and proteins from glioblastoma cell lines revealed the gain of an A20 gene copy number and the overexpression of A20 mRNA and protein in TRAIL-resistant cell lines. Further examination of human glioblastoma tissues confirmed the A20 overexpression in the majority of the tumors. CONCLUSIONS: This study has established the role of the ubiquitin-modifying enzyme A20 in TRAIL resistance and thus identified A20 as a biomarker that can predict TRAIL resistance and as a therapeutic target that can overcome the resistance in glioblastomas.

CB-53. PROTEASOME INHIBITORS SENSITIZE GLIOMA CELLS AND GLIOMA STEM CELLS TO TRAIL-INDUCED APOPTOSIS BY THE CLEAVAGE AND DOWN-REGULATION OF PKC EPSILON

Sarit Kahna¹, Simona Cazacu², Susan Finniss³, Cunli Xiang³, Shlomit Brodie³, Ana Decarvalho⁴, Tom Mikkelsen⁵, and Chaya Brodie⁶; ¹Bar-Ilan University, Ramat-Gan, Israel; ²Henry Ford Hospital, Detroit, MI, USA; ³MI, USA; ⁴Henry Ford Hospital, Detroit, MI, USA; ⁵Detroit, MI, USA; ⁶Henry Ford Hospital, Detroit, MI, USA.

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) induces apoptosis in cancer but not in normal cells and is therefore considered a promising anti-tumor agent. Some cancer cells, however are resistant to the apoptotic effect of TRAIL. Thus, delineating the molecular mechanisms that mediate the resistance of tumor cells to TRAIL is an important clinical endeavor. We have previously reported that the novel protein kinase C (PKC) isoform, PKC epsilon, plays a role in the sensitivity of glioma cells to TRAIL. In this study, we examined the effects of MG-132, a small-molecule inhibitor of the proteasome, on the resistance of glioma and glioma stem cells to TRAIL. We found that some of the glioma cell lines and primary glioma cultures exhibited sensitivity to TRAIL, whereas all the glioma stem cell lines were resistant to it. The combined treatment of MG-132 and TRAIL induced apoptosis in all the resistant glioma cells and glioma stem cells, whereas normal astrocytes and human neural stem cells did not undergo apoptosis in response to this treatment. Similar results were obtained with additional proteasome inhibitors. MG-132 or TRAIL alone did not induce significant changes in the expression or cleavage of PKC epsilon in the TRAIL-resistant cells. In contrast, a combination of both drugs induced some accumulation of the catalytic fragment of PKC epsilon and significantly decreased its expression. The cleavage of PKC epsilon only partially contributed to the decrease in its expression, since a caspase-resistant mutant, PKC epsilon D383A, was still downregulated by the combination of MG-132 and TRAIL treatment. Using real-time PCR, we found that MG-132 and TRAIL significantly decreased the mRNA level of PKC epsilon. Overexpression of PKC epsilon partially protected the cells from the apoptotic effect of MG-132 plus TRAIL, whereas the caspase-resistant mutant PKC epsilon D383A rendered the cells more resistant to the combined treatment. We previously reported that silencing of PKC epsilon decreased the expression of AKT. Similarly, we found that the combined treatment of MG-132 and TRAIL decreased the expression of AKT and that the overexpression of AKT protected the cells from the apoptotic effect of MG-132 and TRAIL. In summary, our results indicate that MG-132 sensitizes glioma cells and glioma stem cells to TRAIL by inducing both cleavage and a decrease of PKC epsilon expression. The catalytic domain of PKC epsilon induces cell apoptosis, and the decrease in the expression of the full-length PKC epsilon reduces glioma cell survival by downregulating the expression of AKT. Combining proteasome inhibitors and TRAIL may be useful therapeutically in the treatment of gliomas and the eradication of glioma stem cells.

CB-54. PHENOTYPIC CHANGES INDUCED BY ANTI-VEGF TREATMENT—A LINK TO RESISTANCE MECHANISMS IN ANTI-ANGIOGENIC THERAPIES?

Stefan Grau¹, Jun Thorsteinsdottir², Joerg-Christian Tonn¹, Roland Goldbrunner¹, Louisa Von Baumgarten¹, and Frank Winkler¹; ¹Ludwig-Maximilians-Universitat Munchen, Munich, Germany; ²AE.

BACKGROUND: Neoangiogenesis is a feature of malignant tumors, including WHO grade IV gliomas. Thus, antiangiogenic therapies are promising adjuvant treatment modalities. Initial clinical and pre-clinical studies have shown that antiangiogenic therapies using anti-vascular endothelial growth factor (VEGF) antibodies (e.g., bevacizumab; trade name, Avastin) are capable of improving progression-free survival in patients with some solid tumors. However, increasing data show that in spite of continuing anti-VEGF therapy, resistance phenomena resulting in secondary re-angiogenesis can occur after a transient reduction and normalization of tumor vascularization. Thus, alternative signaling molecules may be involved in escaping anti-VEGF therapies. METHODS: Human glioma cell lines U87, U373, and U251; brain-derived endothelial cell lines; and freshly isolated CD31-positive endothelial cells from malignant glioma were treated with bevacizumab for different time periods. Cell growth, expression of the lymphangiogenic growth factors VEGF-C and -D, and cellular response to these factors were investigated. RESULTS: Bevacizumab treatment did not reduce proliferation in a short time in any group, but it did decrease proliferation by approximately 10% after longer periods (12 days) of treatment. All cell lines showed significant upregulation of VEGF-D at a protein level after the longer time period. Even more, cells developed reactivity to VEGF-C and -D by increasing proliferation while being unreactive to these substances before bevacizumab treatment. VEGFR3 expression did not increase significantly; however, the phosphorylation of VEGFR3 by VEGF, VEGF-C, and VEGF-D was enhanced in cells treated with bevacizumab. The intracellular response to VEGF, VEGF-C, and VEGF-D changed in a cell-type-specific manner, with a shift from Erk1/2 to p38 and SAP/JNK phosphorylation. CONCLUSION: Treatment with anti-VEGF antibodies in glioma leads to a phenotypic change with the upregulation of VEGF-D and an increased reactivity to VEGF-C and -D, accompanied by intracellular changes in signal transduction. This may represent an escape mechanism of the tumor in anti-angiogenic therapy.

CB-55. ROLE OF TIE2 SIGNALING IN ADHESION AND THE INVASIVE PHENOTYPE OF GLIOMAS

Dan Liu¹, Vanesa Martin², Juan Fueyo³, and Candelaria Gomez-Manzano¹; ¹University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; ²TX, USA; University of Texas M. D. Anderson Cancer Center, TX, USA.

The diffuse, invasive nature of malignant gliomas greatly impedes surgical intervention and contributes to the poor prognosis of glioma patients. Our group recently reported that the expression of tyrosine kinase receptor Tie2 in the non-stromal compartment of surgical glioma specimens was associated with the progression from lower to higher grade tumors. We also showed that Angiopoietin1/Tie2 signaling resulted in upregulation of integrin beta 1 expression and mediated the adhesion of glioma cells to extracellular matrix proteins. These observations suggested that Tie2 is involved in mediating the adhesion and infiltration phenotype of glioma cells. Here, we report that non-stromal Tie2 expression in human gliomas closely surrounded the vascular structures. We also found that the adhesion of U87 MG cells (Tie2-positive) to human umbilical vein endothelial cells (HUVECs) was greatly enhanced upon Ang1 stimulation. The knockdown of Tie2 expression by small interfering RNA (siRNA) prevented the cell-to-cell adhesion of U-87 MG cells. Tie2-mediated adhesion to endothelial cells (ECs) was also confirmed using Tie2-overexpressing isogenic glioma cell lines. Of interest, we observed, in addition to the upregulation of integrin beta 1, the upregulation of N-cadherin in glioma cells following Tie2 activation. Glioma-to-EC adhesion was inhibited by chelating agents, neutralizing antibodies against N-cadherin and integrin beta 1, suggesting that N-cadherin and integrin beta 1 play roles in Tie2-mediated glioma-to-EC adhesion. Of importance, the invasion of U87 cells through Matrigel was enhanced upon Ang-1 treatment. This was inhibited when Tie2 expression was decreased by siRNA, suggesting the contributing role of Tie2 in glioma cell invasion. To assess the biological significance of a Tie2-mediated increase in the adhesion and invasion of Tie2-positive glioma cells, we injected non-tumorigenic, Tie2 negative, glioma cells or non-tumorigenic, Tie2 positive, glioma cells together with ECs into the brains of immunocompromised mice. We found that the interaction of Tie2-positive glioma cells with ECs increased tumor incidence. Importantly, pathological examination revealed the presence of diffusely invasive tumors that were characterized by clusters of cells surrounding glomeruloid vessels, suggesting an in vivo interaction between Tie2-positive glioma cells and ECs, mimicking a "tumor niche" distribution. These results suggest that the activation of Tie2 signaling is involved in the adhesion of glioma cells to stromal components, which in turn might have a critical role in the acquisition of the invasive phenotype of malignant gliomas.

CB-56. THE ROLE OF TEM1 IN BRAIN TUMOR ANGIOGENESIS

Eleanor Carson-Walter¹, Mahlon Johnson², Yang Liu¹, Melissa Whiteman¹, Bethany Winans¹, and Kevin Walter³; ¹Neurosurgery, University of Rochester, Rochester, NY, USA; ²Rochester, NY, USA; ³University of Rochester, Rochester, NY, USA.

INTRODUCTION: Tumor endothelial marker-1 (TEM1, endosialin) is a novel marker of tumor progression expressed by endothelial cells, pericytes, and a subset of tumor stromal cells. We have previously demonstrated that TEM1 is selectively upregulated at the mRNA level in malignant glioma. In this study, we measured the prevalence of TEM1 expression in more than 275 grade II-IV astrocytic and metastatic brain tumors. We demonstrated that TEM1 can be induced in vitro by stimulating growth factor in cultured endothelial cells. We confirmed the upregulation of TEM1 in U87MG intracranial xenografts and investigated the effects of TEM1 knockout on tumor growth and angiogenesis. METHODS: The mRNA expression of TEM1 in clinical tumors and in cultured primary brain endothelial cells was measured by reverse transcriptase and real-time PCR. In situ hybridization and immunohistochemistry were used to confirm cellular localization in tumors. Two microtissue arrays were used for clinical analysis: one contained 275 samples of primary glial tumors and the other contained 40 specimens of normal brain, low-grade brain tumors, glioblastoma, and brain metastasis. Intracranial U87MG glioblastoma xenografts were grown in TEM1 wild-type and knockout nude mice. The two groups of mice were compared for survival with U87MG challenge and for intracranial vascularity. RESULTS: We confirmed the upregulation of TEM1 in a majority of 275 gliomas (79%). Most tumors demonstrated vascular staining for TEM1, while others also contained isolated areas of TEM1 staining within the tumor cells themselves. TEM1 expression was often concentrated at the margin of the tumor. TEM1 expression was absent from normal brain cells but was also highly expressed in the vasculature of brain metastasis. TEM1 was upregulated in cultured primary brain endothelial cells after exposure to the pro-angiogenic growth factors vascular endothelial growth factor, scatter factor/hepatocyte growth factor, and stromal cell-derived factor-1. As in human tumors, TEM1 was induced in the vasculature of intracranial U87MG xenografts, while showing little to no expression in normal mouse brain tissue. We detected no difference in the tumor take or survival between TEM1 wild-type and knockout mice implanted with intracranial xenografts. Intriguingly, we did detect an increase in the number of microvessels within knockout animal tumors compared to the number in wild-type controls. CONCLUSIONS: TEM1 is upregulated in primary and secondary brain tumors and can be induced by angiogenic growth factors. Although our data suggest that TEM1 is not absolutely required for brain tumor growth in one common preclinical model, TEM1 may still serve as an attractive protein for targeting the cerebrovascular compartment in brain tumors.

CB-57. METALLOPROTEASE CLEAVAGE OF EPHRINA1 RELEASES A FUNCTIONAL, SOLUBLE, MONOMERIC LIGAND FOR THE EPHA2 RECEPTOR

Jill Wykosky¹, Enzo Palma¹, Denise Gibo², Sarah Ringler³, Christopher Turner³, and Waldemar Debinski⁴; ¹Neurosurgery, Wake Forest University, Winston-Salem, NC, USA;; ²Neurosurgery, Wake Forest University School of Medicine, Winston-Salem, NC, USA; ³NC, USA; ⁴Wake Forest University, Winston-Salem, NC, USA.

We have found that ephrinA1, a ligand for the EphA2 receptor tyrosine kinase, is expressed at low levels in glioblastoma multiforme (GBM) in contrast to the abundant overexpression of EphA2. Furthermore, exogenous ephrinA1 has a tumor-suppressing role in reversing the malignant features of GBM and other solid tumor cells through the activation and downregulation of EphA2. Previously, ephrinA family proteins have been described as GPI-anchored ligands, whereby a functional ephrin-Eph interaction was thought to require cell-cell contact or the clustering and oligomerization of an exogenous soluble ligand. To shed light on the role of the ephrinA1/EphA2 system in GBM with a focus on the form of ephrinA1 that functionally interacts with EphA2, we investigated the effects of ephrinA1 expression in GBM cells. To do so, we stably transfected U-251 MG GBM cells with ephrinA1 and observed diminished EphA2 levels in confluent cells, supporting the membrane-anchored presence of ephrinA1. Consistent with a tumor-suppressing role for ephrinA1, these cells exhibited profoundly impaired anchorage-independent growth and decreased migration and proliferation. Unexpectedly, when cells were not in contact, we observed the same decrease in EphA2, suggesting the release of a functional, soluble form of ephrinA1. Next, we detected soluble ephrinA1 in the media of U-251 MG-ephrinA1-positive cells and in the media of SK-Br-3, T47D, and ZR75-1 breast carcinoma cells that endogenously expressed the protein. Using gel filtration chromatography, we identified the released ephrinA1 as a monomeric protein. Soluble monomeric ephrinA1 was functional in causing the internalization and downregulation of EphA2 in parental U-251 MG cells, the alteration of GBM cell morphology, the suppression of Ras-MAPK (mitogen-activated protein kinase) signaling, and growth cone collapse in rat embryonic neurons. The treatment of U-251 MG-ephrinA1-positive cells with PI-PLC to shed GPI-anchored proteins released ephrinA1 that was 2 kDa larger than the naturally released ectopic protein. Subsequent to the PI-PLC cleavage of GPI-anchored proteins, these cells released less ephrinA1 over time than untreated cells. Furthermore, a broad-spectrum metalloprotease inhibitor, GM-6001, significantly diminished the release of cleaved ephrinA1 from U-251 MG-ephrinA1-positive cells. Therefore, ephrinA1 is released from the plasma membrane as a soluble, monomeric protein by metalloprotease cleavage at a site that is upstream of the GPI-anchor and is a functional ligand for EphA2 in this form. This work adds another dimension to the current paradigm that requires ephrins to be membrane-anchored or clustered for function and suggests a novel, paracrine role for a soluble monomer of ephrinA1. These findings also have important implications for the design of ephrinA1-based therapeutics and diagnostics targeted to the EphA2 receptor.

CB-58. HGF UPREGULATES CXCR4 EXPRESSION IN GLIOMAS: IMPLICATIONS FOR GLIOMA CELL INVASION IN GBM

Mine Esencay¹, Iva Smirnova², Elizabeth Newcomb², and David Zagzag²; ¹New York University, New York, NY, USA; ²NY, USA.

Invasion is a hallmark of malignant gliomas and is the main reason for therapeutic failure and recurrence of the tumor. CXCR4 is a key chemokine receptor implicated in glioma cell invasion whose expression can be regulated by hypoxia. Here, we report that hypoxia and hepatocyte growth factor promote glioma cell invasion by inducing the expression of CXCR4 through nuclear factor-B (NF-B). Hypoxia and hepatocyte growth factor upregulated CXCR4 protein expression in U87MG and LN229 glioma cell lines. The enhanced expression of CXCR4 was of biological significance, demonstrated by the increased migration of U87MG and LN229 cells towards the CXCR4 ligand stromal cell-derived factor-1 after exposure to hypoxia and pretreatment with hepatocyte growth factor. Hypoxia and hepatocyte growth factor mediated CXCR4 induction, and increased migration was directly regulated by the transcription factor NF-B. Both hypoxia and hepatocyte growth factor activated the expression of NF-B. Following hypoxia and hepatocyte growth factor stimulation, knockdown of this transcription factor resulted in reduced CXCR4 expression and a corresponding decrease in U87MG and LN229 cell migration towards stromal cell-derived factor-1. Taken together, these results suggest that hypoxia and hepatocyte growth factor promote glioma cell invasion by inducing the chemokine receptor CXCR4 through NF-B. These findings will lead to a better understanding of the molecular mechanisms governing glioma invasion and therefore hold significant therapeutic promise.

CB-59. AKT1 AND AKT2 MEDIATE MALIGNANT FUNCTION IN GBM

Anna Joy¹, Ivan Smirnov², Mitsitoshi Nakada³, Jessica Rennert³, Ru-Fang Yeh⁴, Christian Beaudry³, Jean Zenklusen⁵, Howard Fine⁶, Michael Berens³, and Burt Feuerstein⁷; ¹St. Josephs Hospital & Medical Center, Phoenix, AZ, USA; ²CA, USA; ³Translational Genomics Research Institute, Phoenix, AZ, USA; ⁴University of California–San Francisco, San Francisco, CA, USA; ⁵National Institutes of Health, Bethesda, MD, USA; ⁶National Institutes of Health, Bethesda, MD, USA; ⁷St. Josephs Hospital & Medical Center, AZ, USA.

The Akt pathway is implicated in the progression of astrocytic tumors. To better define the Akt pathway as a therapeutic target in glioblastoma multiforme (GBM), we investigated activated Akt isoforms that mediate cellular behavior and that are associated with clinical parameters. We first identified the Akt isoform that is activated (ser473-phosphorylated) in five GBM samples by immunoprecipitating ser473 Akt, then immunoblotting immunoprecipitates with isoform-specific antibodies. The GBM tissue contained predominantly activated Akt2 and some Akt1, but little detectable Akt3. We next analyzed a data set collected at the University of California–San Francisco and The University of Texas M. D. Anderson Cancer Center consisting of the expression profiles of 14 normal and 181 glial tumors for associations of the Akt isoform message with tumor grade and survival. The expression of Akt1 and two messages increased with WHO tumor grade and was associated with shorter survival. In contrast, the Akt3 message decreased with tumor grade and was associated with longer survival. Bioinformatic analysis of expression data suggests that there are multiple subgroups of GBM patients with varying patterns of phosphatidylinositol-3 kinase (PI3K)/Akt pathway expression. Also, siRNA knockdown experiments indicate that Akt2 promotes migration, but Akt3 does not affect it. We have optimized the siRNA knockdown of all three Akt isoforms and are evaluating the effect of knockdown on malignancy-associated functions. Taken together, the data indicate that Akt3 function differs from that of Akt1 and Akt2 and support a role for Akt1 and Akt2 in the malignant behavior of glioma cells. The selective inhibition of Akt1 and/or Akt2 or their downstream effectors may be more effective and less toxic than pan-Akt or -PI3K inhibitors in high-grade glioma. Supported by NIH/NINDS R01 NS042262 (MB), NIH/NINDS R21 NS043446 (MB), Diane and Bruce Halle (BGF), and the Barrow Neurological Foundation (BGF).

CB-60. TUMOR SUPPRESSOR ACTIVITY OF THE DNA DAMAGE RESPONSE IN THE BRAIN

Massimo Squatrito¹ and Eric Holland¹; ¹Memorial Sloan-Kettering Cancer Center, New York, NY, USA.

The maintenance of genomic integrity is essential for embryonic development and adult tissue homeostasis. Defects in the DNA-damage response (DDR) machinery, a network of protein complexes capable of detecting DNA lesions and signaling to downstream effector pathways (cell cycle checkpoints, DNA repair, apoptosis, etc.), are linked to numerous pathological states, including cancers. The checkpoint kinase Chk2 plays a central role in the transduction of the DNA damage signal by phosphorylating a number of distinct cellular substrates. Genetic evidence in humans suggests that Chk2 acts as a tumor suppressor in the brain. A small subset patients with Li-Fraumeni syndrome, a disease characterized by multiple primary neoplasms with increased incidence of glioblastomas (GBMs), exhibited germ-line mutations of Chk2 (with no evidence of p53 mutations). Furthermore, some Chk2-inactivating variants have been recently found in patients with primary GBM. To investigate the role of Chk2 in gliomagenesis, we took advantage of the RCAS/tv-a system, which allows postnatal gene transfer and expression in a cell-type-specific manner. Ntv-a mice that express the TVA receptor under the control of the nestin gene promoter were crossed with Chk2 -/- mice, and the development of platelet-derived growth factor (PDGF)-induced gliomas were followed for the succeeding 3 months. The loss of a single or both copies of the Chk2 gene significantly accelerated PDGF-driven gliomas (P<0.005 and P<0.0001, respectively). Interestingly, Chk2 loss also increased high-grade tumor (GBM) incidence (85% in Chk2 -/- vs. 20% in Chk2 +/+ background, P<0.005). In an increasing number of cancers (most clearly hematopoietic, central nervous system, and breast cancers), different laboratories have identified a subset of tumor cells, named "cancer stem cells" (CSC), that share characteristics with normal stem cells (multilineage differentiation, self-renewal, etc.). These CSCs have been hypothesized to be responsible for tumor relapse due to their high resistance to therapy. The hyperactivation of the DNA damage checkpoint has been recently suggested as a mechanism that induces resistance to irradiation in glioma CSCs; the proposal is that Chk1/2 inhibitors may warrant additional clinical development for glioma treatment. To analyze the role of Chk2 in CSCs in vivo, we are using the ptc +/- model of medulloblastoma, in which the location and the identity of the CSCs in the perivascular niche which has been recently characterized by our group. Preliminary evidence shows that Chk2 -/- medulloblastomas are more resistant to irradiation, showing a higher percentage of nuclear proliferating cell nuclear antigen–positive cells and a lower percentage of cleaved-caspase-3 staining compared to percentages in control tumors. Looking specifically at the perivascular area 6 hours after ionizing radiation, we observed that while the majority of the cells of the perivascular niche in the control tumor show cleaved-caspase-3 staining (cells undergoing apoptosis), the equivalent cells in the Chk2 -/- perivascular niches were cleaved-caspase-3–negative. The data we have collected so far point to an important role for Chk2 in preventing brain tumor formation and in radiation response, indicating the need for a careful examination of the potential risk of pharmacological targeting of Chk2 in these tumors.

CB-61. ELUCIDATING MOLECULAR SIGNALING BY ERBB TYROSINE KINASE RECEPTORS IN GLIOMA CELLS: RULE-BASED MATHEMATICAL MODELING TO IDENTIFY A CONTEXT OF VULNERABILITY

Wendy McDonough¹, Richard Posner², and Michael Berens³; ¹Cancer & Cell Biology, Translational Genomics Research Institute, Phoenix, AZ, USA; ²Bioinformatics, Translational Genomics Research Institute, Phoenix, AZ, USA; ³Translational Genomics Research Institute, Phoenix, AZ, USA.

The ErbB family of tyrosine kinase receptors (ErbB1-4) plays an important role in the tumor progression of several malignancies. The diversity and complexity of the ErbB receptor system is largely due to heterodimerization of receptors, the variety of stimulating ligands, and cross-activation with other transmembrane receptors. Because cellular signal transduction is exceedingly complex (including aberrant gene expression, mutated nodes of signaling, misplaced protein-protein links, and divergent and convergent processing), we believe a mathematical model predicting the dynamic behavior of this system is essential for understanding and predicting vulnerabilities in cancer cells. We have linked proficiencies in quantifying biological endpoints of glioma cell proliferation, migration, survival, and signaling network activation kinetics with innovative rule-based modeling language that depicts the state of signaling nodes en bloc. Our findings demonstrated that when SF767 and U87 glioma cells are co-activated with epidermal growth factor (EGF) and heregulin, pAKT and pErk protein activation increases. Glioma cells whose ErbB3 expression is depleted (siRNA oligonucleotides) show diminished activation of pAKT and pERK compared to intact, dual-Erb1/ErbB3-stimulated cells. However, the knockdown of Gab-1 results in a marked increase in phospho-AKT but not phospho-ERK when stimulated with EGF and heregulin. These results suggest that ErbB3 signaling through phosphatidylinositol-3 kinase occurs independent of Grb2 and Gab-1, that ErbB3 signals preferentially to AKT, and that Akt activation by ErbB1 (EGFR) is dependent on Gab-1. The ErbB-family of receptors may warrant broader inhibition by targeted therapeutics to optimally address glioma cells.

CB-62. INCREASED EXPRESSION OF ATP CITRATE LYASE IS A PUTATIVE GLYCOLYTIC ADAPTATION IN MALIGNANT GLIOMAS

Marie Beckner¹, Ian Pollack², and Jeffrey Kant²; ¹University of Pittsburgh, Pittsburgh, PA, USA; ²PA, USA.

ATP citrate lyase (ACLY) cleaves citric acid, a tricarboxylic acid that is released from inactive mitochondria, thus moderating cytosolic levels of citric acid. Although ACLY is better known for its role in the production of lipids, its control of metabolic acid levels may permit the liberation of glycolytic cells from vascular dependence during invasion. The lack of vascular dependence in glycolytic tumor cells for the removal of metabolic acids is proposed as a malignant feature that is aided by increased expression (and enzyme activity) for ACLY. Our earlier proteomic and functional studies supported the importance of ACLY as a regulator of acidosis in glioblastoma cell lines. In this study, levels of ACLY expression were increased at least 2-fold in 54 of 171 gliomas (Affymetrix-derived data) and were associated with significantly decreased survival in patients (p = 0.0049). The increased expression of a key glycolytic enzyme gene, enolase 1 (ENO1), was also associated with decreased survival (p = 0.0013) in glioma patients. The expression of ACLY and ENO1 were increased at least 2-fold in 40 and 27 glioblastomas (GBMs), respectively, of the 93 GBMs included in the gliomas of the REMBRANDT database on February 12, 2008 (NCI, NINDS, NIH). Chi-square analysis indicated a highly significant association between increased ACLY and ENO1 (chi-square = 23.08, p = 0.0000016, in the database's GBMs). Real-time–quantitative polymerase chain reactions (RQ-PCR) were used to compare the expressions of ACLY and other genes that are candidates for controlling aberrant levels of metabolic acid within glycolytic tumor cells. The increased expression of ENO1 was used as a marker of glycolysis in GBM tissue samples. In addition to ACLY, other candidate genes that were chosen for RQ-PCR analysis by their function, prevalence among GBMs, and associations with decreased survival data in the REMBRANDT database included the carbonic anhydrases 3 and 12 (CA3, CA12), solute carrier family members or monocarboxylic acid transporters (SLC16A3, SLC16A4), lactate dehydrogenases A and C (LDHA, LDHC), hypoxia inducible factor 1A (HIF1A), and glycogen synthase 1 (GYS1), which putatively encodes a mediator of intracellular lactic acid removal (Beckner et al. Lab Invest. 85:1457–70, 2005). Using the delta delta crossing threshold (Ct) method of analysis on real-time data retrieved from an ABI 7500 Prism thermocycler (Applied BioSystems), fold changes in gene expressions within patient samples were found when compared to normal brain RNA (Ambion) that was used as the reference sample with two housekeeping genes [beta actin (ACTB) and glyceraldehyde phosphate dehydrogenase (GAPDH)] used for the normalization of all samples. In frozen tissue obtained from GBMs in five patients, the correlation coefficients of the candidate genes with ENO1 were 0.80, 0.79, 0.59, 0.54, 0.47, 0.33, -0.09, -0.24, and -0.35, for ACLY, CA3, GYS1, LDHC, LDHA, HIF1A, SLC16A3, CA12, and SLC16A4, respectively. The high correlation found for ACLY and ENO1 in the RQ-PCR expression data strongly supports ACLY as a regulator of intracellular metabolic acid removal in the glycolytic cells of malignant gliomas. We gratefully acknowledge support by The Walter L. Copeland Fund for Cranial Research of The Pittsburgh Foundation.

