hes1-protein--human and Brain-Neoplasms

Amplification and high levels of NOTCH ligand expression have been identified in several types of pediatric brain tumors. A phase I trial of weekly MK-0752, an oral inhibitor of gamma-secretase, was conducted in children with recurrent central nervous system (CNS) malignancies to estimate the maximum tolerated dose, dose-limiting toxicities (DLT), pharmacokinetics (PK), and pharmacodynamics of weekly MK-0752.. MK-0752 was administered once weekly at 1000 and 1400 mg/m(2) using a rolling-6 design. PK analysis was performed during the first course. NOTCH and HES expression was assessed by immunohistochemistry and Western blot.. Ten eligible patients were enrolled (median age 8.8 years; range 3.1-19.2) with diagnoses of brain stem glioma (n = 3), ependymoma (n = 2), anaplastic astrocytoma (n = 1), choroid plexus carcinoma (n = 2), medulloblastoma (n = 1), and primitive neuroectodermal tumor (n = 1). Nine were evaluable for toxicity. One DLT of fatigue occurred in the six evaluable patients enrolled at 1000 mg/m(2)/dose. No DLTs were experienced by three patients treated at 1400 mg/m(2)/dose. Non-dose-limiting grade 3 toxicities included lymphopenia, neutropenia, and anemia. Median number of treatment courses was 2 (range 1-10). Two patients continued on therapy for at least 6 months. The median (range) C(max) of MK-0752 was 88.2 μg/mL (40.6 to 109 μg/mL) and 60.3 μg/mL (59.2 to 91.9 μg/mL) in patients receiving 1000 and 1400 mg/m(2)/week, respectively. NOTCH expression was decreased in six of seven patients for whom tissue was available at 24 h post-MK-0752.. MK-0752 is well tolerated and exhibits target inhibition at 1000 and 1400 mg/m(2)/week in children with recurrent CNS malignancies.

Topics: Administration, Oral; Adolescent; Amyloid Precursor Protein Secretases; Area Under Curve; Basic Helix-Loop-Helix Transcription Factors; Benzene Derivatives; Brain Neoplasms; Central Nervous System Diseases; Child; Child, Preschool; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Follow-Up Studies; Gene Expression Regulation; Homeodomain Proteins; Humans; Male; Propionates; Receptor, Notch1; Repressor Proteins; Sulfones; Time Factors; Transcription Factor HES-1; Young Adult

Smarcd1 is a component of an evolutionary conserved chromatin remodeling complex-SWI/SNF, which is involved in transcription factor recruitment, DNA replication, recombination, and repair. Suppression of the SWI/SNF complex required for cellular differentiation and gene regulation may be inducible for cell proliferation and tumorigenicity. However, the inhibitory role of Smarcd1 in human glioblastoma cells has not been well illustrated. Both U87 and U251 human glioblastoma cell lines were employed in the present study. The lentivirus-mediated gene knockdown and overexpression approach was conducted to determine the function of Smarcd1. The protein levels were tested by western blot, and the relative mRNA contents were detected by quantitative real-time PCR. Cell viability was tested by CCK-8 and colony-forming assay. Transwell assays were utilized to evaluate the motility and invasive ability. Flow cytometry was employed to analyze cell cycle and apoptosis. SPSS software was used for statistical analysis. Low expression of Smarcd1 was observed in glioblastoma cell lines and in patients with high-grade glioma. Importantly, the depletion of Smarcd1 promoted cell proliferation, invasion, and chemoresistance, whereas enhanced expression of Smarcd1 inhibited tumor-malignant phenotypes. Mechanistic research demonstrated that overexpression of Smarcd1 decreased the expression of Notch1, while knockdown of Notch1 increased the expression of Smarcd1 through Hes1 suppression. Hence, the crosstalk between Smarcd1 and Notch1, which formed a feedback loop, was crucial in regulation of glioblastoma malignant phenotypes. Furthermore, targeting Smarcd1 could be a potential strategy for human glioblastoma treatment.

