Detailed longitudinal sampling of glioma stem cells in situ reveals Chr7 gain and Chr10 loss as repeated events in primary tumor formation and recurrence.

Intratumoral heterogeneity at the genetic, epigenetic, transcriptomic, and morphologic levels is a commonly observed phenomenon in many aggressive cancer types. Clonal evolution during tumor formation and in response to therapeutic intervention can be predicted utilizing reverse engineering approaches on detailed genomic snapshots of heterogeneous patient tumor samples. In this study, we developed an extensive dataset for a GBM case via the generation of polyclonal and monoclonal glioma stem cell lines from initial diagnosis, and from multiple sections of distant tumor locations of the deceased patient's brain following tumor recurrence. Our analyses revealed the tissue-wide expansion of a new clone in the recurrent tumor and chromosome 7 gain and chromosome 10 loss as repeated genomic events in primary and recurrent disease. Moreover, chromosome 7 gain and chromosome 10 loss produced similar alterations in mRNA expression profiles in primary and recurrent tumors despite possessing other highly heterogeneous and divergent genomic alterations between the tumors. We identified ETV1 and CDK6 as putative candidate genes, and NFKB (complex), IL1B, IL6, Akt and VEGF as potential signaling regulators, as potentially central downstream effectors of chr7 gain and chr10 loss. Finally, the differences caused by the transcriptomic shift following gain of chromosome 7 and loss of chromosome 10 were consistent with those generally seen in GBM samples compared to normal brain in large-scale patient-tumor data sets.

A Core Regulatory Circuit in Glioblastoma Stem Cells Links MAPK Activation to a Transcriptional Program of Neural Stem Cell Identity.

Glioblastoma, the most common primary malignant brain tumor, harbors a small population of tumor initiating cells (glioblastoma stem cells) that have many properties similar to neural stem cells. To investigate common regulatory networks in both neural and glioblastoma stem cells, we subjected both cell types to in-vitro differentiation conditions and measured global gene-expression changes using gene expression microarrays. Analysis of enriched transcription factor DNA-binding sites in the promoters of differentially expressed genes was used to reconstruct regulatory networks involved in differentiation. Computational predictions, which were biochemically validated, show an extensive overlap of regulatory circuitry between cell types including a network centered on the transcription factor KLF4. We further demonstrate that EGR1, a transcription factor previously shown to be downstream of the MAPK pathway, regulates KLF4 expression and that KLF4 in turn transcriptionally activates NOTCH as well as SOX2. These results demonstrate how known genomic alterations in glioma that induce constitutive activation of MAPK are transcriptionally linked to master regulators essential for neural stem cell identify.

Identification of molecular pathways facilitating glioma cell invasion in situ.

Gliomas are mostly incurable secondary to their diffuse infiltrative nature. Thus, specific therapeutic targeting of invasive glioma cells is an attractive concept. As cells exit the tumor mass and infiltrate brain parenchyma, they closely interact with a changing micro-environmental landscape that sustains tumor cell invasion. In this study, we used a unique microarray profiling approach on a human glioma stem cell (GSC) xenograft model to explore gene expression changes in situ in Invading Glioma Cells (IGCs) compared to tumor core, as well as changes in host cells residing within the infiltrated microenvironment relative to the unaffected cortex. IGCs were found to have reduced expression of genes within the extracellular matrix compartment, and genes involved in cell adhesion, cell polarity and epithelial to mesenchymal transition (EMT) processes. The infiltrated microenvironment showed activation of wound repair and tissue remodeling networks. We confirmed by protein analysis the downregulation of EMT and polarity related genes such as CD44 and PARD3 in IGCs, and EFNB3, a tissue-remodeling agent enriched at the infiltrated microenvironment. OLIG2, a proliferation regulator and glioma progenitor cell marker upregulated in IGCs was found to function in enhancing migration and stemness of GSCs. Overall, our results unveiled a more comprehensive picture of the complex and dynamic cell autonomous and tumor-host interactive pathways of glioma invasion than has been previously demonstrated. This suggests targeting of multiple pathways at the junction of invading tumor and microenvironment as a viable option for glioma therapy.

Micro-environment causes reversible changes in DNA methylation and mRNA expression profiles in patient-derived glioma stem cells.

