Brain tumors can arise following deregulation of signaling pathways normally activated during brain development and may derive from neural stem cells. Given the requirement for Hedgehog in non-neoplastic stem cells, we investigated whether Hedgehog blockade could target the stem-like population in glioblastoma multiforme (GBM). We found that Gli1, a key Hedgehog pathway target, was highly expressed in 5 of 19 primary GBM and in 4 of 7 GBM cell lines. Shh ligand was expressed in some primary tumors, and in GBMderived neurospheres, suggesting a potential mechanism for pathway activation. Hedgehog pathway blockade by cyclopamine caused a 40%-60% reduction in growth of adherent glioma lines highly expressing Gli1 but not in those lacking evidence of pathway activity. When GBM-derived neurospheres were treated with cyclopamine and then dissociated and seeded in media lacking the inhibitor, no new neurospheres formed, suggesting that the clonogenic cancer stem cells had been depleted. Consistent with this hypothesis, the stem-like fraction in gliomas marked by both aldehyde dehydrogenase activity and Hoechst dye excretion (side population) was significantly reduced or eliminated by cyclopamine. In contrast, we found that radiation treatment of our GBM neurospheres increased the percentage of these stemlike cells, suggesting that this standard therapy preferentially targets better-differentiated neoplastic cells. Most importantly, viable GBM cells injected intracranially following Hedgehog blockade were no longer able to form tumors in athymic mice, indicating that a cancer stem cell population critical for ongoing growth had been removed. STEM CELLS

Hypoxia promotes the expansion of non-neoplastic stem and precursor cell populations in the normal brain, and is common in malignant brain tumors. We examined the effects of hypoxia on stem-like cells in glioblastoma (GBM). When GBM-derived neurosphere cultures are grown in 1% oxygen, hypoxia-inducible factor 1␣ (HIF1␣) protein levels increase dramatically, and mRNA encoding other hypoxic response genes, such as those encoding hypoxia-inducible gene-2, lysyl oxidase, and vascular endothelial growth factor, are induced over 10-fold. Hypoxia increases the stem-like side population over fivefold, and the percentage of cells expressing CD133 threefold or more. Notch pathway ligands and targets are also induced. The rise in the stem-like fraction in GBM following hypoxia is paralleled by a twofold increase in clonogenicity. We believe HIF1␣ plays a causal role in these changes, as when oxygen-stable HIF1␣ is expressed in normoxic glioma cells CD133 is induced. We used digoxin, which has been shown to lower HIF protein levels in vitro and in vivo, to inhibit the hypoxic response. Digoxin suppressed HIF1␣ protein expression, HIF1␣ downstream targets, and slowed tumor growth in vivo. In addition, pretreatment with digoxin reduced GBM flank xenograft engraftment of hypoxic GBM cells , and daily intraperitoneal injections of digoxin were able to significantly inhibit the growth of established subcutaneous glioblastoma xenografts , and suppressed expression of vascular endothelial growth factor.

Relatively little is known about the molecular changes that promote the formation or growth of pilocytic astrocytomas. We investigated genomic alterations in 25 pilocytic astrocytomas, including 5 supratentorial and 20 posterior fossa tumors, using oligonucleotide array comparative genomic hybridization. Large changes were identified in 7 tumors and included gains of chromosomes 5, 6, and 7 and losses of chromosomes 16, 17, 19, and 22. The most common alteration was a 1.9-MB region of low-level gain at chromosome 7q34 identified in 17 of 20 posterior fossa tumors. In most tumors, the region of gain ended within the BRAF locus and encompassed only exons that encode the BRAF kinase domain. We confirmed copy number increase at the 7q34 locus using quantitative polymerase chain reaction with primers adjacent to the HIPK2, RAB19B, and BRAF genes. Western blot analysis revealed that 3 of 6 pilocytic astrocytomas with 7q34 gain contained high levels of phosphorylated extracellular signal-related kinase (ERK) and nitrogen-activated protein kinase/ERK kinase (MEK), while 1 tumor lacking 7q34 gain and 2 normal brain specimens did not. Immunohistochemical stains of a tissue microarray containing 43 pilocytic astrocytoma identified ERK phosphorylation in 35 (81%). These data indicate that focal gains at chromosome 7q34 and increased BRAF-MEK-ERK signaling are common findings in sporadic pilocytic astrocytomas.

