GENE-07. EPIGENETIC PATHWAYS CONTROLLING HOTAIR EXPRESSION IN GLIOBLASTOMA

Glioblastoma multiforme (GBM) is the most common and aggressive malignant adult brain tumor. Long noncoding RNAs (lncRNAs) have emerged as new players in the cancer paradigm, demonstrating potential roles in both oncogenic and tumor-suppressive pathways. Utilizing single molecule sequencing, we have previously demonstrated that hundreds of lncRNAs, including HOTAIR, are strongly dysregulated in GBM and HOTAIR is critical in GBM cells proliferation. Histone deacetylase inhibitors (HDACi) are small molecules that interfere with histone tail modification, thus altering chromatin structure and epigenetic pathways. However, the mechanistic into HDAC dependent control of HOTAIR expression has remained elusive. We hypothesized that BRD4 that binds to HOTAIR promoter and Gli2 acetylation status could provide insight into regulation of HOTAIR transcription by HDACs. The HOTAIR expression after treatment of HDACi on 2 patient-derived xenografts GBM cells (PDX-10 and PDX-22) were measured via qRT-PCR and the levels of proteins were investigated via Western blot analysis. shRNA constructs targeting individual HDACs (1–7) were used to knockdown HDAC and chromatin immunoprecipitation was performed to assess the protein binding on HOTAIR. From the epigenetic drug library screening, Tricostatin A, NSC-3852, BML-281, Apicidin, M-344, Scriptaid, Oxamflatin and Vorinostat (SAHA) reduced HOTAIR levels more than 50%. HDACi treatments resulted in a dose-dependent decrease in HOTAIR expression in both newly diagnosed and recurrent GBM cells. Both BRD4 RNA and protein levels were also reduced. Furthermore, HDACi treatment resulted in an increase in acetylation and BRD4 binding at HOTAIR gene bodies but a decrease in HOTAIR promoter region. We found that the acetylation of Gli2 after HDACi treatment also prevents its usual occupancy at HOTAIR promoter and subsequent BRD4 recruitment. Importantly, our results unravel a previously unappreciated mechanism through which HDAC inhibition modulate a key regulatory step in GBM cells growth by reducing the recruitment of BRD4 and Gli2 to the HOTAIR promoter.