CB-63. NODES OF VULNERABILITY IN THE TWEAK-R (FN14) SIGNALING NETWORK IN INVASIVE GLIOMA

Nhan Tran¹, Matthew Ennis¹, Wendy McDonough¹, Brock Armstrong¹, Marc Symons², Bodour Salhia¹, Jeffrey Winkles³, and Michael Berens¹; ¹Translational Genomics Research Institute, Phoenix, AZ, USA; ²Feinstein Institute for Medical Research, Manhasset, NY, USA; ³University of Maryland, MD, USA.

Genetic alterations in malignant gliomas affect cell proliferation and cell cycle control and are currently the targets of most chemotherapeutic agents. However, treating advanced glial tumors with adjuvant therapies remains largely unsuccessful because of the inability to effectively target invading cells. We have been investigating whether the multifunctional cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast, growth factor-inducible 14 (Fn14), regulate glioma cell invasive activity. In a previous report, we demonstrated that Fn14 expression was elevated in glioblastoma multiforme (GBM) specimens and was also upregulated in migrating glioma cells in vitro and invading cells in vivo. Moreover, we found that Fn14-induced glioma invasion is dependent on the small GTPase Rac1. Rac1 is a ubiquitous signaling protein operating in many normal tissues; consequently, therapeutic targeting of this protein would likely have toxic effects on normal cells. It is possible that targeting key regulators of Rac1 may be a more effective and specific therapy against invading glioma cells. In this study, we found that Ect2, a guanine nucleotide exchange factor (GEF) for Rho family GTPases, including Rac1 and Cdc42, is overexpressed in GBM and that the overexpression of Ect2 correlates directly with tumor grade and inversely with patient survival. Our data show that Ect2 regulates Rac1 activation downstream of Fn14. The depletion of Ect2 by small interfering RNA (siRNA) duplexes abrogates Fn14-induced Rac1 activation and subsequent glioma cell migration. We also found that TWEAK stimulates the activation of Cdc42 and that this is mediated by Ect2. Interestingly, the depletion of Cdc42 expression impaired TWEAK-induced Rac1 activation and also resulted in a significant reduction of glioma cell migration in vitro. This suggests that Cdc42 is, in part, important for Rac1 activation downstream of the TWEAK-Fn14 signaling pathway and argues for the presence of other Rac-GEFs in the Fn14-Rac1 signaling axis. Currently, we have identified four potential candidate GEFs by proteomics that may activate Rac1 downstream of Cdc42. Validations of the role of these GEFs in relation to Fn14-Rac1–stimulated glioma migration are currently being pursued. Thus, therapy designed to interfere with the function of specific Fn14 signaling elements is likely to target invasive GBM cells.

CB-64. KLF-8 ACTS AS AN SIGNALING MOLECULE FOR INTEGRIN V3 IN GLIOMAS AND IS EXPRESSED GRADE DEPENDENTLY

Oliver Schnell¹, Alexander Romagna¹, Niklas Thon¹, Nikolaus Plesnila¹, Joerg-Christian Tonn¹, and Roland Goldbrunner¹; ¹Neurosurgery, Ludwig-Maximilians-Universitaet Muenchen, Munich, Germany.

OBJECTIVE: The downstream mechanisms used by integrins to promote the proliferation of malignant cells in brain tumors are widely unknown. In the present study, we investigated for the first time pivotal factors of a newly found integrin proliferation pathway that includes the potent transcription factor Krüppel-like factor 8 (KLF-8), which stimulates cyclin D1 and subsequently G1/S-progression of the cell cycle in gliomas of different WHO grades. METHODS: Sections and single tumor cells from primary diagnosed glioblastomas (WHO grade IV, n = 5), anaplastic astrocytomas (WHO grade III, n = 5), low-grade astrocytomas (WHO grade II, n = 5), and normal brain tissue were subjected to immunohistochemical and immunocytochemical staining for integrin alpha(v)beta3 (avb3) and downstream signaling molecules: the phosphorylated focal adhesion kinase (pFAK) and cyclin D1. Isolated tumor cells were stained for the coexpression of integrin avb3 and pFAK, the transcription factor KLF-8, and cyclin D1. To determine whether these molecules are expressed in a WHO-grade–dependent manner, we also performed Western blot analysis in the same specimens. RESULTS: Besides avb3, pFAK, and cyclin D1, the transcription factor KLF-8 was detected in low- and high-grade gliomas. Western blot analysis and immunhistochemistry demonstrated a WHO-grade–dependent enhanced expression of pFAK, KLF-8, and cyclin D1 in high-grade gliomas compared to expression in low-grade glioma and normal brain tissue. On the single cell level, avb3-integrin and the pFAK kinase were found to be co-localized on cell membranes, whereas cyclin D1 and KLF-8 were detected preferentially in the nucleus of isolated cells. CONCLUSION: Our data provide the first description of the potent transcription factor KLF-8 and evidence for a significant grade-dependent expression of downstream integrin signalling molecules in human gliomas. If the strong proliferation-enhancing function of KLF-8 can be confirmed, inhibiting downstream integrin signalling by blocking KLF-8 may represent a novel antiproliferative treatment strategy for malignant brain tumors.

CB-65. MUTANT EGFR SIGNALING IN GLIOBLASTOMA TUMOR MAINTENANCE AND EMERGENCE OF RECEPTOR-INDEPENDENT, TUMOR GROWTH-PROMOTING MECHANISMS

Akitake Mukasa¹, Lynda Chin², Webster Cavenee¹, and Frank Furnari¹; ¹Ludwig Institute-University of California–San Diego, La Jolla, CA, USA; ²Dana Farber Cancer Institute, MA, USA.

Epidermal growth factor receptor (EGFR) gene amplification occurs in approximately 40% of all glioblastomas. Activated EGFR (EGFR^*), consisting of EGFRvIII, de2-7EGFR, or deltaEGFR, which is found in approximately 20–30% of human glioblastomas (GMBs), is the most commonly occurring mutant form and is shown to confer enhanced tumorigenicity. EGFR has been used as a tumor-specific drug target in kinase inhibitor-, antibody-based immunotherapy and immunotoxin approaches, although EGFR^* has not been established as being required for tumor maintenance. Moreover, recent experience with kinase inhibitor therapy indicates that tumors will eventually grow through the therapy, either because of desensitizing mutations in the kinase itself or through the activation of alternate oncogenic pathways. We addressed both of these issues by developing a system in which EGFR^* expression in tumor cells inoculated subcutaneously into nude mice is controlled by doxycycline in their drinking water, allowing the controlled modulation of expression levels. This allowed us to demonstrate that the suppression of EGFR^* attenuates tumor growth and therefore, that it is important for tumor maintenance. Similar to the clinical experience using kinase inhibitors, treated tumors eventually regained aggressive growth after a period of stasis. Interestingly, this characteristic was attained without the re-expression of EGFR^*. To determine how the tumors acquired this ability, we used gene expression microarrays and found that a novel gene, substitute for EGFR^* expression (SE^*E)-1 is highly expressed in the tumors in which EGFR^* had been suppressed and that SE^*E-1 allowed the tumors to escape the need for EGFR^* to maintain tumor growth. SE^*E-1 is also expressed in human GBMs, and the knockdown of its expression in our derived EGFR^*-independent tumors suppressed tumor growth. Taken together, we conclude that EGFR^* is required for tumor maintenance and establishment and that gliomas have a selective pressure in vivo to employ alternative compensatory pathways to acquire aggressiveness. Such alternative pathways function as substitutes for EGFR^* signaling in vivo and should therefore be considered as potential targets for additional therapy.

CB-66. LOSS OF ESTROGEN AND PROGESTERONE SURFACE RECEPTORS DURING TREATMENT OF LEPTOMENINGEAL METASTASIS FROM BREAST CANCER

Jai Grewal¹ and Santosh Kesari²; ¹Neurological Surgery, P.C., Great Neck, NY, USA; ²Dana Farber Cancer Institute, Boston, MA, USA.

In this report, we describe a 61-year-old woman who was diagnosed with estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 receptor (HER2/neu) positive invasive lobular carcinoma in the right breast upon initial biopsy. Following 12 weekly cycles of trastuzumab and navelbine on protocol, she underwent a lumpectomy with a positive sentinel lymph node biopsy. The resected tumor was positive for ER and PR and negative for HER2/neu overexpression. She then underwent a modified radical mastectomy followed by four cycles of Adriamycin and cyclophosphamide. She subsequently received adjuvant radiation therapy followed by 12 cycles of paclitaxel and then adjuvant letrozole. Four years later, she experienced headaches, ataxia, and a seizure. Magnetic resonance imaging (MRI) of the brain and spine revealed non-nodular enhancement of the cerebellar folia, with metastatic adenocarcinoma detected by cerebrospinal fluid (CSF) cytologic analysis. Using immunohistochemistry, malignant CSF cells were found positive for ER and PR and negative for HER2/neu overexpression, concordant with the resected primary breast cancer tissue. There were no other areas of metastatic disease discovered after a systemic work-up. The patient was treated with palliative whole-brain radiation therapy followed by combination capecitabine, exemestane, and intrathecal liposomal cytarabine, resulting in clinical improvement. After 8 months of this regimen, her gait worsened and brain MRI revealed radiological progression consisting of subependymal enhancement in the temporal horns of the lateral ventricles bilaterally. Malignant cells were negative for ER, PR, and HER2/neu by CSF cytology (triple-negative). Capecitabine was switched to pemetrexed, while the other agents were continued. The patient remains alive 1 year after the diagnosis of leptomeningeal disease. During the course of her disease, CSF cytology consistently remained positive. This case illustrates that hormonal receptors may be lost with time in leptomeningeal metastasis from breast cancer. Additionally, in this case, the timing of this change in surface receptor pattern correlated with clinical and radiological progression. The mechanisms responsible for this observation are largely unknown.

CB-67. EXPRESSION OF THE TWIST1 TARGET GENE PERIOSTIN PROMOTES GLIOMA INVASIVENESS IN VITRO AND CORRELATES WITH GLIOMA GRADE AND RECURRENCE

Svetlana A. Mikheeva¹, Andrei M. Mikheev¹, Rob Oxford¹, John-Patrick Maxwell¹, Leila Khorasani¹, and Robert Rostomily¹; ¹Department of Neurological Surgery, University of Washington, Seattle, WA, USA.

The invasive phenotype of human gliomas is a major factor in their poor prognosis and resistance to treatment. Therapy targeted against invasion is desirable, particularly given the association between glioma cell migration and survival, but achieving this goal requires a better understanding of the spectrum of molecular mechanisms that regulate invasion. In a previous study, we identified the basic helix-loop-helix transcription factor, TWIST1, as a gene upregulated in the most malignant grade IV gliomas and that also promotes invasion. To investigate the potential mechanisms of TWIST1 function, we performed microarray analyses of T98G glioma cells with and without TWIST1 overexpression. First, in Matrigel filter assays, we confirmed the pro-invasive function of TWIST1 in T98G by overexpression (85% increase in invasion) and stable knockdown (55% decrease in invasion). Consistent with its pro-invasive function, TWIST1 overexpression was associated with the upregulation of functional groups linked to cell motility (cytoskeleton organization, extracellular matrix proteins, adhesion, regulation of cell motility, and locomotion). Among the individual genes related to cell invasion, we identified and confirmed a 32-fold upregulation of periostin (POSTN) in cells with TWIST1 overexpression. POSTN is a secreted extracellular matrix protein and known transcriptional target of TWIST1 in ostesarcoma cells. Of note, in other cancers, POSTN promotes invasion, metastasis, cell survival and treatment resistance, and angiogenesis. Although POSTN has been identified as a highly upregulated in human glioblastoma multiforme (GBM) by serial analysis of gene expression technique and gene expression microarray studies, its expression and function have never been reported in detail in human gliomas. Therefore, we examined the expression and function of POSTN in human glioma cell lines and its correlation with TWIST1 in human glioma samples and hypothesized that the upregulation of POSTN contributes to TWIST1-mediated invasion in gliomas. Using POSTN recombinant protein we demonstrated that POSTN promotes glioma cell adhesion and migration in a dose-dependent fashion, in part through interactions with alphaV beta3 and alphaV beta5 integrin receptors. SNB19 glioma cells lack detectable endogenous POSTN, but when overexpressed in SNB19, POSTN increased in vitro invasion 90%. In glioma cells with high endogenous levels or forced overexpression, POSTN was detected in both whole cell lysates and secreted into conditioned media. Consistent with this in vitro localization, we detected POSTN in tumor cells at variable levels in all grades, but extracellular accumulation was detected exclusively in grade IV gliomas. With real-time PCR, we found a strong correlation between POSTN and TWIST1 expression in 39 human glioma samples (correlation=0.68, p=001). Moreover, POSTN mRNA expression levels were more than 200-fold higher in grade IV gliomas compared to levels in grade II and III combined. In pairs of recurrent and primary tumors, TWIST1 mRNA levels were modestly increased while POSTN expression increased in 8 of 11 cases from 2.6- to 537-fold. Together, our results indicate that the TWIST1 activation of POSTN may promote the malignant glioma phenotype through enhanced cell motility and invasion and potentially play a role in tumor progression and/or treatment failure.

CB-68. FURIN-MEDIATED CLEAVAGE OF BRAIN ANGIOGENESIS INHIBITOR 1 (BAI1) PRODUCES VASCULOSTATIN-40, A NOVEL SECRETED ANTI-ANGIOGENIC MOLECULE

Sarah Cork¹, Balveen Kaur², Eric Sandberg³, Vladimir E. Belozerov⁴, and Erwin Van Meir¹; ¹Emory University, Atlanta, GA, USA; ²Neurological Surgery, The Ohio State University, Columbus, OH, USA; ³Department of Neurosurgery, Emory University, Atlanta, GA, USA; ⁴GA, USA.

Brain angiogenesis inhibitor 1 (BAI1), a brain-specific G protein-coupled receptor, is an interesting candidate for anti-tumor therapy. We have previously shown that its expression is downregulated or lost in malignant gliomas and that the restoration of BAI1 expression inhibits neovascularization and glioma growth. BAI1 has a large extracellular domain (ECD) that contains five thrombospondin type-1 repeats (TSRs), domains known to exhibit anti-angiogenic activity by engaging the pro-apoptotic CD36 receptor signaling axis in endothelial cells. The BAI1 ECD is proteolytically processed to generate multiple secreted fragments. Here, we show that the primary product is a 40-kDa fragment known as Vasculostatin-40 (Vstat40), which contains one TSR. Vstat40 processing is mediated by the endoprotease furin. The Vstat40 processing site was identified using serial truncations of BAI1 cDNA to generate peptides subsequently processed into Vstat40. This region contains an atypical furin cleavage motif as determined by proprotein cleavage site prediction software. We confirmed furin involvement by showing that Vstat40 processing is abrogated in furin-deficient LoVo cells and that Vstat40 processing is selectively blocked by furin inhibitors. Further, Vstat40 is a potent anti-angiogenic molecule, as indicated by its inhibition of human dermal microvascular endothelial cell (HDMEC) migration and cord formation. Vstat40 did not affect the migration of CD36-negative human umbilical vein endothelial cells (HUVECs), and administration of a CD36-blocking antibody rescued HDMEC migration in the presence of Vstats. Collectively, these results demonstrate that furin mediates the processing of BAI1 into Vstat40, and the anti-angiogenic effects of Vstat40 are significantly mediated via CD36, a receptor previously implicated in the inhibition of tumor angiogenesis.

CB-69. GROWTH FACTOR-MEDIATED INDUCTION OF ANGIOMIR-1 REGULATES GROWTH FACTOR RECEPTOR OVEREXPRESSION ON GLIOMA BLOOD VESSELS

Thomas Wurdinger¹, Bakhos Tannous², Okay Saydam², Johan Skog², Ralph Weissleder³, Xandra Breakefield⁴, and Anna Krichevsky²; ¹Neuro-oncology Research Group, VUmc/Cancer Center Amsterdam and Massachusetts General Hospital/Harvard Medical School, Amsterdam, Netherlands; ²MA, USA; ³Harvard University, MA, USA; ⁴Charlestown, MA, USA.

The formation of new blood vessels by angiogenesis is essential for normal functions and is involved in many disease states. Control of the angiogenic switch in endothelial cells involves changes in levels of pro- and anti-angiogenic molecules acting in concert. The molecular regulation of this angiogenic switch is not well understood. MicroRNAs (miRNAs) can have major regulatory roles in cell physiology by controlling the rates of turnover and the translational efficiency of multiple mRNA molecules. Here, we showed that glioma cells and angiogenic growth factors (including vascular endothelial growth factor, VEGF), induce angiomir-1 in primary human brain microvascular endothelial cells (HBMVECs) in culture, as well as in primary tumor endothelial cells isolated from human brain tumors. Angiomir-1 contributes significantly to the angiogenic switch by directly targeting hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) and inhibiting the HGS-mediated degradation of PDGFR-alpha and VEGFR2. The inhibition by miRNA inhibitors or the induction by precursor forms of angiomir-1 in HBMVECs can turn the angiogenic switch off and on, respectively. To our knowledge, angiomir-1 is the first miRNA to be functionally linked to the angiogenic phenotype, and therefore, the study of this miRNA can give new insights into the role of miRNA regulation in neovascularization. Further, the manipulation of angiomir-1 levels using miRNA antisense molecules or precursor constructs may prove therapeutic in the vast number of diseases in which angiogenesis is a critical component.

CB-70. SHP-2 INHIBITION IN EGFRVIII GLIOBLASTOMAS

Yi Zhan¹ and Donald O'Rourke¹; ¹University of Pennsylvania, Philadelphia, PA, USA.

The protein tyrosine phosphatase (PTPase) SHP-2 plays an important role in RTK-mediated cell proliferation, differentiation, transformation, and cell survival. The deregulation of SHP-2 PTPase activities, such as mutation, expression, or posttranslational modification (including phosphorylation), can contribute to the development of diseases such as cancer. Previously, we showed that a phosphatase-inactive form of SHP-2, SHP-2 C459S, inhibited the transformation of epidermal growth factor receptor (EGFR)vIII human glioblastoma (GBM) cells, and additional studies showed that SHP-2 was essential for EGFRvIII oncogenesis. To further evaluate the importance of SHP-2 in the EGFRvIII phenotype, we employed pharmacologic SHP-2 phosphatase inhibitors in GBM cells of different genotypes. Here, we report that one SHP-2 PTPase inhibitor, NSC-87877, inhibited SHP-2 PTPase activity and growth in soft agar in both wild-type PTEN and PTEN-mutated EGFRvIII cell lines in a dose-dependent manner. Moreover, inhibited cells exhibited a completely nontransformed phenotype in the presence of this inhibitor in the mM range. In contrast, we observed differential effects by erlotinib (Tarceva) on EGFRvIII GBM phenotype. Erlotinib inhibited wild-type PTEN LN229.EGFRvIII, but not PTEN-mutated U87MG.EGFRvIII cell transformation. EGFRvIII expression induced SHP-2 phosphorylation at Tyr542, which increased SHP-2 PTPase activity. Phosphorylated SHP-2 accumulated in the perinuclear region in U87MG.EGFRvIII cells but not in LN229.EGFRvIII cells. Interestingly, this perinuclear staining was enhanced by erlotinib in U87MG. EGFRvIII cells at doses that completely inhibited EGFRvIII tyrosine phosphorylation but not the transformation of U87MG.EGFRvIII cells. Therefore, we combined erlotinib with NSC-87877 and observed an additive inhibitory effect on U87MG.EGFRvIII cell growth. Taken together, these data provide further support that the inhibition of SHP-2 PTPase activity inhibits EGFRvIII GBM transformation regardless of PTEN status. The increased SHP-2 Tyr542 resulting from erlotinib treatment may contribute to resistance that can be overcome by the addition of SHP-2 and EGFR inhibitors. Finally, the use of SHP-2 PTPase inhibitors may represent a potential therapeutic approach for the treatment of human GBMs, in particular, primary GBMs that express the EGFRvIII oncoprotein.

CB-71. SIRPALPHA1 RECEPTORS BLOCK EGFRVIII/SHP-2/SOS-1/MAPK ACTIVATION TO INHIBIT GLIOBLASTOMA CELL MIGRATION

Gurpreet Kapoor¹ and Donald O'Rourke²; ¹Neurosurgery, University of Pennsylvania, Philadelphia, PA, USA; ²Philadelphia, PA, USA.

EGFRvIII is a constitutively active truncated mutant of epidermal growth factor receptor (EGFR), which has been shown to increase the migratory potential of tumor cells and normal fibroblasts. Our previous work has shown that inhibitory signal regulatory protein alpha1 (SIR-Palpha1) receptors interact with SHP-2 to inhibit wild-type (wt) EGFR-mediated tumor migration, survival, and cell transformation (Wu et al., 2000). However, the effects of SIRPalpha1 inhibitory receptors on EGFRvIII-mediated phenotypes are not well understood. The aim of the present work was to investigate the effect of SIRPalpha1 inhibitory receptors on the EGFRvIII signalosome and EGFRvIII-mediated phenotypes. Interestingly, the overexpression of SIRPalpha1 receptors in U87MG/EGFRvIII cells reduced migration but not transformation compared to cells expressing empty vector, as determined using transwell chamber migration assays. Western blot analyses showed decreased p42/44MAPK (mitogen-activated protein kinase), p38MAPK, and JNK phosphorylation in U87MG/EGFRvIII/SIRPalpha cells compared to control cells. However, the phosphorylation status of Akt was not affected in SIRP-overexpressing cells, suggesting that SIRPalpha1 specifically inhibits EGFRvIII-mediated MAPK signaling machinery to downmodulate glioblastoma migration. Recently, we showed that EGFR-mediated signaling in glioblastoma cells is regulated via a critical association between the Gab1 adapter protein and the SHP-2 phosphatase and that SHP-2 is essential for EGFRvIII tumorigenesis (Kapoor et al., 2004). Immunoprecipitation analyses using an anti-SHP-2 antibody showed reduced phosphorylated and total Gab1 but enhanced SIRP/SHP-2 association in U87MG/EGFRvIII/SIRPalpha cells, suggesting that SIRPalpha may interfere with SHP-2 and Gab1 association to downmodulate EGFRvIII-mediated migration. Furthermore, anti-Sos-1 and anti-EGFRvIII immunocomplexes showed a reduced association of SHP-2, EGFRvIII, and Sos-1 in SIRPalpha-overexpressing U87MG/EGFRvIII cells, suggesting that SIRPalpha may inhibit Sos-1 recruitment to the EGFRvIII/SHP-2/Gab1 complex and negatively regulate the Ras/Raf/MEK/MAPK pathway as a mechanism of inhibiting EGFRvIII-mediated migration. Interestingly, SIRPalpha-overexpressing cells showed reduced SHP-2 phosphorylation at the Tyr580 residue, which has previously been shown to positively regulate p44/42MAPK activity. Collectively, our data indicate that SIRPalpha overexpression specifically affects the SHP-2/Sos-1/MAPK activation loop to downmodulate EGFRvIII-mediated migration. Further understanding the molecular interactions between SIRPal-pha inhibitory receptors and the EGFRvIII signalosome may facilitate the identification of novels targets to inhibit EGFRvIII-mediated glioblastoma migration.

CB-72. CD133-NEGATIVE GLIOMA SUBPOPULATIONS GROUPED BY DIFFERENT CELL TYPE MARKERS DISPLAY TUMORGENICITY IN VIVO

Lars Prestegarden¹, Agnete Svendsen², Jian Wang¹, Askland Lasse², Persson Andreas², Sakariassen Per Øystein², Kai Ove Skaftnesmo³, Hovland Randi⁴, and Per Øyvind Enger¹; ¹University of Bergen, Bergen, Norway; ²University of Bergen, Bergen, AE, Norway; ³AE (Europe); ⁴Haukeland Sykehus, Bergen, AE, Norway.

The cancer stem cell hypothesis implies that intratumor heterogeneity is created by rare cancer stem cells. These cells have the capacity to differentiate into various tumor cell types as well as to self-renew. In brain tumors, CD133 has been proposed as a marker for cancer stem cells, although this has been challenged by a series of studies demonstrating tumor formation from CD133-negative glioma cells. To further elucidate the heterogeneity seen in glioblastoma multiforme (GBM), we created lineage-specific lentiviral reporter constructs that expressed GFP under the nestin, glial fibrillary acidic protein (GFAP), or neuron-specific enolase (NSE) promoter. The U373 and U87GBM cell lines were infected with these constructs and separated with the simultaneous removal of CD133-positive cells using fluorescence-activated cell sorting (FACS). In this way, we obtained glioma subpopulations expressing different cell type markers and subsequently implanted these cells in NOD-SCID mice. Finally, we repeated the experiments using specimens from two primary GBM biopsies instead of cell lines. In the cell lines, all glioma cell populations gave rise to tumors at similar rates. In the biopsy specimens, both nestin-positive and nestin-negative cells grew tumors, while none of the lineage-specific cells (GFAP and NSE) produced tumors. We conclude that different glioma subpopulations exhibit tumor-initiating abilities, although glioma cells expressing mature markers may be relatively less tumorigenic than other glioma cell populations.

	EPIDEMIOLOGY

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

 
EP-01. RACIAL/ETHNIC DIFFERENCES IN PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA (PCNSL): A NATIONAL CANCER INSTITUTE (NCI) SURVEILLANCE, EPIDEMIOLOGY AND END RESULTS (SEER) PROGRAM ESTIMATE OF INCIDENCE AND SURVIVAL RATES AMONG UNITED STATES (US) BLACKS, WHITES, AND OTHERS

Jose Pulido¹, Robert Vierkant², Janet Olson³, Lauren Abrey⁴, David Schiff⁵, and Brian O'Neill⁶; ¹Department of Ophthalmology, Mayo Foundation for Medical Education and Research, Rochester, MN, USA; ²Department of Biostatistics, Mayo Foundation for Medical Education and Research, Rochester, MN, USA; ³Department of Epidemiology, Mayo Foundation for Medical Education and Research, Rochester, MN, USA; ⁴Memorial Sloan Kettering Cancer Center, New York, NY, USA; ⁵University of Virginia, Charlottesville, VA, USA; ⁶Department of Neurology, Mayo Foundation for Medical Education and Research, Rochester, MN, USA.

