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SPORE in Brain Cancer Abstract University of Alabama at Birmingham G. Yancey Gillespie, Ph.D., Principal Investigator Revised Summary In spite of over thirty years of effort, the prognosis for patients afflicted with malignant gliomas remains dismal. It is the purpose of this Brain Cancer SPORE to have a positive impact on this unacceptable situation by translating the laboratory-based efforts of four groups of scientists into clinical protocols that address the needs for more effective treatments. The themes that will be investigated in this SPORE include: Anti-Invasion Strategies, Glioma-Host Interactions, Viral and Gene Therapy, and Anti-Angiogenesis Strategies. Four research projects are proposed, each of which has a translational component that has or is expected to lead to a clinical research protocol. These include: 1) "Interferon-Mediated Suppression of MMP-9 Gene Expression and Function in Gliomas" which will examine the role of cytokines and signal transduction pathways in regulating secretion of a pro-invasive enzyme; 2) "Ion Channels and Transporters as Novel, Glioma-specific Targets", which will examine how abnormal functioning of glioma ion channels can be potentially manipulated to develop new treatment modalities, 3) "Viral and Molecular Chemotherapy of Malignant CNS Tumors", which will utilize the extensive expertise in virology available at UAB to develop new gene and viral vector therapies for gliomas, and 4) "The Role of TSP-1 and -2 in the Biology of Gliomas", which will investigate how to utilize fragments of the protein thrombospondin as anti-angiogenic agents. These projects will be supported by five Cores: 1) Administrative (Core A); 2) Human Brain Tumor Tissue (Core B); 3) Clinical Trials (Core C); 4) Brain Tumor Animal Core Facility (Core D); and 5) Biostatistics (Core E). In addition, the SPORE has a Career Development Program directed at developing the careers of two young investigators in brain tumor translational research and a Developmental/Pilot Research Program that has funded over 14 projects. This SPORE has strong institutional commitments from both the University and its Cancer Center, and we have begun to develop active collaborative interactions with the three other Brain Tumor SPOREs nationally.
Interferon-Mediated Suppression of MMP-9 Gene Expression and Function in Gliomas
Glioblastoma multiforme (GBM) is the most malignant and common brain tumor. The diffusively infiltrative nature of GBMs is one of the major causes of mortality in patients afflicted with this form of cancer. Studies to assess the invasiveness of glioma cells in vitro have demonstrated a strong correlation between glioma invasion and high levels of matrix metalloproteinase-9 (MMP-9) expression; in this regard, selective inhibition of MMP-9 abrogates glioma cell invasion in both in vitro and in vivo models. Thus, MMP-9 represents an important therapeutic target for treatment of GBMs. Interferons (IFNs) are multifunctional cytokines that have anti-viral, anti-proliferative, anti-angiogenic and immunomodulatory effects. We have made the novel observation that IFN-? and IFN-? potently inhibit MMP-9 gene expression in human glioma cells. We hypothesize that IFNs have an inhibitory influence on glioma cells, leading to the arrest of tumor cell invasion and angiogenesis via the suppression of MMP-9 expression. We are identifying, for the first time, the molecular mechanisms underlying the in vitro inhibitory effects of IFN-? and IFN-?, and are investigating the involvement of two transcription factors, STAT-1? and CIITA, in this response. These data are furthering our understanding of the regulatory mechanisms of MMP-9 gene transcription and identify important therapeutic targets to abrogate MMP-9 expression. Proposed in vivo studies are validating the effectiveness of IFN suppression of MMP-9. The efficacy of IFN-? and IFN-? gene therapy on the growth, invasion and angiogenic properties of human glioma cells transplanted into the brains of immunocompromised mice are being examined. These studies have revealed important relations between expression of inhibitors of STAT activation and various routes by which STATs are regulated in human gliomas. One of these pathways involves NFkB activation in gliomas. Sulfasalazine, a drug commonly used to treat inflammatory bowel disease, inhibits this pathway and its ability to do so in patients with gliomas is the subject of a clinical trial. This combination of in vitro basic science experiments and translational in vivo studies will lead to a comprehensive understanding of the role of important mediators of invasiveness and angiogenesis in glioma cell biology, and our ability to ameliorate this.
