p53 affects epigenetic signature on SOCS1 promoter in response to TLR4 inhibition.

Suppressor of cytokine signaling (SOCS1) functions as a negative regulator of toll-like receptor (TLR) induced inflammatory signaling. As silencing of SOCS1 is concomitant with elevated TLR4 levels in glioblastoma, we investigated the effect of TLR4 inhibition on SOCS1 expression. Pharmacological inhibition of TLR4 signaling by TAK242 or its siRNA-mediated knockdown in p53 mutant or wild-type glioma cells resulted in either increased or decreased SOCS1 expression and promoter activity, respectively. Genetic manipulation of p53 indicated that SOCS1 expression upon TLR4 inhibition is dependent on p53 mutational status. Increased SOCS1 level was concomitant with diminished nucleosomal occupancy around p53-binding site on SOCS1 promoter. This altered nucleosomal landscape was accompanied by (i) diminished nuclear H3K9me3 and (ii) increased JMJD2A and Brg1 levels. JMJD2A inhibition or ectopic expression of ATPase-deficient BRG1 prevented TAK242 mediated increase in SOCS1 expression. Recruitment of Brg1-p53-JMJD2A complex on p53 binding sites of SOCS1 promoter upon TLR4 inhibition was concomitant with increased SOCS1 expression in p53-mutant cells. The Cancer Genome Atlas (TCGA) dataset indicated an inverse correlation between TLR4 and SOCS1 levels in p53 mutant but not in p53WT GBM. Taken together, p53 mutational status regulates transcriptional plasticity of SOCS1 promoter through differential recruitment of chromatin remodelers and epigenetic regulators in response to TLR4 inhibition.

Mutant Isocitrate Dehydrogenase 1 Disrupts PKM2-ß-Catenin-BRG1 Transcriptional Network-Driven CD47 Expression.

A gain-of-function mutation in isocitrate dehydrogenase 1 (IDH1) affects immune surveillance in gliomas. As elevated CD47 levels are associated with immune evasion in cancers, its status in gliomas harboring mutant IDH1 (IDH1-MT cells) was investigated. Decreased CD47 expression in IDH1-R132H-overexpressing cells was accompanied by diminished nuclear ß-catenin, pyruvate kinase isoform M2 (PKM2), and TCF4 levels compared to those in cells harboring wild-type IDH1 (IDH1-WT cells). The inhibition of ß-catenin in IDH1-WT cells abrogated CD47 expression, ß-catenin-TCF4 interaction, and the transactivational activity of ß-catenin/TCF4. The reverse effect was observed in IDH1-MT cells upon the pharmacological elevation of nuclear ß-catenin levels. Genetic and pharmacological manipulation of nuclear PKM2 levels in IDH1-WT and IDH1-MT cells suggested that PKM2 is a positive regulator of the ß-catenin-TCF4 interaction. The Cancer Genome Atlas (TCGA) data sets indicated diminished CD47, PKM2, and ß-catenin levels in IDH1-MT gliomas compared to IDH1-WT gliomas. Also, elevated BRG1 levels with mutations in the ATP-dependent chromatin-remodeling site were observed in IDH1-MT glioma. The ectopic expression of ATPase-deficient BRG1 diminished CD47 expression as well as TCF4 occupancy on its promoter. Sequential chromatin immunoprecipitation (ChIP-re-ChIP) revealed the recruitment of the PKM2-ß-catenin-BRG1-TCF4 complex to the TCF4 site on the CD47 promoter. This occupancy translated into CD47 transcription, as a diminished recruitment of this complex was observed in glioma cells bearing IDH1-R132H. In addition to its involvement in CD47 transcriptional regulation, PKM2-ß-catenin-BRG1 cross talk affected the phagocytosis of IDH1-MT cells by microglia.

Hexokinase 2 and nuclear factor erythroid 2-related factor 2 transcriptionally coactivate xanthine oxidoreductase expression in stressed glioma cells.

A dynamic network of metabolic adaptations, inflammatory responses, and redox homeostasis is known to drive tumor progression. A considerable overlap among these processes exists, but several of their key regulators remain unknown. To this end, here we investigated the role of the proinflammatory cytokine IL-1ß in connecting these processes in glioma cells. We found that glucose starvation sensitizes glioma cells to IL-1ß-induced apoptosis in a manner that depended on reactive oxygen species (ROS). Although IL-1ß-induced JNK had no effect on cell viability under glucose deprivation, it mediated nuclear translocation of hexokinase 2 (HK2). This event was accompanied by increases in the levels of sirtuin 6 (SIRT6), nuclear factor erythroid 2-related factor 2 (Nrf2), and xanthine oxidoreductase (XOR). SIRT6 not only induced ROS-mediated cell death but also facilitated nuclear Nrf2-HK2 interaction. Recruitment of the Nrf2-HK2 complex to the ARE site on XOR promoter regulated its expression. Importantly, HK2 served as transcriptional coactivator of Nrf2 to regulate XOR expression, indicated by decreased XOR levels in siRNA-mediated Nrf2 and HK2 knockdown experiments. Our results highlight a non-metabolic role of HK2 as transcriptional coactivator of Nrf2 to regulate XOR expression under conditions of proinflammatory and metabolic stresses. Our insights also underscore the importance of nuclear activities of HK2 in the regulation of genes involved in redox homeostasis.

miR-217-casein kinase-2 cross talk regulates ERK activation in ganglioglioma.