INTRODUCTION. PCNSL is a rare primary brain tumor characterized by the perivascular accumulation of neoplastic cells with lymphoid characteristics. PCNSL is seen in two age groups: in younger age groups that are frequently associated with HIV or iatrogenic immunosuppression and in patients over the age of 50 years who generally do not have any other known systemic disease. Approximately 1,000 cases are diagnosed yearly in the United States, and the incidence appears to be increasing over time. The racial and ethnic associations of PCNSL have never been determined. Our clinical impression was that PCNSL was less commonly diagnosed in Blacks than in Whites and rarely in Hispanics, A/PI, and AI/AN. To determine if there was a racial and/or ethnic difference in incidence and survival, we used SEER program data. METHODS. Incident malignancies were classified using the ICD-O (2nd edition) and specified NHL morphologies. The codes queried were only for PCNSL. Data were stratified to those 20–49 years of age and those over 50 years of age. A subgroup analysis reflected the racial groups recognized by SEER. Because minority representation was limited in the initial SEER data from 1973 to 1992, only data for the expanded 13 SEER sites (available beginning in 1992) were used. The age-adjusted incidence rates were calculated using the SEER^*Stat program. Rates were age-adjusted to the 2000 U.S. standard million population and reported per 100,000 person-years. Ninety-five percent confidence intervals (CIs) were calculated based on properties of the Poisson distribution. All analyses were restricted to persons age >20 years. RESULTS. Whites had a PCNSL incidence rate of 0.94 per 100,000 per year (95% CI, 0.90–0.98); among Blacks, the incidence was 1.10 per 100,000 per year (95% CI, 0.98–1.22; Table 1). The AI/AN and A/PI subgroups had a much lower PCNSL incidence rate of 0.51 (95% CI, 0.28–0.74) and 0.64 (95% CI, 0.56–0.72), respectively. A subanalysis of patients aged 20–49 years at diagnosis demonstrated that the PCNSL incidence in Blacks (IR=1.43 [95% CI, 1.27–1.59]) was twice that of similarly aged Whites (IR=0.72 [95% CI, 0.68–0.76]). The AI/AN and A/PI subgroups had rates of 0.58 (95% CI, 0.30–0.86) and 0.21 (95% CI, 0.15–0.27), respectively. For those above the age of 50 years, the incidence ratio between the two racial groups was reversed; the incidence of PCNSL was 1.30 (95% CI, 1.22–1.38) in Whites and 0.56 (95% CI, 0.40–0.72) among Blacks. PCNSL incidence was 0.34 (95% CI, 0–0.70) in AI/AN and 1.31 (95% CI, 1.00–1.53) in A/PI. Survival for all races and all ages at 12 months, 2 years, and 5 years was 33%, 25%, and 16% respectively. Among all persons 20 years and older, survival at 12 months, 24 months, and 60 months among Whites was significantly higher than in Blacks. When stratified into groups of those age 20 to 49 years and those age 50 years or greater, only 12-month survival and 5-year survival were statistically different in the two racial groups (P=0.03 and P=0.05, respectively). CONCLUSIONS. The data presented here are suggestive of a racial influence on incidence and mortality of PCNSL. Implications for research and future directions will be discussed.

EP-02. A COLLABORATIVE STUDY OF THE EPIDEMIOLOGY OF OLIGODENDROGLIAL TUMORS

Bridget Mccarthy¹, Kenneth Aldape², Melissa Bondy², Maryann Butler³, Peter Inskip⁴, Avima Ruder³, Margaret Wrensch⁵, and Faith Davis¹; ¹University of Illinois at Chicago, Chicago, IL, USA; ²The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA; ³National Institute for Occupational Safety and Health, Cincinnati, OH, USA; ⁴NIH/NCI, Bethesda, MD, USA; ⁵University of California, San Francisco, San Francisco, CA, USA.

BACKGROUND: Oligodendroglial tumors are unique subtypes of brain tumors often combined with other miscellaneous glial tumors during data analysis because of the small numbers in any one study. However, individuals with oligodendroglial tumors have different demographic, clinical, and survival characteristics than individuals with other glial histologies, making it important to evaluate risk factors separately. MATERIALS AND METHODS: We collected data on oligodendroglial tumor cases (oligodendrogliomas, anaplastic oligodendrogliomas, and oligoastrocytomas) from five case-control studies of adult gliomas. Two controls per case were frequency-matched on race, gender, age ± 5 years, and study site. We reviewed the questionnaires from each study, and we determined the variables that were common between studies or variables that could be used to create new common variables. We calculated the univariate statistics, and we estimated the odds ratios (ORadj) and 95% confidence intervals (CI) adjusted for age group, gender, race, study site, and year of interview using logistic regression analysis. RESULTS: Overall, 556 cases, including 298 oligodendrogliomas, 130 anaplastic oligodendrogliomas, and 128 oligoastrocytomas (mixed gliomas), and 1,128 controls were compiled. Fifty-three percent of the study participants were male, 90% were Caucasian, and the average age at diagnosis/interview was 44 years. The gender and race frequencies and mean age at diagnosis/interview were similar between cases and controls. In a preliminary data analysis, anaplastic oligodendroglioma cases were more likely than controls to have reported a family history of brain tumors (ORadj = 3.1 [95% CI, 1.4–6.9]). Cases with anaplastic oligodendrogliomas or oligoastrocytomas were less likely than controls to report a history of asthma or allergies combined (ORadj = 0.4 [95% CI, 0.2–0.6] and 0.3 [95% CI, 0.2–0.7], respectively). An inverse relationship was also found between a history of chicken pox and oligodendroglioma (ORadj = 0.7 [95% CI, 0.4–1.0]) or anaplastic oligodendroglioma (ORadj = 0.5 [95% CI, 0.3–0.9]). Oligodendroglioma cases were less likely than controls to have used bottled water or "other" water source compared to a public water source (ORadj = 0.4 [95% CI, 0.2–1.0] and 0.3 [95% CI, 0.1–0.7], respectively). Epilepsy and/or seizures were associated with an increased risk in all three histology groups, but these may be symptoms of a preclinical disease rather than a risk factor. Results were similar when the data were restricted to cases that underwent a pathology review. CONCLUSIONS: Pooling data from several sources has provided an opportunity to investigate etiologic factors in a rare brain tumor subtype. While several factors associated with oligodendroglial tumors are similar to those identified for the broad glioma grouping, some differences in the relationships by histology subtype were apparent. Additional analyses will be presented.

EP-03. AN EPIDEMIOLOGICAL STUDY OF BRAIN CANCER INCIDENCE AMONG A COHORT OF JET ENGINE MANUFACTURING WORKERS: METHODOLOGICAL ISSUES

Jeanine Buchanich¹, Zb Bornemann², Gary Marsh¹, Ada Youk², Frank Lieberman³, Steven Lacey⁴, Kathleen Kennedy⁵, Roger Hancock⁶, and Nurtan Esmen⁴; ¹University of Pittsburgh, Pittsburgh, PA, USA; ²Department of Biostatistics, University of Pittsburgh, Pittsburgh, PA, USA; ³University of Pittsburgh, Pittsburgh, PA, USA; ⁴University of Illinois at Chicago, Chicago, IL, USA; ⁵Environmental and Occupational Health Sciences, University of Illinois at Chicago, Chicago, IL, USA; ⁶University of Illinois at Chicago, Chicago, IL, USA.

INTRODUCTION: In 2002, the University of Pittsburgh and the University of Illinois at Chicago undertook a 7-year exploratory historical cohort and cancer incidence study to investigate a suspected excess of malignant brain cancers at a jet engine manufacturing plant in North Haven, CT. Our cohort included more than 223,000 former and current employees with work experience since 1952 in one or more than eight manufacturing facilities in CT. We evaluated the incidence of benign and malignant central nervous system (CNS) neoplasms, with emphasis on the brain, from 1976 to 2004 and compared the incidence rates to national and regional incidence rates. Our investigation was complemented by a companion exposure assessment project at the University of IL at Chicago, which will characterize the historical work practices and exposures that occurred in each study plant. Ultimately, we will use the work history and exposure data in the cancer incidence study to examine the relationship between CNS neoplasm incidence and the past working environment of the plants. METHODS: Our cancer incidence tracing protocol entailed matching our cohort with cancer registries from 24 states to find incident CNS cases among our cohort members. This presentation will highlight some of the methodological issues involved with undertaking a cancer incidence study of this scope and magnitude. These issues include differences in the application process, institutional review board procedures, requirements for cohort matching, regulations involving the release of data, data availability, and cost. Specific examples from our study will be given. RESULTS and CONCLUSIONS: The authors will present recommendations for improving cancer incidence tracing in the United States to ultimately allow epidemiologists and clinicians greater, and more simplified, access to valuable cancer incidence data. In a later phase of this study, the results of the cancer incidence tracing for the jet engine manufacturing cohort will be presented.

EP-04. GENDER, BIRTH SEASONALITY, AND BIRTH DEFECTS AS RISK FACTORS FOR PEDIATRIC BRAIN TUMORS: A CALIFORNIA CANCER REGISTRY STUDY

Sonia Partap¹, Julie Von Behren², Jane Maclean³, Paul Fisher¹, and Peggy Reynolds²; ¹Stanford University, Stanford, CA, USA; ²Northern California Cancer Center, Berkeley, CA, USA; ³Stanford University, CA, USA.

BACKGROUND: Birth characteristics have been studied as possible risk factors for pediatric brain tumors. Seasonality has also been examined as a proxy for infectious exposure resulting in pediatric brain tumors. In addition, birth defects are thought to be associated with an increased risk for pediatric cancer because of their relationship to genetic syndromes. Studies done in Europe and North America have found that children with birth defects have a 2-fold increase in childhood malignancies, especially in blood and central nervous system (CNS) tumors. Other epidemiologic studies have addressed gender as a risk factor for pediatric brain tumors. However, the majority of these studies have had limited power and did not distinguish between the tumor subtypes. METHODS: We obtained data on 4,070 cases of CNS cancers from the population-based California Cancer Registry. Cases were children aged 0–14 years who were diagnosed between 1988 and 2004 and matched via probabilistic record linkage to a California birth certificate. Four controls were selected for each case from state birth files and matched based on gender and birth year. Cases and controls were screened for noted morphologic defects at birth as recorded on the birth certificates. For the seasonality analyses, we compared the distribution of birth months to the distribution of all births in California in 1998 (n=523, 410 births). RESULTS: Overall, males had more CNS tumors than females (male:female 1.21, P<0.0001).When stratified by tumor subtype, males had a higher incidence of germ cell tumors (145 males:69 females 2.1, P<0.0001) and medulloblastomas (331 males:215 females 1.54, P<0.0001). For astrocytomas, the male to female ratio was not statistically significant at 1.12 (903 males:809 females, P=.18). We found that seasonality (winter, spring, summer, autumn) and month of birth were not significantly different among the cases compared to the population distribution. Preliminary results from the case-control analyses among the youngest cases (ages 0–4 years) showed an increased risk for CNS cancer associated with a nonspecific morphologic birth defect (OR 3.00 [95% CI, 1.07–8.42]) and an even higher association with the presence of a CNS birth defect (OR 5.99 [95% CI, 1.21–29.65]). We are currently expanding this analysis to include older children and to add additional years of case data. CONCLUSION: In this large study of childhood brain tumors, we found an increased risk in males, especially for germ cell tumors and medulloblastomas. We found no clear pattern of seasonality, but we did observe a striking increase in risk associated with birth defects.

EP-05. PATTERNS OF CARE IN ELDERLY PATIENTS WITH OLIGODENDROGLIOMA

Lakshmi Nayak¹, Fabio Iwamoto¹, Anne Reiner², Katherine Panageas¹, Elena Elkin³, and Lauren Abrey⁴; ¹Memorial Sloan-Kettering Cancer Center, New York, NY, USA; ²NY, USA; ³Memorial Sloan-Kettering Cancer Center, NY, USA; ⁴New York, NY, USA.

OBJECTIVE: To evaluate the patterns of care in older patients with oligodendrogliomas from a population-based registry. BACKGROUND: Advanced age has been associated with an inherent poor prognosis and reduced tolerance to treatment in patients with gliomas. Moreover, the elderly glioma population is relatively understudied, and there is no accepted standard treatment for patients with oligodendrogliomas. Consequently, little is known about which treatments are being offered and which factors influence therapeutic decision making in these patients. DESIGN/METHODS: We identified a cohort of patients with oligodendrogliomas who were aged 65 years from the Surveillance, Epidemiology and End Results (SEER) data, which were linked with Medicare claims between 1994 and 2002. SEER, sponsored by the National Cancer Institute, is a consortium of population-based cancer registries covering > 20% of the U.S. population. Medicare is the primary health insurer for 97% of Americans aged 65 years. Among persons aged 65 years in the SEER database, at least 93% have been identified in the Medicare file. RESULTS: A total of 176 patients with oligodendroglioma (52% women) were included; the median overall survival time was 12 months. 77% of the patients were dead, with a median follow-up of 45 months for surviving patients. 57% of the patients underwent resection. Further treatment within 6 months of diagnosis included radiotherapy (RT; 44%), RT and IV chemotherapy (17%), or IV chemotherapy alone (2%); 35% of the patients received neither RT nor IV chemotherapy. The odds ratios of undergoing resection, RT, and IV chemotherapy within 6 months of diagnosis were assessed according to age, comorbidity, sociodemographic characteristics, and year of diagnosis. Unmarried status was the only factor associated with decreased probability of receiving RT (OR=0.33; P=0.02). CONCLUSIONS: Marital status influences the probability of elderly patients with oligodendrogliomas receiving RT. Unmarried patients likely have less social support, and a multidisciplinary approach with social workers and case managers might help facilitate access to treatments such as RT. Compared with similar data for elderly patients with glioblastomas, survival is longer in patients with oligodendrogliomas, suggesting that histologic diagnosis retains prognostic impact in this age group.

EP-06. AN EPIDEMIOLOGICAL STUDY OF BRAIN CANCER MORTALITY AND INCIDENCE AMONG A COHORT OF JET ENGINE MANUFACTURING WORKERS: TOTAL AND CAUSE-SPECIFIC MORTALITY WITH FOCUS ON CNS NEOPLASMS

Gary Marsh¹, Jeanine Buchanich¹, Ada Youk², Michael Cunningham³, Frank Lieberman⁴, Kathleen Kennedy⁵, Steven Lacey⁵, Roger Hancock⁵, and Nurtan Esmen⁵; ¹Biostatistics, University of Pittsburgh, Pittsburgh, PA, USA; ²Biostatistics, University of Pittsburgh, PA, USA; ³University of Pittsburgh, PA, USA; ⁴University of Pittsburgh, Pittsburgh, PA, USA; ⁵University of Illinois at Chicago, Chicago, IL, USA.

INTRODUCTION: In 2002, the University of Pittsburgh undertook a 7-year exploratory historical cohort mortality and cancer incidence study to investigate a suspected cluster of malignant brain cancers at a jet engine manufacturing plant in North Haven, CT. Our investigation included a nested case-control study of brain cancers and is complemented by a companion exposure assessment project at the University of IL at Chicago that will characterize the historical work practices and exposures that occurred in each study plant. Ultimately, we will use the work history and exposure data in our cohort study to examine the relationship between total and cause-specific mortality, with emphasis on benign and maligant central nervous system (CNS) neoplasms and the past working environment of the plants. METHODS: There were 223,894 subjects in our historical cohort study of workers ever employed in one or more of the company's eight CT manufacturing facilities from 1952 to 2001. We determined the vital status through 2004 for 99% of the subjects and the cause of death for 95% of 68,701 identified deaths. We computed the standardized mortality ratios based on U.S. and CT state rates and modeled internal cohort rates to assess relative mortality risks for selected demographic and work history factors, including employment at the facility in North Haven, associated with the index cases and payroll type. Internal comparisons of CNS neoplasms included contributory as well as underlying causes of death. Detailed analyses of CNS neoplasms focused on the diagnostically more accurate 1976–2004 period. RESULTS: Results for total and cause-specific mortality from our first cohort mortality analysis will be presented with focus on CNS neoplasms.

EP-07. MEASURING OCCURENCE AND TIMING OF BRAIN METASTASES USING ADMINISTRATIVE ICD-9 CODES

April Eichler¹ and Elizabeth Lamont²; ¹Neurology, Massachusetts General Hospital, Boston, MA, USA; ²Medicine, Massachusetts General Hospital, Boston, MA, USA.

AIMS: We performed a criterion validation study to determine the accuracy with which International Classification of Diseases, 9th Edition, Clinical Modification (ICD-9-CM) secondary neoplasm diagnosis codes measure 1) the anatomic sites of metastatic cancer involvement and 2) the time to development of metastatic cancer involvement in patients with lung cancer. METHODS: For 100 consecutive patients with newly diagnosed stage IV non–small-cell lung cancer (NSCLC), we merged gold-standard clinical data detailing anatomic sites of metastatic cancer obtained from Massachusetts General Hospital (MGH) charts with MGH inpatient and outpatient billing records. We then compared three ICD-9-CM secondary malignancy codes for brain, bone, and liver to gold-standard data on site of metastatic involvement to determine 1) the sensitivity and specificity of each secondary malignancy code and 2) the timing of their appearance within the administrative data relative to the gold-standard source. RESULTS: Observing patients from diagnosis until death or last follow-up, we found that 48% (48/100) of the patients developed brain metastases, 42% (42/100) developed bone metastases, and 30% (30/100) developed liver metastases at a median follow-up of 43 months within the gold-standard data source. Comparison of these data to MGH administrative billing data from 30 days prior to diagnosis until death or fixed right censoring revealed that more than one occurrence of the ICD-9-CM code for brain metastasis had a sensitivity of 100% (95% CI, 92%–100%) and a specificity of 92% (95% CI, 81%–98%). For bone metastases, the sensitivity was 95% (95% CI, 83%–99%) and the specificity was 97% (95% CI, 73%–93%). For liver metastases, the sensitivity was 77% (95% CI, 58%–90%) and the specificity was 90% (95% CI, 80%–96%). Alternative algorithms requiring 2 or 3 occurrences of ICD-9 secondary neoplasm codes resulted in slight improvement in the area under the receiver operator curve characteristics for brain and bone but not liver metastases. For brain metastases, the median difference in the claims-based date of diagnosis compared with the gold standard was 0 days (interquartile range, 0 to 3.5 days), with 94% falling within 30 days of the gold-standard date of diagnosis. CONCLUSIONS: ICD-9–based algorithms using hospital billing codes may reliably capture the anatomic site and time to development of brain and bone metastases in patients with advanced lung cancer, but these algorithms do not appear to be as reliable for liver metastases in these patients. For elderly Medicare beneficiaries with advanced lung cancer, administrative Medicare claims may be a valid source of population-based information about the incidence of brain metastases.

EP-08. PROGNOSTIC FACTORS IN ADULT AND PEDIATRIC EPENDYMOMA

E. Amirian¹, Michael Scheurer², Terri Armstrong¹, Melissa Bondy¹, and Mark Gilbert³; ¹The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA; ²Baylor College of Medicine, Dan L. Duncan Cancer Center, Houston, TX, USA; ³Houston, TX, USA.

BACKGROUND: Ependymomas are rare central nervous system (CNS) tumors that arise from the ependymal cells that line the ventricular surfaces of the brain, the central canal of the spinal cord, or the filum terminale. They are classified as myxopapillary ependymoma and subependymoma (WHO grade I), ependymoma (WHO grade II), and anaplastic ependymoma (WHO grade III). These tumors account for about 5% of adult intracranial gliomas and up to 10% of childhood CNS tumors. Because of the rarity of these tumors, previous research has been unsuccessful in establishing the importance of usual prognostic factors, such as tumor site and grade, in ependymoma patient survival. Given the controversy surrounding suspected prognostic factors, we sought to identify key demographic and tumor characteristics affiliated with patient survival, using data from the Surveillance, Epidemiology, and End Results (SEER) program of the National Cancer Institute. METHODS: We isolated the ependymoma cases (ICD-O-3 codes 9391–9394) from the full SEER incidence data from 1973 to 2004 (most current data available). Due to the number of cases, we restricted the case definitions to low-grade ependymomas, ICD-O-3 code 9391, and anaplastic ependymomas, ICD-O-3 code 9392 (n=1971 and n=238, respectively). We used Kaplan-Meier curves to visualize survival differences by various factors and multivariate Cox proportional hazards regression models to calculate effect estimates. We also calculated the median survival times and 5-year survival probabilities. We examined both overall survival and brain tumor-specific mortality as outcomes in separate analyses. RESULTS: Individuals with low-grade ependymomas had a consistently better probability of survival over time compared to those with anaplastic ependymomas after adjusting for race and age (P<0.0001). In fact, having an anaplastic tumor conferred an 88% increased risk of mortality over time compared to a low-grade tumor. We also found that having a tumor in the brain conferred 3.70 times the mortality risk of having a tumor in the spinal cord (95% CI, 3.02–4.54) after adjusting for race, age, and tumor grade. However, differences were not apparent between infratentorial and supratentorial tumors. With regard to treatment, the extent of surgery and radiation therapy were both associated with overall survival after controlling for other factors. Individuals who received radiation therapy may have had a slightly higher mortality risk than those who did not (HR 1.15 [95% CI, 0.95–1.39]), whereas individuals who received a gross total resection had a much lower mortality risk than those who received less than a gross total resection (HR 0.54 [95% CI, 0.36–0.83]). Of the demographic variables, age seemed to have the most important effect on survival, with the youngest (<5 years old) and oldest (³60 years old) groups having worse survival. Cause-specific and overall mortality were relatively consistent regarding the effects of tumor grade, tumor site, extent of surgery, and radiation therapy. CONCLUSIONS: Our findings suggest that tumor grade, tumor site, and type of therapy, as well as other clinical and demographic factors, may play an important role in the prognosis of patients with ependymomas. However, more research using larger populations of incident ependymoma cases is warranted, especially for the prognostic value of tumor grade and site.

EP-09. NATIONAL TRENDS IN CENTRAL NERVOUS SYSTEM (CNS) TUMORS IN CHILDREN UNDER 3 YEARS OF AGE AT TIME OF DIAGNOSIS

Daniel Keene¹, Donna Johnston², and the Canadian Pediatric Brain Tumor Consortium³; ¹Department of Pediatrics, Children's Hospital of Eastern Ontario, Ottawa, Ontario, Canada; ²Ottawa, Ontario, Canada; ³Ontario, Canada.

BACKGROUND: Brain tumors in children under 3 years of age are rare. Most reports have been single-institution series or case studies, making it difficult to exam for trends/changes over time. OBJECTIVE: To assess changes in incidence of central nervous system tumors between 1990 and 2005 in children diagnosed in the first 3 years of life. METHODOLOGY: We conducted a national retrospective survey of children under 3 years of age with a diagnosis of CNS tumor. We obtained descriptive data for the demographic variables age, sex, anatomic location of tumor, histological diagnosis, degree of resection, type of therapy, and survival. Based on the Statistics Canada Census, we calculated the yearly age-adjusted incidence rates per 100,000 children. RESULTS: Data were obtained from all provinces of Canada, except Newfoundland,with 579 cases ascertained, resulting in a mean annual age-adjusted incidence of 3.53±0.71 (95% CI, 3.15–3.90)/100,000 children, a supratentorial incidence of 1.64±0.37 (95% CI, 1.45–1.84), infratentorial incidence of 1.70±0.59 (95% CI, 1.39–2.01), and a spinal cord incidence of 0.15±0.11 (95% CI, 0.91–0.21). Incidence rates by major cell line were astrocytic 1.07±0.23 (95% CI, 2.93–3.89), ependymal 0.46±0.28 (95% CI, 0.31–0.61), choroid plexus 0.22±0.12 (95% CI, 0.16–0.28), embryonal 0.95±0.46 (95% CI, 0.70–1.9), neuronal 0.2±0.12 (95% CI, 0.70–1.9), and craniopharyngioma 0.1±0.6 (95% CI, 0.45–0.11). Over the period of the survey, a trend toward an overall increase in incidence for tumors, especially for children between 12 and 24 months of age, infratentorial tumors, ependymal tumors, and embryonal tumors was seen. There was also a significant improvement in survival rates. However, the number of patients undergoing a greater than 90% resection of the tumor did not change significantly (1990–995 = 58.5%, 1996–2000 = 50%; 2001–2005 = 60.7%). CONCLUSIONS: The incidence of brain tumors in this age group remains low.

EP-10. NSAIDS AND EFFECT-MODIFYING INTERACTIONS IN GLIOMA RISK

Bhuma Krishnamachari¹, Bridget Mccarthy², Faith Davis¹, Leslie Stayner³, Donald Hedeker¹, and Kristin Rankin³; ¹University of Illinois at Chicago, Chicago, IL, USA; ²Chicago, IL, USA; ³IL, USA.

INTRODUCTION: Little is known about the impact of the immune system and the many medications on glioma etiology. Research suggests a protective effect on glioma risk with the presence of IgE-related medical conditions, such as allergies, which promote an inflammatory immune profile in humans. Certain commonly used medications may also have a protective effect against gliomas through modulation of the immune system. We hypothesized that antidepressants and nonsteroidal anti-inflammatories (NSAIDs) may be independently protective for glioma risk and that they may interact with each other to further exert a protective effect. We also hypothesized that one or more of these medications may interact with IgE-related conditions to lower glioma risk, thus supporting the idea that immune responses are protective for glioma risk. METHODS: Data were collected as part of an ongoing brain tumor study conducted at Duke University Medical Center and Evanston Northwestern Healthcare. Eligible cases (n=1445) were adults with incident gliomas seen at these referral centers. Multiple controls, up to five friends and/or siblings, were recruited for each case. Data were analyzed separately for sibling and friend control matches: 1) the first analysis consisted of 120 glioma cases and 158 matched sibling controls, and 2) the second analysis included 196 glioma cases and 456 matched friend controls. We investigated the effects of antidepressants, NSAIDS, and IgE conditions (asthma, allergies, or eczema), as well as interactions between these variables. We used conditional logistic regression analysis to generate odds ratios adjusted for age and gender (ORadj) and 95% confidence intervals (CI). Significance was assessed at the alpha=0.05 level. RESULTS: In the matched sibling control analyses, use of NSAIDs alone was protective but not statistically significant (ORadj = 0.61 [95% CI, 0.35–1.06]). Use of antidepressants alone was similarly protective but not statistically significant (ORadj = 0.67 [95% CI, 0.33–1.37). Prior to adjustment, use of NSAIDs and antidepressants combined resulted in a statistically significant reduction in risk compared to use of neither (OR = 0.35 [95% CI, 0.14–0.88]; P=0.03). Adjustment for age and gender resulted in a stronger protective effect than use of either medication alone, although this was no longer statistically significant (ORadj = 0.42 [95% CI, 0.16–1.08]; P=0.07). The protective effect of using NSAIDs alone was similar in the matched friend control analyses; however, the protective effect of antidepressants use alone or use of both NSAIDs and antidepressants was not seen. Although having an IgE-related condition alone was not found to be protective (OR = 0.90 [95% CI, 0.61–1.33]) in the matched friend control analyses, use of NSAIDs in those with IgE-related conditions was protective compared to having neither exposure (OR = 0.55 [95% CI, 0.34–0.88]; P=0.01). CONCLUSIONS: An interaction between NSAIDS and antidepressants was observed in the matched sibling control analyses, but not in the matched friend control analyses. This could be due to fewer differences between individuals in terms of genetic make-up in sibling versus friend controls. Since it is believed that antidepressants are sensitive to genetic background, and because there may be biological evidence of synergy between NSAIDs and antidepressants, these results should be further explored. Additionally, we found a significant interaction between NSAID use and presence of IgE-related conditions in the matched friend control analyses, suggesting that NSAIDs may influence the immune system in a synergistic mechanism with IgE-related conditions that may be protective against glioma development.

EP-11. EXPOSURE RECONSTRUCTION TO INFORM AN EPIDEMIOLOGIC STUDY OF BRAIN CANCER MORTALITY AMONG A COHORT OF JET ENGINE MANUFACTURING WORKERS: AN UPDATE ON FINDINGS TO DATE

Steven Lacey¹, Kathleen Kennedy¹, Roger Hancock¹, Nurtan Esmen¹, Frank Lieberman², Jeanine Buchanich³, Ada Youk³, Michael Cunningham³, and Gary Marsh⁴; ¹Environmental and Occupational Health Sciences, University of Illinois at Chicago, Chicago, IL, USA; ²University of Pittsburgh, Pittsburgh, PA, USA; ³Department of Biostatistics, University of Pittsburgh, Pittsburgh, PA, USA; ⁴University of Pittsburgh, Pittsburgh, PA, USA.

