Identification of ATP citrate lyase as a positive regulator of glycolytic function in glioblastomas.

Glioblastomas, the most malignant type of glioma, are more glycolytic than normal brain tissue. Robust migration of glioblastoma cells has been previously demonstrated under glycolytic conditions and their pseudopodia contain increased glycolytic and decreased mitochondrial enzymes. Glycolysis is suppressed by metabolic acids, including citric acid which is excluded from mitochondria during hypoxia. We postulated that glioma cells maintain glycolysis by regulating metabolic acids, especially in their pseudopodia. The enzyme that breaks down cytosolic citric acid is ATP citrate lyase (ACLY). Our identification of increased ACLY in pseudopodia of U87 glioblastoma cells on 1D gels and immunoblots prompted investigation of ACLY gene expression in gliomas for survival data and correlation with expression of ENO1, that encodes enolase 1. Queries of the NIH's REMBRANDT brain tumor database based on Affymetrix data indicated that decreased survival correlated with increased gene expression of ACLY in gliomas. Queries of gliomas and glioblastomas found an association of upregulated ACLY and ENO1 expression by chi square for all probe sets (reporters) combined and correlation for numbers of probe sets indicating shared upregulation of these genes. Real-time quantitative PCR confirmed correlation between ACLY and ENO1 in 21 glioblastomas (p < 0.001). Inhibition of ACLY with hydroxycitrate suppressed (p < 0.05) in vitro glioblastoma cell migration, clonogenicity and brain invasion under glycolytic conditions and enhanced the suppressive effects of a Met inhibitor on cell migration. In summary, gene expression data, proteomics and functional assays support ACLY as a positive regulator of glycolysis in glioblastomas.

A roadmap for potassium buffering/dispersion via the glial network of the CNS.

Glia use multiple mechanisms to mediate potassium fluxes that support neuronal function. In addition to changes in potassium levels within synapses, these ions are dynamically dispersed through the interstitial parenchyma, perivascular spaces, leptomeninges, cerebrospinal fluid, choroid plexus, blood, vitreous, and endolymph. Neural circuits drive diversity in the glia that buffer potassium and this is reciprocal. Glia mediate buffering of potassium locally at glial-neuronal interfaces and via widespread networked connections. Control of potassium levels in the central nervous system is mediated by mechanisms operating at various loci with complexity that is difficult to model. However, major components of networked glial buffering are known. The role that potassium buffering plays in homeostasis of the CNS underlies some pathologic phenomena. An overview of potassium fluxes in the CNS is relevant for understanding consequences of pathogenic sequence variants in genes that encode potassium buffering proteins. Potassium flows in the CNS are described as follows: K1, the coordinated potassium fluxes within the astrocytic cradle around the synapse; K2, temporary storage of potassium within astrocytic processes in proposed microdomains; K3, potassium fluxes between oligodendrocytes and astrocytes; K4, potassium fluxes between astrocytes; K5, astrocytic potassium flux mediation of neurovasular coupling; K6, CSF delivery of potassium to perivascular spaces with dispersion to interstitial fluid between astrocytic endfeet; K7, astrocytic delivery of potassium to CSF and K8, choroid plexus (modified glia) regulation of potassium at the blood-CSF barrier. Components, mainly potassium channels, transporters, connexins and modulators, and the pathogenic sequence variants of their genes with the associated diseases are described.

Tumor cells from ultrasonic aspirations of glioblastomas migrate and form spheres with radial outgrowth.

Studies of primary cells from malignant brain tumors such as glioblastomas are limited by the small size of surgically resected specimens. However, glioblastomas are also frequently debulked via ultrasonic aspiration. In this study, we examined the functional competence and growth of their aspirated cells. Cells from minced tissue and aspirations were comparable in migration, formation of pseudopodia, development of cellular spheres with radial outgrowth, and neuroectodermal features. Cultures were maintained for more than six weeks without fibroblastic overgrowth. Our observations show that ultrasonically aspirated specimens contain cells useful for studies of tumor migration and growth of tumorspheres.

Glioblastomas with copy number gains in EGFR and RNF139 show increased expressions of carbonic anhydrase genes transformed by ENO1.

