Tumors are caused by a genetic, epigenetic, and developmental defects. Each may trigger deviation from the original cellular developmental program and subsequent imbalances in patterns of growth, maturation, and proliferation may produce tumorigenesis and tumor progression. Once uncontrolled growth has developed into a clinically apparent tumor mass, amplifying cells often independently generate additional genetic mutations and deletions, with a rapid increase in the number of genetic and epigenetic changes (genetic heterogeneity). At the same time, tumor cells interact with genetically intact, reactive host tissues, with variable histopathological results (morphologic heterogeneity). The combination of genetic and morphologic heterogeneity of tumors, in association with developmental deviations, constrain optimal analysis of the complex tumor state. However, recent advances in molecular and pathological techniques provide tools for gaining greater insight into these problems. We take several approaches to gain insight into tumorigenetic mechanisms: we apply molecular diagnostic tools to study tumorigenesis in several inherited and sporadic human tumor conditions and we are developing new technologies that are tailored to the analysis of complex morphologic arrangements as well as technology for diagnostic and therapeutic applications. Our focus is on translational research that links basic science with the clinic. ? ? Glioma progression and glioma differentiation. Selective tissue microdissection was used to obtain pure populations of tumor cells, which we studied using two-dimensional protein gel electrophoresis (2-DGE) and protein sequencing of select target proteins expressed differentially among tumors to distinguish between the two main categories of glioblastomas (GBMs), de novo and secondary GBMs. We isolated and sequenced 11 unique proteins that were differentially expressed in the primary and secondary GBMs and that produced two distinctive proteomic patterns. Thus, the two patterns of GBMs, primary versus secondary, previously distinguished by clinical and genetic differences, can be recognized at the protein level. This may have implications for prognosis and treatment options. We are using the same approach to identify differentially expressed proteins in different stages of glioma formation, as well as within gliomas with different phenotypic expression.? ? Using the above approach, we have discovered that Nuclear Receptor Corepressor (N-CoR) is overexpressed in glioblastoma multiforma (GBM) cells. N-CoR binds to unliganded nuclear receptors such as the retinoid acid receptor and thyroid hormone receptor. When N-CoR forms a complex with Silencing Mediator of Retinoid and Thyroid Hormone Receptors (SMRT), Histone Deacetylase 3 (HDAC3) and retinoic acid receptor, (RAR), transcription of RAR specific target genes is repressed resulting in increased cell proliferation. We also discovered that oxadaic acid acts synergistically with retinoic acid (RA) to inhibit GBM cell growth and increase cell differentiation. Phoshastase-1 inhibitors such as okadaic acid are known to inhibit N-CoR activity. Oxadaic acid is not specifically targeted to N-CoR and is likely to have significant side-effects. Therefore, we seek more specific phosphatase inhibitors from new chemical compounds that affect cell growth of glioblastoma by particularly targeting the Nco-R pathway, but not others.? ? Recently a CRADA between Lixte Biotechonolgy Inc. and NINDS was executed for studying this novel therapeutic paradigm and developing new small chemical compounds. Work was done studying the inhibitory activity of a series of phosphatase inhibitors including norcantharidin, endothal, endothal thioanhydride, and LB-1, a proprietary drug developed and provided by Lixte Biotechnology, Inc. During the recent weeks, protocols for evaluating the new drugs and combinations of drugs for activities in an animal model of human GBM were jointly planned and developed. During recent weeks, protocols for evaluating the new drugs and combinations of drugs for activities in an animal model of human GBM were jointly planned and developed. The preliminary results of the study show an inhibitory effect on tumor growth of the mouse xeno-grafted human GBM with a new compound, LB-1.? ? ? Developmental biology and tumorigenesis. Analagous to other tumor suppressor gene syndromes, tumorigenesis in VHL disease is most commonly initiated by a VHL wild-type deletion in susceptible cells. Several key questions, however, remain unexplained in most, if not all, tumor suppressor gene syndromes: 1) in any organ, only one specific type of tumor occurs; 2) tumorigenesis is restricted to specific sets of organs; and, 3) there is no obvious association between tumor suppressor gene function and tumorigenesis. Our recent studies on the histogenesis of hemangioblastomas reveals evidence that hemangioblastomas represent developmentally-arrested tissue. We have further established developmental effects of pVHL deficiency on central nervous system tissues by the discovery of numerous mesenchymal precursors that precede hemangioblastoma formation. In analogy to embryonal blood island differentiation, blood island formation in hemangioblastomas is associated with transient expression of the erythropoietin (Epo) receptor (EpoR). EpoR expression coincides with expression of Epo secondary to VHL deficiency, which we have recently proposed as a mechanism of tumor progression. This mechanism of tumor progression appears to be applicable to other VHL disease-associated tumors, including renal clear cell carcinoma and endolymphatic sac tumor. ? ? Based on the uniform expression of erythropoietin (EPO) and erythropoietin receptor (EPOR) by VHL associated tumors and previous data indicating a potential EPO driven autocrine loop in these tumors, we attempted to establish the tumor cell line in culture conditions. The maintenance of tumor cells in culture requires an EPO-rich media. Growth of the VHL tumor cells was disrupted by addition of EPO or EPOR antibodies in the culture media. Using EPO rich culture media we routinely cultured the primary tumors and maintained them in culture for other tests. We have characterized and expanded these tumor cells to nucleated erythrocytes and mature erythrocytic progeny by varying the cell culture conditions. These findings further support that the EPO and EPOR functional pathway in these tumors is critical in VHL tumor growth and differentiation. Further, the established culture cell lines will be useful for testing other reagents.

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National Institute of Neurological Disorders and Stroke (NINDS)
Intramural Research (Z01)
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