EPIGENETIC REGULATION OF GBM CELL STEMNESS AND TUMOR PROPAGATING CAPACITY BY OCT4 AND SOX2

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BACKGROUND: (blind field). METHODS: Experiments utilized human glioblastoma neurosphere lines and low passage (<5) primary neurospheres derived from clinical GBM specimens enriched in GBM sphere-forming stem cells. The Adherent GBM cell lines that lack stem-like tumor-propagating cells were used to test phenotype reprogramming by Oct4 and Sox2. Lentiviral and retroviral systems were used for Oct4/Sox2 and micro-RNA gene transfer, respectively. Brain cancer miRNA PCR Arrays (SABiosciences) and quantitative RT-PCR were used to evaluate miRNA expression. OCT4 and Sox2 transcriptional targets were identified and validated by ChIP-PCR and luciferase reporter assays. Micro-RNA promoter methylation was evaluated using bisulfite sequencing. GBM cell stemness was evaluated by sphere-forming assays and analyses of stem cell marker expression by qRT-PCR, immunoblotting and flow cytometry. Tumor xenograft propagating capacity was assayed in immune-deficient mice. RESULTS: In this study, we identify a novel molecular circuit linking reprogramming transcription factors, DNA methylation and microRNA that regulates glioblastoma cell stemness and tumor-propagating capacity. We show that the Oct4 and Sox2 transcription factors induce human glioma cells to transition to a stem-like tumor-propagating state through the direct induction of DNA methyl transferases leading to the methylation-dependent transcriptional regulation of microRNAs. Specifically, miR-148a expression is shown to correlate inversely with GBM cell stemness, to be repressed by Oct4/Sox2-driven DNA methylation, and to inhibit GBM cell stemness and tumor propagation by GBM sphere-forming cells. CONCLUSIONS: These results identify a new methylation-dependent transcriptional axis by which reprogramming transcription factors regulate tumor cell stemness and tumor propagating capacity. Our results also identify miR-148a as a novel tumor-suppressing inhibitor of glioma cell stemness and suggest new approaches for treating GBM by targeting stem-like tumor propagating cell populations. SECONDARY CATEGORY: n/a.

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