An exposure reconstruction to inform an occupational epidemiology study of brain cancer mortality among a cohort of jet engine manufacturing workers is ongoing, and we present an update on the findings here. The overall objective of the exposure analysis was to examine possible occupational risks for increased mortality. The specific objective of this presentation is to describe the data utilized for the estimation of occupational exposures and the general methodology to inform the epidemiology study. A short list of agents of concern was developed for in-depth examination based on carcinogenic plausibility. This was complimented by a method to examine a worker's interaction with a given jet engine part or manufacturing process. A job dictionary is being constructed that contains exposure to part, process, and specific agents by job class over time and will be utilized by the epidemiology team to establish a unique exposure profile for each worker and then to link those profiles to total and cause-specific mortality, with emphasis on malignant and benign CNS neoplasms. The methodology of the overall exposure reconstruction process, exposure estimations developed to date, difficulties encountered, and the means by which the exposure reconstruction findings integrate with the mortality data will be presented.

	EXPERIMENTAL THERAPEUTICS

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

 
ET-02. AFLIBERCEPT (VEGF TRAP) INHIBITS VASCULAR PROLIFERATION AND TUMOR GROWTH AND INCREASES SURVIVAL IN A RAT MODEL OF GLIOBLASTOMA

Katy Linskey¹, Kim Lopez², Aaron Tannenbaum³, Marcela Assanah², Gavin Thurston⁴, Jeffrey Bruce², Steven Rosenfeld², and Peter Canoll²; ¹Columbia University, New York, NY, USA; ²New York, NY, USA; ³NY, USA; ⁴Regeneron, NY, USA.

PURPOSE: Vascular endothelial growth factor (VEGF) is associated with increased vascular proliferation, tumor growth, and a poor prognosis. Thus, VEGF is an attractive candidate target for antiangiogenic therapies for glioblastoma, which expresses high levels of VEGF. In this study, we used aflibercept (VEGF Trap), a competitive inhibitor of VEGF receptors, to treat glioblastoma in a rat model. METHODS AND MATERIALS: A retrovirus expressing PDGF-B-IRES-GFP was injected into the subcortical white matter of adult rats. Treatment included four injections of aflibercept (25 mg/kg/dose SQ) given on days 7, 10, 13, and 16 after the retrovirus injection. Controls were given injections of either Fc or saline. For survival studies, rats were sacrificed at the first signs of tumor morbidity. Survival data were analyzed with the Kaplan-Meier method. In separate short-term experiments, animals were sacrificed 1 day after the last treatment, and tumors were analyzed with immunofluorescence to determine tumor size, vascular density, vascular Ki67 index, and tumor cell Ki67 index (means were determined through an analysis of variance in conjunction with a post-hoc Tukey's test). RESULTS: The median survival time was significantly longer for treated animals than for both sets of controls (aflibercept: 27, PBS: 16, Fc: 16 dpi, P = 0.027). All tumors in the control groups had histologic features of glioblastoma, including vascular proliferation and necrosis. Treated tumors were consistently smaller than control tumors (mean surface area = aflibercept: 8.612, PBS: 25.08, Fc: 20.50 mm2; P = 0.0011) with significantly less vascular proliferation (mean SMA + surface area = aflibercept: 0.4396, PBS: 2.61, Fc: 2.099 mm2; P = 0.004) and necrosis. There was also a significant decrease in the proliferation of tumor cells (mean Ki67 labeling index = aflibercept: 6.297, PBS: 20.85, Fc: 17.24%; P < 0.0001). CONCLUSIONS: Aflibercept provided a significant survival advantage, inhibiting both vascular proliferation and tumor growth. These results suggest that an antiangiogenic therapy, specifically one that neutralizes VEGF, may be effective in human glioblastoma.

ET-03. THE EFFICACY OF TEMOZOLOMIDE IN VITRO AND IN PATIENTS WITH NEWLY DIAGNOSED GBM IS ENHANCED BY ADJUVANT EXPOSURE TO ALTERNATING ELECTRIC FIELDS (TTFIELDS)

Eilon Kirson¹, Vladimir Dbaly², Frantisek Tovarys², Josef Vymazal³, Rosa Schneiderman⁴, Yoram Wasserman⁵, and Yoram Palti¹; ¹NovoCure Ltd., Haifa, Israel; ²Neurosurgery, Na Homolce Hospital, Prague, Czech Republic; ³Radiology, Na Homolce Hospital, Prague, Czech Republic; ⁴Biology, NovoCure Ltd., Haifa, Israel; ⁵Engineering, NovoCure Ltd., Haifa, Israel.

BACKGROUND: Alternating electric fields (TTFields) have been shown to exert anti-tumor activity, destroying cycling cells during mitosis. Preliminary clinical experience suggests efficacy against recurrent glioma; this technique is currently undergoing phase III evaluation in patients with recurrent glioblastoma. In vitro synergy with various chemotherapeutic agents has been shown in breast and lung cancer cell lines (Kirson et al, AACR 2007). METHODS: In the present study, the effects of TTFields alone and in combination with DTIC were tested in human glioma (GBM) cells in vitro (U-87). Subsequently, the effects of TTFields in combination with maintenance temozolomide (TMZ) were tested in an open-label, single-arm, prospective, pilot clinical trial. Ten patients (median age 54 years, KPS 70, gross total tumor resection in 4 patients [40%]) with newly diagnosed GBM were treated with standard TMZ/RT. During the maintenance TMZ administration, TTFields were applied continually (18 hours a day, on average) for up to 18 months. All patients were followed on a monthly basis (including monthly contrast magnetic resonance imaging of the brain). The primary endpoints were feasibility and toxicity, time to disease progression, and overall survival. Results were compared to concurrent and historical controls. RESULTS: In vitro, full additivity of the inhibitory effects of DTIC and TTFields was observed when both treatments were applied concomitantly to GBM cells in culture. This finding was true for all DTIC concentrations tested. In the pilot clinical trial, no device–related, systemic, adverse events were noted throughout the treatment with TTFields (cumulative treatment time more than 170 months; median administration time 12 months). Over time, a mild to moderate skin irritation appeared beneath the electrode gel in all patients. Median time to disease progression was 155 weeks in patients treated with combined TMZ and TTFields, compared to 31 weeks in concurrent control patients treated with TMZ alone (hazard ratio 3.7; 95% confidence interval 2.1–6.7). Half the patients were still progression-free at the end of the trial. Median overall survival was > 40 months in patients treated with combined TMZ and TTFields, compared to 14.7 months reported for historical controls (Stupp et al, 2004). Eight of the 10 patients were still alive at the end of this study. CONCLUSIONS: These promising findings suggest that TTFields can be applied over many months without significant toxicity. The combination with standard TMZ chemotherapy may greatly enhance the anti-tumor effect of TMZ translating into prolonged time to progression and survival.

ET-04. LIGAND-DEPENDENT ACTIVATION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) INDUCES EARLY DRUG RESISTANCE IN BRAIN TUMOR CELLS VIA ACTIVATION OF ITS DOWNSTREAM TARGET GLUTATHIONE S-TRANSFERASE P1

Tatsunori Okamura¹, Simendra Singh¹, Darell Bigner², and Francis Ali-Osman¹; ¹Surgery, Duke University, Durham, NC, USA; ²Pathology, Duke University, Durham, NC, USA.

The EGFR gene is frequently amplified, mutated, and/or overexpressed in malignant gliomas and other human tumors. The resulting aberrant EGFR signaling cascade plays a key role in the development of an aggressive malignant phenotype with a dismal prognosis and high resistance to therapy. To date, however, the essential cellular and molecular mechanisms underlying the association between activated EGFR signaling and drug resistance remain not fully understood. Similar to EGFR, the GSTP1 gene is aberrantly overexpressed in gliomas and other human tumors, and the overexpression is associated with aggressive tumor behavior and poor therapeutic outcome. We recently made the important finding that GSTP1 is a heretofore unrecognized downstream target of EGFR and that, in vitro and in vivo, GSTP1 undergoes EGFR-dependent phosphorylation on tyrosines 3, 7, and 198, resulting in an enhancement of its phase II enzymatic function. In an effort to better understand the impact of EGFR-GSTP1 cross-talk on the drug-resistance phenotype in brain tumors, we examined the effect of the activation of EGFR by its ligand, EGF, on the drug sensitivity of isogenic EGFR and GSTP1+ve and GSTP1–ve malignant brain tumors and a spectrum of malignant glioma cell lines that differ in both EGFR and GSTP1 expression. Our findings demonstrated that ligand activation of EGFR by EGF rapidly induced GSTP1 phosphorylation and a rapid onset of drug resistance in the tumor cells. Using the paired human medulloblastoma cell lines UW228 (which lacks GSTP1 expression due to epigenetic silencing) and UW228-1C (which was engineered to stably express the GSTP1^*C allelic variant), we showed that the induced expression of GSTP1 was associated with increased drug resistance. We also showed that a 5-minute pretreatment with 100 ng/mL of EGF increased the level of drug resistance significantly in the GSTP1+ve cells but not in the parent GSTP1-ve UW228 cells. An increase in drug resistance after short-term exposure to EGF was observed in cells of 4 other malignant glioma cell lines, including U87MG (GBM), MGR1 (AA), and MGR3 (GBM). In the EGFR-activated cells, treatment with the clinically active EGFR inhibitor gefitinib reversed the induced drug resistance. The results with 4 anticancer agents (cisplatin, mitomycin C, doxorubicin and BCNU) further indicated that the mediation of drug resistance by EGFR-GSTP1 interaction differed with the type of drug. Together, these data allowed the conclusion that the induction of drug resistance by the EGFR-GSTP1 crosstalk in brain tumor cells is a rapid event mediated by the signaling cascades of these two proteins and can be reversed by the appropriate use of inhibitors of these signaling pathways. Ongoing studies in our laboratory are directed at achieving further molecular characterization of EGFR-GSTP1-mediated drug resistance, which we believe will help us determine the relevance of this novel resistance mechanism as a determinant of clinical outcome of brain tumor therapy and whether a better understanding of the relationship between EGFR signaling and drug resistance may lead to more optimal use of EGFR and/or GSTP1 inhibitors in targeted and individualized therapy of brain tumors. Supported by grants RO1 CA127872, RO1 CA112519 to FA-O.

ET-05. TUMOR SEQUESTRATION OF LAPATINIB (NABTC 04-01)

John Kuhn¹, Ian Robins², Minesh Mehta³, Howard Fine⁴, Timothy Cloughesy⁵, Patrick Wen⁶, Susan Chang⁷, Lisa Deangelis⁸, Frank Lieberman⁹, David Reardon¹⁰, Lauren Abrey¹¹, Andrew Lassman¹¹, Kenneth Aldape¹², W.K. Alfred Yung¹³, Janet Dancey¹⁴, Kathleen Lamborn⁷, and Michael Prados⁷; ¹University of Texas Health Science Center at San Antonio, San Antonio, TX, USA; ²University of Wisconsin-Madison, Madison, WI, USA; ³University of Wisconsin-Madison, WI, USA; ⁴National Institutes of Health, Bethesda, MD, USA; ⁵University of California, Los Angeles, Los Angeles, CA, USA; ⁶Boston, MA, USA; ⁷University of California, San Francisco, San Francisco, CA, USA; ⁸Memorial Sloan-Kettering Cancer Center, New York, NY, USA; ⁹University of Pittsburgh, Pittsburgh, PA; ¹⁰Durham, NC, USA; ¹¹New York, NY, USA; ¹²The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA; ¹³The University of Texas M. D. Anderson Cancer Center, TX, USA; ¹⁴National Cancer Institute, Bethesda, MD, USA.

BACKGROUND: Lapatinib is a dual, reversible inhibitor of both ErbB1 and ErbB2 tyrosine kinase activity (IC50 10 ng/mL). Also, lapatinib is both a substrate and an inhibitor (IC50 0.025–5 µM) of the blood-brain barrier efflux transporters Pgp and BCRP (Drug Metab Dispos 2008;36:695). We present the tissue distribution results of patients enrolled in NABTC 04-01, a phase II/biomarker study of lapatinib in patients with recurrent glioblastoma multiforme. METHODS: Adult patients who had a KPS equal to or greater than 60, who were not on enzyme-inducing anti-epileptic agents, and who had normal hematologic, metabolic, and cardiac function were eligible for this study. In addition, patients must have been candidates for surgical re-resection at the time of enrollment. Patients were administered 750 mg of lapatinib orally BID for 7 days (time to steady-state) prior to surgery. Blood and tissue samples were obtained at the time of resection. Of note is the fact that the resections usually involved the enhancing component of the tumor, where the blood-brain barrier was most likely disrupted. Lapatinib concentrations were analyzed by LC/MS/MS. Following recovery from surgery, patients received lapatinib in the phase II component of the study. RESULTS: Plasma concentrations at the time of resection averaged 1163 (± 575) ng/mL (n = 15) and tumor tissue 558 (± 566) ng/mL (equivalent to 11.2 µg/g). The tumor-to-plasma ratio ranged from 0.65–39.5 (mean 13.0 ± 11). Tumor concentrations of lapatinib were generally higher than the IC90 values of ErbB2 in xenograft models (N87-110 ng/mL; BT474-209 ng/mL) and ErbB1 IC50 values. CONCLUSION: This is the first study demonstrating tissue distribution of lapatinib in glioma tissue. Tissue sequestration of lapatinib may result from auto-inhibition of the efflux transporters and from the disrupted blood-brain-barrier.

ET-06. INHIBITION OF SP1 EXPRESSION IN GROWING TUMORS BY MITHRAMYCIN A DIRECTLY CORRELATES WITH ITS POTENT ANTIANGIOGENIC EFFECTS IN HUMAN NEUROENDOCRINE TUMOR XENOGRAFT MODELS

Keping Xie¹ and James Yao¹; ¹ The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.

Our previous studies have shown that human carcinoid cells overexpress the pro-angiogenic factor vascular endothelial growth factor A (VEGF) and that transcription factor Sp1 expression correlates with VEGF expression. However, the effect of antiangiogenic therapy on the Sp1/VEGF pathway remains unknown. Treatment with bevacizumab (BVZ), a neutralizing antibody against VEGF, suppressed human carcinoid growth in nude mice in a dose-dependent manner. Gene expression analyses revealed that ineffective doses substantially upregulated the expression of Sp1 and its downstream target genes, including VEGF, EGFR, and PDGFA, in tumor tissues. Treatment with mithramycin A, a Sp1 inhibitor, suppressed the expression of Sp1 and its downstream target genes in both cell culture and tumors growing in nude mice. Combined treatment with BVZ and mithramycin A produced synergistic tumor suppression, which was consistent with suppression of the expression of Sp1 and its downstream target genes. Thus, treatment with BVZ may block VEGF function but activate the pathway of its expression via positive feedback. Given the notion that Sp1 is an important regulator of the expression of multiple angiogenic factors, BVZ-initiated upregulation of Sp1 and subsequent overexpression of Sp1's downstream target genes may profoundly affect the effectiveness of antiangiogenic strategies for human carcinoid tumors.

ET-07. INTRAVENTRICULAR BEVACIZUMAB IS DEVOID OF CLINICAL OR PATHOLOGICAL NEUROTOXICITY IN NEW ZEALAND WHITE RABBITS

Priscilla Brastianos¹, Violette Renard¹, Betty Tyler¹, Daniel Sciubba¹, Susan Eller², Peter Burger³, and Stuart Grossman¹; ¹Oncology, Johns Hopkins University, Baltimore, MD, USA; ²Surgery, Johns Hopkins University, Baltimore, MD, USA; ³Johns Hopkins University, Baltimore, MD, USA.

OBJECTIVE: The leptomeninges and cerebrospinal fluid (CSF) are common sites of metastasis for leukemias and solid tumors. Unfortunately, treatment options for neoplastic meningitis are limited and the prognosis is poor. As tumor cells in the CSF must recruit a vascular supply to survive and multiply, the inhibition of angiogenesis could be an important strategy to prevent the development of clinically significant leptomeningeal metastases. Bevacizumab (BCM) is a widely used, well-tolerated anti-VEGF monoclonal antibody that is effective in a variety of malignancies and that inhibits angiogenesis when placed intraocularly in patients with disorders associated with neovascularization. BCM has not previously been evaluated intrathecally or used in neoplastic meningitis. This study was conducted to investigate clinical and pathologic neurotoxicity of repeated injections of intraventricular bevacizumab in rabbits and to evaluate CSF BCM levels. METHODS: New Zealand white rabbits were randomly assigned to control (n = 4) and treatment (n = 4) groups. All rabbits underwent placement of a subcutaneous reservoir and ventricular catheter (SRVC), with fluoroscopy used to confirm placement of the intraventricular reservoir. Seven days later, the control animals received 0.5 mL of normal saline while the experimental group received 1.5 mg of bevacizumab in 0.5 mL of normal saline injected into their SRVC. The injections were repeated every week in the control and BCM animals for a total of 4 weeks. All animals were examined daily for any sign of neurologic deficits, such as anorexia, seizures, lethargy, cranial nerve palsies, or motor weakness. On day 36, all animals were euthanized and autopsies were performed for gross and histologic examination of the brain. Coronal sections were taken through the catheter entry point into the brain and analyzed by a blinded neuropathologist. RESULTS: Of the 4 rabbits in the control arm, 2 had slightly decreased fecal output 1 day postoperatively, which resolved with metoclopramide and subcutaneous fluid; 1 removed the surgical staples and the intraventricular reservoir on day 2; and 1 had post-operative complications leading to paraparesis and was euthanized (an autopsy report revealed surgical complications). Two control animals had a 1-day history of loose stools, treated with subcutaneous fluids. No neurologic deficits were observed in any of the control animals. Pathologically, 3 control rabbits were evaluated. Chronic inflammation in the choroid plexus of 1 control animal and pathologic findings incident to catheter placement were noted. No other histologic findings were evident. Of the 4 rabbits in the BCM arm, 3 had transient loose stools. No neurologic findings were noted on daily exams. The brains of 4 rabbits were studied at day 36 and had no histologic changes other than those incident to catheter placement. Data on CSF levels of BCM at day 36 will be available for the presentation. CONCLUSION: Repeated injections of intraventricular BCM were associated with no clinical or histologic evidence of neurotoxicity. As intrathecal BCM appears safe, additional studies are under way to determine whether this agent can prevent or delay the clinical onset of leptomeningeal metastases.

ET-08. PROTEASOME INHIBITION IN HIGH-GRADE GLIOMA TREATMENT: A STRATEGY FOR SIMULTANEOUS TUMORICIDAL ACTIVITY, TEMOZOLOMIDE CHEMOSENSITIZATION, AND RADIATION SENSITIZATION

Clark Chen¹, Kennedy Richard², Santosh Kesari³, Andrew Kung³, and Alan D'Andrea³; ¹Harvard University, Boston, MA, USA; ²Dana-Farber Cancer Institute, Boston, MA, USA; ³Boston, MA, USA.

Despite therapeutic advances, malignant gliomas remain among the deadliest of cancers. The current standard of care for malignant gliomas consists of surgical resection followed by therapy with temozolomide (TMZ) and radiation. We reasoned that the identification of agents with simultaneous tumoricidal activity, TMZ-sensitizing activity, and radiosensitizing activity should offer a favorable strategy for therapeutic gain. We undertook an siRNA screen-based strategy using a library consisting of 712 siRNAs directed against 356 DNA repair genes. We identified siRNAs that sensitized the U87 glioma cell line to temozolomide. Simultaneously, another screen was carried out to identify siRNAs with radiation-sensitizing activity. A third screen was conducted to identify siRNAs with tumoricidal activity without TMZ/radiation therapy. We searched for siRNAs that scored strongly positive in all three screens and identified PSMA1, a critical component of the proteasome complex. To exclude "off-target" effects, we recapitulated the findings with pharmacologic inhibitors of proteasome function, including bortezomib (Velcade), MG132, lactacystin, and ALLN. This sensitization was not affected by the overexpression of a dominant negative mutant of p53, epidermal growth factor receptor variant III (EGFRvIII), or O6-methyl-guanine methyl transferase (MGMT)–3 well-studied determinants of glioma resistance to TMZ/radiation therapy. To exclude cell-line specific effects, the TMZ/radiation-sensitizing effect of bortezomib was recapitulated using the T98g, A172, U343, and U373 glioma lines. The sensitizing effect was further observed in 3 OLIG 2+, CD133+ cancer stem cell lines (BT74, BT69, and BT78) derived from patient glioblastoma specimens. The in vivo pertinence of these observations was confirmed using an intracranial U87 xenograft model. In summary, we offer preclinical data supporting proteasome inhibitors as a strategy for malignant glioma therapy. Clinical translation of these findings is facilitated by the number of available proteasome inhibitors and the Food and Drug Administration approval of bortezomib as a treatment for multiple myeloma.

ET-09. A NEW DRUG, ANG1005, A CONJUGATE OF PACLITAXEL AND ANGIOPEP PEPTIDE VECTOR, IS ABLE TO CROSS THE BLOOD-BRAIN BARRIER FOR THE TREATMENT OF BRAIN CANCERS

Reinhard Gabathuler¹, Michel Demeule², Anthony Regina¹, Christian Che¹, Paul Lockman³, Fancy Thomas³, Julie Gaasch³, Helen Thorsheim³, Abedelnasser Abulrob⁴, Quentin Smith³, Danica Stanimirovic⁴, Richard Beliveau⁵, and Jean-Paul Castaigne¹; ¹Angiochem, Inc., Montreal, Quebec, Canada; ²Research, Angiochem, Inc., Montreal, Quebec, Canada; ³Texas Tech University Health Sciences Center, Amarillo, TX, USA; ⁴Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada; ⁵Laboratory of Molecular Medicine, University of Quebec at Montreal, Montreal, Quebec, Canada.

The blood-brain barrier (BBB) is mainly formed by brain capillary endothelial cells, which are closely sealed by tight junctions. This important characteristic provides a natural defense against toxic or infective agents circulating in the blood. Furthermore, brain endothelial cells possess few alternative transport pathways and express high levels of active efflux transport proteins, including P-glycoprotein (Pgp). As a result, the overwhelming majority of small molecules, proteins, and peptides do not cross the BBB. Therefore, the development of a drug delivery system for the brain for the treatment of neurological disorders is of great interest. In the present study, we provide experimental evidence that a new peptide-based drug delivery technology platform (Angiopep) provides a non-invasive and flexible platform for transporting drugs into the central nervous system. The platform consists of a family of peptides derived from a naturally occurring protein capable of crossing the BBB. The angiopeps cross the BBB using a receptor-mediated mechanism involving the low-density lipoprotein receptor-related protein (LRP). The lead carrier peptide (angiopep-2) was evaluated in vivo by in situ brain perfusion with non-invasive optical imaging in mice using radioactively labeled or peptides conjugated with the near-infrared probe Cy5.5. Angiopep-2 peptides were detected very rapidly in the brain parenchyma. Higher fluorescence associated with angiopep-2-Cy5.5 was detected in the brain tumor as compared to the normal brain. Based on this discovery, we have created several new drug entities, the most advanced of which is ANG1005, formed by chemical conjugation of our peptide vector (angiopep-2) to 3 molecules of paclitaxel. In contrast to free paclitaxel, which is normally prevented from reaching the brain by the BBB P-glycoprotein (P-gp) efflux pump, ANG1005 is efficiently transported across the BBB, with an approximately 100–fold higher transport rate compared to free paclitaxel and a 10-fold higher transfer rate than temozolomide (TMZ). Furthermore, ANG1005 is homogenously distributed in rat brains, as measured using in situ brain perfusion. ANG1005 was detected by LC-MS-MS in both normal brain and brain tumors in mice 30 minutes after intravenous injection; brain levels of 700 nM correspond to 2.1 ìM, which is above the therapeutic concentration of paclitaxel. Based upon the higher distribution of ANG1005 in brain tumors, we evaluated the effect of ANG1005 on glioblastoma (U87) xenograft tumor growth in immune-deficient mice. We observed a significant increase (27%) in the survival of mice treated with ANG1005. In a rat glioblastoma (U87) brain orthotopic model, administration of ANG1005 resulted in a shrinking of IC tumors as measured by MRI. Using this platform technology, we can transport small anti-cancer drugs and larger molecules across the BBB. ANG1005 is currently under evaluation in two phase I/II clinical trials for the treatment of primary and secondary brain tumors in humans.

ET-10. HIF-1 INHIBITION BY SIRNA IN AN INTRACRANIAL BRAIN TUMOR MODEL: EFFICACY OF DELIVERY METHOD USING A NOVEL CATIONIC POLYDISULFIDE DELIVERY AGENT

Randy Jensen¹ and David Gillespie²; ¹Society for Neuro-Oncology, Salt Lake City, UT, USA; ²Neurosurgery, Huntsman Cancer Institute, Salt Lake City, UT, USA.

INTRODUCTION: We have previously shown that the expression of hypoxia-inducible factor-1 (HIF-1) increases with increasing grade of gliomas. We have also previously shown that using RNA interference (RNAi) directed against HIF-1 expression resulted in a reduction of tumor growth and angiogenesis in a subcutaneous model. In this study, we apply this same technology to an intracranial model with an emphasis on maximizing delivery methods. METHODS: The glioma cell line U87-LucNeo, with a constitutively active luciferase reporter, was transplanted into the right frontal lobe of mice. The mice were given intratumoral injections of siRNA directed against HIF-1. The siRNA was complexed to a newly engineered cationic polydisulfide delivery agent (MFC). Tumor size was screened by luminescent imaging and tumor cell luciferase activity. Alternatively, mice were given siRNA complexed to MFC through continuously dispensing mini-osmotic pumps that dispensed the siRNA/carrier complex at a rate of 0.25 µL per hour over a period of 14 days. RESULTS: Mice receiving siRNA injections against HIF-1 had an increased lifespan and delayed tumor growth. Treated tumors demonstrated decreased proliferation and microvascular density compared to nontreated controls. The MFC did not demonstrate any toxicity and seemed to provide more efficient delivery of siRNA molecules. CONCLUSION: Our MFC delivery system is safe and effective for the delivery of RNAi into the brain. This RNAi-directed decrease of HIF-1 showed decreased proliferation, microvascular density, and tumor growth and increased survival in an intracranial mouse model.

ET-11. TISSUE CONCENTRATION OF SYSTEMICALLY ADMINISTERED AGENTS IN HUMAN GLIOMAS

Marshall Pitz¹, Arati Desai¹, Stuart Grossman¹, and Jaishri Blakeley¹; ¹Johns Hopkins University, Baltimore, MD, USA.

INTRODUCTION: The blood-brain barrier (BBB) is known to limit the penetration of many anti-neoplastic therapies administered systemically to treat primary brain tumors. Despite this general knowledge, few data are available on intratumoral concentrations of specific agents. As a result, many agents are studied in clinical trials and used in clinical practice even though they may fail to reach therapeutic levels within the tumor. We sought to compile the available human data on simultaneous drug concentrations in the blood and in brain tumors, along with BBB status. METHODS: A systematic review of the literature was conducted for human studies that provided concentrations of anti-neoplastic agents in the blood and in brain tumors (using surgical tissue specimens [ST] or microdialysis [MD]). Each study was assessed for simultaneous brain tumor and blood concentrations, number of sampling time points, number of sampling locations, number of patients, MRI localization of the sample, BBB status, tumor histology, and measurement technique. RESULTS: A total of 27 studies involving 19 anti-neoplastic compounds met our inclusion criteria. Optimal data were available for only 7 agents. The molecular weights (MW), ST vs. MD, and mean tumor tissue to blood concentration ratios (TBR) in contrast-enhancing tumor for these agents were: [1] Intravenous teniposide, MW = 656.66, ST, TBR = 5.20 (van Tellingen, 1997); [2] Intravenous methotrexate, MW = 454, MD, TBR = 0.285 (Blakeley, 2007); [3] Oral temozolomide, MW = 194.15, MD, TBR = 0.073 (Portnow, 2007); [4] Intravenous liposomal daunorubicin, MW = 563.99, ST, TBR = 1.20 (Zuchetti 1999); [5] Intravenous temsirolimus, MW = 1030.30, ST, TBR = 1.43 (Kuhn, 2007); [6] Oral gefitinib, MW = 447, ST, TBR = 26.44 (Hofer, 2006); and [7] Intravenous paclitaxel, MW = 853.93, ST, TBR = 5.14 (Heimans 1994). CONCLUSION: Quantitation of drug concentrations within brain tumors should precede efficacy studies and the empiric use of agents without documented efficacy in clinical trials. The accumulation and objective evaluation of existing data into one source should focus the study and clinical use of anti-neoplastic agents for central nervous system tumors. For those compounds with insufficient information, future studies of drug concentration in human gliomas are needed to advance this fundamental aspect of neuro-oncology.