In vitro and in vivo models are widely used in cancer research. Characterizing the similarities and differences between a patient's tumor and corresponding in vitro and in vivo models is important for understanding the potential clinical relevance of experimental data generated with these models. Towards this aim, we analyzed the genomic aberrations, DNA methylation and transcriptome profiles of five parental tumors and their matched in vitro isolated glioma stem cell (GSC) lines and xenografts generated from these same GSCs using high-resolution platforms. We observed that the methylation and transcriptome profiles of in vitro GSCs were significantly different from their corresponding xenografts, which were actually more similar to their original parental tumors. This points to the potentially critical role of the brain microenvironment in influencing methylation and transcriptional patterns of GSCs. Consistent with this possibility, ex vivo cultured GSCs isolated from xenografts showed a tendency to return to their initial in vitro states even after a short time in culture, supporting a rapid dynamic adaptation to the in vitro microenvironment. These results show that methylation and transcriptome profiles are highly dependent on the microenvironment and growth in orthotopic sites partially reverse the changes caused by in vitro culturing.

Age-specific signatures of glioblastoma at the genomic, genetic, and epigenetic levels.

Age is a powerful predictor of survival in glioblastoma multiforme (GBM) yet the biological basis for the difference in clinical outcome is mostly unknown. Discovering genes and pathways that would explain age-specific survival difference could generate opportunities for novel therapeutics for GBM. Here we have integrated gene expression, exon expression, microRNA expression, copy number alteration, SNP, whole exome sequence, and DNA methylation data sets of a cohort of GBM patients in The Cancer Genome Atlas (TCGA) project to discover age-specific signatures at the transcriptional, genetic, and epigenetic levels and validated our findings on the REMBRANDT data set. We found major age-specific signatures at all levels including age-specific hypermethylation in polycomb group protein target genes and the upregulation of angiogenesis-related genes in older GBMs. These age-specific differences in GBM, which are independent of molecular subtypes, may in part explain the preferential effects of anti-angiogenic agents in older GBM and pave the way to a better understanding of the unique biology and clinical behavior of older versus younger GBMs.

Histone demethylase Jumonji D3 (JMJD3) as a tumor suppressor by regulating p53 protein nuclear stabilization.

Histone methylation regulates normal stem cell fate decisions through a coordinated interplay between histone methyltransferases and demethylases at lineage specific genes. Malignant transformation is associated with aberrant accumulation of repressive histone modifications, such as polycomb mediated histone 3 lysine 27 (H3K27me3) resulting in a histone methylation mediated block to differentiation. The relevance, however, of histone demethylases in cancer remains less clear. We report that JMJD3, a H3K27me3 demethylase, is induced during differentiation of glioblastoma stem cells (GSCs), where it promotes a differentiation-like phenotype via chromatin dependent (INK4A/ARF locus activation) and chromatin independent (nuclear p53 protein stabilization) mechanisms. Our findings indicate that deregulation of JMJD3 may contribute to gliomagenesis via inhibition of the p53 pathway resulting in a block to terminal differentiation.

G-cimp status prediction of glioblastoma samples using mRNA expression data.

Glioblastoma Multiforme (GBM) is a tumor with high mortality and no known cure. The dramatic molecular and clinical heterogeneity seen in this tumor has led to attempts to define genetically similar subgroups of GBM with the hope of developing tumor specific therapies targeted to the unique biology within each of these subgroups. Recently, a subset of relatively favorable prognosis GBMs has been identified. These glioma CpG island methylator phenotype, or G-CIMP tumors, have distinct genomic copy number aberrations, DNA methylation patterns, and (mRNA) expression profiles compared to other GBMs. While the standard method for identifying G-CIMP tumors is based on genome-wide DNA methylation data, such data is often not available compared to the more widely available gene expression data. In this study, we have developed and evaluated a method to predict the G-CIMP status of GBM samples based solely on gene expression data.

Gliomagenesis arising from Pten- and Ink4a/Arf-deficient neural progenitor cells is mediated by the p53-Fbxw7/Cdc4 pathway, which controls c-Myc.

Glioblastoma multiforme is the most common type of primary malignant brain tumor and may arise from a cell with neural stem-like properties. Deregulation of the retinoblastoma, phosphoinositide-3 kinase (PI3K), and p53 pathways are molecular hallmarks of this disease. Recent work has shown that p53(-/-)Pten(-/-) mice form gliomas in a c-Myc-dependent manner. To explore the role of the INK4A/ARF locus and Pten deletions in gliomagenesis, we generated Pten(-/-)Ink4a/Arf(-/-) mouse neural stem cells (mNSC) and such cells were highly proliferative, self-renewing, relatively refractory to differentiation, and induced both low- and high-grade glioma formation in vivo. In contrast to p53(-/-) Pten(-/-) mNSCs, however, Pten(-/-)Ink4a/Arf(-/-) mNSCs do not express appreciable levels of c-Myc in vitro, although glioma stem cells derived from thesecells did. Sequencing of Pten(-/-)Ink4a/Arf(-/-) mNSC-derived tumors revealed spontaneous mutations in Tp53 in vivo with subsequent downregulation of Fbxw7. Expression of p53 mutants in Pten(-/-)Ink4a/Arf(-/-) mNSC or knockdown of Fbxw7 resulted in reexpression of c-Myc with enhanced Pten(-/-)Ink4a/Arf(-/-) mNSC tumorigenecity. We propose that p53 mutations contribute to gliomagenesis by both allowing the overexpression of c-Myc through downregulation of Fbxw7 and by protecting against c-Myc-induced apoptosis.