Purpose BRAF is frequently activated by gene fusion or point mutation in pilocytic astrocytoma, the most common pediatric brain tumor. We investigated the functional effect of constitutive BRAF activation in normal human neural stem and progenitor cells to determine its role in tumor induction in the brain. Experimental Design The constitutively active BRAFV600E allele was introduced into human neurospheres, and its effects on MAPK (mitogen-activated protein kinase) signaling, proliferation, soft agarose colony formation, stem cell phenotype, and induction of cellular senescence were assayed. Immunohistochemistry was used to examine p16INK4a levels in pilocytic astrocytoma. Results BRAFV600E expression initially strongly promoted colony formation but did not lead to significantly increased proliferation. BRAFV600E-expressing cells subsequently stopped proliferating and induced markers of oncogene-induced senescence including acidic β-galactosidase, PAI-1, and p16INK4a whereas controls did not. Onset of senescence was associated with decreased expression of neural stem cell markers including SOX2. Primary pilocytic astrocytoma cultures also showed induction of acidic β-galactosidase activity. Immunohistochemical examination of 66 pilocytic astrocytomas revealed p16INK4a immunoreactivity in the majority of cases, but patients with tumors negative for p16INK4a had significantly shorter overall survival. Conclusions BRAF activation in human neural stem and progenitor cells initially promotes clonogenic growth in soft agarose, suggesting partial cellular transformation, but oncogene-induced senescence subsequently limits proliferation. Induction of senescence by BRAF may help explain the low-grade pathobiology of pilocytic astrocytoma, whereas worse clinical outcomes associated with tumors lacking p16INK4a expression could reflect failure to induce senescence or an escape from oncogene-induced senescence.

Recent studies highlight the importance of BRAF alterations resulting in mitogen activated protein kinase (MAK/ERK) pathway activation in low-grade CNS tumors. We studied 106 low-grade CNS neoplasms in a cohort of primarily pediatric patients to identify the prevalence and clinicopathologic significance of these alterations. Polymerase chain reaction testing identified KIAA1549:BRAF fusions in 51 (48%) tumors overall, including 42 (60%) pilocytic astrocytomas, 4 (17%) unclassifiable low-grade gliomas, 4 (36%) low-grade glioneuronal/neuroepithelial tumors, 0 (of 5) pleomorphic xanthoastrocytomas, 0 (of 4) diffuse astrocytomas (World Health Organization grade II), and 1 (of 3, 33%) pilomyxoid astrocytoma. KIAA1549:BRAF gene fusions confirmed by sequencing included the previously reported ones involving exons 1–16/9–18 (49%), 1–15/9–18 (35%), and 1–16/11–18 (8%) and 2 fusions with novel breakpoints: 1–15/11–18 (6%) and 1–17/10–18 (1%). DNA sequencing identified BRAFV600E mutations in 8% of tumors. BRAFG468A mutations were absent. KIAA1549:BRAF fusions were significantly more frequent in infratentorial (57%) and optic pathway (59%) tumors versus supratentorial (19%) tumors (p = 0.001). We did not identify significantly improved progression-free survival in tumors with fusions. In summary, KIAA1549:BRAF fusions predominate in pilocytic astrocytomas but are also present in some low-grade unclassifiable gliomas and glioneuronal tumors. The prognostic and therapeutic significance of this alteration is unclear and merits further study.

Curcumin is a polyphenolic compound derived from the Indian spice turmeric. We used nanoparticle-encapsulated curcumin to treat medulloblastoma and glioblastoma cells. This formulation caused a dose-dependent decrease in growth of multiple brain tumor cell cultures, including the embryonal tumor derived lines DAOY and D283Med, and the glioblastoma neurosphere lines HSR-GBM1 and JHH-GBM14. The reductions in viable cell mass observed were associated with a combination of G(2)/M arrest and apoptotic induction. Curcumin also significantly decreased anchorage-independent clonogenic growth and reduced the CD133-positive stem-like population. Down-regulation of the insulin-like growth factor pathway in DAOY medulloblastoma cells was observed, providing one possible mechanism for the changes. Levels of STAT3 were also attenuated. Hedgehog signaling was blocked in DAOY cells but Notch signaling was not inhibited. Our data suggest that curcumin nanoparticles can inhibit malignant brain tumor growth through the modulation of cell proliferation, survival and stem cell phenotype.

Purpose: Multiple developmental pathways including Notch, Hedgehog, and Wnt are active in malignant brain tumors such as medulloblastoma and glioblastoma (GBM). This raises the possibility that tumors might compensate for therapy directed against one pathway by upregulating a different one. We investigated whether brain tumors show resistance to therapies against Notch, and whether targeting multiple pathways simultaneously would kill brain tumor cells more effectively than monotherapy.Experimental Design: We used GBM neurosphere lines to investigate the effects of a gamma-secretase inhibitor (MRK-003) on tumor growth, and chromatin immunoprecipitation to study the regulation of other genes by Notch targets. We also evaluated the effect of combined therapy with a Hedgehog inhibitor (cyclopamine) in GBM and medulloblastoma lines, and in primary human GBM cultures.

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