BACKGROUND: Prominence of glycolysis in glioblastomas may be non-specific or a feature of oncogene-related subgroups (i.e. amplified EGFR, etc.). Relationships between amplified oncogenes and expressions of metabolic genes associated with glycolysis, directly or indirectly via pH, were therefore investigated. METHODS: Using multiplex ligation-dependent probe amplification, copy numbers (CN) of 78 oncogenes were quantified in 24 glioblastomas. Related expressions of metabolic genes encoding lactate dehydrogenases (LDHA, LDHC), carbonic anhydrases (CA3, CA12), monocarboxylate transporters (SLC16A3 or MCT4, SLC16A4 or MCT5), ATP citrate lyase (ACLY), glycogen synthase1 (GYS1), hypoxia inducible factor-1A (HIF1A), and enolase1 (ENO1) were determined in 22 by RT-qPCR. To obtain supra-glycolytic levels and adjust for heterogeneity, concurrent ENO1 expression was used to mathematically transform the expression levels of metabolic genes already normalized with delta-delta crossing threshold methodology. RESULTS: Positive correlations with EGFR occurred for all metabolic genes. Significant differences (Wilcoxon Rank Sum) for oncogene CN gains in tumors of at least 2.00-fold versus less than 2.00-fold occurred for EGFR with CA3's expression (p < 0.03) and for RNF139 with CA12 (p < 0.004). Increased CN of XIAP associated negatively. Tumors with less than 2.00-fold CN gains differed from those with gains for XIAP with CA12 (p < 0.05). Male gender associated with CA12 (p < 0.05). CONCLUSIONS: Glioblastomas with CN increases in EGFR had elevated CA3 expression. Similarly, tumors with RNF149 CN gains had elevated CA12 expression. GENERAL SIGNIFICANCE: In larger studies, subgroups of glioblastomas may emerge according to oncogene-related effects on glycolysis, such as control of pH via effects on carbonic anhydrases, with prognostic and treatment implications.

Loss of heterozygosity reveals non-VHL allelic loss in hemangioblastomas at 22q13.

Hemangioblastomas (HBs) are low-grade (World Health Organization grade I/IV) central nervous system (CNS) tumors that frequently contain VHL (3p26) mutations. They occur sporadically and in von Hippel Lindau (VHL) disease. Encoded pVHL aids degradation of hypoxia-inducible factors (HIFs) in the presence of normal oxygen levels. HBs provide an in vivo view of HIF effects within a CNS tumor. Typically, HBs are cystic tumors containing a mural nodule formed by noninvasive, vacuolated stromal cells that are embedded in a network of capillaries. Nine HBs, consecutively resected from 8 patients at our institution during a recent 2-year time span, were evaluated for additional losses of tumor suppressor genes. Non-VHL microsatellites studied for loss of heterozygosity (LOH) are near tumor suppressor genes lost in gliomas, pituitary adenomas, several CNS tumors on 22q, neurofibromatosis 1, and colon carcinomas (13, 2, 2, 1, and 2 markers for each, respectively). LOH in the region of 3p21.3-3p26.3 occurred in 3 of 8 HBs informative for at least 1 marker (D3S1539, D3S2303, or D3S2373). By using 2 markers (D22S417 and D22S532) for 22q13.2, LOH was found in 5 of 8 informative HBs. All 3 HBs with allelic losses near VHL also showed LOH at 22q13.2. No consistent losses were found with markers for 1p34, LMYC, 5q21, 5q32, 9p21, 10q23, 17p13, and 19q13. LOH for the 22q13.2 region in HBs suggests that the loss of another tumor suppressor gene is involved in the pathogenesis of HBs in addition to VHL. Absence of LOH for glioma markers is consistent with the low-grade behavior of HBs.

Giant cell tumor of the skull: a case report and review of the literature.

BACKGROUND: Giant cell tumors are benign lesions that typically occur at the epiphyses of long bones that typically present with pain or swelling. Most data on giant cell tumors in the skull consist of case reports, and many large series of giant cell tumors have no examples in the skull. METHODS: We report a case of giant cell tumor of the skull and review the literature on these lesions. RESULTS: A 24-year-old woman presented with localized tenderness and mild swelling over the left inferior parietal and occipital bones. She was neurologically intact with a nonmobile, tender, palpable mass over the left subocciptal area. A computed tomography (CT) scan showed a radiolucent, expansile, lytic lesion involving the left occipital bone. The patient underwent a left occipital craniectomy with resection of the bone and epidural mass. Permanent histopathologic sections and immunostains revealed a giant cell tumor. CONCLUSIONS: Giant cell tumors are generally benign, locally aggressive lesions for which surgical excision is the treatment of choice. This report contributes to the scarce literature on these tumors in the skull.

