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, and 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 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 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.? ? Developmental biology and tumorigenesis. Analagous to other TSG 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. Current projects on VHL disease-associated hemangioblastomas include a) expression of developmental hemangioblast-associated genes in hemangioblastomas, b) detailed characterization of hemangioblastoma precursor material, c) investigation of the pathomechanisms of hemangioblastoma cyst formation. Representative tissues from different stages are currently investigated with newly developed proteomic techniques for specific stage-associated protein expression.? ? Technology development. a. Development and application of tissue microdissection. Selective tissue microdissection, combined with genetic analysis, has proven to be a unique and powerful experimental approach to identify a) precursor structures in non-hereditary tumor disease, and b) individual histologic components within tumor tissue as either reactive or part of the neoplastic process, and c) to demonstrate single clonality within complex composite tumors, previously thought to represent collision of two separate neoplasms, and metastatic deposits of malignant tumors. These technologies are used to investigate the questions described above, focusing on earliest tumorigenesis of VHL-associated tumors, progression and differentiation of gliomas, and clinical clarification of the pathogenesis of rare neuropathologic disorders. b. Development and application of microdissection combined with proteomic analysis. Over the last 2 years, we have significantly improved proteomic techniques and combined proteomic analysis with exact histologic control via microdissection. This approach is important to type individual types of tumors and for clarification of histogenetic and diagnostic questions. We have developed advanced protocols that allow reliable and consistent protein analysis from minimal amounts of tissue. By application of non-oxidizing silver staining, we are able to reliably resolve proteins and subsequently identify individual proteins by mass spectrometric analysis.
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