Differential Proteomic Expression in Glioblastoma Multiforme (GBM). The lab has developed advanced protocols to enable reliable and consistent protein analysis from minimal amounts of tissue. Tissue microdissection obtains pure populations of brain tumor cells for proteomic and metabolomic analysis. We have observed differences in proteomic patterns among tumors with different clinical phenotypes. The two main categories of GBMs (primary and secondary) were distinguished by their differential expression of 11 target proteins. We are using the same approach to identify differentially-expressed proteins in astrocytomas and oligodendrogliomas. Although tumor histology is the established method of distinguishing tumor types, proteomic analysis may provide additional diagnostic information when histology cannot distinguish between tumor types or the disease course is atypical for the histologic diagnosis. We are investigating with Dr. Xiaoyuan (Shawn) Chen, NIBIB, if different types of brain tumors also have distinguishing metabolomic patterns. Nuclear Receptor Corepressor (N-CoR) Expression in GBM. We found that N-CoR was overexpressed in GBM cells and that nuclear localization of N-CoR was associated with lack of expression of glial fibrillary acidic protein (GFAP) and absent astrocytic differentiation. 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), represses transcription of RAR specific target genes, and increases cell proliferation. This mechanism can be interrupted by okadaic acid and retinoic acid (RA), which inhibit GBM cell growth and increase cell differentiation. Okadaic acid is a protein phosphatase-1 inhibitor that inhibits N-CoR activity but would likely produce significant side-effects because it does not specifically target N-CoR. We are developing other protein phosphatase inhibitors for GBM that specifically target the N-CoR pathway, reduce cell growth, and increase astrocytic differentiation. During the past year we have studied in the mouse xenografted human GBM animal model the inhibitory activity of protein phosphatase 2A (PP2A) inhibitors, LB-100, and LB-102, which were provided by our CRADA partner. These studies showed that these compounds slowed tumor growth. Furthermore, their effects against GBM were enhanced by combining them with radiation or chemotherapy. LB-100 is presently being tested against solid tumors in a Phase 1 clinical trial outside NIH. Our lab is developing other PP2A inhibitors with greater blood-brain barrier penetration and potential effectiveness against glioblastoma. Identification of CNTF Receptor in Brain Tumors as a Potential Diagnostic and Therapeutic Target. We examined the proteome of GBM stem cells using capillary isoelectric focusing (CIEF) with nano-reversed-phase liquid chromatography (nRPLC) peptide separation and mass spectrometry (MS) protein sequencing. CNTF receptor protein consistently appeared in GBM proteomes but not in normal adult brain. Subsequent studies indicated that CNTF receptor expression correlated with the pathologic grade and cellular differentiation of the glioma. CNTF receptor is uniquely expressed in the membrane of GBM stem cells and tissues. We have developed for therapeutic and diagnostic use a CNTF receptor antibody that specifically binds to CNTF receptor in brain tumor. The antibody produced antibody-dependent cellular cytotoxicity (ADCC) in GBM cells. We are currently testing whether CNTF receptor can also be used as an imaging target for GBM cells in the mouse xenografted human GBM model. Developmental Biology and Tumorigenesis of von Hippel-Lindau disease (VHL). Tumorigenesis in VHL is most commonly initiated by loss of heterozygosity (wild-type deletion) in susceptible cells. Several key questions, however, remain unexplained in this, 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 previous studies suggest that hemangioblastoma in VHL represents developmentally-arrested tissue. We discovered that numerous mesenchymal precursors precede hemangioblastoma formation, including blood island formation which is associated with transient expression of the erythropoietin (Epo) receptor (EpoR). EpoR expression coincides with expression of Epo secondary to VHL deficiency. We showed that hemangioblastoma tumor cell culture in EPO-rich media could result in the development of nucleated erythrocytes and mature erythrocytic progeny. These findings provide 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 was developed and tested and shown to inhibit tumor growth in the mouse xenografted VHL tumor model. We recently found that small vessels in hemangioblastoma are indeed of tumor origin and share the same genetic hits seen in