CELL BIOLOGY AND SIGNALING

Abstract

Neuroblastoma, a cancer of the sympathetic nervous system, is the most common extracranial solid tumor in children. MYCN amplification and increased BDNF/TrkB signaling are features of high-grade tumors, yet only ~25% of malignant tumors display those features. Thus, the identification of additional biomarkers and therapeutic targets is essential. Since aminoacylase 1 (ACY1), an amino acid deacetylase, is a putative tumor suppressor in small cell lung and renal cell carcinomas, we investigated whether ACY1 or family members aspartoacylase (ASPA, aminoacylase 2) or aminoacylase 3 (ACY3) could serve a similar function in neuroblastoma. Aminoacylase expression was examined in TrkB-positive, MYCN-amplified SMS-KCNR and TrkB-negative, non-MYCN amplified SK-N-AS and SK-N-SH neuroblastoma cell lines. ACY1 and ASPA exhibited distinct spatial localization in SMS-KCNR and SK-N-SH cells, while ACY3 displayed nuclear expression in all three lines examined. ACY1 was the only aminoacylase whose expression was up-regulated upon neuronal differentiation of SK-N-SH cells in media containing 10% serum. ASPA expression was greater in the least aggressive SK-N-SH line and significantly reduced in the most aggressive SMS-KCNR line. Conversely, ACY3 expression was highly expressed in the most aggressive SMS-KCNR cells. In vivo, aminoacylases are expressed in common sites of neuroblastoma origin. Bioinformatics data mining of Kaplan-Meier survival data revealed that high ACY3 expression is correlated with poor prognosis and that low expression of ACY1 or ASPA is also correlated with poor prognosis, suggesting that the loss of these aminoacylases may contribute to neuroblastoma tumorigenesis. BACKGROUND: Malignant astrocytomas, the most common primary brain tumors, are predominantly fatal with current therapies. In our effort to better understand the biology of astrocytomas, we explored new therapeutic targets. We previously cloned NPAS3, a transcription factor that maps to human chromosome 14. Our principal aim is to comprehend the disease associations of NPAS3, since we recently identified its expression in human astrocytes. We initially identified NPAS3 as an astrocytoma candidate based on the Cancer Genome Project reporting chromosome 14 deletions (with NPAS3) among ~20%-80% of astrocytomas and with >70% of our human astrocytoma panel (n = 433) having aberrant NPAS3 protein expression. Based on the findings from our precursory screen, we next undertook functional analyses of NPAS3 in human astrocytomas. METHODS-RESULTS: After undertaking extensive functional analyses, we now have evidence supporting NPAS3 as an astrocytoma tumor suppressor involved in late-stage tumor progression, based on: 1) Aberrant NPAS3 expression is predominant in surgically resected high-grade astrocytomas compared with low-grade astrocytomas; 2) loss-of-function mutations in NPAS3, which are associated with loss of heterozygosity of the NPAS3 locus, are identified in surgically resected human glioblastomas; 3) absent NPAS3 expression is predominant in malignant human glioma cell lines; 4) over-expressed NPAS3 in malignant glioma cell lines suppresses transformation potential, while converse reduced expression promotes an increase in transformation potential; and 5) a reduced NPAS3 expression (efficiency >90%) in concert with other gliomagenesis genes can transform a well-characterized TERT immortalized human astrocyte cell line and promote the growth of malignant astrocytomas. CONCLUSIONS: Our data provide compelling evidence that the NPAS3 gene is involved in the cause of astrocytomas, with tumor suppressive and late-stage acting progression factor roles. Current research is focused on better understanding NPAS3 in gliomas using other pre-clinical models. Cancer stem cells (CSCs) are capable of unlimited self-renewal and multi-lineage differentiation. We have shown that 2-deoxy-D-glucose (2-DG), a known inhibitor of glycolysis, can inhibit the growth of glioma-derived stem cells (GSC11) under normoxic conditions, and we hypothesize that 2-DG affects the formation of N-glycans by replacing D-mannose in glycosylation processes. We have synthesized 2-DG, D-glucose, and D-mannose labeled with deuterium at C-2 and treated GSC11 cells with these monosaccharides to measure their effects on global N-glycan formation. N-glycans were released with PNGase F and purified over a graphitized carbon cartridge SPE. Oligosaccharides were separated with a TSK-Gel Amide80 column under hydrophilic interaction chromatography conditions and analyzed by positive ion-microelectrospray with an LTQ 14.5 T FT-ICR mass spectrometer 1. Data showed that deuterium-labeled 2-DG was incorporated into the N-glycans, leading to the termination of the extension of the oligosaccharide chain. Comparative glycomic analysis of control, 2-DG-treated, and D-mannose-rescued GSC11 cells revealed a distinct modulation of the N-glycan profile. The levels of all types of