Topics: Animals; Apoptosis; Brain Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chromosomal Proteins, Non-Histone; Down-Regulation; G1 Phase; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Male; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Phenotype; Receptor, Notch1; Signal Transduction; Temozolomide; Transcription Factor HES-1; Transcription, Genetic; Tumor Suppressor Protein p53

Glioma stem cells (GSCs) have been proven to be resistant to various therapeutic strategies, such as temozolomide chemotherapy and radiotherapy, leading to glioma recurrence. Isoliquiritigenin (ISL), a menber of the flavonoids isolated from liquorice has been found to be a potent stimulator of cell differentiation and has potential application for treating various types of cancer including human brain glioma. However, the antitumor activity of ISL on GSCs and the signaling pathway underlying its therapeutic effects are poorly understood. In the present study, GSCs were isolated from SHG44 human glioma cells by serum-free culture and treated with ISL or DAPT (a Notch/γ-secretase inhibitor). It was found that ISL dose-dependently inhibited GSC growth after 72 h of treatment and decreased the formation of tumor spheres. Meanwhile, GSCs differentiated into astrocytes and neurons. Furthermore, these therapeutic effects were accompanied by downregulation of Notch1 and Hes1 at the protein and mRNA levels. Taken together, our results demonstrated that ISL exhibits antitumor effects on GSCs by inhibiting proliferation and inducing differentiation. The therapeutic effect may be related to downregulation of the Notch1 signaling pathway. Application of ISL presents potential benefits for the treatment of human brain glioma.

Topics: Antineoplastic Agents; Brain Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Chalcones; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Glioma; Humans; Neoplastic Stem Cells; Receptor, Notch1; Signal Transduction; Transcription Factor HES-1; Tumor Cells, Cultured

The invasive and lethal nature of Glioblastoma multiforme (GBM) necessitates the continuous identification of molecular targets and search of efficacious therapies to inhibit GBM growth. The GBM resistance to chemotherapy and radiation it is attributed to the existence of a rare fraction of cancer stem cells (CSC) that we have identified within the tumor core and in peritumor tissue of GBM. Since Notch1 pathway is a potential therapeutic target in brain cancer, earlier we highlighted that pharmacological inhibition of Notch1 signalling by γ-secretase inhibitor-X (GSI-X), reduced cell growth of some c-CSC than to their respective p-CSC, but produced negligible effects on cell cycle distribution, apoptosis and cell invasion. In the current study, we assessed the effects of Hes1-targeted shRNA, a Notch1 gene target, specifically on GBM CSC refractory to GSI-X. Depletion of Hes1 protein induces major changes in cell morphology, cell growth rate and in the invasive ability of shHes1-CSC in response to growth factor EGF. shHes1-CSC show a decrease of the stemness marker Nestin concurrently to a marked increase of neuronal marker MAP2 compared to pLKO.1-CSC. Those effects correlated with repression of EGFR protein and modulation of Stat3 phosphorylation at Y705 and S727 residues. In the last decade Stat3 has gained attention as therapeutic target in cancer but there is not yet any approved Stat3-based glioma therapy. Herein, we report that exposure to a Stat3/5 inhibitor, induced apoptosis either in shHes1-CSC or control cells. Taken together, Hes1 seems to be a favorable target but not sufficient itself to target GBM efficaciously, therefore a possible pharmacological intervention should provide for the use of anti-Stat3/5 drugs either alone or in combination regimen.

Topics: Apoptosis; Benzimidazoles; Brain Neoplasms; Carbamates; Cell Differentiation; Cell Proliferation; Dipeptides; ErbB Receptors; Glioblastoma; Humans; Microtubule-Associated Proteins; Neoplasm Invasiveness; Neoplastic Stem Cells; Phosphorylation; Piperidines; Receptor, Notch1; RNA Interference; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor; STAT5 Transcription Factor; Transcription Factor HES-1; Tumor Suppressor Proteins

