Eli E. Bar

A polymeric nanoparticle formulation of curcumin inhibits growth, clonogenicity and stem-like fraction in malignant brain tumors

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 G2/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. See commentary: Indian gold treating cancer in the age of nano

BRAF alterations in primary glial and glioneuronal neoplasms of the central nervous system with identification of 2 novel KIAA1549:BRAF fusion variants.

Abstract 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.

BRAF Activation Induces Transformation and Then Senescence in Human Neural Stem Cells: A Pilocytic Astrocytoma Model

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. Clin Cancer Res; 17(11); 3590–9. ©2011 AACR.

The Notch Target Hes1 Directly Modulates Gli1 Expression and Hedgehog Signaling: A Potential Mechanism of Therapeutic Resistance

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. Results: GBM cells are at least partially resistant to long-term MRK-003 treatment, despite ongoing Notch pathway suppression, and show concomitant upregulation of Wnt and Hedgehog activity. The Notch target Hes1, a repressive transcription factor, bound the Gli1 first intron, and may inhibit its expression. Similar results were observed in a melanoma-derived cell line. Targeting Notch and Hedgehog simultaneously induced apoptosis, decreased cell growth, and inhibited colony-forming ability more dramatically than monotherapy. Low-passage neurospheres isolated from freshly resected human GBMs were also highly susceptible to coinhibition of the two pathways, indicating that targeting multiple developmental pathways can be more effective than monotherapy at eliminating GBM-derived cells. Conclusions: Notch may directly suppress Hedgehog via Hes1 mediated inhibition of Gli1 transcription, and targeting both pathways simultaneously may be more effective at eliminating GBMs cells. Clin Cancer Res; 16(24); 6060–70. ©2010 AACR.

Notch Signaling Promotes Growth and Invasion in Uveal Melanoma

Purpose: To determine whether uveal melanoma, the most common primary intraocular malignancy in adults, requires Notch activity for growth and metastasis. Experimental Design: Expression of Notch pathway members was characterized in primary tumor samples and in cell lines, along with the effects of Notch inhibition or activation on tumor growth and invasion. Results: Notch receptors, ligands, and targets were expressed in all five cell lines examined and in 30 primary uveal melanoma samples. Interestingly, the three lines with high levels of baseline pathway activity (OCM1, OCM3, and OCM8) had their growth reduced by pharmacologic Notch blockade using the γ-secretase inhibitor (GSI) MRK003. In contrast, two uveal melanoma lines (Mel285 and Mel290) with very low expression of Notch targets were insensitive to the GSI. Constitutively active forms of Notch1 and Notch2 promoted growth of uveal melanoma cultures and were able to rescue the inhibitory effects of GSI. MRK003 treatment also inhibited anchorage-independent clonogenic growth and cell invasion and reduced phosphorylation levels of STAT3 and extracellular signal-regulated kinase (Erk)1/2. Suppression of canonical Notch activity using short hairpin RNA targeting Notch2 or CBF1 was also able to reduce tumor growth and invasion. Finally, intraocular xenograft growth was significantly decreased by GSI treatment. Conclusion: Our findings suggest that Notch plays an important role in inducing proliferation and invasion in uveal melanoma and that inhibiting this pathway may be effective in preventing tumor growth and metastasis. Clin Cancer Res; 18(3); 654–65. ©2012 AACR.

MicroRNA profiling in pediatric pilocytic astrocytoma reveals biologically relevant targets, including PBX3, NFIB, and METAP2

Pilocytic astrocytoma (PA) is a World Health Organization grade I glioma that occurs most commonly in children and young adults. Specific genetic alterations have been described in PA, but the pathogenesis remains poorly understood. We studied microRNA (miRNA) alterations in a large cohort of patients with PA. A total of 43 PA, including 35 sporadic grade I PA, 4 neurofibromatosis-1 (NF1)-associated PA, and 4 PA with pilomyxoid features, as well as 5 nonneoplastic brain controls were examined. BRAF fusion status was assessed in most cases. RNA was examined using the Agilent Human miRNA Microarray V3 platform. Expression of miRNA subsets was validated using quantitative real-time PCR (qRT-PCR) with Taqman probes. Validation of predicted protein targets was performed on tissue microarrays with the use of immunohistochemistry. We identified a subset of miRNAs that were differentially expressed in pediatric PAs versus normal brain tissue: 13 miRNAs were underexpressed, and 20 miRNAs were overexpressed in tumors. Differences were validated by qRT-PCR in a subset, with mean fold change in tumor versus brain of -17 (miR-124), -15 (miR-129), and 19.8 (miR-21). Searching for predicted protein targets in Targetscan, we identified a number of known and putative oncogenes that were predicted targets of miRNA sets relatively underexpressed in PA. Predicted targets with increased expression at the mRNA and/or protein level in PA included PBX3, METAP2, and NFIB. A unique miRNA profile exists in PA, compared with brain tissue. These miRNAs and their targets may play a role in the pathogenesis of PA.

