JC Virus T-Antigen Regulates Glucose Metabolic Pathways in Brain Tumor Cells

Recent studies have reported the detection of the human neurotropic virus, JCV, in a significant population of brain tumors, including medulloblastomas. Accordingly, expression of the JCV early protein, T-antigen, which has transforming activity in cell culture and in transgenic mice, results in the development of a broad range of tumors of neural crest and glial origin. Evidently, the association of T-antigen with a range of tumor-suppressor proteins, including p53 and pRb, and signaling molecules, such as β-catenin and IRS-1, plays a role in the oncogenic function of JCV T-antigen. We demonstrate that T-antigen expression is suppressed by glucose deprivation in medulloblastoma cells and in glioblastoma xenografts that both endogenously express T-antigen. Mechanistic studies indicate that glucose deprivation-mediated suppression of T-antigen is partly influenced by 5′-activated AMP kinase (AMPK), an important sensor of the AMP/ATP ratio in cells. In addition, glucose deprivation-induced cell cycle arrest in the G1 phase is blocked with AMPK inhibition, which also prevents T-antigen downregulation. Furthermore, T-antigen prevents G1 arrest and sustains cells in the G2 phase during glucose deprivation. On a functional level, T-antigen downregulation is partially dependent on reactive oxygen species (ROS) production during glucose deprivation, and T-antigen prevents ROS induction, loss of ATP production, and cytotoxicity induced by glucose deprivation. Additionally, we have found that T-antigen is downregulated by the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), and the pentose phosphate inhibitors, 6-aminonicotinamide and oxythiamine, and that T-antigen modulates expression of the glycolytic enzyme, hexokinase 2 (HK2), and the pentose phosphate enzyme, transaldolase-1 (TALDO1), indicating a potential link between T-antigen and metabolic regulation. These studies point to the possible involvement of JCV T-antigen in medulloblastoma proliferation and the metabolic phenotype and may enhance our understanding of the role of viral proteins in glycolytic tumor metabolism, thus providing useful targets for the treatment of virus-induced tumors

Mechanism and significance of metabolic signaling pathways affected by the presence of JCV T-antigen.

<p>JCV T-antigen is downregulated by glucose deprivation in an AMPK-dependent manner. During periods of glucose deprivation, T-antigen inhibits AMPK phosphorylation, which prevents the induction of reactive oxygen species (ROS) and subsequent cytotoxicity. Additionally, T-antigen relieves AMPK-mediated cyclin B1 and cyclin A inhibition, leading to decreased G1 arrest. Glucose deprivation induces both enhanced glycolytic flux to maintain high levels of ATP production as well as enhanced pentose phosphate pathway activation to supply reducing equivalents in the form of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to counteract ROS production produced by glycolysis. T-antigen upregulates transaldolase-1 (TALDO1) expression to shift intermediates from the pentose phosphate pathway towards glycolysis to enhance ATP production and also prevents hexokinase 2 (HK2) upregulation during glucose deprivation. (HK2, hexokinase; G6PDH, glucose 6-phosphate dehydrogenase; 6-PGDH, 6-phoshogluconate dehydrogenase; TKTL, transketolase; PKM2, pyruvate kinase M2; LDH, lactate dehydrogenase; 2-DG, 2-deoxyglucose; 6-AN, 6-aminonicotinamide; OT, oxythiamine).</p

T-antigen prevents glucose deprivation-induced ROS production, reduction in ATP levels, and cytotoxicity.

<p>A. BsB8 cells were incubated with 24 mM N-acetylcysteine (NAC), 1 mM pyruvate, or were untreated and then exposed to glucose deprivation or control medium for 24 hours. Pyruvate was dissolved in a small volume of water and a negligible volume was added to the medium in each well. The expression of T-antigen was assessed in whole-cell extracts. B. Bs1a, Bs1f, or BsB8 cells were exposed to glucose deprivation or control medium for 16 hours, and ROS production was measured using the fluorescent dye, 25 µM carboxy-H₂-DCFDA. Hoechst staining was also performed to label nuclei. C. ROS levels were quantified by calculating the mean pixel intensity in triplicate images acquired. (* = control compared to GD, p<0.05; # = BsB8 compared to Bs1f, p<0.05). D. Bs1a, Bs1f, or BsB8 cells were exposed to glucose deprivation for 16 hours, and ATP levels were then measured. The levels of ATP per cell were measured and were normalized to the total protein present in each sample. E. Bs1a, Bs1f, or BsB8 cells were exposed to glucose deprivation or control medium for 24 hours, and cell viability was measured using Guava ViaCount reagent. (* = control compared to GD, p<0.05; # = BsB8 compared to Bs1a and Bs1f cells, p<0.05). F. Phase-contrast images of cells treated with glucose deprivation in D. C, control; GD, glucose deprivation.</p