ET-12. AN UPDATE ON CORRELATIVE MOLECULAR ENDPOINTS FROM RTOG 0211: PHASE I/II STUDY OF GEFITINIB + RADIATION FOR NEWLY DIAGNOSED GLIOBLASTOMA

A. Chakravarti¹, M. Wang², P. Mischel³, H.I. Robins⁴, A. Guha⁵, M. Machtay⁶, W. Curran⁷, M. Roach⁸, M. Mehta⁴, and A. Dicker⁶; ¹Radiation Oncology, Massachusetts General Hospital, Boston, MA, USA; ²American College of Radiology, Philadelphia, PA, USA; ³University of California, Los Angeles, CA, USA; ⁴University of Wisconsin-Madison, Madison, WI, USA; ⁵University of Toronto, Toronto, Ontario, Canada; ⁶Thomas Jefferson University, Philadelphia, PA, USA; ⁷Emory University Medical School, GA, USA; ⁸University of California, San Francisco, San Francisco, CA, USA.

INTRODUCTION: RTOG 0211 revealed that the addition of gefitinib to radiation therapy was well tolerated, but survival was not significantly improved compared to historical controls (ASTRO 2006). Based on data from a retrospective analysis of recurrent glioblastoma (GBM) treated with epidermal growth factor receptor (EGFR) TKIs, we analyzed whether subgroups with specific molecular signatures were more likely to benefit from anti-EGFR therapies. The present report is the first to evaluate molecular correlates of clinical outcome in newly diagnosed GBM treated with anti-EGFR therapies and represents the most comprehensive correlative analysis performed on GBM patients treated by anti-EGFR therapies. METHODS: Tissue blocks were prospectively collected for 74 of the 148 RTOG 0211 cases. These tissue blocks were used to generate tissue microarrays (TMAs). The predictive values of 12 molecules integral to EGFR signaling (EGFR, pEGFR, EGFRvIII, PTEN, pAKT, pMAPK, pmTor, IGFR1, NFKB, survivin, MGMT, and pSrc) either have been examined or will be examined by the time of the ASTRO meeting. The molecules that have been examined to date are EGFR, EGFRvIII, PTEN, and pAKT. The analysis was performed using the Histo-Rx AQUA platform (a multi-tissue proteomic analysis platform using fluorescence-based image analysis to quantify protein expression in subcellular compartments). In addition, EGFRvIII and PTEN were analyzed using traditional immunohistochemical staining and manual scoring to reproduce the technique that was used in the Mellinghoff New England Journal of Medicine report. RESULTS: Total EGFR, EGFRvIII, and PTEN expression as single markers were not significantly associated with either overall survival (OS) or progression-free survival (PFS) in GBM patients treated with RTOG 0211. Patients with co-expression of EGFRvIII and intact PTEN as determined by manual scoring had an OS of 12 months and a PFS of 5.4 months, compared to 9.5 months OS and 4.7 months PFS in non-co-expressing patients (OS: HR = 1.462 [95% confidence interval (CI): 0.689–3.10]; PFS: HR = 1.836 [95% CI: 0.797–4.23], P = NS (study underpowered to detect significant difference)]. Likewise, AQUA scoring failed to find any significant correlation with outcome in co-expressors of EGFRvIII and PTEN. Patients expressing high levels versus low levels of pAKT had significantly shorter survival times (P = 0.047). CONCLUSIONS: In the upfront setting, activation of AKT signaling appears to be associated with adverse outcome in gefitinib-treated GBM patients. The complete RTOG 0211 correlative data based on the 12-marker panel will be presented at the time of the 2008 ASTRO annual meeting. This publication was supported by grant number RTOG U10 CA21661, CCOP U10 CA37422, Stat U10 CA32115 from the National Cancer Institute; the RTOG seed grant program, Brain Tumor Funders Collaborative Grant (to AC and MM), and NIH/NCI-RO1CA108633 (to AC and PM). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute.

ET-13. EFFECT OF HEMI-GS-NITROXIDE 5-131 ON RADIATION-INDUCED APOPTOSIS IN T98G GLIOMA CELLS

Arlan Mintz¹, Valerian Kagan², Zhentai Huang², and Jianfei Jiang²; ¹Neurosurgery, University of Pittsburgh, Pittsburgh, PA, USA; ²Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA.

INTRODUCTION: Owing to a significant resistance of glioblastoma cells to radiation, local recurrence leading to patient death is common. Attempts at effective dose escalation are limited by radiation-induced neurological damage. We developed an alternative approach that is based on the much higher content of mitochondria in normal brain tissue compared to tumors. We propose to protect radiation-sensitive normal brain by using mitochondrial-targeted scavengers of electrons and free radicals. A novel class of compounds, conjugates of 4-amino-TEMPO and derivates of gramicidin S (hemi-GS-nitroxides), are concentrated in the mitochondria. Based on the differentially higher levels of mitochondria in normal brain, we hope to have a preferential effect on normal brain protection. To ensure that there is no protective effect on tumor cells, we present data on T98G glioma cell lines exposed to hemi-GS-nitroxide 5-131 and radiation. METHODS: T98G cells were seeded in 35-mm dishes at a density of 1x10⁵ cell/dish and allowed to attach overnight. In the control arm, T98G cells were incubated with hemi-GS-nitroxide at concentrations of 0, 5, 10, and 20 µM. In the experimental arm for apoptosis induction, T98G cells were treated with radiation alone or in combination with hemi-GS-nitroxide 5-131 (5, 10, and 20 µM). Cells were gamma-irradiated at a dose of 25 Gy. Nitroxide was removed from cells 6 hours post-irradiation incubation. Cells were harvested for flow cytometry analysis 72 hours post-irradiation using an Annexin V/PI kit (Biovision). Data were completed in triplicate and presented as means ± standard deviation. At the end of the incubation, still-adherent cells were trypsinized and pooled with the cells already detached. RESULTS: In the control cells, hemi-GS-nitroxide did not decrease the PS externalization in dose-dependent fashion. There was a 10% cell death across the range of concentrations used (0, 5, 10, and 20 µM). In the experimental group, the results showed that hemi-GS-nitroxide 1–131 did not protect T98G cells from apoptosis induced by gamma irradiation. There was a 30–40% mean cell death that did not change across concentrations of 0, 5, 10, and 20 µM. CONCLUSION: We plan to protect radiation-sensitive normal brain utilizing mitochondrial-targeted scavengers of electrons and free radicals. Our model predicts that hemi-GS-nitroxides will prevent radiation-induced free radical formation, resulting in reduced mitochondrial-dependent apoptosis of normal brain but not mitochondria-poor tumor cells. Protection of normal brain from radiation-induced apoptosis would allow for dose-escalation trials. In support of this model, we have shown that hemi-GS-nitroxide 5-131 did not demonstrate any protective effect on radiation-induced apoptosis of T98G tumor cells.

ET-14. THE KETOGENIC DIET AS AN ADJUVANT THERAPY FOR MALIGNANT GLIOMAS

Mohammed G. Abdelwahab¹, Do-Young Kim², Heather Milligan², Jong M. Rho², and Adrienne C. Scheck¹; ¹Neuro-Oncology Research, Barrow Neurological Institute, Phoenix, AZ, USA; ²Pediatric Epilepsy, Barrow Neurological Institute, Phoenix, AZ, USA.

The ketogenic diet (KD) is an established, non-pharmacologic treatment for refractory epilepsy. Studies of the neuro-protective effects of this diet have suggested that the resulting increase in ketone bodies (KB) causes a reduction in reactive oxygen species (ROS) and neural protection. Recent studies suggested that metabolic changes induced by the high-fat, low-carbohydrate diet also inhibited the growth of brain tumors in a rodent model and a single published human case report. Our goal is to demonstrate the mechanism(s) of tumor inhibition and to determine whether the anti-proliferative effects of the KD can potentiate the action of chemotherapy and radiation therapy. Cell lines derived from primary and recurrent glioblastomas were grown in Waymouth's MAB 87/3 media with 20% fetal calf serum and treated with a 1:1 mixture of the KBs beta-hydroxybutyrate (BHB) and acetoacetate (AcA) at 2 mM or 10 mM. The apparent cytostatic inhibitory effect of the KB on cell growth was demonstrated using a trypan blue exclusion assay. In addition, cells were treated with 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) within 24 hours of plating to observe the effects of the KB when used in combination with a common chemotherapeutic agent. The addition of BCNU caused an increase in growth inhibition, suggesting that combination therapy may enhance the effects of currently used drug treatments. Biochemical mechanisms were assessed in cultured GL261 cells treated with ketone bodies to determine whether KB altered ROS levels in these cells. Tumor cells had high levels of ROS as determined by 2'7'-dichlorofluorescin diacetate (DCF) fluorescence. The application of either 2 mM or 10 mM ketones resulted in reduction of ROS in these cells. These in vitro results led to the use of an immunocompetent, syngeneic intracranial tumor model in which 105 GL261 cells were stereotactically implanted into the right hemisphere of 10-week old C57Bl/6 mice. Following surgery, animals were fed standard rodent chow for 1 week. Successful GL261 cell implantation was evident the week following surgery, when tumors were visible by magnetic resonance imaging. Animals were then randomized based on tumor size and treated with normal rodent chow or the ketogenic Bio-Serv F3666 diet (Bio-Serv, Frenchtown, NJ). To ensure adequate levels of ketosis, serum BHB levels were checked weekly using the Keto-Site reflectance meter (GDS Diagnostics, Elkhart, IN) on blood obtained from tail clips. Mice were maintained on a 12-hour light-dark cycle, and ketogenic or standard rodent chow was available ad libitum. Animals fed the KD had an increase in median survival of 4 days relative to animals fed standard chow, even in the absence of caloric restriction. To determine whether the KD enhanced the activity of radiation therapy and/or chemotherapy, animals from each dietary treatment group were also given 2 x 2 Gy radiation treatment. Kaplan-Meier analyses confirmed this radiation dose alone was too low to cause any difference in survival. When irradiated and non-irradiated animals were combined for each diet, the effect of the KD yielded a P value of 0.0196 using the Gehan-Breslow-Wilcoxon Test. Experiments are in progress to determine the optimal role of dietary intervention as a concurrent treatment to standard radiation therapy and chemotherapy.

ET-15. THERAPEUTIC EFFICACY OF IL-13-CONJUGATED LIPOSOMAL DOXORUBICIN IN INTRACRANIAL BRAIN TUMOR-BEARING MICE

A. B. Madhankumar¹, Becky Slagle-Webb¹, Jonas M. Sheehan¹, Xinsheng Wang¹, Qing X. Yang², David A. Antonetti³, Pattie Miller², and James R. Connor¹; ¹Department of Neurosurgery, Penn State College of Medicine, M.S. Hershey Medical Center, Hershey, PA, USA; ²Department of Radiology, Pennsylvania State University College of Medicine, Hershey, PA, USA; ³Department of Cellular & Molecular Physiology, Penn State College of Medicine, M.S. Hershey Medical Center, Hershey, PA, USA.

Existing chemotherapies for glioblastoma multiforme either are ineffective at treating the tumor completely or are toxic to normal tissues, limiting potentially effective treatment. We reported earlier that IL-13 receptor-targeted chemotherapies delivered through lipid nanovesicles (liposomes) were effective in targeting glioma tumors in a subcutaneous animal tumor model. In this study, we extrapolated our earlier investigation to an intracranial mouse brain tumor model. Doxorubicin-carrying liposomes of particle sizes ranging from 50 nm to 150 nm were formulated. The charge and size of the nanovesicles were determined to assess their suitability for tumor targeting without any toxicity. The uptake of the IL-13-conjugated liposomal doxorubicin by the glioma tumors was studied initially in unicellular spheroids, 3-dimensional spherical assemblies of cells resembling the in vivo tumors, which were cultured using U87 glioma cells. The ability of these targeted nanoliposomes to cross the blood-brain barrier was studied in an in vitro endothelial cell culture model. We observed cytotoxicity of the transported liposomes in the basal chamber, with the increasing time interval indicating that these liposomes can cross the blood-brain barrier intact. After proving their efficacy in the cell culture model, we performed in vivo experiments in an intracranial brain tumor model in mice. For this study, IL-13-conjugated liposomal doxorubicin (targeted liposomes) and unconjugated liposomes (non-targeted liposomes) were injected into tumor-bearing mice once weekly for 7 weeks (15 mg/kg body weight). The tumor growth and the survival of the mice were monitored weekly. To demonstrate that doxorubicin was entering the tumors, we treated one group of mice with targeted and untargeted liposomal doxorubicin for 3 weeks and then removed the brains. The brains were dissected, sectioned, and fixed on glass slides and immunostained with the IL-13 receptor antibody. These sections were observed under a fluorescence microscope for the endogenous fluorescence of doxorubicin and the immunofluorescence of the IL-13 receptor antibody. Microscopic viewing of the tumor section revealed a significant accumulation of doxorubicin in the tumors when the animals were treated with targeted liposomes. In the therapeutic efficacy studies, we observed a significant reduction in tumor size in the mice treated with the conjugated liposomal doxorubicin compared to mice treated with the untargeted liposomes. Tumor volume decreased from 5 mm³ to 1 mm³ over 6 weeks in mice treated with targeted liposomal doxorubicin, whereas the tumor volume remained almost the same (5 mm³) in mice treated with non-targeted liposomes over the same period.. The mean survival of the mice treated with untargeted liposomes was 23 days, and after 35 days, none of the mice was still alive. In the animals treated with targeted liposomal doxorubicin, the mean survival was much higher, and 4 mice survived even after 150 days of tumor formation. This study suggests that targeted nanovesicles are a viable option for treating brain tumors.

ET-16. A PHASE I TRIAL OF ENZASTAURIN (LY317615) IN PATIENTS WITH RECURRENT GLIOMAS

Teri Kreisl¹, Lyndon Kim¹, Elinzano Heinrich², Cheryl Royce³, Irene Stroud³, Garren Nancy³, Paul Albert⁴, Luna Musib⁵, Donald E. Thornton⁶, and Howard Fine³; ¹National Institutes of Health, Bethesda, MD, USA; ²OH, USA; ³MD, USA; ⁴National Institutes of Health, Bethesda, MD, USA; ⁵Biopharm, Eli Lilly and Company, Indianapolis, IN, USA; ⁶Oncology, Eli Lilly and Company, Indianapolis, IN, USA.

BACKGROUND: Enzastaurin and its metabolites are potent selective inhibitors of PKC-â, which lies in the vascular endothelial growth factor signal cascade. PKC inhibition may also inhibit AKT pathway activation and result in suppression of GSK3-â phosphorylation. Enzastaurin has demonstrated anti-tumor activity and excellent tolerability in previous phase I and phase II studies. This phase I trial explores twice daily dosing in an effort to improve systemic enzastaurin exposure. METHODS: Patients with recurrent glioma were enrolled at two dose levels of enzastaurin, stratified by use of enzyme-inducing anti-epileptic drugs (EIAEDs). A crossover design allowed patients to alternate between daily and twice-daily divided dosing midway through the first 6-week cycle. Patients continuing to subsequent cycles were treated with divided twice-daily dosing. Pharmacokinetic (PK) and GSK3-â sampling were performed. RESULTS: Nineteen patients were enrolled at dose level 1 and treated with 800 mg total daily for patients not taking EIAEDs (N=15) and 1000 mg total daily for patients who were taking EIAEDs (N=6). Dose-limiting toxicities were grade 3 thrombocytopenia (3 pts) and grade 3 transaminitis (2 pts). Due to unacceptable toxicity, a lower dose level then accrued 6 patients, none taking EIAEDs, who received 500 mg total daily dosing. Dose-limiting toxicities occurred in 2 of these patients (1 with grade 3 lymphopenia and 1 with grade 3 thrombocytopenia associated with intracerebral hemorrhage resulting in death. PK data were evaluable for 17 patients. Twice-daily dosing resulted in higher average drug concentrations under steady-state conditions for both dose levels. Correlative studies of GSK3-â activity as a biomarker of AKT pathway inhibition will also be presented. CONCLUSIONS: Twice-daily dosing of enzastaurin increases systemic exposure of the drug but with significant toxicity. Enzastaurin remains a promising drug in the treatment of gliomas and warrants further study using a once-daily administration schedule.

ET-17. ANTI-NEOPLASTON AS2-1 AFFECTS CELL CYCLE CHECKPOINTS, LEADING TO APOPTOSIS IN HUMAN GLIOBLASTOMA CELLS

Sonali Patil¹, Stanislaw Burzynski², Sridhar Chittur³, Emilia Mrowczynski⁴, and Krzysztof Grela¹; ¹Burzynski Research Institute, Stafford, TX, USA; ²Houston, TX, USA; ³Microarray Core Facility, Center for Functional Genomics, Rensselaer, NY, USA; ⁴Burzynski Research Institute, TX, USA.

Brain tumors form because of abnormal, unregulated growth of brain cells that normally would have entered a quiescent resting stage. These cells re-enter the cell cycle due to aberrations in many genes that control cell growth and division. The typical therapy for high-grade tumors includes surgery, radiation, or chemotherapy, but unfortunately these standard treatment options are not curative. Anti-neoplastons are naturally occurring peptides and amino acid derivatives currently being used with positive results in phase II clinical trials for the treatment of several brain tumor types. Phenylacetate (PN) and phenylacetylglutaminate (PG) are the two major components of anti-neoplaston AS2-1 (AS) and are metabolites of phenylbutyrate (PB). Though PN has been well studied, the mechanism of action of PG is not well understood. PN has been reported to cause G1 arrest in several tumor cell lines. Here we report that PG also exerts its effect as an anti-proliferative agent by a similar mechanism. We provide evidence that PG causes G1 blockade and apoptosis. This effect is enhanced when PG and PN are used in combination, as in AS. We performed a screen to detect changes in gene expression in response to PG and PN in U87 glioblastoma cells using the Affymetrix Human Genome plus 2.0 oligonucleotide arrays. Pathway analysis was performed using tools such as DAVID, Onto-express, Genespring, and GenMAPP to identify pathways that show fold enrichment of genes based on the expression data. A significant inhibition of major components of the cell cycle was seen in cells treated with a combination of PG and PN. Prominent genes that were suppressed include CDCs 25A and 25B, cyclins D3 and E, and CDKs 3, 4, and 6. It was clear that the down-regulation of all these genes played an important role in the G1 blockade we observed in U87 cells. Genes of the origin recognition complex (ORC), such as ORC1L and CDC6, were suppressed. Also, several genes of the minichromosome maintenance (MCM) complex, including MCMs 2, 3, 4, 5, 6, and 7, and CDC7 were down-regulated. In addition, we saw suppression of several genes of the G2/M checkpoint, such as cyclins A, B1, and B2, polokinase 1, and CDKs 1 and 2,. PG and PN also affected genes encoding proteins necessary for spindle assembly, such as cohesion complex, securin, MAD2L1, BUB1, and CDC20. This would lead to disruption of mitosis. On the other hand, expression of tumor suppressors such as p21, p53, and GADD45A was activated. Exposure of U87 cells to PG alone caused fewer but significant changes in the expression of critical genes. Expression of GADD45A, p21/CDKN1A, and PPM1A was up-regulated. These genes are involved in the induction of cell cycle arrest and apoptosis. Based on pathway analysis, it was observed that anti-neoplastons affected the expression of more than 40 genes instrumental in the cell cycle in GBM cells. Anti-neoplastons may target multiple levels in the cell cycle and enhance the anti-cancer effect of tumor suppressor genes.

ET-18. THE C282Y VARIANT OF THE HFE GENE IS ASSOCIATED WITH A CANCER PHENOTYPE IN CELLS AND POOR PATIENT OUTCOME

Sang Lee¹, Elana Farace², Becky Slagle-Webb², Jonas Sheehan², and James Connor²; ¹Neurosurgery, Penn State College of Medicine, M.S. Hershey Medical Center, Hershey, PA, USA; ²PA, USA.

Cancer cells have a robust appetite for iron. The HFE protein is part of the cellular iron regulatory mechanism. Therefore, it is logical to hypothesize that polymorphisms in the HFE gene, the most common genetic variant in Caucasians, would impact the phenotype of cancer cells. In this study, we show that a human neuroblastoma cell line carrying the C282Y allelic variant proliferates more rapidly, migrates more extensively, and fails to differentiate upon exposure to retinoic acid compared to the same cell line expressing either the other common allelic variant H63D or wild-type HFE. In an in vivo mouse model, a human glioma cell line carrying the C282Y allele proliferates more rapidly than a glioma cell line with wild-type HFE. We directly determined the effect of HFE on cell proliferation by using siRNA to decrease HFE protein expression. HFE siRNA treatment significantly decreased HFE expression in a dose-dependent manner and inhibited cell proliferation of C282Y-expressing cells. The anti-proliferative effect of HFE siRNA was also demonstrated in vivo. The clinical relevance of these observations was examined by performing a survival analysis on brain tumor patients. Patients whose cancer had the C282Y allele were 3.3-fold more likely to die than patients whose cancer had the wild-type allele. These data provide compelling evidence that intervention strategies and outcome analyses for brain tumor patients and potentially other cancer patients should be stratified according to HFE genotype.

ET-19. CHARACTERIZATION OF A CYTOTOXIC COMPOUND FOR TEMODAR-RESISTANT TUMORS

Sang Lee¹, Becky Slagle-Webb², Jonas Sheehan², and James Connor²; ¹Pennsylvania State University, Hershey, PA, USA; ²PA, USA.

Despite aggressive treatment options, brain tumors almost always recur with fatal consequences. Temodar is the standard alkylation drug for brain tumor chemotherapy. However, as many as 50% of brain tumors are reportedly temodar resistant. Through the drug development program at Penn State Hershey Medical Center, we identified a compound that is cytotoxic to temodar-resistant neuroblastoma and glioma cells. The cells used to screen for this compound carried the C282Y allelic variant of the HFE gene, which we have previously shown conveys temodar resistance to tumor cells. This compound was an analog of thiobarbituric acid. The toxic effect of the identified compound is dose dependent in both temodar-resistant neuroblastoma and glioma cells. The compound was cytotoxic to temodar-resistant glioma cell lines at approximately 4.3 µM of LD50, but temodar-sensitive SW-1088 astrocytoma cells were even more sensitive (LD50: 1.7 µM). At concentrations that were toxic to temodar-resistant glioma cells, the compound was not toxic to normal human astrocytes obtained at autopsy or to rat primary astrocytes. The compound also completely inhibited tumor growth in vivo using an athymic nude mouse subcutaneous tumor model. Notably, the tumor did not come back in the temodar-resistant-glioma-injected nude mice when the compound was discontinued (beyond 7 weeks). The body weight of the animals did not decrease in the animals that received the compound. These data indicate we have identified a new cytotoxic compound that is effective against temodar-resistant tumors both in cell culture and in vivo.

ET-20. INHIBITION OF GLIOBLASTOMA GROWTH IN A HIGHLY INVASIVE NUDE MOUSE MODEL CAN BE ACHIEVED BY TARGETING EGFR BUT NOT VEGFR-2

Katrin Lamszus¹, Tobias Martens², Yvonne Laabs², Hauke Günther², Zhenping Zhu³, and Manfred Westphal⁴; ¹University of Hamburg, Hamburg, Germany; ²Germany, Germany; ³ImClone Systems, NY, USA; ⁴Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

OBJECTIVE: A major shortcoming of traditional mouse models based on xenografted human glioblastoma cell lines is that tumor cells do not invade. Another deficit is that genetic alterations, such as amplification of the epidermal growth factor receptor (EGFR), are typically not maintained in glioma cell lines and xenografts derived thereof. These models are therefore of limited value for preclinical therapeutic studies. To overcome these deficits, we established a highly invasive orthotopic nude mouse model using xenografted short-term spheroid cultures of human glioblastomas. Using this model, we evaluated the effects of monoclonal antibodies against EGFR (cetuximab) and vascular endothelial growth factor receptor-2 (VEGFR-2, antibody DC101). METHODS: Freshly resected glioblastoma tissue was cultured briefly as spheroids in vitro for up to 11 days. Spheroids were injected stereotactically into the striatum of nude mice. When tumors developed histologically, animals were treated for 4 weeks either with continuous interstitial infusion of cetuximab or with intraperitoneal injections of DC101 twice weekly. A novel method had to be developed to determine the extension and density of the highly invasive, diffusely growing tumors. Thirty-six landmark points were defined on H&E-stained sections at 6 different coronal levels with 6 different areas each (eg, striatum, corpus callosum, thalamus). Image analysis was employed to assess tumor cell densities at these landmark areas. RESULTS: Highly invasive xenografts were obtained from 9 different glioblastomas. Of 7 different xenograft cases treated with cetuximab, 3 responded to treatment with significant tumor growth inhibition, whereas 4 did not. All responsive tumors were derived from glioblastomas exhibiting EGFR gene amplification as well as expression of the truncated EGFRvIII receptor variant. EGFR amplification and expression of EGFRvIII were maintained in mouse xenografts as determined by FISH analysis and RT-PCR, respectively. In contrast, none of the non-responsive tumors displayed EGFR amplification or expression of EGFRvIII. In responding tumors, the proportion of apoptotic cells was greatly increased, whereas the fraction of proliferating cells was decreased. In addition, all responsive tumors expressed p-Akt, but only 2 of 3 tumors expressed PTEN. None of 4 xenograft cases treated with DC101 responded to treatment, and quantification of intratumoral blood vessels showed that the diffusely invading tumors grew largely independent of angiogenesis. CONCLUSIONS: This is the first study showing that inhibition of invasive glioblastoma growth can be achieved in vivo using interstitial delivery of an anti-EGFR antibody. Importantly, tumor responsiveness depended on the presence of amplified and/or mutated EGFR, which appears to sensitize tumors to treatment. In contrast, antiangiogenic treatment with DC101 was not effective against the diffusely invading tumors, which grew obviously independent of neovascularization.

ET-21. CONVECTION-ENHANCED DELIVERY OF NANOLIPOSOMAL TOPOTECAN AND GADODIAMIDE IN RODENT INTRACRANIAL BRAIN TUMOR XENOGRAFTS AND NORMAL BRAIN

Matthias Luz¹, Millicent Dugich-Djordjevic², John Bringas³, Piotr Hadaczek³, Greg Johnson⁴, Amy Grahn⁵, Simon Eastman⁶, and Krystof Bankiewicz³; ¹MedGenesis Therapeutix Inc., Mannheim, Germany; ²Neurocore, San Diego, CA, USA; ³Neurosurgery, Brain Tumor Research Center, University of California, San Francisco, San Francisco, CA, USA; ⁴MedGenesis Therapeutix Inc., Victoria, British Columbia, Canada; ⁵MedGenesis Therapeutix Inc., Lake Bluff, IL, USA; ⁶Burnaby, British Columbia, Canada.