A phase I/II trial of enzastaurin in patients with recurrent high-grade gliomas.

Enzastaurin, a potent inhibitor of protein kinase C-beta, inhibits angiogenesis and has direct cytotoxic activity against glioma cells in preclinical studies. Patients with recurrent high-grade gliomas were stratified by histology and use of enzyme-inducing antiepileptic drugs (EIAEDs). Patients on EIAED were treated on the phase I dose-escalation portion of the trial with evaluation of serum pharmacokinetics as the primary endpoint. Patients not on EIAED were treated on the phase II portion of the trial with radiographic response and progression-free survival (PFS) as primary objectives. Patients in phase I received enzastaurin 525-900 mg/d. Phase II patients received 500 or 525 mg/d. One hundred and eighteen patients were accrued to this trial. Therapy was well tolerated with thrombosis, thrombocytopenia, hemorrhage, and elevated alanine aminotransferase as the most commonly observed drug-associated grade 3 or higher toxicities. Patients on EIAED had serum enzastaurin exposure levels approximately 80% lower than those not on EIAED. Dose escalations up to 900 mg/d did not substantially increase serum exposure levels and a maximally tolerated dose was never reached. Twenty-one of 84 evaluable patients (25%) experienced an objective radiographic response. The 6-month PFS was 7% for patients with glioblastoma and 16% for patients with anaplastic glioma. Phosphorylation of glycogen synthase kinase-3 in peripheral blood mononuclear cells was identified as a potential biomarker of drug activity. Enzastaurin has anti-glioma activity in patients with recurrent high-grade glioma, but does not appear to have enough single-agent activity to be useful as monotherapy.

A phase I trial of enzastaurin in patients with recurrent gliomas.

PURPOSE: Enzastaurin is a selective inhibitor of protein kinase C beta. Prior phase I studies did not show increased drug exposures with escalating once daily administration. Limits from gastrointestinal absorption may be overcome by twice daily dosing, potentially improving antitumor effects. EXPERIMENTAL DESIGN: We conducted a phase I dose escalation study in 26 patients with recurrent malignant glioma, stratified by use of enzyme-inducing antiepileptic drugs, to investigate whether divided twice daily dosing results in higher exposures compared with once daily dosing. Phosphorylated glycogen synthase 3 beta was analyzed as a potential biomarker of enzastaurin activity. RESULTS: Enzastaurin was poorly tolerated at all dose levels evaluated (500, 800, and 1,000 mg total daily), with thrombocytopenia and prolonged QTc as dose-limiting toxicities. The average drug concentration of enzastaurin under steady-state conditions was doubled by twice daily dosing compared with daily dosing [1.990; 90% confidence interval (CI), 1.450-2.730]. Additionally, geometric mean ratios doubled with 800 versus 500 mg dosing for both daily (2.687; 90% CI, 1.232-5.860) and twice daily regimens (1.852; 90% CI, 0.799-4.292). Two patients achieved long-term benefit (over 150 weeks progression free). CONCLUSIONS: Higher and more frequent dosing of enzastaurin resulted in improved drug exposure but with unacceptable toxicity at the doses tested. Phosphorylated glycogen synthase 3 beta may be a useful biomarker of the biological activity of enzastaurin. Enzastaurin has activity in a subset of malignant glioma patients and warrants continued study in combination with other agents using a maximal once daily dose of 500 mg.

Correlation analysis between single-nucleotide polymorphism and expression arrays in gliomas identifies potentially relevant target genes.