Low-level amplification of oncogenes correlates inversely with age for patients with nontypical meningiomas.

BACKGROUND: This study sought to identify genes in nontypical meningiomas with gains in copy number (CN) that correlate with earlier age of onset, an indicator of aggressiveness. METHODS: Among 94 adult patients, 91 had 105 meningiomas that were histologically confirmed. World Health Organization grades I (typical), II (atypical), and III (anaplastic) were assigned to tumors in 76, 14, and 1 patient, respectively. Brain invasion indicated that two World Health Organization grade I meningiomas were biologically atypical. DNA from 15 invasive/atypical/anaplastic meningiomas and commercial normal DNA were analyzed with multiplex ligation dependent probe amplification. The CN ratios (fold differences from normal) for 78 genes were determined. The CN ratio was defined as [tumor CN]/[normal CN] for each gene to normalize results. RESULTS: Characteristic gene losses (CN ratio < 0.75) occurred in >50% of the invasive/atypical/anaplastic meningiomas at 22q11, 1p34.2, and 1p22.1 loci. Gains (CN ratio ≥ 2.0) occurred in each tumor for 2 or more of 19 genes. Each of the 19 genes' CN ratio was ≥ 2.0 in multiple tumors, and their collective sums (up to 49.1) correlated inversely with age (r = -0.72), minus an outlier. In patients ≤ 55 versus >55 years, 5 genes (BIRC2, BRAF, MET, NRAS, and PIK3CA) individually exhibited significantly higher CN ratios (P < 0.05) or a trend for them (P < 0.09), with corrections for multiple comparisons, and their sums correlated inversely with age (r = -0.74). CONCLUSIONS: Low levels of amplification for selected oncogenes in invasive/atypical/anaplastic meningiomas were higher in younger adults, with the CN gains potentially underlying biological aggressiveness associated with early tumor development.

Rapid detection of high-level oncogene amplifications in ultrasonic surgical aspirations of brain tumors.

BACKGROUND: Genomic tumor information, such as identification of amplified oncogenes, can be used to plan treatment. The two sources of a brain tumor that are commonly available include formalin-fixed, paraffin-embedded (FFPE) sections from the small diagnostic biopsy and the ultrasonic surgical aspiration that contains the bulk of the tumor. In research centers, frozen tissue of a brain tumor may also be available. This study compared ultrasonic surgical aspiration and FFPE specimens from the same brain tumors for retrieval of DNA and molecular assessment of amplified oncogenes. METHODS: Surgical aspirations were centrifuged to separate erythrocytes from the tumor cells that predominantly formed large, overlying buffy coats. These were sampled to harvest nuclear pellets for DNA purification. Four glioblastomas, 2 lung carcinoma metastases, and an ependymoma were tested. An inexpensive PCR technique, multiplex ligation-dependent probe amplification (MLPA), quantified 79 oncogenes using 3 kits. Copy number (CN) results were normalized to DNA from non-neoplastic brain (NB) in calculated ratios, [tumor DNA]/[NB DNA]. Bland-Altman and Spearman rank correlative comparisons were determined. Regression analysis identified outliers. RESULTS: Purification of DNA from ultrasonic surgical aspirations was rapid (<3 days) versus FFPE (weeks) and yielded greater amounts in 6 of 7 tumors. Gene amplifications up to 15-fold corresponded closely between ultrasonic aspiration and FFPE assays in Bland-Altman analysis. Correlation coefficients ranged from 0.71 to 0.99 using 3 kit assays per tumor. Although normalized CN ratios greater than 2.0 were more numerous in FFPE specimens, some were found only in the ultrasonic surgical aspirations, consistent with tumor heterogeneity. Additionally, CN ratios revealed 9 high-level (≥ 6.0) gene amplifications in FFPE of which 8 were also detected in the ultrasonic aspirations at increased levels. The ultrasonic aspiration levels of these amplified genes were also greater than 6.0 CN ratio, except in one case (3.53 CN ratio). Ten of 17 mid-level (≥3.0 & <6.0 CN ratio) amplifications detected in FFPE were also detected as being increased (≥ 2.0 CN ratio) in the aspirations. CONCLUSIONS: Buffy coats of centrifuged ultrasonic aspirations contained abundant tumor cells whose DNA permitted rapid, multiplex detection of high-level oncogene amplifications that were confirmed in FFPE. VIRTUAL SLIDES: http://www.diagnosticpathology.diagnomx.eu/vs/1883718801686466.