Convection-enhanced delivery (CED) of highly stable nanoliposomal agents encapsulating chemotherapeutic drugs has previously been effective against intracranial rodent brain xenografts. To advance CED further by understanding targeted infusion distribution, a paramagnetic agent infused with the therapeutic agent is desired. This study tested a newly developed non-PEGylated nanoliposome containing the topoisomerase I inhibitor topotecan (topoCED) and a nanoliposome containing the neuroimaging agent gadodiamide (gadoCED). The results demonstrated that tissue pharmacokinetics of topoCED (0.5 mg/mL) following a single 20 µL CED administration per hemisphere showed a substantial area under the concentration time curve (AUC) of 153.8 µg·day/g of brain tissue and a half-life of approximately 1 day. The AUC and half-life of topoCED far exceeded that of free topotecan, indicating longer drug release kinetics from the liposome, a desirable characteristic for CED. Using the in vivo U87MG intracranial rodent xenograft model, a clear and consistent survival advantage as compared to untreated controls was demonstrated with the co-administration of two dose levels of topoCED (1.0 mg/mL and 0.5 mg/mL) and gadoCED (1.15 mg/mL), given as two sequential CED infusions 3 days apart. The findings showed both topoCED dose levels resulted in a highly statistically significant increase in overall survival (P < 0.0001) when compared to controls. Median survival was increased by 65% for the high-dose group (33.0 days versus 20 days) and by 48% for the low-dose group (29.5 days versus 20 days) over the control group. In the low-dose group, the effect size was slightly more moderate than in the high-dose group, suggestive of a dose/concentration-dependent effect. Supporting the efficacy results, the in vitro 50% inhibitory concentration for topoCED (0.8 µM) showed excellent potency and was well below the brain tissue concentration range of 1.24–146.4 µM over the first 96 hours, based on the pharmacokinetic results. GadoCED alone or co-infused with topoCED did not appear to result in additive or synergistic cytotoxicity, even at the very high concentration of 200 µM. Also, the combination of topoCED and gadoCED was shown to co-convect well in U87MG intracranial rodent xenograft tumor tissue or naïve brain tissue, with a correlation coefficient range of 0.97–0.99. TopoCED at higher concentrations (1.6 mg/mL) co-infused with gadoCED (1.15 mg/mL) in naïve brain tissue resulted in no evidence of histopathological changes within targeted regions that were attributable to either agent. The combined positive results of tissue pharmacokinetics, efficacy, cytotoxicity, co-convection, and lack of toxicity of topoCED in a clinically meaningful dose range and in combination with an ideal matched-liposome paramagnetic agent, gadoCED, implicates further clinical applications of this therapy in the treatment of malignant glioma.

ET-22. INHIBITION OF XIAP FACILITATES TRAIL-INDUCED APOPTOSIS AND IMPROVES SURVIVAL IN A BIOLUMINESCENT GLIOMA XENOGRAFT MODEL

Vinay K. Puduvalli¹, Frederick F. Lang¹, and Yuanfang Liu¹; ¹ The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.

Overexpression of phosphorylated Akt, a characteristic of glioblastoma multiforme, enhances the viability of malignant cells by activating survival pathways and inhibiting pro-apoptotic factors. X-linked inhibitor of apoptosis (XIAP) is a downstream target of Akt that can potently inhibit caspase 3 and caspase 9 and abrogate both death receptor and mitochondrial pathways of apoptosis. By activating the death receptor pathway, TNF-related apoptosis-inducing ligand (TRAIL) potently induces apoptosis in gliomas, an effect that can be inhibited by XIAP. We have demonstrated that Ad-sTRAIL, an adenoviral vector that expresses soluble TRAIL (sTRAIL), can induce apoptosis in gliomas and increase survival in an intracranial glioma xenograft model. We hypothesized that inhibition of XIAP could enhance TRAIL-induced apoptosis and improve the survival advantage provided by Ad-sTRAIL. To test this hypothesis, U251HF and SNB19 glioma cells were treated with Ad-XAF1 (an adenoviral construct expressing the endogenous XIAP inhibitor XAF1) or with Compound #12 (a chemical inhibitor of XIAP) and relevant controls and exposed to soluble TRAIL. Inhibition of XIAP significantly enhanced sTRAIL-induced apoptosis in vitro. Using a novel ex-vivo organotypic human glioma slice model, we observed a similar increase in the degree of cytotoxicity with the combination of Ad-sTRAIL and Ad-XAF1. Next, we examined the efficacy of this combination in a mouse intracranial glioma xenograft model with tumors derived from implanted U251HF-Luc cells that constitutively express luciferase and can be monitored by bioluminescent imaging. Intratumoral injection of Ad-EGFP, Ad-XAF1, Ad-sTRAIL, or Ad-XAF1+Ad-sTRAIL alone along with appropriate controls was performed biweekly for 3 weeks. Change in tumor size was measured as a function of quantitative bioluminescent imaging, and survival was recorded. Tumors treated with the combination of Ad-XAF1 and Ad-sTRAIL showed a progressive decrease in bioluminescence compared with other treatment conditions. Correspondingly, a statistically significant increase in survival was seen in animals treated with Ad-XAF1+Ad-sTRAIL compared with either agent alone or controls. Our results show that the efficacy of Ad-sTRAIL was significantly improved by inhibition of XIAP, suggesting a potential locoregional strategy using these agents for treatment of malignant gliomas.

ET-23. INTRATUMORAL CONCENTRATION OF IMATINIB MESYLATE IN PATIENTS WITH HIGH-GRADE GLIOMA: PRELIMINARY RESULTS FROM STUDY J0623

Matthias Holdhoff¹, Jeffrey Supko², Gary Gallia³, Jon Weingart⁴, Alfredo Quinones-Hinojosa⁴, Alessandro Olivi⁴, and Stuart Grossman⁵; ¹Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA; ²Massachusetts General Hospital, Boston, MA, USA; ³Neurosurgery, Johns Hopkins University, Baltimore, MD, USA; ⁴Neurosurgery, Johns Hopkins University, Baltimore, MD, USA; ⁵Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA.

BACKGROUND: Imatinib mesylate is a selective inhibitor of the platelet-derived growth factor (PDGF) receptor tyrosine kinase, which plays a role in tumorigenesis in a subset of gliomas that overexpress this receptor. Previous data suggested that imatinib barely crosses the blood-brain barrier. However, little is known about its brain tumor penetration when there is pre-existing blood-brain barrier disruption. METHODS: Patients eligible for this study had recurrent high-grade glioma (grade III or grade IV) and were scheduled for repeat resection. Imatinib mesylate was administered orally (600 mg) for 7 days prior to surgery. At the time of surgery, 3 tumor samples were obtained from areas of increased contrast enhancement on magnetic resonance imaging (MRI), determined intraoperatively by MRI/wand imaging. Two plasma samples were obtained before and after resection. The imatinib concentration in the tumor and plasma samples was determined by mass spectroscopy, and the tumor:plasma ratio was used to assess the drug's entry into the tumor. RESULTS: Analysis from the first patient demonstrated that in areas of intense contrast enhancement on MRI/wand imaging, the intratumoral concentration of imatinib was significantly higher than the plasma concentration; the tumor:plasma ratios were 2.27 and 1.56. In an area of less intense enhancement, the tumor:plasma ratio was 0.70. All of the measured areas within the tumor had a plasma:tumor ratio of greater than 0.1, which was the predefined threshold value for a therapeutic concentration. To date, 3 patients have been enrolled in the study. Imatinib levels in tumor and plasma from the second and third patients are will be presented at this conference, as will data from additional patients accrued to the study. CONCLUSION: These data suggest that imatinib can cross the blood-tumor barrier in high-grade glioma and that the intratumoral concentration can even exceed the plasma concentration. There appears to be a correlation between contrast enhancement and intratumoral concentration of imatinib in these tumors.

ET-24. SREBP1/ACC LIPOGENESIS PATHWAY EMERGE AS POTENTIAL THERAPEUTIC TARGET IN EGFRVIII EXPRESSION GLIOMA

Deliang Guo¹, Robert Prins², Isabel Hildebrandt³, Julie Dang⁴, Horacio Soto⁵, Johannes Czernin³, Andrew Watson⁶, Caius Radu³, Timothy Cloughesy⁵, and Paul Mischel⁴; ¹Pathology and Laboratory Medicine, University of California, Los Angeles, Los Angeles, CA, USA; ²Neuro-Surgery, University of California, Los Angeles, Los Angeles, CA, USA; ³Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA, USA; ⁴Pathology and Laboratory Medicine, University of California, Los Angeles, Los Angeles, CA, USA; ⁵University of California, Los Angeles, Los Angeles, CA, USA; ⁶Medicine, University of California, Los Angeles, Los Angeles, CA, USA.

The constitutively active mutant form of epidermal growth factor receptor (EGFR), EGFRvIII, is a key regulator of PI3K/mTOR signaling in glioblastoma patients (Choe et al, Cancer Res 2003; Mellinghoff et al, New Engl J Med 2005). We previously demonstrated that patients whose tumors express this constitutively active oncogene and who have retained the PTEN tumor suppressor protein are more likely to have a favorable response to EGFR kinase inhibitors (Mellinghoff et al, New Engl J Med 2005). However, PTEN loss is common in glioblastoma patients, and patients with EGFRvIII-expressing, PTEN-deficient tumors appear to be a subset of patients that is particularly difficult to treat. The potential reliance of these tumors on PI3K/mTOR signaling raises the possibility of therapeutic intervention. However, recent work from our group raised the possibility that direct mTOR inhibition by rapamycin may promote feedback activation, leading to clinical resistance (Cloughesy et al, PLoS Med 2008). Therefore, we determined whether AMPK-mediated inhibition of mTOR signaling could potentially provide an effective alternative. Here we demonstrate, using a strategic panel of isogenic U87 glioblastoma cells, that EGFRvIII enhances AICAR-mediated growth arrest of glioblastoma cells. We performed a series of functional and biochemical studies to show that doses of AICAR sufficient to achieve growth arrest did not result in complete inhibition of S6 phosphorylation (or Akt feedback activation), raising the possibility that AICAR mediated inhibition of cholesterol and fatty acid synthesis may contribute to AMPK-mediated growth arrest. Performing biochemical analysis of a panel of glioblastoma autopsy samples with tumor and matched contralateral normal brain and immunohistochemical analysis of a tissue microarray of 163 glioblastoma samples with matched normal brain, we showed that acetyl Co-Enzyme A carboxylase alpha (ACC) and the active nuclear formed sterol regulatory element-binding protein-1 (SREBP-1), key mediators of fatty acid synthesis, are elevated in glioblastomas. Furthermore, we demonstrated by thin layer chromatography that EGFRvIII/EGFR promoted fatty acid synthesis. Consistent with the key role for inhibition of fatty acid synthesis in mediating AICAR's growth inhibitor effect, we demonstrated that supplementation with mevalonate and palmitate (end products of HMG-CoA reductase and ACC) largely rescued the AICAR-mediated growth arrest, suggesting that the growth arrest was mediated by AICAR's effects on lipogenesis and cholesterol biosynthesis through these enzymes. Furthermore, the HMG-CoA reductase inhibitor atorvastatin and the ACC inhibitor TOFA potently synergized to promote apoptosis in EGFRvIII-expressing glioblastoma cells but not in non-EGFRvIII-expressing glioblastoma cells, and these effects were rescued by supplementation with mevalonate and palmitate. Finally, we showed that EGFRvIII-expressing glioblastoma cells showed a greatly enhanced sensitivity to apoptosis when treated with the SREBP inhibitor 25-hydroxycholesterol. In vivo, we demonstrate by microPET using 18F-FDG that AICAR treatment promoted dramatic tumor shrinkage and decreased glucose metabolism in EGFRvIII–expressing U87 glioma cells but not wild-type U87 cells. These data strongly suggest that EGFRvIII-expressing cells have enhanced reliance on lipogenesis and glucose uptake and raise the possibility that patients with EGFRvIII-expressing, PTEN-deficient tumors, a group that is particularly to treat, could potentially benefit from pharmacologic activation of AMPK or targeting of key lipid regulators SREBP, ACC, and HMG-CoA reductase.

ET-25. COMBINED TREATMENT WITH HISTONE DEACETYLASE INHIBITORS ENHANCES CYTOTOXIC ACTIVITY OF ANTI-DR5/TRAIL-R2 MONOCLONAL ANTIBODIES IN HUMAN GLIOMA CELLS

Motoo Nagane¹, Saki Shimizu², Eiji Mori², Shiro Kataoka², and Yoshiaki Shiokawa²; ¹Department of Neurosurgery, Kyorin University Faculty of Medicine, Tokyo, Japan; ²Japan.

BACKGROUND: TNF-related apoptosis-inducing ligand (TRAIL/Apo2L), a member of the TNF family, induces apoptosis through binding to its cognate death receptors, preferentially DR5/TRAIL-R2 in glioma cells. However, resistance to TRAIL could hamper its clinical utility, necessitating strategies to overcome resistance. Inhibitors of histone deacetylase (HDAC) have been shown to inhibit cell cycle progression and induce apoptosis in tumor cells and thereby are expected to enhance TRAIL sensitivity. MATERIALS AND METHODS: Fully human anti-human DR5 monoclonal antibodies (mAbs) E11 and KMTR2 and soluble FLAG-tagged TRAIL were used either singly or in combination with an HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), to treat a panel of human glioma cell lines. Cytotoxicity was determined by MTT assay. Cell surface expression was analyzed by flow cytometry. Whole cellular protein expression levels were determined by Western blot. RESULTS: Treatment of human glioma cells with SAHA resulted in induction of acetylated histone H3 and suppressed cellular viability, with IC50 values mostly 2–10 mM. Combination treatment of SAHA and either soluble TRAIL or anti-DR5 mAb E11 at sublethal concentrations synergistically induced cytotoxicity in 8 of 10 glioma cell lines. In contrast, combination of SAHA and anti-DR4 mAb did not exhibit such effects. SAHA treatment upregulated DR5 cell surface expression in some cell lines but downregulated expression of c-FLIPL, an intrinsic cellular apoptosis inhibitor acting at the death-inducing signaling complex, in the majority of cell lines. SiRNA-mediated suppression of c-FLIPL expression resulted in enhancement of cellular sensitivity to TRAIL treatments. CONCLUSIONS: HDAC inhibitor SAHA sensitized human glioma cells to TRAIL-based therapy, and its effect was mediated, at least in part, through modulation of expression of apoptosis-related genes. These results could provide a potential therapeutic strategy against intractable malignant gliomas.

ET-26. PRECLINICAL ANALYSIS OF DEPOCYT THERAPY FOR INTRACEREBRAL TUMORS

Jeffrey Olson¹, Zhaobin Zhang², Constantinos Hadjipanayis³, and Pamela New⁴; ¹Emory University, Atlanta, GA, USA; ²Dept. of Neurosurgery, Emory University, Atlanta, GA, USA; ³Neurosurgery, Emory University, Atlanta, GA, USA; ⁴Houston, TX, USA.

INTRODUCTION: Though many systemically delivered agents have had disappointing efficacy in intrinsic cerebral malignancies, some may be resurrected by using direct delivery. Intracerebral/intratumoral delivery has been explored, but it has become less appealing since the well-done study IL13-PE38QQR was unable to show efficacy. However, opportunities with other agents remain. Intravenous cytarabine has a poor track record for intrinsic malignancies of the brain. DepoCyt is liposomally encapsulated cytarabine that is approved by the U.S. Food and Drug Administration for intrathecal treatment of leptomeningeal malignancy. Its use for treatment of tumors within the brain has not been explored. METHODS: DepoCyt was studied (1) for its effect on GL261, U87MG, and 9L cell lines by colorimetric proliferation studies in vitro over cytarabine concentrations of 0.01–1000 µM, (2) by HPLC separation and mass spectrometric quantitative pharmacokinetic analysis of injected intracranial tumors, and (3) for efficacy in established intracranial models of the above-mentioned cells using C57BL/6 mice, nude rats (Cr:NIH-rnu), and the Fisher 344 rat, respectively. Seven days after implantation, animals were treated with 10 µL doses by (1) a single injection, (2) a 25-minute infusion, (3) 3 separate injections (on days 7, 9, and 11 after tumor implantation), or (4) a control injection. RESULTS: DepoCyt concentration destroys liposomal encapsulation, but it can be diluted. In vitro, it inhibits cell proliferation with a half-maximal inhibitory concentration (IC50) estimated at 0.05 µM in the GL261 cell line, 0.5 µM in the 9L cell line, and 5 µM in the U87MG cell line. Direct intracerebral injection causes no toxicity over 12 months as measured by behavior, weight, and histology. After intratumoral injection, mean cytarabine concentration was 50.48 µM after 2 days, 22.2 µM after 5 days, and 13.99 µM after 6 days. In each tumor model and DepoCyt administration regimen, mean survival was superior to control injections. With the GL261 model, comparison of control to the single injection yielded a P value of 0.21 (with Student's t-test for unpaired data used in all comparisons). With the 25-minute infusion, the P value was 0.046, and for animals receiving the 3 separate injections, the P value was 0.005. In the U87MG model, the superiority of the survival of the animals receiving a single injection yielded a P value of 0.08. For the 25-minute infusion, the P value was 0.007, and for those receiving the 3 separate injections, the P value was 0.001. For the 9L model, comparing control treatment to single injection yielded a P value of 0.03. For the 25-minute infusion, the P value was 0.02, and for those receiving the 3 separate injections, the P value was 0.002. CONCLUSION: DepoCyt is safe and efficacious in these intracranial brain tumor models. By direct delivery, it reaches intratumoral concentrations greater than the IC50 measured in vitro for at least 6 days. This use of DepoCyt may be of clinical value and warrants (1) further evaluation of intracranial cytarabine kinetics after injection and (2) refinement of delivery methods to optimize its effect and assess its potential for use in humans.

ET-27. CYCLOPHOSPHAMIDE INCREASES THE INCIDENCE OF BREAST CANCER BRAIN METASTASES IN A RAT MODEL

Edward Neuwelt¹, D. Thomas Dickey², Matthew Hunt², Y. Jeffrey Wu², Patricia Steeg³, and Leslie Muldoon⁴; ¹Oregon Health & Science University, Portland, OR, USA; ²OR, USA; ³MD, USA; ⁴Portland, OR, USA.

INTRODUCTION: We assessed the effect of treatment with cyclophosphamide (CTX) on metastatic formation in the female nude rat after intra-arterial infusion of MDA-MB-231BR human breast cancer cells expressing high levels of HER2. METHODS: MDA-MB-231BR-HER2 cells (0.5–5 x 106 cells) were infused into the right internal carotid artery in female nude rats. The experimental groups were: 1) no pretreatment (n = 6); 2) pretreatment with CTX (100 mg/kg) IP 24 hours before infusion (n = 19); and 3) CTX pretreatment and a second dose of CTX 14 days post-infusion (n = 6). The rats were closely monitored for changes in body weight, behavior, and neurological function. Some animals were scanned by 3T MR using T1-weighted sequences with gadolinium contrast. Animals were sacrificed by 8 weeks post-infusion, and coronal vibratome sections were stained by immunohistochemistry for cytokeratins (Cam5.2). RESULTS: The brains of the 6 rats receiving no CTX developed no tumors even with the highest tumor cell dose. The single-dose CTX group exhibited single intracerebral tumors in 4 of 19 cases. The double-dose CTX treatment resulted in multiple brain tumors in 6 of 6 animals at 8 weeks post-infusion. The tumors were found to be in the millimeter size range, larger than those in the mouse model, and were scattered throughout the brain. CONCLUSIONS: The development of breast cancer brain metastases after intra-arterial infusion in the nude rat was found to be inconsistent. Pretreatment with CTX resulted in an increase in tumor metastases. Adding a second dose of CTX 14 days post-infusion resulted in tumor growth in 6 of 6 animals. A consistent model of metastatic breast cancer will allow us to pursue the investigation of a range of imaging and treatment options. This study also brings up the possibility that CTX or other treatments that compromise immune responses could put patients at risk of increased metastatic disease.

ET-28. EGFRVIII AND C-MET PATHWAY INHIBITORS SYNERGIZE AGAINST PTEN-NULL/EGFRVIII+ GLIOBLASTOMA XENOGRAFTS INDEPENDENT OF AKT AND MAPK INHIBITION

Bachchu Lal¹, C. Rory Goodwin², Yingying Sang³, Catherine Foss⁴, Katherine Cornet¹, Sameena Muzamil¹, Martin Pomper⁴, Jin Kim⁵, and John Laterra⁶; ¹Neurology, Johns Hopkins University and Kennedy Krieger Institute, Baltimore, MD, USA; ²Neuroscience, Johns Hopkins University, Baltimore, MD, USA; ³Kennedy Krieger Institute, Baltimore, MD, USA; ⁴Radiology, Johns Hopkins University, Baltimore, MD, USA; ⁵Galaxy Biotech, Inc., Mountain View, CA,; ⁶Johns Hopkins University, Baltimore, MD, USA.

Receptor tyrosine kinase (RTK) systems, such as hepatocyte growth factor (HGF) and its receptor c-Met and epidermal growth factor receptor (EGFR), are responsible for the malignant progression of multiple solid tumors. Recent research shows that these RTK systems co-modulate overlapping oncogenic downstream signaling pathways. This paper investigates how EGFRvIII, a constitutively active EGFR deletion mutant, alters tumor growth and signaling responses to anti-HGF therapy. We previously reported that systemic delivery of a neutralizing anti-HGF mAb (L2G7) inhibits growth of HGF-expressing intracranial glioma xenografts. We now show that L2G7 potently inhibits Akt and MAPK activation in PTEN-null/HGF+/c-Met+/EGFRvIII-U87 glioma xenografts (U87wt). Isogenic EGFRvIII+ U87 xenografts (U87-EGFRvIII) remained responsive to single–agent, neutralizing, anti-HGF mAb therapy, albeit with substantially reduced efficacy. The diminished responsiveness of U87-EGFRvIII xenografts to HGF:c-Met pathway inhibition was associated with no MAPK pathway inhibition and with reduced Akt inhibition. HGF:c-Met and EGFRvIII inhibitors synergized to produce substantial anti-tumor effects (they inhibited tumor cell proliferation, enhanced apoptosis, arrested tumor growth, and prolonged animal survival) against both subcutaneous and orthotopic U87-EGFRvIII xenografts. The dramatic response to combining HGF:c-Met and EGFRvIII pathway inhibitors in U87-EGFRvIII xenografts occurred in the absence of Akt and MAPK inhibition. These findings suggest that the c-Met and EGFRvIII pathways act together to unmask a downstream signaling pathway, the oncogenic effects of which are not predicted by Akt or MAPK activation. This "RTK pathway switching" provides new insights relevant to oncogenic RTK pathway targeting and reveals potential limitations of certain biochemical biomarkers to predict the efficacy of RTK inhibition in genetically diverse cancers.

ET-29. EVALUATION OF CYTOCHROME C (CYT C) AND SUBUNIT IV OF CYT C OXIDASE (COX IV) LEVELS IN NORMAL HUMAN BRAINS AND GLIOBLASTOMA SPECIMENS

Arlan Mintz¹, Zhentai Huang², Jianfei Jiang², and Valerian Kagan²; ¹Neurosurgery, University of Pittsburgh, Pittsburgh, PA, USA; ²Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA.

INTRODUCTION: Initial radiation-induced tumor cell death in glioblastoma is eventually overwhelmed by local recurrence, leading to patient death. Radiation-induced neurological damage limits effective dose escalation. Apoptosis, a mitochondrial-dependent process, is the dominant mechanism of normal cell death from radiation. Tumors have developed different mechanisms of resistance to apoptosis, including minimization of mitochondrial respiration dependence and successful proliferation in hypoxia. To further expound these differences in sensitivity to pro-apoptogens (eg, radiation and chemotherapy), we performed quantitative estimates of mitochondrial content in paired specimens of human gliomas and adjacent normal brain tissue. Cytochrome c (cyt c) and cyt c oxidase subunit IV (COX IV) were used as mitochondrial markers. METHODS: Glioblastoma fragments and paired adjacent normal brain specimens obtained from cortical surface during approach to subcortical tumors comprised 5 paired tissue samples. Control was normal brain from a resected temporal lobe epilepsy specimen. Specimens were homogenized in RIPA buffer and centrifuged at 10,000 x g for 10 minutes. The supernatants were separated on 15% SDS-PAGE, transferred onto a nitrocellulose membrane, and probed with antibodies against cyt c, COX IV, or actin (loading control); this was followed by horseradish peroxidase-coupled detection. The protein band profile was analyzed by densitometry. Cyt c and COX IV levels were analyzed by Western blotting. Protein amounts were presented by the percentage of ratio of COX IV or cyt c over actin from each sample compared to that of 1 normal brain tissue (Epilepsy-1) chosen as the standard in every membrane. RESULTS: Concentration of mitochondria in glioblastoma and its paired adjacent normal brain tissue was assessed using levels of 2 mitochondrial proteins, cytochrome c (intermembrane space) and COX IV (inner mitochondrial membrane), and examined by Western blot. The average ratio of cyt c to actin in normal brain samples and glioblastoma specimens was 258.1 and 49.3, separately. The amount of cyt c was approximately 5.2-fold lower in glioblastoma specimens compared to normal brain tissues. The COX IV average ratio compared to actin was 181.9 in normal brain tissues and 11.9 in glioblastoma specimens. Therefore, the content of COX IV was about 15.2-fold lower in glioblastoma specimens compared to normal brain tissues. CONCLUSION: Using levels of two mitochondrial proteins, cyt c (intermembrane space) and COX-IV (inner mitochondrial membrane), we quantitatively analyzed mitochondrial content from paired specimens of tumor and adjacent brain. The paired specimens provided an important internal control and were compared to a normal epilepsy specimen. We found a 5.2-fold decrease in cyt c and a 15.2-fold decrease in COX IV in glioblastoma compared to matched control normal brain. Based on the differentially much higher content of mitochondria in normal brain vs. tumors, we are developing an approach to protect radiation-sensitive normal brain using mitochondrial-targeted scavengers of electrons and free radicals. A novel class of compounds, conjugates of 4-amino-TEMPO and derivates of gramicidin S (hemi-GS-nitroxide), can selectively target mitochondria. Preliminary data suggests prevention of radiation-induced free radical formation results in reduced apoptosis of normal brain but not mitochondria-poor tumor cells. Selective protection of normal brain will therefore allow for dose-escalation trials.

ET-30. REDUCTION OF OXIDATIVE STRESS WITHIN GBM TUMOR AND STEM CELLS HALTS PROPAGATION AND CELL DIVISION

Paul Clark¹, Hirak Basu², Daniel Treisman³, and John Kuo⁴; ¹Department of Neurological Surgery, University of Wisconsin-Madison, Madison, WI, USA; ²Comprehensive Cancer Center, University of Wisconsin-Madison, Madison, WI, USA; ³Neurological Surgery, University of Wisconsin-Madison, Madison, WI, USA; ⁴Department of Neurological Surgery, University of Wisconsin-Madison, Madison, WI, USA.