Primary brain tumors are a major cause of cancer mortality in the United States. Therapy for gliomas, the most common type of primary brain tumors, remains suboptimal. The development of improved therapeutics will require greater knowledge of the biology of gliomas at both the genomic and transcriptional levels. We have previously reported whole genome profiling of chromosome copy number alterations (CNA) in gliomas, and now present our findings on how those changes may affect transcription of genes that may be involved in tumor induction and progression. By calculating correlation values of mRNA expression versus DNA copy number average in a moving window around a given RNA probe set, biologically relevant information can be gained that is obscured by the analysis of a single data type. Correlation coefficients ranged from -0.6 to 0.7, highly significant when compared with previous studies. Most correlated genes are located on chromosomes 1, 7, 9, 10, 13, 14, 19, 20, and 22, chromosomes known to have genomic alterations in gliomas. Additionally, we were able to identify CNAs whose gene expression correlation suggests possible epigenetic regulation. This analysis revealed a number of interesting candidates such as CXCL12, PTER, and LRRN6C, among others. The results have been verified using real-time PCR and methylation sequencing assays. These data will further help differentiate genes involved in the induction and/or maintenance of the tumorigenic process from those that are mere passenger mutations, thereby enriching for a population of potentially new therapeutic molecular targets.

Glycogen synthase kinase-3 inhibition induces glioma cell death through c-MYC, nuclear factor-kappaB, and glucose regulation.

Glycogen synthase kinase 3 (GSK3), a serine/threonine kinase, is involved in diverse cellular processes ranging from nutrient and energy homeostasis to proliferation and apoptosis. Its role in glioblastoma multiforme has yet to be elucidated. We identified GSK3 as a regulator of glioblastoma multiforme cell survival using microarray analysis and small-molecule and genetic inhibitors of GSK3 activity. Various molecular and genetic approaches were then used to dissect out the molecular mechanisms responsible for GSK3 inhibition-induced cytotoxicity. We show that multiple small molecular inhibitors of GSK3 activity and genetic down-regulation of GSK3alpha/beta significantly inhibit glioma cell survival and clonogenicity. The potency of the cytotoxic effects is directly correlated with decreased enzyme activity-activating phosphorylation of GSK3alpha/beta Y276/Y216 and with increased enzyme activity inhibitory phosphorylation of GSK3alpha S21. Inhibition of GSK3 activity results in c-MYC activation, leading to the induction of Bax, Bim, DR4/DR5, and tumor necrosis factor-related apoptosis-inducing ligand expression and subsequent cytotoxicity. Additionally, down-regulation of GSK3 activity results in alteration of intracellular glucose metabolism resulting in dissociation of hexokinase II from the outer mitochondrial membrane with subsequent mitochondrial destabilization. Finally, inhibition of GSK3 activity causes a dramatic decrease in intracellular nuclear factor-kappaB activity. Inhibition of GSK3 activity results in c-MYC-dependent glioma cell death through multiple mechanisms, all of which converge on the apoptotic pathways. GSK3 may therefore be an important therapeutic target for gliomas. Future studies will further define the optimal combinations of GSK3 inhibitors and cytotoxic agents for use in gliomas and other cancers.

Epigenetic-mediated dysfunction of the bone morphogenetic protein pathway inhibits differentiation of glioblastoma-initiating cells.

Despite similarities between tumor-initiating cells with stem-like properties (TICs) and normal neural stem cells, we hypothesized that there may be differences in their differentiation potentials. We now demonstrate that both bone morphogenetic protein (BMP)-mediated and ciliary neurotrophic factor (CNTF)-mediated Jak/STAT-dependent astroglial differentiation is impaired due to EZH2-dependent epigenetic silencing of BMP receptor 1B (BMPR1B) in a subset of glioblastoma TICs. Forced expression of BMPR1B either by transgene expression or demethylation of the promoter restores their differentiation capabilities and induces loss of their tumorigenicity. We propose that deregulation of the BMP developmental pathway in a subset of glioblastoma TICs contributes to their tumorigenicity both by desensitizing TICs to normal differentiation cues and by converting otherwise cytostatic signals to proproliferative signals.

Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines.

The concept of tumor stem cells (TSCs) provides a new paradigm for understanding tumor biology, although it remains unclear whether TSCs will prove to be a more robust model than traditional cancer cell lines. We demonstrate marked phenotypic and genotypic differences between primary human tumor-derived TSCs and their matched glioma cell lines. Unlike the matched, traditionally grown tumor cell lines, TSCs derived directly from primary glioblastomas harbor extensive similarities to normal neural stem cells and recapitulate the genotype, gene expression patterns, and in vivo biology of human glioblastomas. These findings suggest that TSCs may be a more reliable model than many commonly utilized cancer cell lines for understanding the biology of primary human tumors.

Decreased expression of hypothalamic neuropeptides in Huntington disease transgenic mice with expanded polyglutamine-EGFP fluorescent aggregates.

