Candidate: The Principal Investigator of this Research Career Award is an Assistant Professor of Neurosurgery at the University of Pittsburgh. The purpose of this K08 award is to provide the PI with sufficient training in molecular biology to transition from a mentored to an independent clinician scientist. Both the Pl's research interests and clinical practice focus on translational brain tumor research. Environment: Research will be performed at the University of Pittsburgh, within laboratory space dedicated to the PI, augmented by resources and space provided by the project mentor. Project collaborators are drawn from the University of Pittsburgh and outside institutions. The training plan involves formal training in bioinformatics, molecular biological techniques, animal models of cancer, and translational research. Research Plan: The project will identify glioma endothelial marker genes (GEMS) that are up-regulated in brain endothelium in response to signaling from brain tumors. Candidate GEMS are identified through Serial Analysis of Gene Expression (SAGE), a quantitative technique to measure mRNA expression levels, on endothelial cells purified from clinical brain tumor and normal brain specimens. Candidate GEMS determined from SAGE are further examined against a panel of representative human brain tumor specimens using in situ hybridization and immunohistochemistry. GEMS that are up-regulated within the endothelium across a spectrum of brain tumors are investigated for biological relevance. GEMS expression is quantitatively assessed using 3-D fibrin gel models of in vitro angiogenesis and in vivo using human brain tumor xenografts in athymic mice. Using these models, expression of individual GEMS are silenced using expression plasmids coding for short-hairpin RNA (shRNA) targeted against the gene of interest. The biologic response to GEMS inhibition is measured by MTT assay, TUNEL, microtubule formation, tumor xenograft growth, and vascular index. GEMS whose inhibition blocks angiogenesis are suitable for preclinical testing as gene therapy targets using the shRNA constructs developed as part of the project or as targets for small molecule inhibitors. In this fashion, using bioinformatics and high throughput screening, the project will elucidate new genes (GEMS) associated with brain tumor angiogenesis and rapidly adapt these discoveries to design novel therapies for malignant glioma. ? ?
