In recent years, important advances have been achieved in the understanding of the molecular biology of glioblastoma multiforme (GBM); thus, complex genetic alterations and genomic profiles, which recurrently involve multiple signaling pathways, have been defined, leading to the first molecular/genetic classification of the disease. In this regard, different genetic alterations and genetic pathways appear to distinguish primary (eg, EGFR amplification) versus secondary (eg, IDH1/2 or TP53 mutation) GBM. Such genetic alterations target distinct combinations of the growth factor receptor-ras signaling pathways, as well as the phosphatidylinositol 3-kinase/phosphatase and tensin homolog/AKT, retinoblastoma/cyclin-dependent kinase (CDK) N2A-p16(INK4A), and TP53/mouse double minute (MDM) 2/MDM4/CDKN2A-p14(ARF) pathways, in cells that present features associated with key stages of normal neurogenesis and (normal) central nervous system cell types. This translates into well-defined genomic profiles that have been recently classified by The Cancer Genome Atlas Consortium into four subtypes: classic, mesenchymal, proneural, and neural GBM. Herein, we review the most relevant genetic alterations of primary versus secondary GBM, the specific signaling pathways involved, and the overall genomic profile of this genetically heterogeneous group of malignant tumors.

Glioblastomas are cytogenetically heterogeneous tumors that frequently display alterations of chromosomes 7, 9p, and 10q. We used high-density (500K) single-nucleotide polymorphism arrays to investigate genome-wide copy number alterations and loss of heterozygosity in 35 primary glioblastomas. We focused on the identification and detailed characterization of alterations involving the most frequently altered chromosomes (chromosomes 7, 9, and 10), the identification of distinct prognostic subgroups of glioblastomas based on the cytogenetic patterns of alteration for these chromosomes, and validation of their prognostic impact in a larger series of tumors from public databases. Gains of chromosome 7 (97%), with or without epidermal growth factor receptor (EGFR) amplification, and losses of chromosomes 9p (83%) and 10 (91%) were the most frequent alterations. Such alterations defined five different cytogenetic groups with a significant effect on patient survival; notably, EGFR amplification (29%) was associated with a better survival among older patients, as confirmed by multivariate analysis of a larger series of glioblastomas from the literature. In addition, our results provide further evidence about the relevance of other genes (eg, EGFR, CDKN2A/B, MTAP) in the pathogenesis of glioblastomas. Altogether, our results confirm the cytogenetic heterogeneity of glioblastomas and suggest that their stratification based on combined assessment of cytogenetic alterations involving chromosomes 7, 9, and 10 may contribute to the prognostic evaluation of glioblastomas. ( J Mol Diagn 2011, 13: 634 -647;

BackgroundGlioblastoma multiforme (GBM) displays multiple amplicons and homozygous deletions that involve relevant pathogenic genes and other genes whose role remains unknown.MethodologySingle-nucleotide polymorphism (SNP)-arrays were used to determine the frequency of recurrent amplicons and homozygous deletions in GBM (n = 46), and to evaluate the impact of copy number alterations (CNA) on mRNA levels of the genes involved.Principal FindingsRecurrent amplicons were detected for chromosomes 7 (50%), 12 (22%), 1 (11%), 4 (9%), 11 (4%), and 17 (4%), whereas homozygous deletions involved chromosomes 9p21 (52%) and 10q (22%). Most genes that displayed a high correlation between DNA CNA and mRNA levels were coded in the amplified chromosomes. For some amplicons the impact of DNA CNA on mRNA expression was restricted to a single gene (e.g., EGFR at 7p11.2), while for others it involved multiple genes (e.g., 11 and 5 genes at 12q14.1–q15 and 4q12, respectively). Despite homozygous del(9p21) and del(10q23.31) included multiple genes, association between these DNA CNA and RNA expression was restricted to the MTAP gene.ConclusionsOverall, our results showed a high frequency of amplicons and homozygous deletions in GBM with variable impact on the expression of the genes involved, and they contributed to the identification of other potentially relevant genes.

Abstract. Glioblastomas (GBM) are the most lethal subtype of astrocytomas, with a mean patient survival rate of 12 months after diagnosis. The gold standard treatment of GBM, which includes surgery followed by the combination of radiotherapy and chemotherapy with temozolomide (TMZ), increases the survival rate to 14.6 months. The success of TMZ appears to be limited by the occurrence of chemoresistance that allows glioma cells to escape from death signaling pathways. However, the mechanism of TMZ action is yet to be clarified although some controversial results have been reported. Therefore, our aim was to evaluate the occurrence of apoptosis and autophagy in glioma cells treated with TMZ and to correlate TMZ action with the survival pathways Pi3K/ Akt and ERK1/2 MAP kinase. Cell proliferation was evaluated by incorporation of bromodeoxyuridine. Apoptosis was studied by flow cytometry as well as by fluorescence confocal microscopy in order to evaluate the sub G0/G1 percentage of cells and chromatin condensation. The expression of the autophagy-associated protein, LC3, as well as Akt and ERK1/2 was performed by Western blotting. In TMZ-treated GBM cells the expression of LC3, the autophagy-associated protein was increased and only a reduced percentage of cells underwent apoptosis. In addition, we showed that the phosphorylation status of Pi3K/Akt and ERK1/2 MAP kinase was maintained during the treatment with TMZ, suggesting that glioma cells escape from TMZ-induced cell death due to these signaling pathways. The chemoresistance of U-118 cells to TMZ was partially eradicated when cells were simultaneously treated with specific inhibitors of Pi3K/Akt and ERK1/2 MAP kinase signaling pathways and TMZ. Therefore, we hypothesized that in order to induce glioma cell death it is essential to evaluate the activation of the survival pathways and establish a combined therapy using TMZ and inhibitors of those signaling pathways.

The lncRNA HOTAIR has been implicated in several human cancers. Here, we evaluated the molecular alterations and upstream regulatory mechanisms of HOTAIR in glioma, the most common primary brain tumors, and its clinical relevance. HOTAIR gene expression, methylation, copy-number and prognostic value were investigated in human gliomas integrating data from online datasets and our cohorts. High levels of HOTAIR were associated with higher grades of glioma, particularly IDH wild-type cases. Mechanistically, HOTAIR was overexpressed in a gene dosage-independent manner, while DNA methylation levels of particular CpGs in HOTAIR locus were associated with HOTAIR expression levels in GBM clinical specimens and cell lines. Concordantly, the demethylating agent 5-Aza-2′-deoxycytidine affected HOTAIR transcriptional levels in a cell line-dependent manner. Importantly, HOTAIR was frequently co-expressed with HOXA9 in high-grade gliomas from TCGA, Oncomine, and our Portuguese and French datasets. Integrated in silico analyses, chromatin immunoprecipitation, and qPCR data showed that HOXA9 binds directly to the promoter of HOTAIR. Clinically, GBM patients with high HOTAIR expression had a significantly reduced overall survival, independently of other prognostic variables. In summary, this work reveals HOXA9 as a novel direct regulator of HOTAIR, and establishes HOTAIR as an independent prognostic marker, providing new therapeutic opportunities to treat this highly aggressive cancer.