Pediatric Glioblastoma Cell Research Articles

Overexpression and constitutive nuclear localization of cohesin protease Separase protein correlates with high incidence of relapse and reduced overall survival in glioblastoma multiforme.

Separase, an enzyme that cleaves the chromosomal cohesin during mitosis, is overexpressed in a wide range of human epithelial cancers of breast, bone and prostate (Meyer et al., Clin Cancer Res 15(8):2703-2710, 2009). Overexpression of Separase in animal models results in aneuploidy and tumorigenesis. We have examined the expression and localization of Separase protein in adult and pediatric glioblastoma and normal brain specimens. Immunofluorescence microscopy and Western blot analysis showed significant overexpression of Separase in all adult and a subset of pediatric glioblastoma cells. Tumor status and patient survival strongly correlate with the mislocalization of Separase into the nucleus throughout all stages of the cell cycle. Unlike exclusively nuclear localization in mitotic control cells, glioblastoma samples have a significantly higher number of resting (interphase) cells with strong nuclear Separase staining. Additionally, patient survival analysis demonstrated a strong correlation between overexpression of Separase protein in adult glioblastoma and a high incidence of relapse and reduced overall survival. These results further strengthen our hypothesis that Separase is an oncogene whose overexpression induces tumorigenesis, and indicate that Separase overexpression and aberrant nuclear localization are common in many tumor types and may predict outcome in some human malignancies.

MerTK inhibition is a novel therapeutic approach for glioblastoma multiforme

Glioblastoma is an aggressive tumor that occurs in both adult and pediatric patients and is known for its invasive quality and high rate of recurrence. Current therapies for glioblastoma result in high morbidity and dismal outcomes. The TAM subfamily of receptor tyrosine kinases includes Tyro3, Axl, and MerTK. Axl and MerTK exhibit little to no expression in normal brain but are highly expressed in glioblastoma and contribute to the critical malignant phenotypes of survival, chemosensitivity and migration. We have found that Foretinib, a RTK inhibitor currently in clinical trial, inhibited phosphorylation of TAM receptors, with highest efficacy against MerTK, and blocked downstream activation of Akt and Erk in adult and pediatric glioblastoma cell lines, findings that are previously unreported. Survival, proliferation, migration, and collagen invasion were hindered in vitro. Foretinib treatment in vivo abolished MerTK phosphorylation and reduced tumor growth 3-4 fold in a subcutaneous mouse model. MerTK targeted shRNA completely prevented intracranial and subcutaneous glioma growth further delineating the impact of MerTK inhibition on glioblastoma. Our findings provide additional target validation for MerTK inhibition in glioblastoma and demonstrate that robust MerTK inhibition can be achieved with the multi-kinase inhibitor Foretinib as an innovative and translational therapeutic approach to glioblastoma.

BUB1 and BUBR1 inhibition decreases proliferation and colony formation, and enhances radiation sensitivity in pediatric glioblastoma cells

Glioblastoma (GBM) is a very aggressive and lethal brain tumor with poor prognosis. Despite new treatment strategies, patients' median survival is still lower than 1 year in most cases. The expression of the BUB gene family has demonstrated to be altered in a variety of solid tumors, pointing to a role as putative therapeutic target. The purpose of this study was to determine BUB1, BUB3, and BUBR1 gene expression profiles in glioblastoma and to analyze the effects of BUB1 and BUBR1 inhibition combined or not with Temozolomide and radiation in the pediatric SF188 GBM cell line. For gene expression analysis, 8 cell lines and 18 tumor samples were used. The effect of BUB1 and BUBR1 inhibition was evaluated using siRNA. Apoptosis, cell proliferation, cell cycle kinetics, micronuclei formation, and clonogenic capacity were analyzed after BUB1 and BUBR1 inhibition. Additionally, combinatorial effects of gene inhibition and radiation or Temozolomide (TMZ) treatment were evaluated through proliferation and clonogenic capacity assays. We report the upregulation of BUB1 and BUBR1 expression and the downregulation of BUB3 in GBM samples and cell lines when compared to white matter samples (p < 0.05). Decreased cell proliferation and colony formation after BUB1 and BUBR1 inhibition were observed, along with increased micronuclei formation. Combinations with TMZ also caused cell cycle arrest and increased apoptosis. Moreover, our results demonstrate that BUB1 and BUBR1 inhibition sensitized SF188 cells to γ-irradiation as shown by decreased growth and abrogation of colony formation capacity. BUB1 and BUBR1 inhibition decreases proliferation and shows radiosensitizing effects on pediatric GBM cells, which could improve treatment strategies for this devastating tumor. Collectively, these findings highlight the potentials of BUB1 and BUBR1 as putative therapeutic targets for glioblastoma treatment.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 5640: Picropodophyllin downregulates p53 and increases the Warburg effect in pediatric glioblastoma cells.