Albumin marks pseudopodia of astrocytoma cells responding to hepatocyte growth factor or serum.

It is well accepted that dysfunction in the blood brain barrier (BBB) allows permeation of albumin from the bloodstream into astrocytic brain tumors, especially glioblastomas, the most aggressive astrocytomas. In vitro, bovine serum albumin (BSA) aids functional cell assays by maintaining cytokines and growth factors in solution and delivering its cargo of fatty acids. Earlier, we showed that BSA was prominent in lysates prepared from pseudopodia formed by U87 astrocytoma cells. The present studies investigated the association of albumin with pseudopodia formed by U87 and LN229 astrocytoma cells. With hepatocyte growth factor (HGF) stimulation, cell migration was enhanced and BSA, especially its dimerized form, was prominent in pseudopodia compared to unmigrated cells on one-dimensional gels and immunoblots. When lysates were equalized for levels of glyceraldehyde-3-phosphate dehydrogenase, the rise for BSA levels in pseudopodia vs migrated cells was comparable or greater than levels noted for established pseudopodial proteins, beta-actin and ezrin. The increase for dimerized BSA in pseudopodia compared to unmigrated cells was greater than the rise in levels of beta-actin, ezrin, HGF, and phosphorylated Met when pseudopodia were harvested from filters with 1 mum pores using either cell line. Fluorescein (F)-labeled BSA co-localized with HGF on actin-rich cellular protrusions and with CM-DiI labeled pseudopodial plasma membranes. The F-BSA highlighted small, individual pseudopodial profiles more so than complex pseudopodial networks (reticulopodia) or unmigrated cells. Labeled human serum albumin also decorated pseudopodia preferentially. Albumin's association with pseudopodia may help to explain its selective accumulation in astrocytomas in vivo. The leaky BBB permits serum albumin to enter the microenvironment of astrocytomas thus allowing their invasive cells contact with serum albumin as a source of fatty acids that would be useful for remodeling cell membranes in pseudopodia. Thus, albumin potentially aids and marks invasion as it accumulates in these tumors.

Proteomic characterization of harvested pseudopodia with differential gel electrophoresis and specific antibodies.

Malignant gliomas (astrocytomas) are lethal tumors that invade the brain. Invasive cell migration is initiated by extension of pseudopodia into interstitial spaces. In this study, U87 glioma cells formed pseudopodia in vitro as cells pushed through 3 microm pores of polycarbonate membranes. Harvesting pseudopodia in a novel two-step method provided material for proteomic analysis. Differences in the protein profiles of pseudopodia and whole cells were found using differential gel electrophoresis (DIGE) and immunoblotting. Proteins from two-dimensional (2D) gels with M(R)'s of 20-100 kDa and pI's of 3.0-10.0 were identified by peptide mass fingerprinting analysis using mass spectrometry. For DIGE, lysates of pseudopodia and whole cells were each labeled with electrophilic forms of fluorescent dyes, Cy3 or Cy5, and analyzed as mixtures. Analysis was repeated with reciprocal labeling. Differences in protein distributions were detected by manual inspection and computer analysis. Topographical digital maps of the scanned gels were used for algorithmic spot matching, normalization of background, quantifying spot differences, and elimination of artifacts. Pseudopodial proteins in Coomassie-stained 2D gels included isoforms of glycolytic enzymes as the largest group, seven of 24 proteins. Peptide mass fingerprint analysis of DIGE gels demonstrated increased isoforms of annexin (Anx) I, AnxII, enolase, pyruvate kinase, and aldolase, and decreased mitochondrial manganese superoxide dismutase and transketolase in pseudopodia. Specific antibodies showed restricted immunoreactivity of the hepatocyte growth factor (HGF) alpha chain to pseudopodia, indicating localization of its active form. Met (the HGF receptor), actin, and total AnxI were increased in pseudopodial lysates on immunoblots. Increased constituents of the pseudopodial proteome in glioma cells, identified in this study as actin, HGF, Met, and isoforms of AnxI, AnxII, and several glycolytic enzymes, represent therapeutic targets to consider for suppression of tumor invasion.

Glycolytic glioma cells with active glycogen synthase are sensitive to PTEN and inhibitors of PI3K and gluconeogenesis.