INTRODUCTION: Oxidative stress resulting in generation of reactive oxygen species (ROS) has been implicated in glioblastoma multiforme (GBM) oncogenesis and may contribute to cancer progression and recurrence. Decreasing high ROS within GBM may be a viable therapeutic approach. Recent evidence shows that a small fraction of GBM cells exhibits stem-like properties and can efficiently initiate tumor xenografts. These GBM stem cells are likely critical for recurrence after standard therapies. We tested whether ROS reduction halts proliferation of GBM tumor and stem cells. METHODS: Three lines of human GBM tumor cells (BT22, BT33, BT12.1) and cancer stem cells (GBM CSC) were isolated and validated in vitro and in vivo under institution-approved protocols for stem cell and tumor xenograft initiation properties. CPC-410 (a gift from Colby Pharmaceutical) is a novel anti-oxidant nitroxide analog molecule that acts via mitochondrial-specific electron spin trapping to reduce ROS generation without interfering with normal electron transport chain function. Cellular ROS levels were quantified using a dichlorofluorescein (DCF) assay. GBM-derived stem and tumor cell dose response to CPC-410 was measured using a DNA quantitation assay; cell proliferation was measured via BrdU incorporation. CPC-410-treated U87 glioma, normal human fetal neural stem cells (hNSC), and human astrocyte (NHA) lines cultured in standard conditions served as comparison controls. RESULTS: (1) Elevated ROS was observed in all tested GBM lines. ROS levels were measured by DCF assay, normalized to cellular DNA content, and expressed as percent increase from normal astrocyte ROS levels (U87: 129% ± 34%; BT22T: 143% ± 12%; BT33T: 136% ± 29%; P < 0.05). CPC-410 administration eliminated ROS elevations. (2) CPC-410 inhibited propagation of 3 primary GBM lines and U87 glioma at 50% of the effective dose against normal astrocytes (IC50 [µM], NHA: 0.51 ± 0.13; BT22T: 0.33 ± 0.18; BT33T: 0.19 ± 0.07; U87 glioma: 0.23 ± 0.09; P < 0.05). CPC-410 inhibited DNA replication in GBM cell lines compared to astrocytes in BrdU incorporation assays (at 1 µM CPC-410 [% BrdU+], NHA: 21% ± 8 %; BT22T: 1% ± 2 %; BT33T: 0.6% ± 1 %; U87 glioma: 4% ± 5 %; P < 0.05). (3) GBM CSC lines were isolated, cultured in stem cell media and validated for tumor xenograft formation in SCID mice. CPC-410 treatment significantly inhibited GBM CSC propagation compared to hNSC control (IC50 [µM], hNSC: 0.34 ± 0.14; BT22SC: 0.22 ± 0.11; BT33SC: 0.13 ± 0.10; BT12.1SC: 0.20 ± 0.11, respective to above parental tumor lines). CPC-410 also decreased GBM CSC cell division, as observed via BrdU incorporation assays. CONCLUSION: GBM-derived stem cell and tumor cell growth was significantly and selectively inhibited by the novel mitochondrial superoxide inhibitor CPC-410, suggesting that oxidative stress reduction is a well-tolerated, promising anti-GBM strategy.

ET-31. RADIATION SENSITIVTY IN HUMAN ASTROCYTES, T98G CELLS, AND HUMAN BRAIN MICROVASCULAR ENDOTHELIAL CELLS

Arlan Mintz¹, Zhentai Huang², Jianfei Jiang², and Valerian Kagan³; ¹Neurosurgery, University of Pittsburgh, Pittsburgh, PA, USA; ²Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA; ³Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA.

INTRODUCTION: Current therapy for malignant gliomas includes post-operative radiation therapy combined with chemotherapy. DNA damage has been considered the principal cause of cell death by ionizing radiation and alkylating chemotherapy agents. The effect of radiation on central nervous system toxicity depends on radiation-induced cell death of normal cellular elements. Strategies designed to protect the brain from radiation-induced apoptosis will be most efficacious on those cellular elements that have the greatest radiation sensitivity. We tested cellular elements (human astrocytes and brain microvascular endothelial cells) and a tumor cell line with various doses of radiation to assess radiation sensitivity. METHODS: Primary human astrocytes, brain mircovascular endothelial cells, and human glioblastoma T98G cells were seeded in 35-mm dishes at a density of 1x10⁵ cells per dish and allowed to attach overnight. Cells were then irradiated at different doses. Cells were harvested for flow cytometry analysis 72 hours post-irradiation using an Annexin V/PI kit (Biovision). RESULTS: The data were obtained in triplicate experiments and presented as means +/- standard deviation. Analysis of cell death by externalization of phosphatidylserine (PS) revealed that human brain microvascular endothelial cells were most sensitive to radiation-induced apoptosis. Around 70% of endothelial cells (vs. 11% in untreated cells) were annexin V-FITC positive after 24 hours post-irradiation incubation. In contrast, only about 5% and 8% of human astrocytes and T98G cells, separately, exposed PS on the surface under the same conditions. Further radiation dose-dependent and time course results showed that human astrocytes were resistant to PS externalization. Only about 10% of human astrocytes showed annexin V-FITC positive after 72 hours post-irradiation incubation. DISCUSSION: Normal brain tissue surrounding malignant gliomas is exposed to radiation as part of the irradiation field and results in central nervous system toxicity. Our newly developed approach includes protection of radiation-sensitive normal brain using mitochondria-targeted scavengers of electrons and free radicals by capitalizing on a selective increase in mitochondria in normal brain as compared to tumor. The toxicity to radiation-induced apoptosis was found to differ among different cell types. Human brain microvascular endothelial cells were very sensitive to radiation-induced apoptosis (70%) compared to only 5%-8% of human astrocytes and T98G cells, after 24 hours post-irradiation. Endothelial cells may therefore represent an important target for protective strategies aimed at dose escalation to more effectively eradicate tumor cells.

ET-32. PRECLINICAL STUDY OF THE HSP-90 INHIBITOR IPI-504 IN MALIGNANT GLIOMA

Daniela Bota¹, Stephen Keir², Kelsey Albert¹, Emmanuel Normant³, and Henry Friedman²; ¹University of California, Irvine, Orange, CA, USA; ²Duke University, Durham, NC, USA; ³Infinity Pharmaceuticals, Inc., Cambridge, MA, USA.

There is an unmet clinical need for better therapies targeting the uniformly lethal malignant gliomas. Novel agents are developed in an attempt to affect specific molecular mechanisms involved in abnormal signaling and resistance to apoptosis of the malignant glioma cells while having minimal effect on the normal neural stem cells involved in cognition. One of the more innovative therapeutic targets is the molecular chaperone heat shock protein 90 (Hsp90). Inhibition of this essential protein leads to degradation of multiple oncogenic proteins, such as Raf-1, ErbB2, and Akt, resulting in cell-cycle arrest and apoptosis. Infinity agent IPI-504 is an Hsp90 inhibitor currently in phase II clinical development that has shown activity in multiple preclinical cancer models, including models of hematologic malignancies and solid tumors such as lung, breast, and ovarian cancers. IPI-504 is currently being studied in several different clinical settings. No data are yet available on the activity of this agent in malignant gliomas. We have examined response to IPI-504 treatment in 2 glioma cell lines (D54-MG and U251) and their derived lines that are resistant to temozolomide and temozolomide plus O6-benzyl guanine (D54-MG TR and OTR and U251 TR and OTR). Both parent cell lines and the resistant derived lines showed high sensitivity to low doses of IPI-504 (all the EC50 values were under 60 nM). In comparison with the malignant glioma lines, the normal neural stem cell lines showed an EC50 value of 3 µM, which suggest a large therapeutic index. This is especially important for translating this treatment in the clinical arena, where many of the current therapies are inducing cancer-related cognitive dysfunction that greatly affect the patient's quality of life. Based on these very encouraging data, we have initiated animal studies in malignant glioma xenograft models, and the results will be available at the time of presentation.

ET-33. TREATMENT OF A XENOGRAFT MODEL OF GLIOBLASTOMA MULTIFORME WITH YEAST CYTOSINE DEAMINASE PRODRUG THERAPY

Laura Serwer¹, Marieke Van Der Aa¹, Limin Cao¹, Virginia Platt², Katherine Jerger¹, Leanna Lagpacan¹, and Francis Szoka¹; ¹Biopharmaceutical Sciences, University of California, San Francisco, San Francisco, CA, USA; ²Joint Graduate Group in Bioengineering, University of California, Berkeley and San Francisco, San Francisco, CA, USA.

Enzyme prodrug therapy is a drug delivery strategy that combines local delivery of a nontoxic enzyme with systemic delivery of a nontoxic prodrug. When the prodrug interacts with the enzyme, active drug is generated, producing a higher concentration of active drug locally and a lower concentration of the active drug systemically. We have taken advantage of the robust activity of yeast cytosine deaminase (yCD) to convert the nontoxic prodrug 5-fluorocytosine (5-FC) into the toxic chemotherapeutic drug 5-fluorouracil (5-FU). Here we compared the efficacy of the native thermolabile yCD to a more thermostable mutated yCD in a xenograft model of glioblastoma multiforme (GBM). Additionally, we addressed the effect of the treatment date on survival, treating at either 7 or 12 days after tumor implantation. We used a modified version of the human U87 GBM cell line that expresses luciferase implanted in congenitally athymic Rnu/Rnu rats. On day 0, the rats were injected with 1.5x10⁶ cells in the right caudate putamen. On or after day 7, animals were assayed for luciferase activity. Those without luciferase activity were assumed to lack a tumor and were excluded from the study. On either day 7 or 12, 4.2U of yCD was delivered intratumorally by convection-enhanced delivery (CED). 1U of enzyme is able to convert 1 µmol of 5-FC to 5-FU in 1 minute. CED applies positive pressure over a prolonged infusion time to increase the distribution of the protein. Beginning on the day of yCD treatment, 50 mg of 5-FC was delivered by oral gavage twice a day for 5 consecutive days, for a total dose of 500 mg per rat. 5-FC is highly bioavailable and able to penetrate the central nervous system. Average day of death for untreated animals in the model was day 28. To assess the validity of the model, we conducted mock control studies where athymic rats underwent a tumoring surgery and a subsequent CED surgery, both times receiving only saline. The animals did not suffer any long-term effects from either surgery. We also conducted a dosing study in which the yCD enzyme was administered in increasing amounts and the delivery of 5-FC was kept constant. We found that low doses of the enzyme (3–17U) were very well-tolerated, with slight weight loss experienced only at the highest dose tested (33U). Administering the native thermolabile yCD in combination with 5-FC on day 12 gave a small but not significant increase in overall survival (P = 0.077) compared to control animals receiving 5-FC only. Using the thermostable yCD on day 12 increased significance slightly (P = 0.05) as compared to controls. Treating with the thermostable yCD and 5-FC on day 7, however, dramatically increased the significance of overall survival (P < 0.013). Studies with a fluorescent surrogate protein for yCD suggested that the enzyme was cleared from the brain within 48 hours. We are currently following up on this data using immunohistochemistry and enzyme activity assays to determine both the localization and pharmacokinetics of the thermostable yCD in the rodent brain. Work supported by NIH CA107268–01.

ET-34. ONCOLYTIC HSV THERAPY SENSITIZES GLIOMA CELLS TO INTEGRIN ANTAGONIST CILENGITIDE IN VITRO AND IN VIVO

Kazuhiko Kurozumi¹, Jayson Hardcastle², Christopher Alvarez-Breckenridge³, Jeffrey Kutsikovich³, E.A. Chiocca³, and Balveen Kaur⁴; ¹Neurological Surgery, Ohio State University, Columbus, OH, USA; ²Ohio State University, Columbus, OH, USA; ³Columbus, OH, USA; ⁴Society for Neuro-Oncology, Columbus, OH, USA.

Oncolytic viral therapy exploits the natural or engineered propensity of some viruses to replicate in cancer cells, causing their lytic destruction with minimal damage to non-neoplastic tissue. We have been investigating oncolytic HSV-1 (OV) therapy-induced changes in tumor microenvironment and have uncovered a significant induction of CYR61 gene expression (8.94-fold, P = 0.001) in tumors treated with OV in vivo. CYR61 is a secreted extra cellular matrix protein that can bind to and activate integrin ávâ3 and ávâ5 on glioma and endothelial cells. Further CYR61 mRNA and protein were induced upon infection using different HSV-1-derived OVs in multiple human glioma lines and primary human tumor-derived glioma cells in vitro and in orthotopic human gliomas grown in nude mice in vivo. Increased CYR61 in the ECM of breast cancer cells has also been shown to activate an autocrine loop, resulting in upregulation of its own receptor ávâ3 and increased sensitization to integrin anatgonists (Menendez JA et al, Oncogene 2005). Consistent with this, we found that glioma cells stably overexpressing CYR61 were much more sensitive to cell killing by integrin antagonist cilengitide than control cells (overexpressing, LD50 = 146.5 ng/mL versus control, LD50 = 521.1 ng/mL, P < 0.05). Therefore, we hypothesized that OV treatment, which induces secreted CYR61, would sensitize them to integrin antagonists such as cilengitide. We detected synergistic killing of glioma cells treated with OV and cilengitide compared to cells treated with either agent alone in vitro (Chou-Talalay combination indices = 0.48–0.49). These results suggested that treatment of glioma with OV followed by treatment with cilengitide would enhance therapeutic efficacy. We tested this hypothesis in human glioma cells (U87ÄEGFR) grown as intracranial tumors in vivo. Briefly, 7 days after tumor cell implantation, mice were treated with OV or PBS by direct intratumoral injection. Five days after OV treatment, mice were injected systemically with a single dose of cRGD (5 mg/kg) or PBS. Mice treated with OV and cilengitide survived significantly longer than mice treated with OV alone (median survival: control, 12 days; OV, 16 days; cRGD + OV, 25 days; P < 0.001 between control and cRGD + OV; P < 0.01 between OV and cRGD + OV). These results indicated that OV therapy is a sensitizer to cilengitide and reveals the potential of combining OV treatment with cilengitide to improve therapeutic outcome.

ET-35. ADJUNCTIVE THERAPY INCREASES EFFICACY OF M002, AN ONCOLYTIC HSV-1 EXPRESSING IL-12, IN PRECLINICAL MODELS OF NERVOUS SYSTEM TUMORS

David Bauer¹, Daniel Harmon¹, Xiaojia Zheng², Sreelatha Meleth³, G. Gillespie⁴, Jackie Parker⁵, and James Markert¹; ¹Neurosurgery, University of Alabama at Birmingham, Birmingham, AL, USA; ²Physiology, University of Alabama at Birmingham, Birmingham, AL, USA; ³Hematology/Oncology, University of Alabama at Birmingham, Birmingham, AL, USA; ⁴Neurosurgery, University of Alabama at Birmingham, Birmingham, AL, USA; ⁵Pediatric Infectious Disease, University of Alabama at Birmingham, Birmingham, AL, USA.

INTRODUCTION: The prognosis of glioblastoma multiforme is abysmal, despite multimodality therapy. Tumors develop uncontrolled growth by evading host immune response. Recent studies show a possible synergistic effect with multimodality therapy including oncolytic, attenuated human herpes simplex virus type 1 (HSV-1), radiation, and cyclophosphamide (CPA). The virus used, M002, is a gamma1-34.5-deleted oncolytic HSV-1 that expresses murine interleukin-12. CPA is a nitrogen mustard alkylating agent that has immunomodulatory and immunosuppressive effects. METHODS: Tumor cells were grown in vitro in DMEM-F12 media with 7% FBS and L-glutamine. A total of 5x10⁶ cells (in 0.2 mL serum-free medium) were injected into the right flank of each anesthetized mouse. Once the tumors approached 150 mm³ in volume, mice were divided into three cohorts with equal mean tumor volumes. Each cohort received 2 mg of CPA by intraperitoneal injection. Two days later, either saline or 1x10⁷ plaque-forming units (PFU) of M002 was injected into the tumor. The tumors were irradiated with 10 Gray gamma radiation 6 to 8 hours later. Mice were assessed daily, and tumor volume was recorded every tw2o days. The average tumor volume was calculated for each mouse for each time point. Average tumor volume and average percent increase in volume were calculated for each cohort and graphed versus time on a Cartesian plane. A Kaplan-Meier survival curve was made comparing groups. The log-rank test was used to compare cohorts, and the difference of least mean squares was used to compare cohorts at each timepoint. Mice were euthanized when the tumor grew greater than 25 mm in any one direction or if a mouse became moribund. RESULTS: For this study, two separate flank tumor models in immunocompetent mice were evaluated: the A/J/N2A model and the B6D2F1/4C8 model. For each experiment, there were 10 mice per cohort and a total of 6 cohorts, which were: i) saline only control, ii) M002, iii) CPA and M002, iv) M002 and radiation, v) CPA and radiation, and vi) M002 combined with CPA and radiation. The log-rank test found a difference among cohorts in both models (P < 0.0001). We used the difference of least mean squares to compare cohorts at each time-point. Our findings are summarized as follows: 1. In the A/J/N2A model, the addition of radiation did not increase the anti-tumor effect of CPA and M002. M002 and CPA both significantly retarded tumor growth, and in combination, M002 and CPA produced an increased anti-tumor response. 2. In the B6D2F1/4C8 model, M002, radiation, and CPA alone all provided a significant anti-tumor effect. There was a significant benefit when these modalities were used in combination. All three together provided a still-increased anti-tumor effect. A possible explanation for our data is the p38 MAP kinase pathway. Phosphorylation of p38 increases viral replication. In the more radioresistant A/J/N2A model, p38 may not be phosphorylated, and in the more radiosensitive B6D2F1/4C8 model, p38 may be phosphorylated. We are currently exploring this mechanism of interaction in our models. CONCLUSIONS: The oncolytic virus M002 and CPA significantly retard tumor growth in both tumor models. Radiation provides additional benefit in the more radiosensitive B6D2F1/4C8 model, but it does not provide additional benefit in the A/J/N2A model.

ET-36. ANGIOTENSIN-(1–7) INHIBITS THE GROWTH OF HUMAN GLIOBLASTOMA XENOGRAFTS IN NUDE MICE

Glenn Lesser¹, Kyle Carver², Steven Newton², E. Ann Tallant², and Patricia Gallagher²; ¹Wake Forest University Health Sciences, Winston-Salem, NC, USA; ²NC, USA.

Despite aggressive combined modality therapy, the median survival of patients with newly diagnosed glioblastoma multiforme (GBM) remains poor, and few effective therapies are available for disease recurrence. Recent data suggest that antiangiogenic strategies may be particularly useful for this highly vascular tumor. The purpose of this study was to determine whether angiotensin-(1–7) [Ang-(1–7)], an endogenous peptide hormone that activates the AT(1–7) receptor mas, is an effective targeted therapy for GBM. We initially demonstrated that Ang-(1–7) significantly reduced the growth of human GBM U87, U13,8 and U373 cells. All three cell lines contained mRNA for mas, suggesting that this AT(1–7) receptor may mediate the anti-proliferative response to Ang-(1–7). More importantly, Ang-(1–7) significantly decreased the proliferation of human GBM U87 in vivo in a mouse xenograft model. Athymic mice with tumors resulting from injection of human U87 cells were treated for 18 days with either saline or 1000 µg/kg Ang-(1–7), delivered by subcutaneous injection every 12 hours. The average volume of the tumors from mice treated with the heptapeptide was approximately 3-fold less than the size of the tumors from control animals (586.1 ± 94.5 mm³ versus 1845.5 ± 238.1 mm³; n = 6, P < 0.05). The decrease in tumor proliferation in the animals administered Ang-(1–7) also was reflected in the reduction in size as compared to the tumors from saline-treated mice (1.31 ± 0.12 g versus 2.45 ± 0.23 g; n = 6, P < 0.05). An associated decrease in both cyclooxygenase 2 (COX-2) and prostaglandin E synthase (PGES-1) mRNA was observed in tumors from Ang-(1–7)-injected mice when compared to saline-treated controls (COX-2: 1.00 ± 0.06 versus 0.55 ± 0.07 relative gene expression; PGES-1: 1.03 ± 0.08 versus 0.40 ± 0.06; n = 6, P < 0.05). A time-dependent reduction in both COX-2 and PGES-1 mRNAs also was observed in parent U87 cells following treatment with 100 nM Ang-(1–7) for up to 8 hours. The heptapeptide had no effect on COX-1 or PGI synthase mRNA in xenograft tumors or U87 cells, suggesting that Ang-(1–7) may decrease the concentration and alter the ratio of proliferative and anti-proliferative prostaglandins to reduce GBM growth. In addition, a marked decrease in vascular endothelial growth factor (VEGF) mRNA was demonstrated in the human U87 xenografts from mice administered Ang-(1–7) compared to tumors from the saline control animals (1.05 ± 0.07 versus 0.59 ± 0.09; n = 6, P < 0.05). Furthermore, 4-hour and 8-hour treatment of the U87 parent cells with 100 nM Ang-(1–7) also caused a 25% and 50% reduction, respectively, in VEGF mRNA, suggesting that the heptapeptide may reduce tumor growth by inhibiting angiogenesis. Taken together, these data suggest that Ang-(1–7) may be a first-in-class compound for the treatment of GBM, providing combination therapy as a selective COX-2/PGES-1 and angiogenic inhibitor, targeting a specific AT(1–7) receptor mas.

ET-37. TARGETED OPENING OF THE BLOOD BRAIN BARRIER BY PHOTOCHEMICAL INTERNALIZATION

Henry Hirschberg¹, Michelle Zhang², David Chighvinadze², H. Michael Gach³, Francisco Uzal⁴, Even Angell-Petersen⁵, and Steen Madsen⁶; ¹Beckman Laser Institute, Irvine, CA, USA; ²Health Physics, University of Nevada, Las Vegas, NV, USA; ³Research Imaging Facility, Nevada Cancer Institute, NV, USA; ⁴University of California, Davis, CA, USA; ⁵Norwegian Radium Hospital, Norway; ⁶Health Physics, University of Nevada, Las Vegas, NV, USA.

INTRODUCTION: Eradication of the cells infiltrating normal brain from malignant brain tumors poses a significant clinical challenge that is unlikely to be solved using conventional treatment regimens. This is due to the fact that these migratory cells are protected by the blood-brain barrier (BBB), which prevents the delivery of anti-tumor agents. We have evaluated the ability of photochemical internalization (PCI) to selectively open the BBB in a localized tissue volume. This would permit access of chemotherapeutic agents to brain tumor cells residing in the resection cavity wall but limit their penetration into normal brain remote from the site of illumination. PCI is a new technology to improve the use of macromolecules in cancer therapy in a site-specific manner. The concept is based on the use of specially designed photosensitizers that localize preferentially in the membranes of endocytic vesicles. Upon light activation, the photosensitizer interacts with ambient oxygen, causing vesicular membrane damage and releasing encapsulated drug into the cell cytosol. In this way, macromolecules encapsulated by the vesicles will reach the cytosol and exert their biologic activity instead of being degraded by lysosomal hydrolases. The PCI-based relocation and activation of the macromolecules has the advantage of minimal side effects since the effect is localized to the area exposed to light. MATERIALS AND METHODS: PCI treatment was performed employing the photosensitizer AlPcS2a for the PCI-mediated delivery of Clostridium perfringens prototoxin (Cl p) into the capillary endothelial cells of the BBB. Cl p is an agent known to open the BBB. Nontumor-bearing inbred Fisher rats were treated with a sublethal light fluence level and a nontoxic dose of Cl p given either intraperitoneally or directly into the brain. Post-contrast T1 magnetic resonance imaging scans were used to monitor the degree of BBB disruption, which can be inferred from the intensity and size of the contrast agent visualized. F98 tumor cells were implanted into the brains of some animals, and these were treated 24 hours later with PCI-mediated BBB opening followed by intravenous administration of bleomycin. RESULTS: PCI delivery of Cl p at fluence levels of 1J demonstrated significant contrast in a tissue volume surrounding the point at which light was delivered. No effect on the BBB was observed if 1J of light was given in the absence Cl p or with Cl p and photosensitizer in the absence of light. The survival of animals implanted with F98 tumor cells was significantly extended following PCI BBB opening and bleomycin therapy compared to nontreated controls. CONCLUSION: PCI-delivered Cl p was highly effective in opening the BBB in a limited region of the brain with either intracranial or intraperitoneal administration. Intraperitoneal administration opened the BBB in a larger volume and for a longer time interval compared to intracranial injection. PCI-mediated BBB opening clearly increased the efficacy of bleomycin therapy in tumor-bearing rats.

ET-38. COMBINED MODALITY THERAPY WITH RADIATION, TEMOZOLOMIDE, AND PI3-KINASE INHIBITORS FOR THE POTENTIAL TREATMENT OF MALIGNANT GLIOMAS

Gautam Prasad¹, Sabine Mueller¹, Xiaodong Yang², C. David James¹, Mitchel Berger¹, Michael Prados¹, and Daphne Haas-Kogan¹; ¹University of California, San Francisco, San Francisco, CA, USA; ²CA, USA.

PURPOSE: Targeting phosphoinositide 3-kinase (PI3K) is a promising approach to the treatment of gliomas. Demonstrating a synergy of radiation and temozolomide is a key step toward producing an experimental agent with the potential to impact survival in glioma patients. We sought to evaluate the ability of targeted inhibition of PI3K/mTOR to cooperate with radiation therapy and temozolomide and induce synergistic anti-neoplastic effects in well-characterized glioma xenograft cell lines. METHODS: We used human GBM specimens that have been serially passaged in nude mice and that have known well-characterized genotypes. These xenografts maintain amplification and overexpression of epidermal growth factor receptor (EGFR) when it is present in the resected primary tumor. Cell viability in vitro was assessed using the CellTiter-Glo assay (Promega), which quantifies ATP in viable cells. One day after plating xenograft cells in 96-well plates (6 identical samples per condition), we added Exelixis XL765, a dual PI3K/mTOR inhibitor, and/or temozolomide to the cells followed by 8 Gy of ionizing radiation (XRT) administered 30 minutes later using a Cesium-137 irradiator. Samples were then incubated for 72 hours prior to analysis. Viability was tested after treatment with a single agent (either XL765, temozolomide, or XRT), two agents (in all combinations), or tri-modality therapy. RESULTS: Seven xenografts with various EGFR and PTEN genotypes were treated with escalating doses of XL765 administered as a single agent. XL765 showed anti-neoplastic activity in all xenografts examined, with IC50 values ranging from 1.5 µM to 10 µM. The lowest IC50 (1.5 µM) was observed in an EGFR wild-type (wt) amplified, PTEN wt xenograft, and the highest IC50 (10 µM) was observed in an EGFR wt amplified, PTEN null xenograft. Three xenografts, GBM6 (EGFRvIII, PTEN wt), GBM8 (EGFR wt, PTEN null), and GBM14 (EGFR null, PTEN null), were tested using tri-modality therapy. In GBM6 and GBM14, tri-modality therapy was superior to treatment with any single agent or combination of two agents (P < 0.05 for all comparisons). In GBM8, tri-modality therapy was superior to treatment with any single agent, XL765+XRT, or temozolomide+XRT (P < 0.05 for all comparisons), but it was not significantly different than XL765+temozolomide. CONCLUSIONS: XL765, a dual PI3K/mTOR inhibitor, showed anti-neoplastic activity as a single agent in vitro against gliomas. Encouraging activity was observed when it was combined with either radiation or temozolomide and when administered as part of a tri-modality therapy. The effects of molecular genotype on response to XL765 are being explored further.

ET-39. IMMUNE AND GENE THERAPIES FOR CANCER METASTATIC TO THE BRAIN

Amy Lin¹, Michelle Hickey¹, Barbara Mueller¹, Kazunori Haga², Noriyuki Kasahara², and Carol Kruse¹; ¹Sidney Kimmel Cancer Center, San Diego, CA, USA; ²David Geffen School of Medicine, Los Angeles, CA, USA.

Patients with advanced cancers of many types, including lung, breast, skin, and prostate, often exhibit brain metastases at late stage. Current treatments for brain metastases are ineffective, making the need for new therapeutic strategies most urgent. An immunotherapy strategy employing intracranial placement of alloreactive cytotoxic T lymphocytes (alloCTL) that recognize the human leukocyte antigens (HLA) of the tumor host has shown promise preclinically in a rat model utilizing 9L gliosarcoma. Additionally, a gene therapy approach utilizing prodrug therapy following tumor transduction with tumor-selective replication competent retroviral (RCR) vectors carrying a suicide gene also showed promise preclinically in a solid tumor model. We will present our findings of each individual therapy in other models of metastatic brain cancer. The functionalities of human alloCTL and RCR vectors were evaluated in multiple cancer cell lines in vitro. BCM2 BG2 cells with known propensity to migrate to the brain expressed HLA that could be further upregulated upon exposure to interferon-gamma. Human alloCTL, generated by mixed lymphocyte tumor reaction using irradiated BCM2 BG2 cells, displayed cytotoxicity to the relevant target cells in a HLA-restricted manner. With RCR vectors, we showed that those with a GFP marker gene were able to infect and replicate in BCM2 BG2 cells, and those that were chronically infected with RCR vector maintained cell surface HLA expression. These data suggest that alloCTL are functionally capable of recognizing and injuring cancer cells capable of forming brain metastases and that RCR vectors retain their functional capacity to spread within those cancer cells. We will present our findings with the individual treatment modalities and the combination of the two in vivo against single and multiple metastatic cancer foci in the brain.