Abstract Picropodophyllin (PPP) is reported to be a potent and selective inhibitor of tyrosine phosphorylation of insulin-like growth factor-1 receptor (IGF-1R) [Girnita et al., Clin Cancer Res 12:1383-91, 2006]. We previously showed increases in glucose uptake and lactate production in paediatric glioblastoma KNS42 cells after 24h of PPP-treatment [Wong Te Fong et al., Proc Mol Tar Ther B61, 2012]. These findings are unexpected for IGF-1R inhibition. Upregulation of c-Myc or downregulation of p53 are known to upregulate glycolysis in cancer [DeBerardinis et al., Cell Metab 7:11-20, 2008a; Kawauchi et al., Nat Cell Bio 30(9): 1792-5, 2007]. In order to identify the molecular mechanism that drives our previously observed upregulation of glycolysis following PPP treatment, we examine the changes in glucose metabolism in PPP-treated cells over a 96h time-course and relate these metabolic changes to c-Myc and p53 expression. KNS42 cells were treated for 6, 24, 48 and 96h with 2.4μM (5xIC50) of PPP. Culture media from the PPP-treated and control cells were analyzed by 1H Magnetic Resonance Spectroscopy (MRS) to examine the effect of PPP on glucose uptake and lactate excretion. pErk, pAkt, c-Myc, p53 and β-actin (loading control) protein expressions were examined by Western blot (WB). No change in cell number was found after 6h of PPP treatment but ∼50% of PPP-treated cells remained by 24h when compared to controls (p&amp;lt;0.001) and similar numbers of treated cells were also observed at 48 and 96h, indicating that a cohort of cells was initially killed by PPP (the doubling time of KNS42 is ∼30h) and that the remaining cell population survived the treatment. WB showed decreases in pErk (from 6h) and pAkt (from 24h) expression in PPP-treated cells, consistent with the mechanism of drug action. 1H MRS analysis of culture media showed significant increases in glucose uptake (p&amp;lt;0.01) and lactate excretion (P&amp;lt;0.0001) in PPP-treated cells at 24, 48 and 96h, and these metabolic changes are associated with decreased expression of p53 at these time-points. No significant metabolic changes were found in PPP-treated cells at 6h and p53 was unchanged compared to controls at this time-point. Reduced expression of c-Myc was found in PPP-treated cells from 24h. Our data indicate that it is p53 downregulation and not changes in c-Myc expression that drives the upregulation of glycolysis in KNS42 cells from 24h of PPP-treatment, and that these cellular and metabolic adaptations help cellular survival during treatment, suggesting a possible mechanism of resistance to PPP. Downregulation of p53 may drive the upregulation of glycolysis, representing a possible mechanism of resistance to PPP treatment in KNS42 cells. We acknowledge the support received from the CRUK and EPSRC Cancer Imaging Centre in association with the MRC and Department of Health (England) grant C1060/A10334, NHS funding to the NIHR Biomedical Research Centre. Citation Format: Anne-Christine LF Wong Te Fong, Chris Jones, John R. Griffiths, Martin O. Leach, Yuen-Li Chung. Picropodophyllin downregulates p53 and increases the Warburg effect in pediatric glioblastoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5640. doi:10.1158/1538-7445.AM2013-5640

Abstract B61: Picropodophyllin (PPP) increases glucose metabolism and lactate production in paediatric glioblastoma cells