Gangliogliomas (GGs) are the most commonly diagnosed long-term epilepsy-associated tumors (LEATs). Although molecular characterizations of brain tumors have identified few novel biomarkers among the LEATs, mechanisms of pathogenesis remain poorly understood. In this study, global microarray-based microRNA (miRNA) expression profile on a set of 9 GGs indicated 66 miRNAs to be differentially expressed in GG as compared to normal brain. The differences validated by qRT-PCR indicated microRNA-217 to be the most downregulated. Through insilico analysis, ERK1/2 and casein kinase (CK-2α) were predicted to be miR-217 regulated. As decreased miR-217 expression was concomitant with upregulated ERK1/2 and CK-2α levels in GG; the interplay between these molecules was investigated in primary human neural precursor cells to mimic the glioneuronal characteristics of these tumors. miR-217 over-expression-mediated decrease in pERK, CK-2α, and mGluR1 levels was accompanied with increase in glycogen accumulation. Importantly, increase in miR-217 levels upon CK-2α inhibition indicated inverse correlation between the two. Inhibition of CK-2α also decreased ERK and mGluR1 levels. By demonstrating, for the first time, the existence of miR-217-CK-2 cross talk and its effects on known epileptogenic factors, these findings provide a unique insight into the pathogenesis of ganglioglioma. By highlighting the role of CK-2 in affecting miR-217/ERK/mGluR1 interplay, this study suggests that targeting CK-2 may afford a novel strategy aimed at LEATs. KEY MESSAGES: Global microarray of ganglioglioma indicates downregulation of miR-217. Decreased miR-217 expression is concomitant with elevated CK-2α and Erk levels. Inverse correlation between miR-217 and CK-2α in primary human neural precursors. miR-217 agomir or CK-2α inhibition decreases pERK and mGluR1 levels. CK-2α affects miR-217/ERK/mGluR1 interplay in long-term epilepsy-associated tumors.

Telomerase reverse transcriptase (TERT) - enhancer of zeste homolog 2 (EZH2) network regulates lipid metabolism and DNA damage responses in glioblastoma.

Elevated expression of enhancer of zeste homolog 2 (EZH2), a histone H3K27 methyltransferase, was observed in gliomas harboring telomerase reverse transcriptase (TERT) promoter mutations. Given the known involvement of TERT and EZH2 in glioma progression, the correlation between the two and subsequently its involvement in metabolic programming was investigated. Inhibition of human telomerase reverse transcriptase either pharmacologically or through genetic manipulation not only decreased EZH2 expression, but also (i) abrogated FASN levels, (ii) decreased de novo fatty acid accumulation, and (iii) increased ataxia-telangiectasia-mutated (ATM) phosphorylation levels. Conversely, diminished TERT and FASN levels upon siRNA-mediated EZH2 knockdown indicated a positive correlation between TERT and EZH2. Interestingly, ATM kinase inhibitor rescued TERT inhibition-mediated decrease in FASN and EZH2 levels. Importantly, TERT promoter mutant tumors exhibited greater microsatellite instability, heightened FASN levels and lipid accumulation. Coherent with in vitro findings, pharmacological inhibition of TERT by costunolide decreased lipid accumulation and elevated ATM expression in heterotypic xenograft glioma mouse model. By bringing TERT-EZH2 network at the forefront as driver of dysregulated metabolism, our findings highlight the non-canonical but distinct role of TERT in metabolic reprogramming and DNA damage responses in glioblastoma.

SIRT6 regulated nucleosomal occupancy affects Hexokinase 2 expression.

To understand the molecular association between inflammation and dysregulated metabolism in glioblastoma, the effect of IL-1ß on Hexokinase 2 (HK2) expression was investigated. IL-1ß induced HK2 expression was accompanied by heightened SIRT6 and MZF1 levels. IL-1ß mediated overall decrease in chromatin compactness on HK2 promoter involved diminished nucleosomal occupancy around the most labile region bearing MZF1 sites. Importantly, SIRT6 and MZF1 served as negative regulators of HK2. Ectopic SIRT6 induced formation and recruitment of MZF1-SIRT6 complex to MZF1 site was concomitant with increased nucleosomal occupancy. The function of SIRT6 as co-repressor of MZF1 was inconspicuous in cells treated with IL-1ß alone, as IL-1ß-induced HIF-1α prevented SIRT6 availability for interaction with MZF1. Taken together, SIRT6 over-expression establishes a condition whereby reconfiguration of the HK2 promoter chromatin structure makes it receptive to interaction with MZF1/SIRT6 complex, thereby favouring a regulatory state conducive to diminished transcription.