Prolonged Inhibition of Glioblastoma Xenograft Initiation and Clonogenic Growth following In Vivo Notch Blockade

Purpose: To examine the effects of clinically relevant pharmacologic Notch inhibition on glioblastoma xenografts. Experimental Design: Murine orthotopic xenografts generated from temozolomide-sensitive and -resistant glioblastoma neurosphere lines were treated with the γ-secretase inhibitor MRK003. Tumor growth was tracked by weekly imaging, and the effects on animal survival and tumor proliferation were assessed, along with the expression of Notch targets, stem cell, and differentiation markers, and the biology of neurospheres isolated from previously treated xenografts and controls. Results: Weekly MRK003 therapy resulted in significant reductions in growth as measured by imaging, as well as prolongation of survival. Microscopic examination confirmed a statistically significant reduction in cross-sectional tumor area and mitotic index in a MRK003-treated cohort as compared with controls. Expression of multiple Notch targets was reduced in the xenografts, along with neural stem/progenitor cell markers, whereas glial differentiation was induced. Neurospheres derived from MRK003-treated xenografts exhibited reduced clonogenicity and formed less aggressive secondary xenografts. Neurospheres isolated from treated xenografts remained sensitive to MRK003, suggesting that therapeutic resistance does not rapidly arise during in vivo Notch blockade. Conclusions: Weekly oral delivery of MRK003 results in significant in vivo inhibition of Notch pathway activity, tumor growth, stem cell marker expression, and clonogenicity, providing preclinical support for the use of such compounds in patients with malignant brain tumors. Some of these effects can persist for some time after in vivo therapy is complete. Clin Cancer Res; 19(12); 3224–33. ©2013 AACR.

Dse1 may control cross talk between the pheromone and filamentation pathways in yeast.

The filamentous/invasive growth pathway is activated by nutrient limitation in the haploid form of the yeast Saccharomyces cerevisiae, whereas exposure to mating-pheromone causes cells to differentiate into gametes. Although these two pathways respond to very different stimuli and generate very different responses, they utilize many of the same signaling components. This implies the need for robust mechanisms to maintain signal fidelity. Dse1 was identified in an allele-specific suppressor screen for proteins that interact with the pheromone-responsive Gβγ, and found to bind both to a Gβγ-affinity column, and to the shared MEKK, Ste11. Although overexpression of Dse1 stimulated invasive growth and transcription of both filamentation and mating-specific transcriptional reporters, deletion of DSE1 had no effect on these outputs. In contrast, pheromone hyper-induced transcription of the filamentation reporter in cells lacking Dse1 and in cells expressing a mutant form of Gβ that exhibits diminished interaction with Dse1. Thus, the interaction of Dse1 with both Gβ and Ste11 may be designed to control cross talk between the pheromone and filamentation pathways.

Notch signaling activation in pediatric low-grade astrocytoma.

Abstract Pilocytic astrocytoma (PA) is the most common primary brain tumor in children; various signaling pathways have been implicated in its biology. The Notch signaling pathway has been found to play a role in the development, stem cell biology, and pathogenesis of several cancers, but its role in PA has not been investigated. We studied alterations in Notch signaling components in tumor tissue from 18 patients with PA and 4 with other low-grade astrocytomas to identify much needed therapeutic targets. We found that Notch pathway members were overexpressed at the mRNA (NOTCH1, NOTCH2, HEY1, HEY2) and protein (HES1) levels in PAs at various anatomic sites compared with non-neoplastic brain samples. These changes were not associated with specific BRAF alterations. Inhibiting the Notch pathway in the pediatric low-grade astrocytoma cell lines Res186 and Res259 using either RNA interference or a &ggr;-secretase inhibitor resulted in variable, but significant, reduction in cell growth and migration. This study suggests a potential role for Notch signaling in pediatric low-grade astrocytoma tumorigenesis and that Notch signaling may be a viable pathway therapeutic target.

Abstract 4141: Notch signaling: A new potential target in the treatment of uveal melanoma

Uveal melanoma is the most common primary intra-ocular malignancy in adults, and causes significant mortality due to its propensity to metastasize. Our aim is to investigate the role of Notch signaling in promoting uveal melanoma proliferation and invasion. We examined five established uveal melanoma cell lines, and found using qPCR that the Notch 1-2-3 receptors, Jag1-2 ligands and the pathway target Hes1 were expressed to varying degrees in all lines. We then blocked Notch signaling using the gamma-secretase inhibitor (GSI) MRK003, and found that only three of the lines (OCM1, OCM3, OCM8) had their growth inhibited by the drug. Interestingly, these three lines had significantly higher levels of Hes1 mRNA as compared to the two lines resistant to GSI treatment (Mel 285, Mel 290). GSI treatment induced a dosedependent reduction of anchorage-independent clonogenic growth, with 50% inhibition in OCM1, 70% in OCM3, and 40% in OCM8, as well as a significant reduction in Hes1 mRNA and protein levels. Apoptosis, as measured by cleavage and activation of caspase-9, was also induced. Finally, we observed inhibition of cellular invasion in GSI treated cultures using transwell migration and scratch assays. Preliminary studies of snap-frozen primary tumors indicate that Notch pathway components are expressed in primary uveal melanoma in vivo at levels as high or higher than those in our cell line models. In addition, oligonucleotide microarray analysis showed a significant increase in Jag2 and Notch3 expression levels in primary uveal melanomas that metastasized as compared to those that did not. Our findings suggest that the Notch pathway plays an important role in inducing cellular proliferation and invasion in uveal melanoma, and that inhibiting this pathway using pharmacological agents may be effective in preventing tumor growth and metastasis.