Huntington disease is caused by polyglutamine (polyQ) expansion in huntingtin. Selective and progressive neuronal loss is observed in the striatum and cerebral cortex in Huntington disease. We have addressed whether expanded polyQ aggregates appear in regions of the brain apart from the striatum and cortex and whether there is a correlation between expanded polyQ aggregate formation and dysregulated transcription. We generated transgenic mouse lines expressing mutant truncated N-terminal huntingtin (expanded polyQ) fused with enhanced green fluorescent protein (EGFP) and carried out a high-density oligonucleotide array analysis using mRNA extracted from the cerebrum, followed by TaqMan RT-PCR and in situ hybridization. The transgenic mice formed expanded polyQ-EGFP fluorescent aggregates and this system allowed us to directly visualize expanded polyQ aggregates in various regions of the brain without performing immunohistochemical studies. We show here that polyQ-EGFP aggregates were intense in the hypothalamus, where the expression of six hypothalamic neuropeptide mRNAs, such as oxytocin, vasopressin and cocaine-amphetamine-regulated transcript, was down-regulated in the transgenic mouse brain without observing a significant loss of hypothalamic neurons. These results indicate that the hypothalamus is susceptible to aggregate formation in these mice and this may result in the down-regulation of specific genes in this region of the brain.

Co-chaperone CHIP associates with expanded polyglutamine protein and promotes their degradation by proteasomes.

A major hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions of the disease proteins that are ubiquitinated and often associated with various chaperones and proteasome components. But, how the polyglutamine proteins are ubiquitinated and degraded by the proteasomes are not known. Here, we demonstrate that CHIP (C terminus of Hsp70-interacting protein) co-immunoprecipitates with the polyglutamine-expanded huntingtin or ataxin-3 and associates with their aggregates. Transient overexpression of CHIP increases the ubiquitination and the rate of degradation of polyglutamine-expanded huntingtin or ataxin-3. Finally, we show that overexpression of CHIP suppresses the aggregation and cell death mediated by expanded polyglutamine proteins and the suppressive effect is more prominent when CHIP is overexpressed along with Hsc70.

Dosage-dependent over-expression of genes in the trisomic region of Ts1Cje mouse model for Down syndrome.

Down syndrome (DS) is the most common chromosomally caused form of mental retardation and is caused by trisomy of chromosome 21. The over-expression of genes located on the trisomic region has been assumed to be responsible for the phenotypic abnormalities of DS, but this hypothesis has not been confirmed fully and the very existence of gene dosage effects has been called into question. We have therefore investigated global gene expression profiles in Ts1Cje, a mouse model for DS that displays learning deficits and has a segmental trisomy of chromosome 16 orthologous to a segment of human chromosome 21 spanning from Sod1 to Znf295. DNA microarray analyses of six Ts1Cje and six normal littermate (2N) mouse brains at postnatal day 0 with probe sets representing approximately 11,300 genes revealed that the number of expressed genes and their identities in Ts1Cje mice were almost same in 2N mice. Notably, the expression levels of most genes in the trisomic region were increased approximately 1.5-fold, and the top 24 most consistently over-expressed genes in the Ts1Cje mice were all located in the trisomic region. In contrast, the expression levels of genes on other chromosomes or the euploid region of chromosome 16 were largely the same (1.0-fold) in Ts1Cje and 2N mice. These results indicate that the genes in the trisomic region of Ts1Cje are over-expressed in a dosage-dependent manner and are implicated in the molecular pathogenesis of DS.

Gem GTPase and tau: morphological changes induced by gem GTPase in cho cells are antagonized by tau.

A series of observations have indicated that tau, one of the major microtubule-associated proteins, is involved in neuronal cell morphogenesis and axonal maintenance. Tau is also the major component of paired helical filaments found in brains affected by Alzheimer's disease. To explore an as yet unidentified role of tau in vivo, approximately 11,000 mRNAs were profiled from tau-deficient mouse brains and compared with those from control brains at the same ages. The expression of Gem GTPase, a small GTP-binding protein of the ras superfamily, was significantly increased in the brains of tau-deficient mice at 8 weeks of age. Because Gem GTPase is a negative regulator of the Rho-Rho kinase pathway for cytoskeletal organization, this protein was transiently overexpressed in Chinese hamster ovary cells that do not express tau. Overexpression of Gem GTPase induced a marked elongation of Chinese hamster ovary cells, and simultaneous expression of tau eliminated this effect, although tau did not bind directly to Gem GTPase. This anti-elongation activity of tau was attributed to its microtubule-binding domain, and homologous domains of microtubule-associated proteins 2 and 4 exhibited similar antagonistic activities. Taken together, the present results indicate that the level of Gem GTPase and its cell elongation activity are modulated by tau and suggest that tau may be involved in a Gem GTPase-mediated signal transduction pathway.