The Role Of Glutamate In Glioma Growth
In the normal brain, glial cells extend processes into the perisynaptic space of essentially all excitatory synapses in the brain. This space is narrow, and consequently, glutamate released at synapses can readily reach concentrations of hundreds of micromoles in the synaptic and perisynaptic space. Glial cells express two potent Na+-dependent glutamate transporters, GLAST and GLT-1, which tightly regulate perisynaptic glutamate concentrations. Any significant spillage of glutamate from the synaptic space causes extensive excitotoxic neural injury. Using a combination of biochemical and immunohistological assays we examined expression and function of GLAST and GLT-1 in established human glioma cell lines, including STTG-1, D-54MG, D-65MG, U-373MG, U-138MG, U-251MG, CH-235MG. All glioma cells studied showed impaired glutamate uptake, with maximum transport rates of less than 5% of normal astrocytes. Moreover, rather than removing glutamate from the extracellular fluid, glioma cells released large amounts of glutamate resulting in elevations of [Glu]o in excess of 100 mM within hours in a space which is 103-fold larger than the cellular volume. Exposure of cultured hippocampal neurons to glioma-conditioned medium elicited sustained [Ca2+]i elevations that were followed by widespread neuronal death. Similarly, coculturing of hippocampal neurons and glioma cells, either with or without direct contact, resulted in neuronal death. These data suggest that growing glioma tumors may actively kill surrounding neuronal cells through release of glutamate. This glutamate release may also be, in part, responsible for tumor-associated seizures that occur frequently in conjunction with glioma. Using antibodies to GLAST and GLT-1 we stained acute glioma biopsies and found that GLT-1 expression was completely lost in gliomas. GLAST on the other hand was mislocalized to the cell nucleus. Further studies revealed that the principle pathway for glutamate release by gliomas is through a cystine-glutamate exchanger, identified as system Xc. Interestingly, this exchanger can be inhibited by a group of phenylglycine derivatives including the metabotropic glutamate receptor agonist/antagonist (S)-4-Carboxyphenylglycine (S-4CPG), which potently and selectively inhibits glutamate release from glioma cells and prevents neurotoxicity. Moreover, we have shown that sulfasalazine, a drug used to treat inflammatory bowel disease is an effective blocker of system Xc and induces glioma cell apoptosis. Since our data suggested that neurotoxic release of glutamate by gliomas may be prevented by sulfasalazine or phenylglycine derivatives, we have instituted clinical trials to determine whether these may be useful as adjuvant treatment for brain tumors.
Radiation Enhancement of Viral Molecular Chemotherapy of Malignant Gliomas
This multidisciplinary group of investigators has several years' experience working together designing and characterizing viral vector approaches to gene therapy of malignant brain tumors. A major focus has been producing and testing both non-replicative and replicative adenovirus (Ad) and conditionally replicative herpes simplex virus (HSV) vectors that express foreign gene products within infected tumor cells. These studies have been conducted at both the in vitro and in vivo levels to demonstrate proof-of-principle, safety and efficacy in experimental mouse models of intracranial gliomas. We have conducted Phase I and III clinical trials using retrovirus, Ad and HSV administered intratumorally in patients with malignant gliomas. This project proposes to design and deploy effective viral vector therapies of malignant glioma by utilizing rational combinations of foreign gene-viral vectors, oncolytic virus and irradiation, defined by additive, synergistic or antagonistic interactions determined for these various modalities. Aim 1 seeks to optimize the timing and dose of irradiation to achieve greater viral replication and spread and/or enhanced foreign gene expression in glioma cells and in intracranial experimental gliomas in athymic nude mice. Aim 2 will develop and characterize both replicative HSV and replicative Ad that express the pro-drug converting enzyme cytosine deaminase (CD) and optimize its use in intracranial preclinical models of malignant gliomas in combination with systemic 5-flurocytosine. Other genetic constructs (uracil phosphoribosyl transferase, mutant version of CD) and drugs (dihydropyrimidine inhibitors) that facilitate appropriate 5-FU incorporation into host cell DNA synthesis pathways will also be tested to improve the therapeutic effect. Further, the radiation sensitization properties of 5-FU will be characterized to achieve a greater anti-glioma effect. Aim 3 will combine findings in Aims 1 and 2 to test strategies that rationally combine intratumoral viral vector injection, systemic pro-drug administration and low dose external beam irradiation to achieve the most effective and safe anti-glioma therapy (ies). Aim 4 will translate our findings in preclinical models for brain tumor therapy into pilot and Phase I clinical trials in patients with malignant gliomas. Our findings have led to a currently ongoing Phase I trial to determine the safety of a candidate HSV injected intratumorally followed in 24 hrs with a single fraction of radiation.
