The Long Non-Coding RNA HOXA-AS2 Promotes Proliferation of Glioma Stem Cells and Modulates Their Inflammation Pathway Mainly through Post-Transcriptional Regulation.

Glioblastomas represent approximatively half of all gliomas and are the most deadly and aggressive form. Their therapeutic resistance and tumor relapse rely on a subpopulation of cells that are called Glioma Stem Cells (GSCs). Here, we investigated the role of the long non-coding RNA HOXA-AS2 in GSC biology using descriptive and functional analyses of glioma samples classified according to their isocitrate dehydrogenase (IDH) gene mutation status, and of GSC lines. We found that HOXA-AS2 is overexpressed only in aggressive (IDHwt) glioma and GSC lines. ShRNA-based depletion of HOXA-AS2 in GSCs decreased cell proliferation and altered the expression of several hundreds of genes. Integrative analysis revealed that these expression changes were not associated with changes in DNA methylation or chromatin signatures at the promoter of the majority of genes deregulated following HOXA-AS2 silencing in GSCs, suggesting a post-transcriptional regulation. In addition, transcription factor binding motif enrichment and correlation analyses indicated that HOXA-AS2 affects, directly or indirectly, the expression of key transcription factors implicated in GCS biology, including E2F8, E2F1, STAT1, and ATF3, thus contributing to GCS aggressiveness by promoting their proliferation and modulating the inflammation pathway.

Widespread overexpression from the four DNA hypermethylated HOX clusters in aggressive (IDHwt) glioma is associated with H3K27me3 depletion and alternative promoter usage.

In human, the 39 coding HOX genes and 18 referenced noncoding antisense transcripts are arranged in four genomic clusters named HOXA, B, C, and D. This highly conserved family belongs to the homeobox class of genes that encode transcription factors required for normal development. Therefore, HOX gene deregulation might contribute to the development of many cancer types. Here, we study HOX gene deregulation in adult glioma, a common type of primary brain tumor. We performed extensive molecular analysis of tumor samples, classified according to their isocitrate dehydrogenase (IDH1) gene mutation status, and of glioma stem cells. We found widespread expression of sense and antisense HOX transcripts only in aggressive (IDHwt) glioma samples, although the four HOX clusters displayed DNA hypermethylation. Integrative analysis of expression, DNA methylation, and histone modification signatures along the clusters revealed that HOX gene upregulation relies on canonical and alternative bivalent CpG island promoters that escape hypermethylation. H3K27me3 loss at these promoters emerges as the main cause of widespread HOX gene upregulation in IDHwt glioma cell lines and tumors. Our study provides the first comprehensive description of the epigenetic changes at HOX clusters and their contribution to the transcriptional changes observed in adult glioma. It also identified putative 'master' HOX proteins that might contribute to the tumorigenic potential of glioma stem cells.

HOX gene cluster (de)regulation in brain: from neurodevelopment to malignant glial tumours.

HOX genes encode a family of evolutionarily conserved homeodomain transcription factors that are crucial both during development and adult life. In humans, 39 HOX genes are arranged in four clusters (HOXA, B, C, and D) in chromosomes 7, 17, 12, and 2, respectively. During embryonic development, particular epigenetic states accompany their expression along the anterior-posterior body axis. This tightly regulated temporal-spatial expression pattern reflects their relative chromosomal localization, and is critical for normal embryonic brain development when HOX genes are mainly expressed in the hindbrain and mostly absent in the forebrain region. Epigenetic marks, mostly polycomb-associated, are dynamically regulated at HOX loci and regulatory regions to ensure the finely tuned HOX activation and repression, highlighting a crucial epigenetic plasticity necessary for homeostatic development. HOX genes are essentially absent in healthy adult brain, whereas they are detected in malignant brain tumours, namely gliomas, where HOX genes display critical roles by regulating several hallmarks of cancer. Here, we review the major mechanisms involved in HOX genes (de)regulation in the brain, from embryonic to adult stages, in physiological and oncologic conditions. We focus particularly on the emerging causes of HOX gene deregulation in glioma, as well as on their functional and clinical implications.

Transcriptional alterations in glioma result primarily from DNA methylation-independent mechanisms.

In cancer cells, aberrant DNA methylation is commonly associated with transcriptional alterations, including silencing of tumor suppressor genes. However, multiple epigenetic mechanisms, including polycomb repressive marks, contribute to gene deregulation in cancer. To dissect the relative contribution of DNA methylation-dependent and -independent mechanisms to transcriptional alterations at CpG island/promoter-associated genes in cancer, we studied 70 samples of adult glioma, a widespread type of brain tumor, classified according to their isocitrate dehydrogenase (IDH1) mutation status. We found that most transcriptional alterations in tumor samples were DNA methylation-independent. Instead, altered histone H3 trimethylation at lysine 27 (H3K27me3) was the predominant molecular defect at deregulated genes. Our results also suggest that the presence of a bivalent chromatin signature at CpG island promoters in stem cells predisposes not only to hypermethylation, as widely documented, but more generally to all types of transcriptional alterations in transformed cells. In addition, the gene expression strength in healthy brain cells influences the choice between DNA methylation- and H3K27me3-associated silencing in glioma. Highly expressed genes were more likely to be repressed by H3K27me3 than by DNA methylation. Our findings support a model in which altered H3K27me3 dynamics, more specifically defects in the interplay between polycomb protein complexes and the brain-specific transcriptional machinery, is the main cause of transcriptional alteration in glioma cells. Our study provides the first comprehensive description of epigenetic changes in glioma and their relative contribution to transcriptional changes. It may be useful for the design of drugs targeting cancer-related epigenetic defects.