Abstract
Glioblastoma (GBM) contains rare glioma stem-like cells (GSCs) with capacities of self-renewal, multi-lineage differentiation, and resistance to conventional therapy. Drug-induced differentiation of GSCs is recognized as a promising approach of anti-glioma therapy. Accumulating evidence suggests that unique properties of stem cells depend on autophagy. Here we demonstrate that BIX01294, an inhibitor of a G9a histone methyltransferase (introducing H3K9me2 and H3K27me3 repressive marks) triggers autophagy in human glioma cells. Pharmacological or genetic inhibition of autophagy decreased LC3-II accumulation and GFP-LC3 punctation in BIX01294-treated cells. GSCs-enriched spheres originating from glioma cells and GBM patient-derived cultures express lower levels of autophagy related (ATG) genes than the parental glioma cell cultures. Typical differentiation inducers that upregulate neuronal and astrocytic markers in sphere cultures, increase the level of ATG mRNAs. G9a binds to the promoters of autophagy (LC3B, WIPI1) and differentiation-related (GFAP, TUBB3) genes in GSCs. Higher H3K4me3 (an activation mark) and lower H3K9me2 (the repressive mark) levels at the promoters of studied genes were detected in serum-differentiated cells than in sphere cultures. BIX01294 treatment upregulates the expression of autophagy and differentiation-related genes in GSCs. Pharmacological inhibition of autophagy decreases GFAP and TUBB3 expression in BIX01294-treated GSCs suggesting that BIX01294-induced differentiation of GSCs is autophagy-dependent.
Highlights
Glioblastoma (GBM, WHO grade IV glioma) is the most frequent, primary malignant brain tumor in adults and remains incurable despite aggressive treatments[1]
We propose a model in which BIX01294 inhibits G9a histone methyltransferase in GSCs, which results in reduction of the repressive H3K9me[2] marks and the increase in the activation H3K4me[3] marks at the promoters of autophagy- and differentiation-related genes
We demonstrated for the first time, that BIX01294 reduces H3K9me[2] and H3K27me[3] levels and induces autophagy in glioma cells
Summary
Glioblastoma (GBM, WHO grade IV glioma) is the most frequent, primary malignant brain tumor in adults and remains incurable despite aggressive treatments[1]. GBMs contain a rare population of glioma stem-like cells (GSCs, called glioma-initiating cells) with capacities of self-renewal, multi-lineage differentiation, and resistance to conventional chemotherapy and radiotherapy. SOX2, OCT4 and NANOG participate in maintaining self-renewal, proliferation, survival, and multi-lineage differentiation potential of embryonic and somatic stem cells and GSCs7. Epigenome-wide mapping of chromatin states in GBMs identified four core transcription factors, such as POU3F2 ( called OCT7, BRN2), SOX2, SALL2, and OLIG2, which are able to reprogram differentiated tumor cells into GSCs8. The differentiated cells loose long-term self-renewal potential in vitro and fail to propagate tumors in vivo[8], suggesting that induction of GSC differentiation may be a good strategy to eliminate GSCs in GBM9. Upon autophagy induction G9a dissociated from the promoters of these genes, which reduced the H3K9me[2] repressive histone modifications and increased the H3K9ac active histone marks[28]
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