Abstract Introduction: Picropodophyllin (PPP) is of interest as an anti-cancer agent, as it has been shown to induce growth inhibition and apoptosis in a variety of human cancer cells and xenografts. Recently, PPP was found to have dramatic antitumor effects in human glioblastoma cells and in subcutaneous and intracerebral xenografts and reported to be an insulin-like growth factor-1 receptor inhibitor; however, its full mechanisms of action remain unclear. Aims: To examine the effect of PPP on glucose metabolism in cancer cells and to use hyperpolarized [1-13C]pyruvate 13C-magnetic resonance spectroscopy (MRS) and conventional 1H-MRS to measure lactate formation/production in real-time and in steady state, respectively, in order to develop a non-invasive biomarker of response for PPP treatment in a pediatric glioblastoma cell line. Methods: Pediatric glioblastoma KNS42 cells were treated for 24 hours with 2.4μM (5×IC50) of PPP. Real-time 13C exchange from hyperpolarized [1-13C] pyruvate to lactate was performed by DNP-13C MRS. Culture media and extracts from the PPP-treated and control KNS42 cells were analyzed by 1H-MRS to examine the effect of PPP on glucose metabolism and lactate production. Lactate dehydrogenase (LDH) expression was examined by western blot and LDH activity was also measured enzymatically. Cellular protein content and size were also evaluated. Results and Discussion: The rate of real-time 13C exchange from pyruvate to lactate measured by DNP-13C MRS showed a significant increase in the PPP-treated group (P = 0.027 versus control group). These DNP changes in PPP-treated KNS42 cells are consistent with the significant increase in intracellular lactate (P = 0.003) and lactate excretion to the culture media (P &amp;lt; 0.001). This increase in lactate production is also consistent with significantly elevated LDH activity (P = 0.003) in the PPP-treated group. However, no change in LDH expression was observed in the PPP-treated group. Significant increases in intracellular glucose (P = 0.006) and glucose uptake (P = 0.008) were also found in PPP-treated KNS42 cells. These changes indicate an up-regulation of glucose metabolism following PPP treatment and a subsequent increase in lactate production. Twenty-four hours PPP treatment caused a significant decrease in cell number when compared to controls (∼40%, P = 0.001). No significant difference in cellular protein concentration and size were found in PPP-treated cells when compared to controls, indicating that the observed increases in glucose metabolism, lactate production and LDH activity are due to changes in cellular metabolism following PPP treatment and not from changes in cellular size or protein concentration. The increase in lactate production following PPP treatment found in this study is an unusual observation, as most cancer targeting-treatments cause a decrease in lactate production. Hence, the use of DNP and 13C MRS to measure lactate production could provide a more specific non-invasive biomarker of response for PPP treatment. Conclusions: PPP treatment resulted in increased glucose metabolism and lactate production in KNS42 cells, as shown by both real time and steady-state measurements. These changes have potential as specific biomarkers of response for PPP treatment. We acknowledge the support received from the CRUK and EPSRC Cancer Imaging Centre with the MRC and Department of Health (England) grant C1060/A10334, NHS funding to the NIHR Biomedical Research Centre.

Abstract 2501: Inhibition of the PI3K pathway potentiates temozolomide effects in pediatric glioblastoma and results in alterations in glucose and choline metabolism detected by MRS

