G. Yancey Gillespie

Inhibition of Cystine Uptake Disrupts the Growth of Primary Brain Tumors

Glial cells play an important role in sequestering neuronally released glutamate via Na+-dependent transporters. Surprisingly, these transporters are not operational in glial-derived tumors (gliomas). Instead, gliomas release glutamate, causing excitotoxic death of neurons in the vicinity of the tumor. We now show that glutamate release from glioma cells is an obligatory by-product of cellular cystine uptake via system \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{x}_{\mathrm{c}}^{-}\) \end{document}, an electroneutral cystine-glutamate exchanger. Cystine is an essential precursor for the biosynthesis of glutathione, a major redox regulatory molecule that protects cells from endogenously produced reactive oxygen species (ROS). Glioma cells, but not neurons or astrocytes, rely primarily on cystine uptake via system \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{x}_{\mathrm{c}}^{-}\) \end{document} for their glutathione synthesis. Inhibition of system \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{x}_{\mathrm{c}}^{-}\) \end{document} causes a rapid depletion of glutathione, and the resulting loss of ROS defense causes caspase-mediated apoptosis. Glioma cells can be rescued if glutathione status is experimentally restored or if glutathione is substituted by alternate cellular antioxidants, confirming that ROS are indeed mediators of cell death. We describe two potent drugs that permit pharmacological inhibition of system \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{x}_{\mathrm{c}}^{-}\) \end{document}. One of these drugs, sulfasalazine, is clinically used to treat inflammatory bowel disease and rheumatoid arthritis. Sulfasalazine was able to reduce glutathione levels in tumor tissue and slow tumor growth in vivo in a commonly used intracranial xenograft animal model for human gliomas when administered by intraperitoneal injection. These data suggest that inhibition of cystine uptake into glioma cells through the pharmacological inhibition of system \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{x}_{\mathrm{c}}^{-}\) \end{document} may be a viable therapeutic strategy with a Food and Drug Administration-approved drug already in hand.

CD133 Is a Marker of Bioenergetic Stress in Human Glioma

Mitochondria dysfunction and hypoxic microenvironment are hallmarks of cancer cell biology. Recently, many studies have focused on isolation of brain cancer stem cells using CD133 expression. In this study, we investigated whether CD133 expression is regulated by bioenergetic stresses affecting mitochondrial functions in human glioma cells. First, we determined that hypoxia induced a reversible up-regulation of CD133 expression. Second, mitochondrial dysfunction through pharmacological inhibition of the Electron Transport Chain (ETC) produced an up-regulation of CD133 expression that was inversely correlated with changes in mitochondrial membrane potential. Third, generation of stable glioma cells depleted of mitochondrial DNA showed significant and stable increases in CD133 expression. These glioma cells, termed rho 0 or ρ0, are characterized by an exaggerated, uncoupled glycolytic phenotype and by constitutive and stable up-regulation of CD133 through many cell passages. Moreover, these ρ0 cells display the ability to form “tumor spheroids” in serumless medium and are positive for CD133 and the neural progenitor cell marker, nestin. Under differentiating conditions, ρ0 cells expressed multi-lineage properties. Reversibility of CD133 expression was demonstrated by transfering parental mitochondria to ρ0 cells resulting in stable trans-mitochondrial “cybrid” clones. This study provides a novel mechanistic insight about the regulation of CD133 by environmental conditions (hypoxia) and mitochondrial dysfunction (genetic and chemical). Considering these new findings, the concept that CD133 is a marker of brain tumor stem cells may need to be revised.

Systemic anti-hepatocyte growth factor monoclonal antibody therapy induces the regression of intracranial glioma xenografts.

PURPOSE: Hepatocyte growth factor (HGF) and its receptor Met are involved in the initiation, progression, and metastasis of numerous systemic and central nervous system tumors. Thus, an anti-HGF monoclonal antibody (mAb) capable of blocking the HGF-Met interaction could have broad applicability in cancer therapy. EXPERIMENTAL DESIGN: An anti-HGF mAb L2G7 that blocks binding of HGF to Met was generated by hybridoma technology, and its ability to inhibit the various biological activities of HGF was measured by in vitro assays. The ability of L2G7 to inhibit the growth of tumors was determined by establishing s.c. and intracranial xenografts of human U87 and U118 glioma cell lines in nude mice, and treatment with 100 microg of L2G7 or control given i.p. twice per week. RESULTS: MAb L2G7 strongly inhibited all biological activities of HGF measured in vitro, including cell proliferation, cell scattering, and endothelial tubule formation. Treatment with L2G7 completely inhibited the growth of established s.c. xenografts in nude mice. Moreover, systemic administration of L2G7 from day 5 induced the regression of intracranial U87 xenografts and dramatically prolonged the survival of tumor-bearing mice from a median of 39 to >90 days. L2G7 treatment of large intracranial tumors (average tumor size, 26.7 mm(3)) from day 18 induced substantial tumor regression (control group, 134.3 mm(3); L2G7 treated group, 11.7 mm(3)) by day 29 and again prolonged animal survival. CONCLUSIONS: These findings show that blocking the HGF-Met interaction with systemically given anti-HGF mAb can have profound antitumor effects even within the central nervous system, a site previously believed to be resistant to systemic antibody-based therapeutics.

