Marc H. Symons - US grants

[unreadable] DESCRIPTION (provided by applicant): The migratory and invasive properties of tumor cells are critical for metastasis, which is the main cause of treatment failure for cancer patients. The signaling mechanisms that control cell migration and invasion largely remain to be elucidated however. The long-term goal of this proposal is to elucidate the signaling pathways that are activated by the small GTPase Rac1 in the control of cell migration and invasion and to use this information to identify novel drug targets for cancer therapy. The phosphatidylinositol phosphatase synaptojanin 2 (SJ2) is a novel Rac effector that is required for the formation of lamellipodia and invadopodia and for tumor cell migration and invasion. Furthermore, SJ2 binds to Grb2 and cortactin, two proteins that have been implicated in the control of the actin cytoskeleton. Grb2 activates N-WASP, which stimulates actin nucleation, whereas cortactin promotes actin filament branch formation. The objective of this application is to determine the molecular mechanisms that underlie the role of SJ2 in lamellipodia and invadopodia formation and tumor cell migration and invasion. The central hypothesis of this proposal is that SJ2 contributes to tumor cell migration and invasion by acting downstream of Rac1 to coordinate the activities of N-WASP and cortactin. To accomplish the goals of this application, the following specific aims will be pursued: 1) To determine whether Rac1 regulates SJ2 by localizing SJ2 to lamellipodia and invadopodia. 2) To test the hypothesis that SJ2 regulates cell migration and invasion and the formation of lamellipodia and invadopodia by stimulating cortactin-dependent actin filament branching. 3) To test the hypothesis that synaptojanin 2 regulates cell migration and invasion and the formation of lamellipodia and invadopodia by regulating Grb2/N-WASP-dependent actin nucleation. The molecular analysis of the functions of SJ2 in the formation of lamellipodia and invadopodia and tumor cell migration and invasion will significantly expand our current understanding of the role of Rac1 in malignant transformation. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Glioblastoma multiforme is an extremely aggressive cancer, with a mean patient survival time of less than one year. A critical problem in the treatment of these brain tumors is the extensive infiltration of individual tumor cells into adjacent brain tissue. These invading cells are highly resistant to radiation and chemotherapy and currently, there are no anti-invasive therapies available. Members of the Rho family of small GTPases contribute to glioblastoma cell invasion in vitro. The long-term goal of this proposal is to dissect Rho GTPase-mediated signaling pathways that are critical for glioma invasion and to use this information to identify novel drug targets for therapeutic intervention against brain tumor dispersal. Rho proteins are activated by guanine nucleotide exchange factors (GEFs). The objective of this proposal is two-fold: to identify in a genome-wide fashion RhoGEFs that are necessary for glioma invasion and to provide clinical information about the activation state of these RhoGEFs in brain tumor tissue. The central hypothesis of this proposal is that glioblastoma dispersal is caused in large part by the hyperactivation of a relatively small subset of RhoGEFs. To accomplish the goals of this application, the following specific aims will be pursued: 1) to determine in a genome-wide fashion which RhoGEFs are required for glioma invasion in vitro. RhoGEFs that contribute to glioma invasion in vitro will be identified by screening a focused library of small interfering RNAs (siRNAs) directed against all RhoGEFs in the human genome using a 96-well format invasion assay. RhoGEF hits will be validated using an organotypic ex vivo brain slice invasion assay. 2) To determine which of the invasion-validated RhoGEFs are hyperactive in malignant versus low grade glioma using a novel RhoGEF pull-down assay. RhoGEF expression levels during glioma progression will also be determined using immunohistochemistry or in situ hybridization. In addition, using a novel assay that monitors the activation state of specific Rho GTPases in tissues, the hypothesis will be tested that at least some Rho GTPases are hyperactive in malignant versus low grade glioma. It is expected that the approaches outlined in this proposal will identify a significant number of RhoGEFs as novel glioma invasion genes. It also is anticipated that this study will yield significant clinical information on these RhoGEFs that will allow prioritization of these invasion genes for animal model and in depth mechanistic studies. This application addresses a key goal of the Brain Tumor Progress Review Group, conducted in 2000 by the NINDS and NCI, namely to increase our understanding of brain tumor dispersal, which is a critical problem in the treatment of brain tumors. We anticipate that the proposed research will identify a number of invasion genes that represent novel potential therapeutic targets. We also expect that we will obtain significant clinical information that will aid in the validation of these invasion genes. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This proposal is a request for a Leica LMD7000 laser capture microdissection system. This instrument allows for the isolation of single cells or small groups of cells from thin sections of frozen or formalin-fixed paraffin-embedded archival tissue, with the purpose of collecting RNA, DNA or proteins. It enables the investigator to analyze a specific cell population out of a complex tissue environment, with applications including DNA genotyping, RNA transcript profiling, proteomics and signal transduction analysis. This proposal features highly clinically relevant projects from 15 NIH-supported scientists. These projects include a number of studies that focus on the role of cytokines and the endothelium in the control of sepsis, several cancer/hyperplasia-related studies on HPV-induced respiratory papillomas, chronic lymphocytic leukemia, brain tumors and other tumors, the regulation of the blood-brain barrier, pulmonary hypertension, glomerulosclerosis and alcohol addiction. A more basic study on B cell differentiation is also included. Thus, the acquisition of the LCM microscope will significantly enhance the outcomes of over 18 NIH-funded grants. Moreover, the LCM technology should contribute significantly to the long-range research goal of The Feinstein Institute: the translation of the understanding of biological mechanisms to novel treatments and diagnostics. In addition, it will strengthen and expand existing collaborations between the Feinstein Institute and the various clinical departments of the North Shore-Long Island Jewish Health System and maximize the use of its Tissue Donation Program. The LCM system will be housed in the Light Microscopy Core Facility (LMCF) of the Feinstein Institute. Maintenance of the instrument and technical guidance and training of investigators will be supported by the current management of the LMCF. The PI of this application, who also is the LMCF supervisor, in conjunction with the Light Microscopy and Core Services Advisory Committees, will oversee the utilization of the LCM system. LCM-based efforts will be further supported by a pathologist at the North Shore University Hospital, who serves as a consultant, in addition to a number of other collaborating pathologists. A financial plan is in place to recover maintenance costs. Utilization of the LCM system by NIH-funded investigators will be prioritized and efforts will be made to introduce LCM technology and its application to new investigators.