Glioblastoma multiforme (GBM) are the most common primary malignant brain tumor in adults, with a median survival of about one year. This poor prognosis is attributed primarily to therapeutic resistance and tumor recurrence after surgical removal, with the root cause suggested to be found in glioblastoma stem cells (GSCs). Using glial fibrillary acidic protein (GFAP) as a reporter of astrocytic differentiation, we isolated multiple clones from three independent GSC lines which express GFAP in a remarkably stable fashion. We next show that elevated expression of GFAP is associated with reduced clonogenicity in vitro and tumorigenicity in vivo. Utilizing this in vitro cell-based differentiation reporter system we screened chemical libraries and identified the non-depolarizing neuromuscular blocker (NNMB), Atracurium Besylate, as a small molecule which effectively induces astroglial but not neuronal differentiation of GSCs. Functionally, Atracurium Besylate treatment significantly inhibited the clonogenic capacity of several independent patient-derived GSC neurosphere lines, a phenomenon which was largely irreversible. A second NNMB, Vecuronium, also induced GSC astrocytic differentiation while Dimethylphenylpiperazinium (DMPP), a nicotinic acetylcholine receptor (nAChR) agonist, significantly blocked Atracurium Besylate pro-differentiation activity. To investigate the clinical importance of nAChRs in gliomas, we examined clinical outcomes and found that glioma patients with tumors overexpressing CHRNA1 or CHRNA9 (encoding for the AChR-α1 or AChR-α9) exhibit significant shorter overall survival. Finally, we found that ex-vivo pre-treatment of GSCs, expressing CHRNA1 and CHRNA9, with Atracurium Besylate significantly increased the survival of mice xenotransplanted with these cells, therefore suggesting that tumor initiating subpopulations have been reduced.

Topics: Animals; Astrocytes; Atracurium; Basic Helix-Loop-Helix Transcription Factors; Blotting, Western; Brain Neoplasms; Cell Differentiation; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Glial Fibrillary Acidic Protein; Glioblastoma; Homeodomain Proteins; Humans; Mice, Nude; Microscopy, Fluorescence; Neoplastic Stem Cells; Neuromuscular Blocking Agents; Receptors, Cholinergic; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factor HES-1; Xenograft Model Antitumor Assays

Glioblastoma is the most common and aggressive primary brain tumor in adults, with a poor prognosis because of its resistance to radiotherapy and chemotherapy. Merlin/NF2 (moesin-ezrin-radixin-like protein/neurofibromatosis type 2) is a tumor suppressor found to be mutated in most nervous system tumors; however, it is not mutated in glioblastomas. Merlin associates with several transmembrane receptors and intracellular proteins serving as an anchoring molecule. Additionally, it acts as a key component of cell motility. By selecting sub-populations of U251 glioblastoma cells, we observed that high expression of phosphorylated Merlin at serine 518 (S518-Merlin), NOTCH1 and epidermal growth factor receptor (EGFR) correlated with increased cell proliferation and tumorigenesis. These cells were defective in cell-contact inhibition with changes in Merlin phosphorylation directly affecting NOTCH1 and EGFR expression, as well as downstream targets HES1 (hairy and enhancer of split-1) and CCND1 (cyclin D1). Of note, we identified a function for S518-Merlin, which is distinct from what has been reported when the expression of Merlin is diminished in relation to EGFR and NOTCH1 expression, providing first-time evidence that demonstrates that the phosphorylation of S518-Merlin in glioblastoma promotes oncogenic properties that are not only the result of inactivation of the tumor suppressor role of Merlin but also an independent process implicating a Merlin-driven regulation of NOTCH1 and EGFR.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Brain Neoplasms; Cell Line, Tumor; Cyclin D1; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Homeodomain Proteins; Humans; Mice; Mice, Nude; Neurofibromin 2; Phosphorylation; Receptor, Notch1; Transcription Factor HES-1

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 γ-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.

Topics: Adolescent; Antineoplastic Agents; Astrocytoma; Basic Helix-Loop-Helix Transcription Factors; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Child; Child, Preschool; Core Binding Factors; Cyclic S-Oxides; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Immunoglobulin J Recombination Signal Sequence-Binding Protein; Male; Receptors, Notch; Signal Transduction; Thiadiazoles; Transcription Factor HES-1; Young Adult