Mechanism of Inhibition of Angiogenesis by Thrombospondin-1 and -2
Angiogenesis is characteristic of anaplastic gliomas, and it likely contributes to the rapid growth and highly invasive nature of these tumors resulting in the short survival of afflicted patients. Thrombospondin (TSP)-1 and -2 are downregulated in anaplastic gliomas contributing to the pro-angiogenic tumor microenvironment. The anti-angiogenic effect of TSP-1 and -2 is attributed to their type 1 repeat domain, as recombinant type 1 repeat domain and specific peptides derived from that domain that bind to the CD36 receptor replicate the anti-angiogenic effect. TSP-1 and -2, and type 1 repeat peptides from TSP-1 and -2 that bind to the CD36 receptor are thought to act by inducing apoptosis of activated endothelial cells. Data from other investigators studying non-brain endothelial cells suggests that TSP-1 and type 1 repeat peptides from TSP-1 and -2 induce the so called "extrinsic" or death receptor-mediated pathway of apoptosis. Our preliminary data indicate that TSP-1 and type 1 repeat peptides from TSP-1 inhibit capillary tube formation of human and mouse brain microvascular endothelial cells, and induce apoptosis of these cells in vitro. Furthermore, a type 1 repeat peptide for TSP-1 that binds to the CD36 receptor inhibits tumor growth in an intracerebral xenograft model of malignant astrocytoma, and results in a significant increase in apoptosis in the tumor. Our goal is to dissect the apoptotic signaling pathway(s) induced by TSP-1 and type 1 repeat peptides from TSP-1 that bind to the CD36 receptor. We will achieve this goal by completing the following three aims: 1) determine the mechanism by which CD36 receptor engagement with TSP-1 or type 1 repeat peptides promotes apoptosis of brain microvascular endothelial cells in vitro; 2) determine the in vivo molecular requirements for TSP-1 induced apoptosis of brain microvascular endothelial cells in an intracerebral mouse model of malignant glioma using mice-deficient in specific apoptosis-associated genes; and 3) determine if peptides from the type 1 repeat domain of TSP-1 can be used as therapeutic agents in two animal models of malignant glioma and in a Phase I clinical trial in patients with malignant glioma. These studies will identify new therapeutic agents that enhance pro-apoptotic effects of type 1 repeat peptides from TSP-1 and 2, or specifically promote apoptosis of tumor-associated brain microvascular endothelial cells. Research to date has led to the design and initiation of a dose-escalation Phase I clinical trial of a specific peptide of TSP-1, GDGVITRIR, supplied by Abbott Laboratories, in conjunction with radiation therapy in patients with newly diagnosed GBM.
Administrative Core
The objective of the Administrative Core is to provide centralized management support for the research projects of Specialized Program of Research Excellence (SPORE) in Brain Cancer. The administrative Core will oversee operations and budgetary issues for SPORE investigators. It will coordinate the organization of the various oversight committees that will periodically monitor the progress of individual SPORE projects, including the External Advisory committee and the Executive Committee. It will manage oversight and recruitment activities that are a part of the Developmental Projects and Career Development Programs. The Administrative Core will ensure continued integration of the Brain Cancer SPORE with the UAB Comprehensive Cancer Center. This will occur through: 1) periodic meetings of the Principal Investigator and Co-Investigator with the Cancer Center staff, and 2) coordinating the relocation of the Neuro-Oncology Program to laboratory and administrative space within the Cancer Center. Finally, the Administrative Core will be responsible for supporting interactions with the other SPOREs that are active at UAB, as well as with the other Brain Tumor SPOREs.
Brain Tumor Tissue Core
The Brain Tumor Tissue (BTT) core facility will assist each of the Project Leaders in the SPORE application in their efforts to investigate the role of various molecules (proteins, mRNA, DNA) in brain tumor proliferation, invasion and angiogenesis. The BTT will also function to confirm and validate the role of various therapeutic agents by providing a repository for human normal brain and malignant glioma tissues and cells. In all instances, tissues collected for this tissue bank will be waste tissues considered as not needed for diagnostic purposes and all records will be encoded so that patient confidentiality is assiduously preserved. The principal objective of this core facility will be to obtain and archive fresh tissue samples from brain tumor patients operated at University Hospital (UAB), and The Children's Hospital (TCH) for primary or recurrent disease. It will also perform routine DNA/RNA extractions and PCR/RT-PCR analyses for specific marker and standard housekeeping genes. In addition, quality of archived tissues and cells will be monitored by performing routine histochemical and imunohistochemical studies on tissue preserved in OCT blocks. Tissue collection, archiving, use and results will be fully integrated with a computerized database basic demographic and treatment response data on all brain tumor (or normal brain tissue donor) patients treated and entered into the computerized database using encoding techniques to ensure patient confidentiality. A secondary objective will be to prepare explant cultures of brain tumors and cultures of normal astrocytes and microvascular endothelial cells for project investigators. In addition, the personnel in the core facility will perform a limited number of molecular biology and histologic studies on tissue based on specific genes of specific interest currently investigated by program Project members. Finally, the BTT core facility staff will work with each Project member to utilize our database for correlative analyses based on extent of evlauable treatment response and gene expression.
