Aspartate-ß-hydroxylase (ASPH): A potential therapeutic target in human malignant gliomas.

BACKGROUND: Despite therapeutic advances, survival with glioblastoma multiforme (GBM) remains below 15 months from diagnosis due to GBM's highly infiltrative nature which precludes complete surgical resection. Patient outcomes could potentially be improved by targeting genes and pathways that drive neoplastic cell motility and invasiveness, including hypoxia-inducible factor-1 (HIF-1α), NOTCH, and aspartate-ß-hydroxylase (ASPH). METHODS: Human astrocytoma biopsy specimens (n = 37), WHO Grades II-IV, were analyzed for levels and distributions of ASPH and HIF-1α immunoreactivity by immunohistochemical staining, and ASPH, Notch, JAG, HES1, HEY1 and HIF1α mRNA expression by quantigene multiplex analysis. The effects of small molecule inhibitors on ASPH's catalytic activity, cell viability and directional motility were examined in vitro in established GBM cell lines and primary tumor cells from an invasive mouse model of GBM. RESULTS: The highest grade astrocytoma, i.e. GBM was associated with the highest levels of ASPH and HIF1α, and both proteins were more abundantly distributed in hypoxic compared with normoxic regions of tumor. Furthermore, mining of the TCGA database revealed higher levels of ASPH expression in the mesenchymal subtype of GBM, which is associated with more aggressive and invasive behavior. In contrast, lower grade astrocytomas had low expression levels of ASPH and HIF1α. In vitro experiments demonstrated that small molecule inhibitors targeting ASPH's catalytic activity significantly reduced GBM viability and directional motility. Similar effects occurred in GBM cells that were transduced with a lentiviral sh-ASPH construct. CONCLUSION: This study demonstrates that increased ASPH expression could serve as a prognostic biomarker of gliomas and may assist in assigning tumor grade when biopsy specimens are scant. In addition, the findings suggest that GBM treatment strategies could be made more effective by including small molecule inhibitors of ASPH.

Medulloblastoma outcome is adversely associated with overexpression of EEF1D, RPL30, and RPS20 on the long arm of chromosome 8.

BACKGROUND: Medulloblastoma is the most common malignant brain tumor of childhood. Improvements in clinical outcome require a better understanding of the genetic alterations to identify clinically significant biological factors and to stratify patients accordingly. In the present study, we applied cytogenetic characterization to guide the identification of biologically significant genes from gene expression microarray profiles of medulloblastoma. METHODS: We analyzed 71 primary medulloblastomas for chromosomal copy number aberrations (CNAs) using comparative genomic hybridization (CGH). Among 64 tumors that we previously analyzed by gene expression microarrays, 27 were included in our CGH series. We analyzed clinical outcome with respect to CNAs and microarray results. We filtered microarray data using specific CNAs to detect differentially expressed candidate genes associated with survival. RESULTS: The most frequent lesions detected in our series involved chromosome 17; loss of 16q, 10q, or 8p; and gain of 7q or 2p. Recurrent amplifications at 2p23-p24, 2q14, 7q34, and 12p13 were also observed. Gain of 8q is associated with worse overall survival (p = 0.0141), which is not entirely attributable to MYC amplification or overexpression. By applying CGH results to gene expression analysis of medulloblastoma, we identified three 8q-mapped genes that are associated with overall survival in the larger group of 64 patients (p < 0.05): eukaryotic translation elongation factor 1D (EEF1D), ribosomal protein L30 (RPL30), and ribosomal protein S20 (RPS20). CONCLUSION: The complementary use of CGH and expression profiles can facilitate the identification of clinically significant candidate genes involved in medulloblastoma growth. We demonstrate that gain of 8q and expression levels of three 8q-mapped candidate genes (EEF1D, RPL30, RPS20) are associated with adverse outcome in medulloblastoma.

A novel role for extracellular signal-regulated kinase 5 and myocyte enhancer factor 2 in medulloblastoma cell death.