Hypoxia is a hallmark of solid tumors including glioblastoma (GBM). Its synergism with Notch signaling promotes progression in different cancers. However, Notch signaling exhibits pleiotropic roles and the existing literature lacks a comprehensive understanding of its perturbations under hypoxia in GBM with respect to all components of the pathway. We identified the key molecular cluster(s) characteristic of the Notch pathway response in hypoxic GBM tumors and gliomaspheres. Expression of Notch and hypoxia genes was evaluated in primary human GBM tissues by q-PCR. Clustering and statistical analyses were applied to identify the combination of hypoxia markers correlated with upregulated Notch pathway components. We found well-segregated tumor-clusters representing high and low HIF-1α/PGK1-expressors which accounted for differential expression of Notch signaling genes. In combination, a five-hypoxia marker set (HIF-1α/PGK1/VEGF/CA9/OPN) was determined as the best predictor for induction of Notch1/Dll1/Hes1/Hes6/Hey1/Hey2. Similar Notch-axis genes were activated in gliomaspheres, but not monolayer cultures, under moderate/severe hypoxia (2%/0.2% O2). Preliminary evidence suggested inverse correlation between patient survival and increased expression of constituents of the hypoxia-Notch gene signature. Together, our findings delineated the Notch-axis maximally associated with hypoxia in resected GBM, which might be prognostically relevant. Its upregulation in hypoxia-exposed gliomaspheres signify them as a better in-vitro model for studying hypoxia-Notch interactions than monolayer cultures.

Topics: Basic Helix-Loop-Helix Transcription Factors; Brain Neoplasms; Cell Hypoxia; Cluster Analysis; Glioblastoma; Homeodomain Proteins; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Kaplan-Meier Estimate; Osteopontin; Phosphoglycerate Kinase; Principal Component Analysis; Prognosis; Real-Time Polymerase Chain Reaction; Receptors, Notch; Signal Transduction; Transcription Factor HES-1; Tumor Cells, Cultured; Up-Regulation

Glioblastoma multiforme (GBM) is the most common adult malignant glioma with poor prognosis due to the resistance to radiotherapy and chemotherapy, which might be critically involved in the repopulation of cancer stem cells (CSCs) after treatment. We had investigated the characteristics of cancer stem-like side population (SP) cells sorted from GBM cells, and studied the effect of Honokiol targeting on CSCs. GBM8401 SP cells possessed the stem cell markers, such as nestin, CD133 and Oct4, and the expressions of self-renewal related stemness genes, such as SMO, Notch3 and IHH (Indian Hedgehog). Honokiol inhibited the proliferation of both GBM8401 parental cells and SP cells in a dose-dependent manner, the IC50 were 5.3±0.72 and 11±1.1 μM, respectively. The proportions of SP in GBM8401 cells were diminished by Honokiol from 1.5±0.22% down to 0.3±0.02% and 0.2±0.01% at doses of 2.5 μM and 5 μM, respectively. The SP cells appeared to have higher expression of O6-methylguanine-DNA methyltransferase (MGMT) and be more resistant to Temozolomide (TMZ). The resistance to TMZ could be only slightly reversed by MGMT inhibitor O6-benzylguanine (O6-BG), but markedly further enhanced by Honokiol addition. Such significant enhancement was accompanied with the higher induction of apoptosis, greater down-regulation of Notch3 as well as its downstream Hes1 expressions in SP cells. Our data indicate that Honokiol might have clinical benefits for the GBM patients who are refractory to TMZ treatment.

Topics: Antineoplastic Agents; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Biphenyl Compounds; Brain Neoplasms; Cell Proliferation; Dacarbazine; Dose-Response Relationship, Drug; Down-Regulation; Drug Synergism; Drug Therapy, Combination; Glioblastoma; Homeodomain Proteins; Humans; Lignans; Neoplastic Stem Cells; Receptor, Notch3; Receptors, Notch; Temozolomide; Transcription Factor HES-1; Tumor Cells, Cultured

