Abstract PR04: The role of long noncoding RNA HIF1A-AS2 in hypoxic environment of glioblastoma
Authors: Mineo Marco , Ricklefs Franz , Lyons Shawn S. , Ivanov Pavel , Chiocca E. Antonio , Godlewski Jakub , Bronisz Agnieszka 
Published: May 2016

Purpose: While multiple protein-coding genes are known to play a crucial role in the formation and progression of glioblastoma multiforme (GBM), the role of long-non-coding RNAs (lncRNAs) in these cascades remains to be fully characterized. Considering the fundamental roles of hypoxia in the cellular and microenvironmental complexity and unexplored function of lncRNAs in GBM pathobiology, the identification of novel lncRNAs and their target pathways that drive the adaptation to hypoxic niche is crucial for better understanding of the development and progression of this highly heterogeneous brain tumor. Transcriptome profiling of GBM have revealed the presence of four clearly distinguishable subtypes, variably expressed in individual cells within a tumor. This finding may have potential clinical implications, including subtype-specific rearrangements of transcriptional programs related to oncogenic signaling, growth and hypoxia. However, the subtype-specific role of lncRNAs in tumorigenic potential of GBM subtypes remains largely unknown.

Materials and Methods: The brain tissue samples including GBM tumors and non-pathological tissue adjacent to the tumor were collected. GBM-derived primary stem cells (GSCs) classified by transcriptome analysis as proneural (P) and mesenchymal (M), were collected and exposed to hypoxic conditions. Using custom designed Nanostring platform analysis followed by qPCR, the expression patterns of 70 cancer-related lncRNAs were characterized. The in situ hybridization and qPCR analysis was used to validate sub-cellular localization of selected lncRNA. RNA immunoprecipitation (RIP) and mass-spectroscopy (MS) was performed to map RNA-protein interaction and identified targets were validated by Western blotting. The global GSC transcriptome profiling upon lncRNA de-regulation was performed using Pan Cancer Nanostring platform. The knock-down and overexpression strategies in GSC were used to characterize cellular and molecular phenotypes in vitro and in in vivo intracranial GBM model.

Results: We identified lncRNA HIF1A-AS2 (Hypoxia Inducible Factor 1 alpha - antisense 2) as one of the most deregulated in GBM comparing to the matched adjacent tissue. Despite lack of difference in the hypoxia-dependent activation of HIF1A protein between P and M GSC, HIF1A-AS2 was specifically upregulated in M GSC in vitro and in vivo. The deregulation of expression of HIF1A-AS2 had a broad effect on autophagy-related signaling network regulated in response to hypoxia in M but not P GSC. The IGF2BP2 and DHX9 were identified as direct protein partners of this lncRNA. The deregulation of HIF1A-AS2 affected the recruitment of IGF2BP2 to its mRNA targets (e.g. HMG1) and stability of DHX9 protein (but not mRNA), resulting in deregulation of its target genes (e.g FOSL1). Downregulation of HIF1A-AS2 in vivo led to de-regulation of its downstream effectors and resulted in survival benefit of mice bearing M GSC-originated tumors.

Conclusion: P and M GSC respond differently to hypoxic stress by induction of autophagy to maintain their homeostasis and viability and this mechanism is controlled by HIF1A-AS2.

This abstract is also presented as Poster A16.

Citation Format: Marco Mineo, Franz Ricklefs, Shawn S. Lyons, Pavel Ivanov, E. Antonio Chiocca, Jakub Godlewski, Agnieszka Bronisz. The role of long noncoding RNA HIF1A-AS2 in hypoxic environment of glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr PR04.