Loss of protein inhibitors of activated STAT-3 expression in glioblastoma multiforme tumors: implications for STAT-3 activation and gene expression.

Purpose: STATs activate transcription in response to numerous cytokines, controlling proliferation, gene expression, and apoptosis. Aberrant activation of STAT proteins, particularly STAT-3, is implicated in the pathogenesis of many cancers, including GBM, by promoting cell cycle progression, stimulating angiogenesis, and impairing tumor immune surveillance. Little is known about the endogenous STAT inhibitors, the PIAS proteins, in human malignancies. The objective of this study was to examine the expression of STAT-3 and its negative regulator, PIAS3, in human tissue samples from control and GBM brains. Experimental Design: Control and GBM human tissues were analyzed by immunoblotting and immunohistochemistry to determine the activation status of STAT-3 and expression of the PIAS3 protein. The functional consequence of PIAS3 inhibition by small interfering RNA or PIAS3 overexpression in GBM cells was determined by examining cell proliferation, STAT-3 transcriptional activity, and STAT-3 target gene expression. This was accomplished using [3H]TdR incorporation, STAT-3 dominant-negative constructs, reverse transcription-PCR, and immunoblotting. Results and Conclusions: STAT-3 activation, as assessed by tyrosine and serine phosphorylation, was elevated in GBM tissue compared with control tissue. Interestingly, we observed expression of PIAS3 in control tissue, whereas PIAS3 protein expression in GBM tissue was greatly reduced. Inhibition of PIAS3 resulted in enhanced glioblastoma cellular proliferation. Conversely, PIAS3 overexpression inhibited STAT-3 transcriptional activity, expression of STAT-3–regulated genes, and cell proliferation. We propose that the loss of PIAS3 in GBM contributes to enhanced STAT-3 transcriptional activity and subsequent cell proliferation.

Oncolytic viruses: clinical applications as vectors for the treatment of malignant gliomas.

Gene therapy using viral vectors for the treatment of primary brain tumors has proven to be a promising novel treatment modality. Much effort in the past has been placed in utilizing replication-defective viruses to this end but they have shown many disadvantages. Much recent attention has been focused on the potential of replication-competent viruses to discriminatingly target, replicate within, and destroy tumor cells via oncolysis, leaving adjacent post-mitotic neurons unharmed. The engineered tumor-selective herpes simplex-1 virus (HSV-1) mutants G207 and HSV1716 have completed Phase I investigations in the treatment of recurrent high-grade glioma. The results of these clinical trials are reviewed here. This review also aims to examine the manipulation and development of other viruses for the treatment of malignant glioma, including Newcastle disease virus, reovirus, poliovirus, vaccinia virus, and adenoviruses, in particular the adenovirus mutant ONYX-015.

Glucose metabolism heterogeneity in human and mouse malignant glioma cell lines

SummaryThe current study examined specific bioenergetic markers associated with the metabolic phenotype of several human and mouse glioma cell lines. Based on preliminary studies, we hypothesized that glioma cells would express one of at least two different metabolic phenotypes, possibly acquired through progression. The D-54MG and GL261 glioma cell lines displayed an oxidative phosphorylation (OXPHOS)-dependent phenotype, characterized by extremely long survival under glucose starvation, and low tolerance to poisoning of the electron transport chain (ETC). Alternatively, U-251MG and U-87MG glioma cells exhibited a glycolytic-dependent phenotype with functional OXPHOS. These cells displayed low tolerance to glucose starvation and were resistant to a ETC blocker. Moreover, these cells could be rescued in low glucose conditions by oxidative substrates (e.g., lactate, pyruvate). Finally, these two phenotypes could be distinguished by the differential expression of LDH isoforms. OXPHOS-dependent cells expressed both LDH-A and -B isoforms whereas glycolytic-dependent glioma cells expressed only LDH-B. In the latter case, LDH-B would be expected to be essential for the use of extracellular lactate to fuel cell activities. These observations raise the possibility that the heterogeneity in glucose metabolism and, in particular, the sole expression of LDH-B, might identify an important biological marker of glioma cells that is critical for their progression and that might afford a new target for anticancer drugs.

Metabolism and Metabolic Actions of 6-Methylpurine and 2-Fluoroadenine in Human Cells