microRNA-9 is a key regulator of neuronal development aberrantly expressed in brain malignancies, including medulloblastoma. The mechanisms by which microRNA-9 contributes to medulloblastoma pathogenesis remain unclear, and factors that regulate this process have not been delineated.. Expression and methylation status of microRNA-9 in medulloblastoma cell lines and primary samples were analysed. The association of microRNA-9 expression with medulloblastoma patients' clinical outcome was assessed, and the impact of microRNA-9 restoration was functionally validated in medulloblastoma cells.. microRNA-9 expression is repressed in a large subset of MB samples compared with normal fetal cerebellum. Low microRNA-9 expression correlates significantly with the diagnosis of unfavourable histopathological variants and with poor clinical outcome. microRNA-9 silencing occurs via cancer-specific CpG island hypermethylation. HES1 was identified as a direct target of microRNA-9 in medulloblastoma, and restoration of microRNA-9 was shown to trigger cell cycle arrest, to inhibit clonal growth and to promote medulloblastoma cell differentiation.. microRNA-9 is a methylation-silenced tumour suppressor that could be a potential candidate predictive marker for poor prognosis of medulloblastoma. Loss of microRNA-9 may confer a proliferative advantage to tumour cells, and it could possibly contribute to disease pathogenesis. Thus, re-expression of microRNA-9 may constitute a novel epigenetic regulation strategy against medulloblastoma.

Topics: Adult; Aged; Basic Helix-Loop-Helix Transcription Factors; Brain Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cerebellum; CpG Islands; Epigenesis, Genetic; Female; Fetus; Gene Silencing; Homeodomain Proteins; Humans; Male; Medulloblastoma; MicroRNAs; Prognosis; Promoter Regions, Genetic; Transcription Factor HES-1

Glioma stemness has been recognized as the most important reason for glioma relapse and drug resistance. Differentiation of glioma stem cells (GSCs) has been implicated as a novel approach to target recurrent glioma. However, the detailed molecular mechanism involved in the differentiation of GSCs has not yet been elucidated. This study identified CPEB1 as the key modulator that induces the differentiation of GSCs at the post-transcriptional level. Gain and loss of function experiments showed that CPEB1 expression reduced sphere formation ability and the expression of stemness markers such as Nestin and Notch. To elucidate the detailed molecular mechanism underlying the action of CPEB1, we investigated the interacting ribonome of the CPEB1 complex using a Ribonomics approach. CPEB1 specifically suppressed the translation of HES1 and SIRT1 by interacting with a cytoplasmic polyadenylation element. The expression profile of CPEB1 negatively correlated with overall survival in glioma patients. Overexpression of CPEB1 decreased the number of GSCs in an orthotopically implanted glioma animal model. These results suggest that CPEB1-mediated translational control is essential for the differentiation of GSCs and provides novel therapeutic concepts for differentiation therapy.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Brain Neoplasms; Cell Differentiation; Down-Regulation; Female; Glioma; HEK293 Cells; Heterografts; Homeodomain Proteins; Humans; Mice; Mice, Inbred BALB C; mRNA Cleavage and Polyadenylation Factors; Neoplastic Stem Cells; Sirtuin 1; Transcription Factor HES-1; Transcription Factors; Transcription, Genetic; Transfection

Notch signaling has been demonstrated to have a central role in cancer stem-like cells (CSLCs) in glioblastoma multiforme (GBM). We have recently demonstrated the inhibitory effect of arsenic trioxide (ATO) on CSLCs in glioblastoma cell lines. In this study we used neurosphere recovery assay that measured neurosphere formation at three time points to assess the capacity of the culture to repopulate after ATO treatment. Our results provided strong evidence that ATO depleted CSLCs in GBM, and inhibited neurosphere recovery and secondary neurosphere formation. ATO inhibited the phosphorylation and activation of AKT and STAT3 through Notch signaling blockade. These data show that the ATO is a promising new approach to decrease glioblastoma proliferation and recurrence by downregulation of Notch pathway.

Topics: Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Basic Helix-Loop-Helix Transcription Factors; Brain Neoplasms; Cell Line; Cell Proliferation; Gene Expression Regulation, Neoplastic; Glioblastoma; Homeodomain Proteins; Humans; Neoplastic Stem Cells; Oxides; Phosphorylation; Proto-Oncogene Proteins c-akt; Receptors, Notch; Signal Transduction; STAT3 Transcription Factor; Transcription Factor HES-1