Clinical Trials Core
The overall purpose of the Clinical Trials Core is to provide the infrastructure and expertise to aid the investigators of the SPORE into translating their laboratory-based findings into clinical protocols. The Specific Aims for this Core are: 1) to provide for optimal support of investigators in this project to undertake clinical research involving patients with CNS malignancies, 2). to provide patients with CNS malignancies and referring physicians access to clinical research treatments developed by investigators of this project, and 3). to facilitate the introduction of protocols derived from the translational research of this SPORE to a national cooperative consortium of brain tumor clinical centers (NABTT). The Core is directed by Dr. L. Burton Nabors, who provides overall leadership and administration of the Core and oversees its interactions with the Comprehensive Cancer Center, the General Clinical Research Center, and other institutional components that can support the Core's operations. He will be aided in this regard by the Clinical Trials Core Committee, whose membership included the Clinical Co-Investigators of each project, the other Core Directors, and members with expertise in radiation oncology and neuropsychology. This committee will meet monthly to review the status of translational progress in each project, and will assist Dr. Nabors in the writing and implementation of clinical protocols resulting from this translational research. Dr. Nabors has acquired Supplemental Grant support from the SPORE Program for one Phase I clinical trial and currently, the Clinical Trials Core is conducting 3 Phase I trials developed from SPORE-based research.
Brain Tumor Animal Models Core Facility
This core facility assists each of the Project Leaders in the SPORE application in their efforts to test, in relevant animal brain tumor model systems, the preclinical safety and efficacy of various therapies designed to achieve an improved anti-glioma effect. This core facility centralizes all animal experimentation associated with this SPORE, standardizing surgical and animal handling techniques and minimizing the chance for trivial interferences that could hamper comparative analysis. Tumor volume, tumor mass and survival statistics are collected where appropriate. Normal and tumor tissues are collected and submitted to each investigator or are processed in this core for gene expression or histopathologic analyses. Project 1 tests the capacity of adenoviral vector-delivered IFN? to modulate MMP-9 expression, glioma migration and angiogenesis in human gliomas in the brains of scid mice. Project 2 determines whether specific inhibitors of ion channel function block human glioma migration and growth in the brains of scid or nude mice. Project 3 defines the ability of low doses of irradiation to synergize with herpesvirus oncolytic activity and adenoviral based-prodrug "molecular chemotherapy" in both human gliomas in nude mice and inducible gliomas in transgenic models. Project 4 uses syngeneic gliomas in immunocompetent mice to determine the function of TSP-1 fragments and human gliomas in scid mice as well as inducible gliomas in a transgenic model to test the capacity of thrombospondin-derived peptides to elicit and effective anti-angiogenic response. The Core coordinates with the 8.5T Small Animal NMR Facility for all NMR imaging and spectroscopic studies of tumor bearing mice involved in these preclinical evaluations. Finally, the Core acquires and evaluates specific transgenic models for suitability for preclinical toxicity and efficacy analyses for each of the modalities proposed by the individual Projects within this SPORE.
Biostatistics/Bioinformatics Core
The primary objective of the Biostatistics Core is to provide centralized statistical services and collaborative research support for the research projects of the Specialized Program of Research Excellence (SPORE) for Brain Cancer. The Biostatistics Core serves as the focal point from which SPORE investigators and career development candidates can draw statistical expertise for the design, management and analysis of their research projects. The specific aims of the Biostatistics Core are to: (1) coordinate and manage statistical activities in the SPORE to ensure that investigators have ready access to statistical consultation and support; (2) provide statistical expertise in study design including endpoint definition, sample size determination and power calculation, randomization procedures, date collection form design, and plans for report generation, interim reviews and final analysis; (3) provide the SPORE; providing state-of-the-art computing facilities with up-to-date software for data management, statistical analysis, and network communications; and developing and managing a website for the SPORE; and (4) provide statistical analysis for SPORE projects using contemporary statistical and computing methodologies. |
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