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Glioblastoma (GBM) harbors subpopulations of therapy-resistant tumor-initiating cells (TICs) that are self-renewing and multipotent. To understand the regulation of the TIC state, we performed an image-based screen for genes regulating GBM TIC maintenance and identified ZFHX4, a 397 kDa transcription factor. ZFHX4 is required to maintain TIC-associated and normal human neural precursor cell phenotypes in vitro, suggesting that ZFHX4 regulates differentiation, and its suppression increases glioma-free survival in intracranial xenografts. ZFHX4 interacts with CHD4, a core member of the nucleosome remodeling and deacetylase (NuRD) complex. ZFHX4 and CHD4 bind to overlapping sets of genomic loci and control similar gene expression programs. Using expression data derived from GBM patients, we found that ZFHX4 significantly affects CHD4-mediated gene expression perturbations, which defines ZFHX4 as a master regulator of CHD4. These observations define ZFHX4 as a regulatory factor that links the chromatin-remodeling NuRD complex and the GBM TIC state., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (©2012 AACR.)

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.

CD133+ populations of human glioblastoma multiforme (GBM) cells are reportedly enriched for tumor stem cells (TSCs) or tumor-initiating cells (TICs). Approximately 40% of freshly isolated GBM specimens, however, do not contain CD133+ tumor cells, raising the possibility that CD133 may not be a universal enrichment marker for GBM TSCs/TICs. Here we demonstrate that stage-specific embryonic antigen 1(SSEA-1/LeX)+ GBM cells fulfill the functional criteria for TSC/TIC, since (1) SSEA-1+ cells are highly tumorigenic in vivo, unlike SSEA-1- cells; (2) SSEA-1+ cells can give rise to both SSEA-1+ and SSEA-1- cells, thereby establishing a cellular hierarchy; and (3) SSEA-1+ cells have self-renewal and multilineage differentiation potentials. A distinct subpopulation of SSEA-1+ cells was present in all but one of the primary GBMs examined (n = 24), and most CD133+ tumor cells were also SSEA-1+, suggesting that SSEA-1 may be a general TSC/TIC enrichment marker in human GBMs.

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.