Notch pathway plays critical role in stem cell maintenance and angiogenesis, as well as cell fate decisions of cancer. However, concrete mechanisms of notch pathway regulation in glioma were not well known, especially mediated by microRNAs. In this study, we identified a brain-specific miRNA, miR-524-5p, which was associated with the pathological grade and overall survival of gliomas. Restorated expression of miR-524-5p in glioma suppressed cell proliferation and invasion both in vitro and in vivo. Using bioinformatics and biological approaches, we found that Jagged-1 and Hes-1, two key components of notch pathway, were direct targets of miR-524-5p. Knocking down of Jagged-1 or Hes-1 partially phenocopied miR-524-5p re-expression, whereas forced expression of Jagged-1 or Hes-1 reversed the effects of miR-524-5p on proliferation and invasion of glioma. Moreover, miR-524-5p levels in glioma samples were inversely correlated with Jagged-1 and Hes-1 and their overexpressions were associated with poor survival. Thus, we have identified that miR-524-5p behaves as a tumor suppressor by negatively targeting Jagged-1 and Hes-1 and provides an additional option to inhibit this oncogene in gliomas.

Topics: Animals; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Biomarkers, Tumor; Blotting, Western; Brain Neoplasms; Calcium-Binding Proteins; Cell Movement; Cell Proliferation; Chromatin Immunoprecipitation; Female; Gene Expression Profiling; Genes, Tumor Suppressor; Glioma; Homeodomain Proteins; Humans; Immunoenzyme Techniques; Intercellular Signaling Peptides and Proteins; Jagged-1 Protein; Luciferases; Membrane Proteins; Mice; Mice, Nude; MicroRNAs; Oligonucleotide Array Sequence Analysis; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Serrate-Jagged Proteins; Transcription Factor HES-1

Medulloblastoma (MDB) is the most common brain malignancy of childhood. It is currently thought that MDB arises from aberrantly functioning stem cells in the cerebellum that fail to maintain proper control of self-renewal. Additionally, it has been reported that MDB cells display higher endogenous Notch signaling activation, known to promote the survival and proliferation of neoplastic neural stem cells and to inhibit their differentiation. Although interaction between hypoxia-inducible factor-1α (HIF-1α) and Notch signaling is required to maintain normal neural precursors in an undifferentiated state, an interaction has not been identified in MDB. Here, we investigate whether hypoxia, through HIF-1α stabilization, modulates Notch1 signaling in primary MDB-derived cells. Our results indicate that MDB-derived precursor cells require hypoxic conditions for in vitro expansion, whereas acute exposure to 20% oxygen induces tumor cell differentiation and death through inhibition of Notch signaling. Importantly, stimulating Notch1 activation with its ligand Dll4 under hypoxic conditions leads to expansion of MDB-derived CD133(+) and nestin(+) precursors, suggesting a regulatory effect on stem cells. In contrast, MDB cells undergo neuronal differentiation when treated with γ-secretase inhibitor, which prevents Notch activation. These results suggest that hypoxia, by maintaining Notch1 in its active form, preserves MDB stem cell viability and expansion.

Topics: AC133 Antigen; Antigens, CD; Basic Helix-Loop-Helix Transcription Factors; Brain Neoplasms; Cell Differentiation; Cell Proliferation; Cell Survival; Glycoproteins; Homeodomain Proteins; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Intermediate Filament Proteins; Medulloblastoma; Neoplastic Stem Cells; Nerve Tissue Proteins; Nestin; Peptides; Polymerase Chain Reaction; Receptor, Notch1; Receptors, Notch; Signal Transduction; Transcription Factor HES-1; Tumor Cells, Cultured

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.. 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.. 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.. 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.

Topics: Antineoplastic Combined Chemotherapy Protocols; Basic Helix-Loop-Helix Transcription Factors; Brain Neoplasms; Cell Line, Tumor; Cyclic S-Oxides; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Glioblastoma; Hedgehog Proteins; Homeodomain Proteins; Humans; Receptors, Notch; Signal Transduction; Thiadiazoles; Transcription Factor HES-1; Transcription Factors; U937 Cells; Veratrum Alkaloids; Zinc Finger Protein GLI1

Altered Notch signaling seems linked with medulloblastoma (MB) formation and resveratrol exhibits anti-medulloblastoma effects. However, the influence of resveratrol in Notch signaling of MB cells has not been described. This issue was addressed here by checking Notch1 and Notch2 statuses in three MB cell lines with and without resveratrol treatment. Notch1 and Notch2 were detected in the cytoplasm of three cell lines under normal condition, which were up-regulated by resveratrol along with differentiation, apoptosis and enhanced Hes1 nuclear translocation. Nevertheless, blockage of Notch enzymatic cleavage with gamma-seacretase inhibitors, DAPT and L-685,458, neither interrupted resveratrol-caused cellular events nor affected MB cell growth. These results demonstrate that Notch signaling has little relevance with resveratrol-induced differentiation and apoptosis and may not be a universal critical factor of MB cells.