T-antigen downregulation during glucose deprivation is associated with AMPK-mediated cell cycle control.

<p>A. BsB8 cells were exposed to glucose deprivation for 24 hours, and the expression of T-antigen and cell cycle regulators was monitored by western blot. B. BsB8 cells were exposed to the indicated doses of AICAR, and the expression of T-antigen and cell cycle regulators was measured by western blot. C. BsB8 cells were treated with 5 uM Compound C (CC) or DMSO (D) and exposed to glucose deprivation for 24 hours, and the expression of cell cycle regulators was monitored by western blot. D. Cell cycle analysis using Guava cell cycle reagent was performed in cells from C. (*, p<0.05). E. BsB8 cells were transfected with CMV-cyclin E or empty vector, and T-antigen expression was monitored by western blot. C, control; GD, glucose deprivation.</p

JCV T-antigen is downregulated by glucose deprivation in <i>ex vivo</i> glioblastoma xenograft slice cultures.

<p>A. HJC-2 brain xenografts were sectioned and cultured <i>in vitro</i> and were then treated with control medium or glucose deprivation, with and without rescue from glucose deprivation by subsequent incubation in control medium, or were untreated for various time-points and were harvested for whole tissue lysates. Subsequently, western blot analysis for T-antigen expression was performed. B. Hematoxylin and eosin (H&E) staining as well as immunohistochemical analysis for T-antigen expression were performed in tissue samples obtained from the xenograft slice cultures described in A. GD, glucose deprivation.</p

LAB-METABOLIC PATHWAYS

Glioblastoma continues to rank among the most lethal primary human tumors. Despite treatment with the most rigorous surgical, chemotherapeutic, and radiation regimens, the median survival is just 12-15 months after diagnosis for patients with glioblastoma. One feature of glioblastoma associated with poor prognosis is the degree of hypoxia and expression levels of hypoxia-inducible factor-1 α (HIF-1α). HIF-1α expression allows metabolic adaptation to low oxygen availability, partly through upregulation of vascular endothelial growth factor (VEGF) and increased tumor angiogenesis. In this study, we demonstrate an induced level of astrocyte-elevated gene-1 (AEG-1) in high-grade as compared to low-grade astrocytomas and association of AEG-1 with necrotic areas in glioblastoma. AEG-1 was recently demonstrated to be an oncogene that can induce angiogenesis in glioblastoma. Results from in vitro studies show that AEG-1 is induced by hypoxia in a HIF-1α-dependent manner and that PI3K inhibition abrogates AEG-1 induction during hypoxia. Furthermore, we show that AEG-1 is induced by glucose deprivation and that prevention of intracellular reactive oxygen species (ROS) accumulation prevents this induction. Additionally, AEG-1 knockdown results in increased, whereas AEG-1 overexpression results in decreased, ROS production and glucose deprivation-induced cytotoxicity, indicating that AEG-1 induction is necessary for cells to survive this type of stress. Moreover, AEG-1 modulates the expression of glycolytic enzymes in glioblastoma cells in vitro and in vivo and regulates the expression of these enzymes as well as glycolytic flux during metabolic stress, such as glucose deprivation. The AEG-1-induced glycolytic profile in glioblastoma cells is also modulated by glycolytic inhibition. Studies in nude mice demonstrate that AEG-1 knockdown reduces the growth of glioblastoma xenografts and also promotes chemosensitivity to glycolytic inhibition in vitro. These findings identify a novel role for AEG-1 in the regulation of glycolysis in glioblastoma and indicate that anti-glycolytic therapies may be useful in treating malignancies that demonstrate AEG-1-overexpression