Expression of the neurotrophin-3 receptor, tyrosine kinase C (TrkC), is associated with favorable prognosis in medulloblastoma patients. This may be due to increased tumor apoptosis induced by TrkC activation. Neurotrophin-3/TrkC-induced apoptosis is inhibited by the mitogen-activated protein (MAP) kinase (MAPK) pharmacologic antagonists SB203580 and PD98059. In addition to extracellular signal-regulated kinase (ERK)-1/2, PD98059 also inhibits the more recently identified neurotrophin-responsive MAPK, ERK5 (big MAPK 1). In the present study, we investigate the contribution of ERK5 and its target myocyte enhancer factor 2 (MEF2) to neurotrophin-3/TrkC-induced medulloblastoma cell death. Neurotrophin-3 not only enhanced ERK5 phosphorylation but also significantly enhanced the transcriptional activity of MEF2, a specific target of ERK5. Overexpression of both ERK5 and MEF2 induced a statistically significant increase in cell death of neurotrophin-3-responsive and nonresponsive medulloblastoma cell lines (Daoy-trkC and Daoy) and primary cultures of patched heterozygous mouse medulloblastomas. Only those cells expressing MAP/ERK kinase 5 (MEK5) plus ERK5 or MEF2 constructs underwent apoptosis, indicating that overexpression of either is sufficient to induce medulloblastoma cell death. Expression of a dominant-negative MEF2 or small interfering RNA for the ERK5 activator, MEK5, significantly inhibited neurotrophin-3-induced cell death. The dominant-negative MEF2 construct also blocked MEK5/ERK5-induced cell death, supporting a role for MEF2 downstream of ERK5. Co-immunoprecipitation studies revealed direct interaction of phosphorylated ERK5 with MEF2 in response to neurotrophin-3. Our investigation of the mechanism of neurotrophin-3/TrkC-induced apoptosis has identified a novel role for both MEK5/ERK5 and MEF2 in cell death, suggesting that these molecules can be exploited to induce apoptosis in both TrkC-expressing and non-expressing medulloblastoma cells.

Requirement of Tyr-992 and Tyr-1173 in phosphorylation of the epidermal growth factor receptor by ionizing radiation and modulation by SHP2.

The epidermal growth factor receptor (EGFR) is activated by ionizing radiation (IR) in many human carcinomas, mediating a cytoprotective response and subsequent radioresistance. The underlying molecular mechanisms remain to be understood, and we propose here a specific role for the Tyr-992 residue of EGFR and examine its regulation by the phosphatase, SHP2. The -fold increase in phosphorylation of Tyr-992 in response to IR is twice that seen with ligand (EGF) binding. Mutation of Tyr-992 blocked completely IR-induced EGFR phosphorylation and reduced activation of the downstream signaling molecule, phospholipase Cgamma. IR has previously been demonstrated to inhibit activity of protein-tyrosine phosphatases. Following protein-tyrosine phosphatase inhibition by sodium vanadate both EGFR expressing Chinese hamster ovary (CHO) and A431 exhibited up to an 8-fold increase in the basal level of Tyr-992 phosphorylation, significantly higher than that seen with Tyr-1173, Tyr-1068, and total EGFR Tyr. CHO cells expressing a SHP2 mutant also demonstrated up to an 8-fold increase in the basal level of Tyr-992 phosphorylation. In this study we show the unique association of SHP2 with EGFR in response to IR, with up to a 2.5-fold increase in the direct association of endogenous SHP2 with EGFR-wt in response to 2 gray of IR in both CHO and A431 cells. Mutation of Tyr-992 abolished this response. In conclusion we have identified several differentially activated Tyr residues, one of which is not only more sensitive to activation by IR, translating into differential activation of downstream signaling, but uniquely modulated by the phosphatase SHP2.

Medulloblastoma tumorigenesis diverges from cerebellar granule cell differentiation in patched heterozygous mice.

Medulloblastoma is a cerebellar tumor that can arise through aberrant activation of Sonic hedgehog (Shh) signaling, which normally regulates cerebellar granule cell proliferation. Mutations of the Shh receptor PATCHED (PTCH) are associated with medulloblastomas, which have not been found to have loss of PTCH heterozygosity. We address whether patched (Ptc) heterozygosity fundamentally alters granule cell differentiation and contributes to tumorigenesis by increasing proliferation and/or decreasing apoptosis in Ptc+/- mice. Our data show that postnatal Ptc+/- mouse granule cell precursor growth is not globally altered. However, many older Ptc+/- mice display abnormal cerebellar regions containing persistently proliferating granule cell precursors. Since fewer Ptc+/- mice form medulloblastomas, these granule cell rests represent a developmentally disrupted, but uncommitted stage of tumorigenesis. Although Ptc+/- mouse medulloblastomas express neurodevelopmental genes, they diverge from granule cell differentiation in their discordant coexpression of postmitotic markers despite their ongoing growth. Like human medulloblastomas, mouse tumors with reduced levels of the neurotrophin-3 receptor, trkC/Ntrk3, display decreased apoptosis in vivo, illustrating the role of TrkC in regulating tumor cell survival. These results indicate that Ptc heterozygosity contributes to tumorigenesis by predisposing a subset of granule cell precursors to the formation of proliferative rests and subsequent dysregulation of developmental gene expression.

Molecular biology of medulloblastoma therapy.

Molecular biological studies have created a new understanding of medulloblastomas, revealing key cell signaling pathways that promote tumor growth. These studies have identified molecular markers that can serve as prognostic indicators and which will provide therapeutic targets in the very near future. In contrast to conventional histological techniques, molecular biological methods are designed to detect highly unstable molecules degraded by conventional tissue collection and fixation protocols. Consequently, a new approach to tissue collection is required for molecular analysis as we enter the next era of brain tumor therapy.