Topics: Active Transport, Cell Nucleus; Amyloid Precursor Protein Secretases; Antineoplastic Agents; Antioxidants; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Brain Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cytoplasm; Enzyme Inhibitors; Homeodomain Proteins; Humans; Medulloblastoma; Receptor, Notch1; Receptor, Notch2; Receptors, Notch; Resveratrol; Signal Transduction; Stilbenes; Transcription Factor HES-1; Up-Regulation

Polypyrimidine tract binding protein (PTB) is expressed in developing mammalian astrocytes, absent in mature adult astrocytes, and aberrantly elevated in gliomas. It is unclear whether PTB is a coincidental marker of tumor progression or a significant mediator of tumorigenesis. In developing Drosophila, the absence of the PTB homolog, hephaestus, results in increased Notch activity. Since Notch is a well-known inducer of glial cell fate, we determined whether overexpression of PTB in glial cell tumors provides a selective growth advantage by inhibiting activated Notch (Notch1IC)-mediated differentiation. To do this, we performed an immunohistochemical analysis for expression of PTB, activated Notch1 (Notch1IC), Hes1 (a Notch target), and GFAP on an extensive human tissue microarray that included 246 gliomas, 10 gliosarcomas, and 10 normal brains. Statistically significant PTB overexpression was seen in all glioma grades, with the highest increase in grade IV tumors. Notch1IC was also abnormally expressed in gliomas except in a subset of grade IV tumors in which it was absent. This decrease in Notch1IC was not associated with increased PTB expression. We conclude that PTB, and Notch1 serve as independent and functionally unlinked markers of glioma progression.

Topics: Basic Helix-Loop-Helix Transcription Factors; Biomarkers, Tumor; Brain Neoplasms; Glial Fibrillary Acidic Protein; Glioma; Homeodomain Proteins; Humans; Immunoenzyme Techniques; Neoplasm Staging; Polypyrimidine Tract-Binding Protein; Receptor, Notch1; Tissue Array Analysis; Transcription Factor HES-1

The role of Notch signaling in tumorigenesis can vary; Notch1 acts as an oncogene in some neoplasms, and a tumor suppressor in others. Here, we show that different Notch receptors can have opposite effects in a single tumor type. Expression of truncated, constitutively active Notch1 or Notch2 in embryonal brain tumor cell lines caused antagonistic effects on tumor growth. Cell proliferation, soft agar colony formation, and xenograft growth were all promoted by Notch2 and inhibited by Notch1. We also found that Notch2 receptor transcripts are highly expressed in progenitor cell-derived brain tumors such as medulloblastomas, whereas Notch1 is scarce or undetectable. This parallels normal cerebellar development, during which Notch2 is predominantly expressed in proliferating progenitors and Notch1 in postmitotic differentiating cells. Given the oncogenic effects of Notch2, we analyzed its gene dosage in 40 embryonal brain tumors, detecting an increased copy number in 15% of cases. Notch2 gene amplification was confirmed by fluorescence in situ hybridization in one case with extremely high Notch2 mRNA levels. In addition, expression of the Notch pathway target gene Hes1 in medulloblastomas was associated with significantly shorter patient survival (P = 0.01). Finally, pharmacological inhibition of Notch signaling suppresses growth of medulloblastoma cells. Our data indicate that Notch1 and Notch2 can have opposite effects on the growth of a single tumor type, and show that Notch2 can be overexpressed after gene amplification in human tumors.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Brain Neoplasms; Cell Division; Cell Line, Tumor; Cerebellar Neoplasms; Gene Dosage; Homeodomain Proteins; Humans; Medulloblastoma; Mice; Neuroectodermal Tumors, Primitive; Receptor, Notch1; Receptor, Notch2; Receptors, Cell Surface; Signal Transduction; Transcription Factor HES-1; Transcription Factors