ET-40. METRONOMIC CHEMOTHERAPY FOR RODENT GLIOMA MODEL

Akihide Kondo¹, Marcelo Soares², Veena Rajaram³, Stewart Goldman⁴, and Tadanori Tomita⁵; ¹Children's Memorial Research Center, Chicago, IL, USA; ²Children's Memorial Research Center, IL, USA; ³Children's Memorial Hospital, Plaza, IL, USA; ⁴Chicago, IL, USA; ⁵Children's Memorial Hospital, Chicago, IL, USA.

OBJECTIVE: Low-dose continuous chemotherapy (ie, metronomic chemotherapy) has been a focus of clinical trials, as it may work for tumors that have acquired resistances to various agents, and it may have potentially favorable toxicity profiles. However, the literature has not clearly shown efficacy or histologic response with this type of therapy for patients with brain tumors, especially in protocols using a single, common chemotherapeutic agent. In this experiment, we investigated the time course of tumor growth and associated histologic changes in a rodent glioma model using low-dose and long-term administration of carboplatin. METHODS: To visualize tumor responses to metronomic sequential chemotherapy, we used an in vivo biologic imaging technique with a rat gliosarcoma model. This rodent and cell line have had a role of preclinical experimental modeling for human glioblastoma. Rat 9L gliosarcoma cells stably expressing the luciferase gene after genetic procedures were implanted in the rat right striatum. Animals were randomly divided to 4 groups. Carboplatin was administrated intraperitoneally to group 1 (4 mg) and group 2 (2.4 mg) daily for 28 days. Group 3 received intraperitoneally administrated carboplatin (10 mg) every 2 weeks for 28 days, and rats in group 4 had no treatment. Tumor growth was observed by luminescence strength 15 minutes after intraperitoneal injection of luciferine potassium salt using a Xenogen system (Caliper Life Science, Hopkinton, MA), and these signals were tracked for response to each chemotherapy once or twice a week. To observe the vascularity of tumors and proliferation of tumor cells, immunohistochemical assessment was carried out on brain specimens, which were excised from rats after euthanasia at certain periods. RESULTS: Distinguishing growth curves for tumors were observed in corresponding chemotherapy groups. Immunohistochemical assessment revealed a significant differences in the vascularity of tumors between the control group and the metronomic therapy groups. CONCLUSION: We found that metronomic chemotherapy using carboplatin could have a potential ability to control tumor growth in our rodent glioma model.

ET-41. MOTEXAFIN GADOLINIUM ENHANCES ALA-PDT EFFICACY IN HUMAN GLIOMA SPHEROIDS

Steen Madsen¹, Marlon Mathews², Rogelio Sanchez³, Chung-Ho Sun⁴, Even Angell-Petersen⁵, and Henry Hirschberg⁶; ¹Health Physics, University of Nevada, Las Vegas, Las Vegas, NV, USA; ²University of California, Irvine, Orange, CA, USA; ³Beckman Laser Institute, University of California, Irvine, CA,; ⁴Beckman Laser Institute and Medical Clinic, University of California, Irvine, Irvine, CA, USA; ⁵Norwegian Radium Hospital, Norway; ⁶Beckman Laser Institute, University of California, Irvine, Irvine, CA, USA.

The prognosis for patients with glioblastoma multiforme (GBM) is poor. Even with the best available treatments, consisting of surgical resection followed by postoperative fractionated radiotherapy combined with temozolomide chemotherapy, median survival is less than 15 months. Since approximately 80% of patients develop tumor recurrence within 2 cm of the original tumor site, the aim of postoperative therapy is to eliminate the nests of tumor cells remaining in the margins of the resection cavity. Photodynamic therapy (PDT) has been investigated as a postoperative treatment following surgical tumor resection. PDT is a local form of treatment in which a tumor-localizing photosensitizer is activated by light of a specific wavelength. The resultant photochemical and photobiological events result in singlet oxygen-induced photodamage to tumor tissues. PDT with endogenous protoporphyrin IX (PpIX), a potent photosensitizer induced by the exogenous administration of 5-aminolevulinic acid (ALA), has been established for routine clinical treatments of several pre-malignancies and cancers. The rapid attenuation of light in the brain is a significant limitation of PDT, since it constrains the effective treatment depth to the immediate vicinity of the resection margin. One approach to extending treatment volumes is to use redox active drugs that enhance the efficacy of PDT, thus lowering the light fluence required to obtain a cytotoxic effect. Motexafin gadolinium (MGd) is an expanded porphyrin that has been shown to enhance the cytotoxic potential of ionizing radiation by causing redox stress in tumor cells. This compound was evaluated as a potential PDT-enhancing agent in an in vitro system consisting of human glioma spheroids. Specifically, the ability of MGd to potentiate ALA-PDT toxicity was evaluated at low light fluences using spheroid growth and migration assays. Results from the present work showed that MGd was capable of enhancing the cytotoxic effects of ALA-PDT. The combination therapy resulted in significant growth suppression compared to ALA-PDT-treated spheroids. In addition, the combination of MGd and ALA-PDT resulted in an enhanced inhibitory effect on cellular migration compared to ALA-PDT. In some cases, near complete inhibition of cellular migration was observed. These studies suggest that MGd may be useful as a PDT enhancer in the management of brain tumors. This bears further investigation in animal models.

ET-42. NOVEL REPLICATION-COMPETENT HERPES SIMPLEX VIRUS AND THERAPY OF EXPERIMENTAL GLIOBLASTOMA MULTIFORME

Costas Hadjipanayis¹, Revaz Machaidze², Erwin Van Meir³, and Neal Deluca⁴; ¹Emory University School of Medicine, Atlanta, GA, USA; ²GA, USA; ³Emory University, Atlanta, GA, USA; ⁴PA, USA.

Herpes simplex virus 1 (HSV-1) recombinant vectors are currently being tested in the therapy of patients with glioblastoma multiforme (GBM). The ability to target rapidly dividing tumor cells while sparing normal brain forms the basis of viral-based therapy of brain cancer. We describe a novel replication-competent HSV-1 construct, d8-10, with a gene deletion restricting growth of the virus in neurons while permitting its growth in dividing cells. The gene deletion corresponds to a polyserine tract present within the immediate-early (IE) gene, ICP4, that is conserved in other neurotropic viruses. ICP4 is essential for HSV-1 replication. Infection of multiple established human GBM cell lines was performed with d8-10 to determine cell survival and growth of the virus. Administration of the virus by convection-enhanced delivery (CED) was performed in immunocompromised mice implanted with intracranial human GBM tumors to determine the effect on tumor growth and efficacy of animal treatment. In addition, animal toxicity was determined after intracranial viral infusion. Harvesting and staining of the brains of animals was performed to determine the pattern of in vivo infection of implanted tumors and the surrounding brain. The d8-10 virus was able to grow effectively and kill multiple established human GBM cell lines over a 6-day period. The d8-10 construct can be administered by CED in mice implanted with GBM. No toxicity occurred in animals and a tumor response was found in treated animals. Animal survival studies were also performed.

ET-43. TUMOR SUPPRESSION THROUGH P53-MEDIATED GALECTIN-3 SECRETION AND APOPTOSIS INDUCTION

Sok-Hyong Lee¹, Fatima W. Khawaja², Abdessamad Zerrouqi¹, Narra Devi³, and Erwin Van Meir¹; ¹Emory University, Atlanta, GA, USA; ²Neurosurgery, Emory University, Atlanta, GA, USA; ³Department of Neurosurgery, Emory University, Atlanta, GA, USA.

Tumor suppressor p53, which is activated by DNA-damaging agents such as ultraviolet light, radiation, and chemotherapy, prevents tumor growth through a variety of mechanisms. We recently found that the activation of p53 can mediate a novel bystander effect, eg, the killing of adjacent cells through the induction of apoptosis. This p53 bystander effect is mediated through the secretion of a soluble factor in the culture medium of wild-type p53 cells and is absent when conditioned media of p53-null cells is used. We determined that the soluble factor is galectin 3 (Gal3) through proteomics, antibody, siRNA neutralization assays, Gal3-deficient cells. Galectins are a family of animal lectins, which bind beta-galactose moieties. Gal3 is the only chimeric family member of galectins and consists of a collagen-like N-terminal region and a C-terminal carbohydrate recognition domain (CRD), which has storing binding affinity for lactose and N-acetyl-lactosamine. We found the apoptotic activity of p53-induced extracellular Gal3: a variety of tumor cell types (malignant glioma, breast, colon, prostate, lung) were sensitive to Gal3-mediated killing, while normal cells (fibroblast, endothelial) were not affected. We demonstrated that conditional sGal3 induction (tet-on system) in pre-established glioma xenografts strongly inhibited tumor growth without overt toxicity in mice. Furthermore, we found that Gal3-mediated tumor cell killing was neutralized with lactose, a Gal3-CRD binding ligand, and that Gal3-mediated apoptosis was inhibited by treatment with a caspase 9 inhibitor (LEHD) but not by a caspase 8 inhibitor (IETD). In summary, our data demonstrate that Gal3 is secreted by the activation of p53 and induces adjacent tumor cell killing through CRD-mediated ligand binding and induction of apoptosis. These findings are important for our understanding of p53 tumor suppressor activity and suggest that Gal3 can be exploited as a therapeutic agent for cancer.

ET-44. SYSTEMIC ADMINISTRATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) ANTIBODY (BEVACIZUMAB) INCREASES THE ANTI-TUMOR ACTIVITY OF TEMOZOLOMIDE AGAINST INTRACRANIAL GLIOMA

Caroline Berenguer¹, Olivier Chinot², Pierre-Marie Martin³, and L'Houcine Ouafik⁴; ¹U911 - CRO2, INSERM, Marseille, France; ²Service de neurochirurgie, Assistance Publique Hôpitaux de Marseille-Université de la Méditerranée, Marseille, France; ³Laboratoire de transfert d'oncologie biologique, Assistance Publique Hôpitaux de Marseille, Marseille, AE (Europe), France; ⁴U911-CRO2 Equipe 4, Université de la Méditerranée / Assistance Publique Hôpitaux de Marseille, Marseille, France.

PURPOSE: Tumor angiogenesis is mediated by tumor-secreted angiogenic growth factors that interact with their surface receptors expressed on endothelial cells. Vascular endothelial growth factor (VEGF) and its receptor play an important role in vascular permeability and tumor angiogenesis. Temozolomide (TMZ) is a DNA methylating agent that has shown promising anti-tumor activity in recent clinical trials against high-grade gliomas, metastatic melanoma, and brain lymphoma. In this study, we tested whether systemic administration of bevacizumab (Avastin), a monoclonal antibody against VEGF, could enhance the efficacy of TMZ against glioblastoma multiforme in the brain. EXPERIMENTAL DESIGN: An orthothopic xenograft of the human U87 cell line (105) was implanted in nude mice with the coordinate (A, +0; L, -3; D, -3). Animals were treated with TMZ (100 mg/kg intraperitoneally) + Avastin (200 mg/mouse) using a schedule of TMZ for 5 days + Avastin 2 times a week or Avastin + TMZ for 5 days + Avastin 2 times a week. The efficacy of drug treatment was assessed by the increase in mouse survival. RESULTS: Combined treatment of TMZ + Avastin significantly increased survival in tumor-bearing mice with respect to untreated controls or to groups treated with either TMZ or Avastin only. Interestingly, systemic administration of Avastin shortly before TMZ administration significantly increased survival compared to TMZ + Avastin. CONCLUSIONS: These data indicate that systemic administration of Avastin significantly enhances the anti-tumor efficacy of TMZ against glioblastoma multiforme.

ET-45. ACTIVITY OF TANDUTINIB (MLN0518) IN XENOGRAFT MODELS OF GLIOMA

Barbara Hibner¹, Natasha Iartchouk², Robbie Robertson³, Ribo Guo⁴, Michael Smith⁴, Daniel Bradley⁴, Jennifer Terkelson⁴, Juliet Williams⁴, and Matthew Silva⁵; ¹Millennium Pharmaceuticals Inc., Cambridge, MA, USA; ²Cancer Pharmacology, Millennium Pharmaceuticals Inc., Cambridge, MA, USA; ³Cancer Pharmacology, Millennium Pharmaceuticals Inc., MA, USA; ⁴MA, USA; ⁵Cambridge, MA, USA.

Dysregulated autocrine and paracrine PDGF stimulation is thought to contribute to both the early transformation events and the maintenance of glioma tumorigenesis and angiogenesis. Virtually all glioma cell lines and surgical specimens express the PDGF A and PDGF B ligands, and PDGF receptors are typically expressed both in both glioma tumor cells and in the endothelial cells of the tumor vasculature. Tandutinib (MLN0518) is a potent, ATP-competitive and reversible inhibitor of Type III receptor tyrosine kinases (RTKs). In vitro, tandutinib inhibits Flt3, cKIT, and PDGFRÒ, with a median IC50 of approximately 30 nM. In cellular assays, tandutinib inhibited the autophosphorylation of these receptors, with an IC50 of approximately 200 nM. Tandutinib is quite selective; of the other receptor tyrosine kinases tested, only CSF-1R was inhibited, with a cellular IC50 of 3.4 µM. Because of its activity against PDGFR and cKIT and its ability to cross the blood-brain barrier, tandutinib may be an effective treatment for glioma. In vitro, tandutinib demonstrates a dose-dependent inhibition of the autophosphorylation of the rat C6 glioma PDGFRÒn by Western blot. Treatment of nude mice bearing the C6 tumor cell line implanted subcutaneously with a single dose of tandutinib delivered by oral (PO), intraperitoneal (IP) or subcutaneous (SC) administration resulted in > 50% inhibition of PDGFRÒ phosphorylation through 10 hours. Tandutinib demonstrated anti-tumor activity when dosed by PO, SC, or IP routes of administration. The maximum anti-tumor effect was achieved with SC dosing on a twice-daily (BID) schedule. This is consistent with mouse PK data, which shows a greater exposure with SC dosing compared to PO or IP dosing. When given for 10 days on an SC BID schedule, the anti-tumor activity in the C6 model was dose dependent, with a tumor growth inhibition (TGI) of 73% for 5 mg/kg, 88% for 12 mg/kg, and 89% for 20 mg/kg. These doses were well tolerated, with minimal body weight loss (< 5%). Tandutinib showed additive anti-tumor effects when given concurrently with temozolomide (TMZ) for 7 days in the C6 model. In this study, tandutinib at 20 mg/kg SC BID gave a TGI of 79%. TMZ at 100 mg/kg PO QD gave a TGI of 62%; both treatments were statistically significant compared to vehicle control. The combination treatment gave an additive TGI of 91%. TMZ treatment was associated with significant body weight loss (14.5%); however, this did not increase with the combination (14%). Tandutinib showed efficacy in the C6 intra-cerebral model, which was monitored in vivo by magnetic resonance imaging (MRI). A 41% reduction in tumor volume was measured in the treated group as compared to vehicle control. Subsequent studies of tumor perfusion and permeability using dynamic contrast-enhanced MRI are being explored to describe the antiangiogenic activity of tandutinib treatment.

ET-46. INVESTIGATION OF A TISSUE-SPECIFIC PROMOTER GENE THERAPY APPROACH FOR TREATMENT OF METASTATIC BRAIN MELANOMA: TYROSINASE PROMOTER-DRIVEN EXPRESSION OF TOXINS (SAPORIN, AMPHINASE, OR SMALL GLOBULAR PROTEIN) SPECIFICALLY TARGETS MELANOMA CELLS

James Wang¹, Mike Chen¹, and Yufang Yin²; ¹City of Hope, Duarte, CA, USA; ²CA, USA.

Brain metastasis is a dreaded occurrence in melanoma patients. Conventional therapies such as surgery, radiation, and chemotherapy have limited efficacy. In this study, we investigate a gene therapy approach that couples a tissue-specific promoter, the tyrosinase promoter, with a toxin. Tyrosinase is expressed in melanoma cells but not in normal brain tissue. Therefore, we hypothesized that a construct with tyrosinase promoter (TyrP)-driven expression of toxins (saporin, amphinase, or small globular protein) will specifically cause suicide of melanoma cells in the milieu of the brain. The effect of adding 1 to 4 enhancer elements (HE) was also investigated. HE-TyrP-toxin, CMV-toxin (positive control), or an empty plasmid were transiently transfected into a panel of 4 human melanoma cell lines and 2 non-melanoma cell lines. Significant cytotoxicity was evident with all three toxins. Constructs with HE-TyrP were cytotoxic only in the melanoma cell lines. Additional HE elements did not significantly increase toxicity or specificity. In conclusion, TyrP-based gene therapy for metastatic brain melanoma appears promising and warrants further investigation.

ET-47. THE PHARMACOKINETICS AND PHARMACODYNAMICS OF THE PDGFR INHIBITOR TANDUTINIB (MLN0518) IN MOUSE XENOGRAFT MODELS OF GLIOBLASTOMA

Arijit Chakravarty¹, Robbie Robertson², Natasha Iartchouk³, Zheng Lu¹, Johnny Yang¹, Juliet Williams⁴, Ribo Guo⁴, Michael Smith⁴, Matthew Silva⁴, Mark Milton⁴, Mark Manfredi⁴, and Barbara Hibner¹; ¹Millennium Pharmaceuticals Inc., Cambridge, MA, USA; ²Cancer Pharmacology, Millennium Pharmaceuticals Inc., MA, USA; ³Cancer Pharmacology, Millennium Pharmaceuticals Inc., Cambridge, MA, USA; ⁴Cambridge, MA, USA.

The prognosis for patients with glioblastoma remains bleak, despite numerous clinical trials in the past 20 years. The molecular heterogeneity of glioblastoma tumors and the frequent development of resistance pose particular challenges to the treatment of this disease. Because gliomas are highly vascularized and depend on new blood vessels for an adequate blood supply, targeting the process of angiogenesis provides an attractive alternative to the direct targeting of tumor cells. Tandutinib (MLN0518) is an ATP-competitive and reversible inhibitor of Type III receptor tyrosine kinases (RTKs). In vitro, tandutinib inhibits Flt3, cKIT, and PDGFR, with a median IC50 of approximately 30 nM. In cellular assays, tandutinib inhibited the autophosphorylation of these receptors, with an IC50 of approximately 200 nM. Tandutinib is quite selective; of the other receptor tyrosine kinases tested, only CSF-1R was inhibited under these test conditions, with a cellular IC50 of 3.4 µM. Due to tandutinib's activity against PDGFR and cKIT, both of which are implicated in angiogenesis, tandutinib may be an effective treatment for glioma. Preclinical studies presented here demonstrated that the concentration of tandutinib in the tumor was 15- to 30-fold greater than the corresponding plasma concentration of tandutinib in a subcutaneous (SC) nude mouse model of rat C6 glioma. After a single dose, the concentrations of tandutinib in the mouse plasma and brain were in the same range, with evidence of brain accumulation over time. These data suggest that tandutinib is able to cross the blood-brain barrier in mice. The pharmacodynamics of tandutinib in the SC rat C6 glioma model were evaluated using quantitative Western blot analysis of PDGFR-beta phosphorylation. Tandutinib potently inhibited PDGFR-beta autophosphorylation for 4–8 hours after a 20 mg/kg SC dose, with a gradual return to baseline levels by 24 hours post-dose. Tandutinib suppressed tumor growth in the SC rat C6 glioma model with doses of 12 mg/kg and 20 mg/kg BID for 10 days given SC. In in vitro proliferation tests, however, tandutinib did not inhibit the proliferation of the C6 cell line, suggesting that tandutinib is acting as an antiangiogenic therapy in this model. Tandutinib also inhibited phosphorylation of PDGFR after a single dose at 20 mg/kg SC in an intracranial model of rat C6 glioma. Semi-compartmental modeling was used to determine the IC90 of the pPDGFR inhibition in the SC rat C6 glioma model. The plasma concentrations observed at the efficacious dose level were consistent with the predicted IC90/IC50 values.

ET-48. ACTIVE FURIN PROTEASE LEVELS MAY BE A BETTER PREDICTOR OF GBM CELLS' SENSITIVITY TO MEMBRANE RECEPTOR-TARGETED RECOMBINANT CYTOTOXINS THAN THE LEVELS OF A TARGETED RECEPTOR

Tiefu Liu¹ and Waldemar Debinski¹; ¹Neurosurgery, Society for Neuro-Oncology, Winston-Salem, NC, USA.

Molecularly targeted recombinant cytotoxins containing bacterial toxins have demonstrated potential to improve the outcome of patients with GBM. For example, an interleukin 13 (IL13)-based cytotoxin containing a derivative of Pseudomonas exotoxin A, hIL13-PE38QQR, demonstrated prominent and highly significant prolongation of progression-free survival when compared with standard of care in patients with recurrent GBM. In general, cell killing by the recombinant cytotoxins is approximately proportional to the levels of the targeted receptor. However, there is a considerable discrepancy between the IC50s for the cytotoxins seen in GBM cells and the number of targeted receptor sites. In our recent studies, we noted that the levels of furin, a protease that is responsible for intracellular maturation of the toxin portion in a cytotoxin, might better correlate with the cytotoxin's effect. Thus, we have examined a panel of nine established cell lines, including 6 human GBM cell lines (U-251, U-373, SNB-19, A-172, DBTRG, and T98G), murine glioma cell lines, and a normal glial cell line. These cells exhibit varying expression levels of IL13RÑ2, a target receptor for IL13-based cytotoxins in GBM cells. In the present study, we found that these cell lines also exhibited varying levels of activated furin. We treated these cells with a new generation of mutated IL13-based diphtheria toxin fusion protein (DT-IL13QM) to establish the IC50s in these cells. The cells were either non-responders (3 cell lines) or were susceptible to the cytotoxin, with the IC50s spanning two logs of cytotoxin concentrations. We analyzed the levels of IL13RÑ2 and furin in all the cells by Western blotting. We found a statistically significant correlation between activated furin levels and the responsiveness to the cytotoxin (P = 0.028). Our data suggest that in addition to the necessary presence of a targeted receptor for a recombinant cytotoxin in GBM cells, the level of activated furin is an important predictor of the cytotoxin's killing effect. This finding suggests that susceptibility to the cytotoxin also depends to a large extent on efficient processing of the toxin molecules in the endocytic compartment before release into the cytosol of a cell. Our findings raise an interesting possibility of upregulating the activity of furin during the treatment of GBM with recombinant cytotoxins to increase their anti-tumor efficacy and/or adjusting the amount of administered cytotoxins according to the patient's own furin activity.

ET-49. CYTOTOXICITY OF SORAFENIB IN MELANOMA CELL LINES SUGGESTS A POTENTIAL ROLE FOR CONVECTION-ENHANCED DELIVERY OF SORAFENIB INTO METASTATIC BRAIN MELANOMAS

Yufang Yin¹, James Wang¹, Richard Jove², and Mike Chen¹; ¹City of Hope, Duarte, CA, USA; ²City of Hope, Duarate, CA, USA.

Metastatic melanoma of the brain is resistant to standard therapies and is associated with a very poor prognosis. Our aim in this study was to determine, in vitro, the feasibility of treating metastatic brain melanomas with convection-enhanced delivery of sorafenib and concurrent systemic chemotherapy. Convection-enhanced delivery is a form of direct intraparenchymal injection in which bulk flow is generated in extracellular channels to distribute therapeutic agents such as sorafenib. Sorafenib, normally thought of as a multikinase inhibitor, has recently been shown to disrupt stat3 dimerization. Stat3, a transcription factor associated with immunomodulation and proliferation, is constitutively active in malignant melanomas, particularly those in the brain. Inhibiting stat3 in melanoma tumor models induces tumor cell death and tumor regression. Therefore, we hypothesized that sorafenib would result in cell death and that combining sorafenib with another chemotherapeutic agent would be synergistic. The expression of total and phospho stat3 in 4 different melanoma cell lines and in normal brain tissue was determined. The toxicity of sorafenib alone and in combination with temozolomide or melphalan was tested in the melanoma cell lines A2058, HTB72, HTB65, and B16. Analysis of stat3 expression indicated that stat3 was expressed and phosphorylated in the melanoma cell lines, whereas there was little stat3 expression in normal brain tissue. Treatment with sorafenib alone resulted in significant cytotoxicity. Sorafenib in combination with temozolomide or melphalan resulted in cell line-specific synergism that was modest. Collectively, these data suggest that sorafenib warrants further investigation for direct delivery in metastatic brain melanomas.

ET-50. A WINDOW FOR THERAPY: TIE2 AS A NEW ACCOMPLICE IN THE MULTIDRUG-RESISTANCE PHENOTYPE OF GLIOMAS

Vanesa Martin¹, Marta Alonso¹, Jing Xu¹, Dan Liu¹, Juan Fueyo¹, and Candelaria Gomez-Manzano¹; ¹ The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.

Malignant gliomas invariably recur after therapy by exhibiting a multidrug-resistance phenotype. The identification of brain tumor stem cells (BTSCs) has been a defining moment in cancer research. According to the BTSC model, those cells exhibit the capacity to repopulate tumors after treatment. Therefore, eradication of both tumor bulk and the cancer stem-cell compartment may be essential to achieve stable, long-lasting remission and even a cure. In order to efficiently target gliomas, the mechanisms of drug resistance in both the tumor bulk and the BTSCs need to be elucidated. We have previously identified abnormal high expression of the Tie2 receptor in non-stromal cells within glioma tumors in correlation with tumor malignancy. Here we also described the expression of Tie2 in glioma-derived stem cells. Several lines of evidence indicate that Tie2 promotes chemoresistance in normal stem cells, mediating the repopulation of the bone marrow after genotoxic stress. Regarding this, Tie2 expression in both tumor and BTSCs in gliomas suggests a possible role of Tie2 in the chemoresistance of these tumors. Thus, we studied the role of Tie2 activation in drug resistance by testing the cytotoxicity of several chemotherapeutic drugs in a panel of human glioma cells and Tie2-expressing BTSCs. Human glioma cells expressing Tie2 and Tie2-expressing BTSCs were more resistant to a panel of chemotherapeutic agents with different mechanisms of action upon stimulation with angiopoietin 1 (Ang1), the natural Tie2 ligand, while Tie2 negative cells didn't show any prosurvival response under Ang1 stimulation. The chemoresistance of those cells was Tie2-dependent, as demonstrated by experiments in downmodulation of Tie2 by siRNA or by transferring Tie2 cDNA to Tie2-negative cells. We further showed that Tie2 pathway activation regulates the expression of ABCG2 and MRP2/ABCC2 transporters. However, no modification in the levels of MDR1/P-glycoprotein or MRP1/ABCC1 was observed. Functional studies showed that the Tie2 active pathway was related with drug efflux, assessed by using selected substrates and inhibitors of the ATP-binding cassette transporters. Of interest, inhibition of PI3K/Akt activity partially abrogated Tie2-mediated upregulation of ABCG2 and ABCC2. These results were consistent with induction of Akt phosphorylation upon Ang1 stimulation in Tie2-expressing cells. Taken together, our results indicate that Tie2 activation may play an important role in multidrug resistance in gliomas through Akt activation and upregulation of ABC transporters, both in the tumor bulk and in BTSCs, opening a new field for the search of more effective therapies to avoid until-now-inevitable brain tumor recurrence.