N-glycans were decreased (by ~4-fold) in 2-DG-treated GSC11 cells compared with control cells. In contrast, N-glycan synthesis in GSC11 cells could be rescued to almost "normal control" levels by adding exogenous D-mannose. D-mannose rescue of 2-DG-treated GSC11 cells drastically reduced the incorporation of 2-DG into the N-glycans. These results indicate that 2-DG can interfere with biochemical transformations of D-mannose and that such interference might contribute to the overall antitumor effects of 2-DG. (1 Schaub T M, Hendrickson C L, Horning S, Quinn J P, Senko M W, and Marshall A G. High performance mass spectrometry: Fourier transform ion cyclotron resonance at 14.5 Tesla. Anal. Chem. 2008, 80, 3985-3990.) Glioblastoma (GBM) is the most common and most intractable brain malignancy in adults. Patients with GBM have a dismal prognosis, with a median survival of 12-14 months. Epidermal growth factor receptor (EGFR) and its constitutively activated variant EGFRvIII are linked to GBM resistance to therapy; the mechanisms underlying this association, however, are still unclear. Also unclear are the mechanisms underlying the resistance of GBM to EGFR-targeted monotherapy and combination therapy. We report here that in GBM cell lines, xenografts, and primary specimens (N = 101), EGFR and EGFRvIII paradoxically co-express with p53-upregulated modulator of apoptosis (PUMA), a proapoptotic member of the Bcl-2 family of proteins primarily located on the mitochondria, unlike other BH3-only proteins. Mitochondrial PUMA is known to bind to and antagonize antiapoptotic Bcl-2/Bcl-xL/Mcl-1 and also to associate with and activate the apoptotic executor Bax, together leading to apoptotic response upon appropriate stress. Our results showed that both EGFR and EGFRvIII bind to PUMA constitutively and under apoptotic stress, subsequently sequestering PUMA in the cytoplasm. EGFR siRNA-mediated expression knockdown relocates PUMA from the cytoplasm onto the mitochondria. The EGFR-PUMA interaction is independent of epidermal growth factor (EGF)-induced EGFR activation and is sustained under treatment with an EGFR kinase inhibitor, Iressa. Although GBM cells express several proapoptotic members of the Bcl-2 protein family, our results indicate that PUMA is essential for therapy-induced apoptosis and thus for drug sensitivity. Importantly, we found that Bcl-2/Bcl-xL/Mcl-1 inhibitors (BH3 mimetics) that mimic PUMA's antiapoptotic activity sensitize EGFR- and EGFRvIII-expressing GBM cells to Iressa. Collectively, we uncovered a novel kinase-independent function of EGFR and EGFRvIII that contributes to GBM resistance to EGFR kinase inhibition and apoptosis-inducing agents and also provides a rationale for targeting kinase-dependent and -independent activities of EGFR as a novel combination therapy for GBM. PURPOSE: The monoterpene perillyl alcohol (POH), a Ras inhibitor with the potential capacity to arrest gliomagenesis, is being used in a phase I/II clinical trial in adults with recurrent malignant glioma. The present study aimed to investigate the efficacy of the intranasal administration of POH and the survival rate in patients with recurrent glioblastoma (GBM) in comparison with a historical control group of GBM patients. PATIENTS AND METHODS: The study included 89 adults with recurrent GBM who received daily intranasal administration of 440 mg POH and 52 matched GBM patients as the historical control group. RESULTS: The 6-month progression-free survival (stable disease) rate was 48.3% for POH-treated patients, with a significant (p = 0.0001) survival advantage compared with the untreated historical control group. The median survival time for patients with secondary GBM was 11.2 months, longer (p = 0.0002) than for patients with primary GBM (5.9 months). Age-adjustment multivariate analysis showed a significant difference (p = 0.0002) in the survival rate between primary and secondary GBM patients. Patients with tumors localized in deep regions (e.g., thalamus, basal ganglia) survived longer (p = 0.0083) than those with tumors in lobar regions. Radiographic improvement and reduction of corticosteroid dosage (36%) was further associated with a delay in progression. CONCLUSION: Intranasal administration of POH increased the overall survival of patients with recurrent GBM compared with historical controls, especially of patients with secondary GBM and those with tumor localized in deep regions of the brain, without clinical evidence of side effects for more than a year. INTRODUCTION: Loss of function mutations and deletions in the neurofibromin tumor suppressor gene underlie neurofibromatosis type 1 (NF1), which, with a birth incidence of 1 in 3000, is the most common inherited tumor-predisposing syndrome in humans. While the molecular mechanisms that contribute to the neoplastic manifestations have been attributed to Ras-GTPase activating protein (GAP) activity mediated through the GAP related domain (GRD) of NF1, there is no definite co