Abstract Introduction: Children with high grade-glioma have a poor outcome with a 5-year survival rate of less than 20%. The use of adjuvant temozolomide (TMZ) has now been well accepted as a standard of care in glioblastoma. However, it does not seem to improve survival in children. We have recently shown that resistance to TMZ in pediatric glioma cell lines can be reversed by PI3K pathway inhibition. The aim of this study was to identify whether combination of PI3K pathway inhibition with TMZ would result in metabolic changes detectable by magnetic resonance spectroscopy (MRS), and potentially can be used as a noninvasive method of monitoring response in early stage clinical trials in children with glioma. Methods: The MGMT-independent TMZ-resistant pediatric glioblastoma cell line KNS42 was treated for 48h with dual PI3K/mTOR inhibitor PI-103 (2xGI50), TMZ (2xGI50) or with the combination of both (each at 2xGI50). Control and treated cells were extracted and the aqueous fractions were analyzed by 1H MRS. Results and Discussion: The number of treated KNS42 cells was significantly reduced relative to their controls, to 79±8% and 65±10% (P&amp;lt;0.02) following treatment with PI-103 and TMZ respectively. Combination of PI-103 and TMZ further reduced the number of treated vs. control cells (down to 54±6%, P&amp;lt;0.01). 1H MRS analysis showed that PI-103 treatment caused a decrease in lactate, phosphocholine (PC) and total choline (tCho) levels down to 48±17%, 52±11%, 64±14% (P&amp;lt;0.02) respectively. In contrast, treament with TMZ resulted in an increase in glycerolphosphocholine (GPC) up to 176±43% (P=0.05). Combination of PI-103 with TMZ retained the combined metabolic effects of both agents including a decrease in the levels of lactate (49±12%, P&amp;lt;0.01) and PC (71±11%, P&amp;lt;0.02) while an increase in GPC (190±40%, P&amp;lt;0.03) levels. tCho was normalized by the opposing changes in PC and GPC and hence was similar to controls. To explore potential mechanisms underlying the detected metabolic changes, immunoblotting was used to assess treatment effects on protein levels of enzymes involved in glycolysis and choline metabolism. The decrease in PC levels was associated with a decrease in the protein levels of Choline kinase (ChoK), the enzyme responsible for choline phosphorylation into PC. ChoK was not affected by TMZ single treatment leading to no change in PC levels. Furthermore, a decrease in hexokinase II protein was detected following PI-103 treatment and the combination, but not with TMZ alone, suggesting a mechanism for depletion of lactate. Conclusions: Our results show that using MRS we can detect distinct biomarkers following treatment with PI-103 compared to TMZ. Choline metabolites and lactate are potential noninvasive biomarkers for monitoring response to PI3K/mTOR inhibitors both as single agents and in combination with cytotoxic therapy in children with glioma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2501. doi:1538-7445.AM2012-2501

MGMT-Independent Temozolomide Resistance in Pediatric Glioblastoma Cells Associated with a PI3-Kinase–Mediated HOX/Stem Cell Gene Signature

Sensitivity to temozolomide is restricted to a subset of glioblastoma patients, with the major determinant of resistance being a lack of promoter methylation of the gene encoding the repair protein DNA methyltransferase MGMT, although other mechanisms are thought to be active. There are, however, limited preclinical data in model systems derived from pediatric glioma patients. We screened a series of cell lines for temozolomide efficacy in vitro, and investigated the differential mechanisms of resistance involved. In the majority of cell lines, a lack of MGMT promoter methylation and subsequent protein overexpression were linked to temozolomide resistance. An exception was the pediatric glioblastoma line KNS42. Expression profiling data revealed a coordinated upregulation of HOX gene expression in resistant lines, especially KNS42, which was reversed by phosphoinositide 3-kinase pathway inhibition. High levels of HOXA9/HOXA10 gene expression were associated with a shorter survival in pediatric high-grade glioma patient samples. Combination treatment in vitro of pathway inhibition and temozolomide resulted in a highly synergistic interaction in KNS42 cells. The resistance gene signature further included contiguous genes within the 12q13-q14 amplicon, including the Akt enhancer PIKE, significantly overexpressed in the KNS42 line. These cells were also highly enriched for CD133 and other stem cell markers. We have thus shown an in vitro link between phosphoinositide 3-kinase-mediated HOXA9/HOXA10 expression, and a drug-resistant, progenitor cell phenotype in MGMT-independent pediatric glioblastoma.

EGFRvIII Deletion Mutations in Pediatric High-Grade Glioma and Response to Targeted Therapy in Pediatric Glioma Cell Lines

The epidermal growth factor receptor (EGFR) is amplified and overexpressed in adult glioblastoma, with response to targeted inhibition dependent on the underlying biology of the disease. EGFR has thus far been considered to play a less important role in pediatric glioma, although extensive data are lacking. We have sought to clarify the role of EGFR in pediatric high-grade glioma (HGG). We retrospectively studied a total of 90 archival pediatric HGG specimens for EGFR protein overexpression, gene amplification, and mutation and assessed the in vitro sensitivity of pediatric glioma cell line models to the small-molecule EGFR inhibitor erlotinib. Amplification was detected in 11% of cases, with corresponding overexpression of the receptor. No kinase or extracellular domain mutations were observed; however, 6 of 35 (17%) cases harbored the EGFRvIII deletion, including two anaplastic oligodendrogliomas and a gliosarcoma overexpressing EGFRvIII in the absence of gene amplification and coexpressing platelet-derived growth factor receptor alpha. Pediatric glioblastoma cells transduced with wild-type or deletion mutant EGFRvIII were not rendered more sensitive to erlotinib despite expressing wild-type PTEN. Phosphorylated receptor tyrosine kinase profiling showed a specific activation of platelet-derived growth factor receptor alpha/beta in EGFRvIII-transduced pediatric glioblastoma cells, and targeted coinhibition with erlotinib and imatinib leads to enhanced efficacy in this model. These data identify an elevated frequency of EGFR gene amplification and EGFRvIII mutation in pediatric HGG than previously recognized and show the likely necessity of targeting multiple genetic alterations in the tumors of these children.