Increased glycolysis is characteristic of malignancy. Previously, with a mitochondrial inhibitor, we demonstrated that glycolytic ATP production was sufficient to support migration of melanoma cells. Recently, we found that glycolytic enzymes were abundant and some were increased in pseudopodia formed by U87 glioma (astrocytoma) cells. In this study, we examined cell migration, adhesion (a step in migration), and Matrigel invasion of U87 and LN229 glioma cells when their mitochondria were inhibited with sodium azide or limited by 1% O(2). Cell migration, adhesion, and invasion were comparable, with and without mitochondrial inhibition. Upon discovering that glycolysis alone can support glioma cell migration, unique features of glucose metabolism in astrocytic cells were investigated. The ability of astrocytic cells to remove lactate, the inhibitor of glycolysis, via gluconeogenesis and incorporation into glycogen led to consideration of supportive genetic mutations. Loss of phosphatase and tensin homolog (PTEN) releases glycogenesis from constitutive inhibition by glycogen synthase kinase-3 (GSK3). We hypothesize that glycolysis in gliomas can support invasive migration, especially when aided by loss of PTEN's regulation on the phosphatidylinositol-3 kinase (PI3K)/Akt pathway leading to inhibition of GSK3. Migration of PTEN-mutated U87 cells was studied for release of extracellular lactic acid and support by gluconeogenesis, loss of PTEN, and active PI3K. Lactic acid levels plateaued and phosphorylation changes confirmed activation of the PI3K/Akt pathway and glycogen synthase when cells relied only on glycolysis. Glycolytic U87 cell migration and phosphorylation of GSK3 were inhibited by PTEN transfection. Glycolytic migration was also suppressed by inhibiting PI3K and gluconeogenesis with wortmannin and metformin, respectively. These findings confirm that glycolytic glioma cells can migrate invasively and that the loss of PTEN is supportive, with activated glycogenic potential included among the relevant downstream effects.

Extracellular angio-associated migratory cell protein plays a positive role in angiogenesis and is regulated by astrocytes in coculture.

The extracellular form of angio-associated migratory cell protein (AAMP), a recently discovered protein, plays a positive role in angiogenesis and can be regulated by astrocytes. Angiogenic activities are inhibited by an affinity-purified, polyclonal antibody generated to recombinant AAMP. Inhibition of endothelial cell tube formation was previously shown and now endothelial cell migration assays using this antibody show dose-dependent inhibition (75%) of endothelial cell migration. Also, antisense inhibition has been used to determine the effects of reducing total AAMP (extracellular and intracellular forms). An AAMP-specific antisense oligonucleotide that targets a region near its amino terminus, anti-MES, inhibits (45%) total AAMP production by bovine aortic endothelial cells (BAECs), compared to a negative control oligonucleotide. Paradoxically, comparable use of antisense-MES results in a 27% increase in BAEC motility. Decreased cellular production of total AAMP (via antisense) that results in an increase of endothelial migration contrasts with antibody inhibition of extracellular AAMP that decreases migration. This indicates compartment-specific roles for AAMP in angiogenesis. Transwell cocultures of human astrocytes and BAECs increase (53%) the amount of extracellular AAMP found associated with endothelial cells. Therefore, regulation of extracellular AAMP by astrocytes is hypothesized to aid in angiogenesis of the nervous system. Extracellular AAMP's positive role may be either as a promoter or as a permissive protein in this process.

Intracranial extramedullary hematopoiesis associated with pilocytic astrocytoma: a case report.

Intracranial EMH is only occasionally found in primary brain tumors (mostly hemangioblastomas) and, to our knowledge, this is the first case of EMH associated with an astrocytoma. Intracranial extramedullary hematopoiesis (EMH) is described in a 29-year-old man with a recurrent pilocytic astrocytoma in the tectal region. Special stains confirmed the identities of erythroid, myeloid and megakaryocytic cells. The patient had no evidence of a predisposing bone marrow disorder or systemic EMH. Although the presence of multinucleated and blastic cells associated with a low-grade brain neoplasm is unusual, recognition of hematopoietic lineages allows EMH to be readily identified. Another tumor resection after a year of follow-up confirmed the absence of malignant progression in this recurrent astrocytoma. The small number of cases describing intracranial EMH in the absence of systemic hematologic abnormalities are correlated with the findings in this case. The low incidence of intracranial EMH indicates that cells with hematopoietic potential are seldom exposed to a supportive microenvironment within the central nervous system. However, intracranial EMH should be included as a potential, ancillary diagnosis when considering brain lesions. This may be particularly true if medical therapies involving growth factors or stem cells are found to promote hematopoiesis.