other VHL tumor cells. The molecular mechanism responsible for VHL tumorigenesis is being pursued further. Loss of VHL protein activity has been found to dysregulate hypoxia-inducible factors (HIF), which are transcription factors controlling iron metabolism, erythropoiesis, and angiogenesis. We recently identified novel gain-of-function mutations in HIF-2a in paragangliomas and somatostatinomas associated with polycythemia. Mutations in HIF protein had not previously been identified in any cancer. Functional analysis indicated that the mutations decreased hydroxylation of HIF-2a by prolyl hydroxylase (PHD), reducing HIF-2a affinity to VHL, HIF-2a degradation, and consequently increasing HIF-2a half-life, which led to up-regulation of hypoxia-related genes EPO, VEGFA, GLUT1, and END1 in tumors. The findings showed for the first time that HIF mutations are involved in tumor development. The unique HIF-2a mutations identified will importantly impact the study of the cellular mechanism of oxygen-sensing signaling in normal, hypoxic and pseudo-hypoxic conditions. Identifying the molecular mechanism responsible for loss of functional activity in mutated proteins in von Hippel-Lindau (VHL) disease and neurofibromatosis type 2 (NF2). Deficiency of a single protein resulting from a gene mutation is a frequent cause of inherited diseases. However, the mechanism responsible for the mutant protein losing function is not explained. We recently discovered that in VHL and NF2, degradation of mutated VHL protein and NF2 protein (merlin), respectively, is more rapid than for normal (wild type) protein, leading to quantitative deficits in these proteins even though they retain much of their intrinsic functional activity. Further study showed that increased protein degradation resulted from protein instability arising during protein synthesis. Subsequent studies indicated that the mutant protein folded incorrectly and that modulation of specific molecular mediators of protein folding could restore functional activity to the mutated protein. These findings suggest that these mediators could be used as therapeutic targets to increase function of the mutant protein in patients with VHL and NF2. We have recently identified and developed chemical compounds, LB205 and Vorinostat, which actively modulate these mediators. A clinical pilot study examining the effects of Vorinostat on central nervous system hemangioblastoma in VHL disease will be initiated within the next year.

Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2013
Total Cost
$1,295,755
Indirect Cost
City
State
Country
Zip Code
Absinta, Martina; Nair, Govind; Filippi, Massimo et al. (2014) Postmortem magnetic resonance imaging to guide the pathologic cut: individualized, 3-dimensionally printed cutting boxes for fixed brains. J Neuropathol Exp Neurol 73:780-8
Jochmanova, Ivana; Yang, Chunzhang; Zhuang, Zhengping et al. (2013) Hypoxia-inducible factor signaling in pheochromocytoma: turning the rudder in the right direction. J Natl Cancer Inst 105:1270-83
Lu, Jie; Frerich, Jason M; Turtzo, L Christine et al. (2013) Histone deacetylase inhibitors are neuroprotective and preserve NGF-mediated cell survival following traumatic brain injury. Proc Natl Acad Sci U S A 110:10747-52
Yang, Chunzhang; Rahimpour, Shervin; Yu, Albert C H et al. (2013) Regulation and dysregulation of astrocyte activation and implications in tumor formation. Cell Mol Life Sci :
Brady, Roscoe O; Yang, Chunzhang; Zhuang, Zhengping (2013) An innovative approach to the treatment of Gaucher disease and possibly other metabolic disorders of the brain. J Inherit Metab Dis 36:451-4
Taieb, David; Yang, Chunzhang; Delenne, Blandine et al. (2013) First report of bilateral pheochromocytoma in the clinical spectrum of HIF2A-related polycythemia-paraganglioma syndrome. J Clin Endocrinol Metab 98:E908-13
Pacak, Karel; Jochmanova, Ivana; Prodanov, Tamara et al. (2013) New syndrome of paraganglioma and somatostatinoma associated with polycythemia. J Clin Oncol 31:1690-8
Yang, Chunzhang; Rahimpour, Shervin; Lu, Jie et al. (2013) Histone deacetylase inhibitors increase glucocerebrosidase activity in Gaucher disease by modulation of molecular chaperones. Proc Natl Acad Sci U S A 110:966-71
Nduom, Edjah K; Yang, Chunzhang; Merrill, Marsha J et al. (2013) Characterization of the blood-brain barrier of metastatic and primary malignant neoplasms. J Neurosurg 119:427-33
Lu, Ying; Wang, Weimin; Wang, Junchen et al. (2013) Overexpression of Arginine Transporter CAT-1 Is Associated with Accumulation of L-Arginine and Cell Growth in Human Colorectal Cancer Tissue. PLoS One 8:e73866

Showing the most recent 10 out of 11 publications