Pediatric glioblastoma cell line shows different patterns of expression of transmembrane ABC transporters after in vitro exposure to vinblastine

Resistance to drug is a major cause of treatment failure in pediatric brain cancer. The multidrug resistance (MDR) phenotype can be mediated by the superfamily of adenosine triphosphate-binding cassette (ABC) transporters. The dynamics of expression of the MDR genes after exposure to chemotherapy, especially the comparison between pediatric brain tumors of different histology, is poorly described. To compare the expression profiles of the multidrug resistance genes ABCB1, ABCC1, and ABCG2 in different neuroepithelial pediatric brain tumor cell lines prior and following short-term culture with vinblastine. Immortalized lineages from pilocytic astrocytoma (R286), anaplasic astrocytoma (UW467), glioblastoma (SF188), and medulloblastoma (UW3) were exposed to vinblastine sulphate at different schedules (10 and 60 nM for 24 and 72 h). Relative amounts of mRNA expression were analyzed by real-time quantitative polymerase chain reaction. Protein expression was assessed by immunohistochemistry for ABCB1, ABCC1, and ABCG2. mRNA expression of ABCB1 increased together with augmenting concentration and time of exposure to vinblastine for R286, UW467, and UW3 cell lines. Interestingly, ABCB1 levels of expression diminished in SF188. Following chemotherapy, mRNA expression of ABCC1 decreased in all cell lines other than glioblastoma. ABCG2 expression was influenced by vinblastine only for UW3. The mRNA levels showed consistent association to protein expression in the selected sets of cell lines analyzed. The pediatric glioblastoma cell line SF188 shows different pattern of expression of multidrug resistance genes when exposed to vinblastine. These preliminary findings may be useful in determining novel strategies of treatment for neuroepithelial pediatric brain tumors.

Functional expression of bombesin receptor in most adult and pediatric human glioblastoma cell lines; role in mitogenesis and in stimulating the mitogen-activated protein kinase pathway

Functional bombesin receptors were identified in most human glioblastoma cell lines examined (∼85% of lines). Bombesin stimulated the release of intracellular Ca 2+ in human adult (U-373MG, D-247MG, U-118MG, U-251MG, D-245MG, U-105MG, D-54MG, A-172MG, and D-270MG lines) and pediatric (SJ-S6 and SJ-G2 lines) glioblastoma cell lines. Stimulation of the glioblastoma cell line U-373MG with bombesin or gastrin-releasing peptide (GRP) induced mitogenesis, measured by [ 3H]thymidine incorporation into DNA, and stimulated the tyrosine phosphorylation of the mitogen-activated protein (MAP) kinases (Erk1 and Erk2). The stimulation of the MAP kinase phosphorylation in U-373MG cells was time- and peptide concentration-dependent. Both bombesin and GRP showed similar potencies in stimulation of intracellular Ca 2+ release and activation of the MAP kinase pathway in U-373MG cells, whereas neuromedin B (NMB) peptide was less potent. Bombesin and GRP induced the release of cytosolic Ca 2+ in a concentration-dependent manner. Because bombesin and GRP were more potent than NMB peptide in increasing the cytosolic Ca 2+ levels in U-373MG cells, we concluded that the BB 2 subtype (also known as GRP-preferring receptor subtype) of the bombesin receptor is expressed in this cell line. The bombesin receptor antagonist ([Leu 13- ψ(CH 2NH)Leu 14]bombesin) blocked bombesin induced Ca 2+ release and attenuated MAP kinase activation in U-373MG cells demonstrating that bombesin is acting through a receptor-dependent mechanism. This study indicates that functional bombesin receptors are widely expressed in human glioblastoma cell lines.