Activation of purine nucleoside analogs by Escherichia coli purine nucleoside phosphorylase (PNP) is being evaluated as a suicide gene therapy strategy for the treatment of cancer. Because the mechanisms of action of two toxic purine bases, 6-methylpurine (MeP) and 2-fluoroadenine (F-Ade), that are generated by this approach are poorly understood, mechanistic studies were initiated to learn how these compounds differ from agents that are being used currently. The concentration of F-Ade, MeP, or 5-fluorouracil required to inhibit CEM cell growth by 50% after a 4-hr incubation was 0.15, 9, or 120 microM, respectively. F-Ade and MeP were also toxic to quiescent MRC-5, CEM, and Balb 3T3 cells. Treatment of CEM, MRC-5, or Balb 3T3 cells with either F-Ade or MeP resulted in the inhibition of protein, RNA, and DNA syntheses. CEM cells converted F-Ade and MeP to F-ATP and MeP-ribonucleoside triphosphate (MeP-R-TP), respectively. The half-life for disappearance of HeP-ribonucleoside triphosphate from CEM cells was approximately 48 hr, whereas the half-lives of F-ATP and ATP were approximately 5 hr. Both MeP and F-Ade were incorporated into the RNA and DNA of CEM cells. These studies indicated that the mechanisms of action of F-Ade and MeP were quite different from those of other anticancer agents, and suggested that the generation of these agents in tumor cells by E. coli PNP could result in significant advantages over those generated by either herpes simplex virus thymidine kinase or E. coli cytosine deaminase. These advantages include a novel mechanism of action resulting in toxicity to nonproliferating and proliferating tumor cells and the high potency of these agents during short-term treatment.

Lyn kinase activity is the predominant cellular SRC kinase activity in glioblastoma tumor cells.

Cellular Src activity modulates cell migration, proliferation, and differentiation, and recent reports suggest that individual members of the Src family may play specific roles in these processes. As we have found that Lyn, but not Fyn, activity promotes migration of glioblastoma cells in response to the cooperative signal generated by platelet-derived growth factor receptor beta and integrin alpha(v)beta3, we compared the activity and expression of Lyn and Fyn in glioblastoma (grade IV) tumor biopsy samples with that in anaplastic astrocytoma (grade III) tumors, nonneoplastic brain, and normal autopsy brain samples. Lyn kinase activity was significantly elevated in glioblastoma tumor samples. Notably, the Lyn kinase activity accounted for >90% of pan-Src kinase activity in glioblastoma samples but only approximately 30% of pan-Src kinase activity in the other groups. The levels of phosphorylation of the autophosphorylation site were consistent with significantly higher Lyn activity in glioblastoma tumor tissue than nonneoplastic brain. Although the normalized levels of Lyn protein and the relative levels of Lyn message were significantly higher in glioblastoma samples than nonneoplastic brain, the normalized levels of Lyn protein did not correlate with Lyn activity in the glioblastoma samples. There was no significant difference in the normalized levels of c-Src and Fyn protein and message in the glioblastoma and nonneoplastic brain. Immunostaining revealed that Lyn is located primarily in the glioblastoma cells in the tumor biopsies. These data indicate that Lyn kinase activity is significantly elevated in glioblastoma tumors and suggest that it is the Lyn activity that promotes the malignant phenotype in these tumors.

Acquisition of Temozolomide Chemoresistance in Gliomas Leads to Remodeling of Mitochondrial Electron Transport Chain

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of high-grade gliomas. Acquired chemoresistance is a severe limitation to this therapy with more than 90% of recurrent gliomas showing no response to a second cycle of chemotherapy. Efforts to better understand the underlying mechanisms of acquired chemoresistance to TMZ and potential strategies to overcome chemoresistance are, therefore, critically needed. TMZ methylates nuclear DNA and induces cell death; however, the impact on mitochondria DNA (mtDNA) and mitochondrial bioenergetics is not known. Herein, we tested the hypothesis that TMZ-mediated alterations in mtDNA and respiratory function contribute to TMZ-dependent acquired chemoresistance. Using an in vitro model of TMZ-mediated acquired chemoresistance, we report 1) a decrease in mtDNA copy number and the presence of large heteroplasmic mtDNA deletions in TMZ-resistant glioma cells, 2) remodeling of the entire electron transport chain with significant decreases of complexes I and V and increases of complexes II/III and IV, and 3) pharmacologic and genetic manipulation of cytochrome c oxidase, which restores sensitivity to TMZ-dependent apoptosis in resistant glioma cells. Importantly, human primary and recurrent pairs of glioblastoma multiforme (GBM) biopsies as well as primary and TMZ-resistant GBM xenograft lines exhibit similar remodeling of the ETC. Overall these results suggest that TMZ-dependent acquired chemoresistance may be due to a mitochondrial adaptive response to TMZ genotoxic stress with a major contribution from cytochrome c oxidase. Thus, abrogation of this adaptive response may reverse chemoresistance and restore sensitivity to TMZ, providing a strategy for improved therapeutic outcomes in GBM patients.

Intertumoral Heterogeneity within Medulloblastoma Subgroups

While molecular subgrouping has revolutionized medulloblastoma classification, the extent of heterogeneity within subgroups is unknown. Similarity network fusion (SNF) applied to genome-wide DNA methylation and gene expression data across 763 primary samples identifies very homogeneous clusters of patients, supporting the presence of medulloblastoma subtypes. After integration of somatic copy-number alterations, and clinical features specific to each cluster, we identify 12 different subtypes of medulloblastoma. Integrative analysis using SNF further delineates group 3 from group 4 medulloblastoma, which is not as readily apparent through analyses of individual data types. Two clear subtypes of infants with Sonic Hedgehog medulloblastoma with disparate outcomes and biology are identified. Medulloblastoma subtypes identified through integrative clustering have important implications for stratification of future clinical trials.