Differential Proteomic Expression in Glioblastoma Multiforme (GBM). Pure populations of GBM cells were obtained by selective tissue microdissection. These cells were analyzed by two-dimensional protein gel electrophoresis (2-DGE), as well as protein sequencing. These molecular techniques identified 11 select target proteins that were expressed differentially among GBMs that permitted distinguishing between the two main categories of GBMs (primary and secondary). These proteins produced two distinctive proteomic patterns indicating that primary and secondary GBMs are distinguishable at the protein level. Further, we are using the same approach to identify differentially expressed proteins in astrocytomas and oligodendrogliomas. Several specific protein markers have been identified and they are proven to be useful for differential pathology diagnosis. Nuclear Receptor Corepressor (N-CoR) Expression in GBM. We have found that N-CoR is overexpressed in GBM cells. Studies indicate that the subcellular localization and presence of N-CoR is related to the differentiation state of GBM cells. Cells expressing nuclear N-CoR do not express glial fibrillary acidic protein (GFAP), which is consistent with astrocytic differentiation. Cells with cytoplasmic N-CoR or loss of N-CoR express GFAP. N-CoR binds to unliganded nuclear receptors such as the retinoid acid receptor and thyroid hormone receptor. When N-CoR forms a complex with histone deacetylase 3 (HDAC3) and retinoic acid receptor (RAR), it acts as an inhibitory mediator of retinoid and thyroid hormone receptors (SMRT) and transcription of RAR specific target genes is repressed resulting in increased cell proliferation. Further, a synergistic interaction between oxadaic acid and retinoic acid (RA) inhibits GBM cell growth and increases cell differentiation. N-CoR activity is inhibited by phoshastase-1 inhibitors, including okadaic acid. Oxadaic acid is not specifically targeted to N-CoR and is likely to have significant side-effects. We believe these findings provide critical information for enhancing current understanding of glioma biology which may lead to new therapeutic options for patients. Therefore, we seek more specific phosphatase inhibitors from new chemical compounds that affect cell growth and differentiation in GBM by specifically targeting the N-CoR pathway, but not others. Recently, we have been studying this novel therapeutic paradigm and analyzing new small chemical compounds. Work is currently in progress studying the inhibitory activity of a series of phosphatase inhibitors and HDAC inhibitors. LB1, a phosphatase inhibitor and LB 2, an HDAC inhibitor were developed and provided by our CRADA partner. During the last year, protocols for evaluating the new drugs and combinations of drugs for activities in an animal model of human GBM were completed. The results of the study show an inhibitory effect on tumor growth of the mouse xeno-grafted human GBM with both new compounds, LB1 and LB2. Furthermore, LB1 in combination with radiation or chemo-agents enhances the radiation and chemotherapeutic effects on GBM. We are currently preparing all the necessary tests and materials such as animal toxicity and formulation of the LB1 compound for clinical trial. Identification of CNTF Receptor in Brain Tumors as Potential Diagnostic and Therapeutic Target. We used a more recently developed proteomic technique to distinguish the proteome of GBM stem cells by capillary isoelectric focusing (CIEF) with nano-reversed-phase liquid chromatography (nRPLC) peptide separation and MS/MS protein sequencing. We have found that CNTF receptor protein consistently appeared in GBM proteomes but not in normal adult brain. Subsequent studies indicated that its expression correlates with the pathologic grades of the glioma and their cell differentiation. Taking CNTF receptor as an unique expression character in GBM stem cells and tissues and its membrane localization with large portion of protein extra-cellular distribution, we further studied antibody-dependent cellular cytotoxicity (ADCC) in GBM cells. The preliminary results of the study showed an inhibition of tumor cell growth and strong cellular cytotoxicity effect. We are planning to develop a CNTF receptor antibody specifically binding to CNTF receptor in brain tumor for therapeutic use, as well as potential molecular imaging marker. We are currently testing whether CNTF receptor can be used as an imaging target for GBM cells in mouse xeno-grafted human GBM model. Developmental Biology and Tumorigenesis of von Hippel-Lindau disease (VHL). Analagous to other tumor suppressor gene syndromes, tumorigenesis in VHL is most commonly initiated by loss of heterozygosity (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 revealed 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 proteins in hemangioblastomas, b) detailed characterization of hemangioblastoma precursor material, c) investigation of the vessel cell origin of hemangioblastoma by genetic analysis to address the critical questions whether the tumor cell participating directly to the angiogenesis. We have completed the study and the results indicated that the small vessel in hemangioblastoma is indeed derived from tumor origin and they shared same genetic hits as seen in other VHL tumor cells. Based on the uniform expression of EPO and 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 cultured the primary tumors and maintained them in culture for in vitro testing. 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. A new small molecule EPO analog has been developed and work is currently in progress studying the inhibitory activity of EPO analog on VHL associated tumor. The preliminary results of the study indicate an inhibitory effect on tumor growth of the mouse xeno-grafted VHL tumor with the new small molecule compound.

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