The Long Non-coding RNA HIF1A-AS2 Facilitates the Maintenance of Mesenchymal Glioblastoma Stem-like Cells in Hypoxic Niches

Abstract

Long-non-coding RNAs (lncRNAs) have an undefined role in the pathobiology of glioblastoma multiforme (GBM). These tumors are genetically and phenotypically heterogeneous with transcriptome subtype-specific GBM stem-like cells (GSCs) that adapt to the brain tumor microenvironment, including hypoxic niches. We identified hypoxia inducible factor 1 alpha-antisense RNA 2 (HIF1A-AS2) as a subtype-specific hypoxia inducible lncRNA, up-regulated in mesenchymal GSCs. Its deregulation affects GSC growth, self-renewal and hypoxia-dependent molecular reprogramming. Amongst the HIF1A-AS2 interactome, IGF2BP2 and DHX9 were identified as direct partners. This association was needed for maintenance of expression of their target gene, HMGA1. Down-regulation of HIF1A-AS2 led to delayed growth of mesenchymal GSC tumors, survival benefits, and impaired expression of HMGA1 in vivo. Our data demonstrate that HIF1A-AS2 contributes to GSCs’ speciation and adaptation to hypoxia within the tumor microenvironment, acting directly through its interactome/targets and indirectly by modulating responses to hypoxic stress depending on the subtype-specific genetic context.

MicroRNA-Mediated Dynamic Bidirectional Shift between the Subclasses of Glioblastoma Stem-like Cells

“…GSC subclass stratification, initially performed by gene signature (Mao et al, 2013; Mineo et al, 2016; Ricklefs et al, 2016), was further validated by analysis of subclass-enriched proteins and gene microarray, categorizing them as either proneural-like (P) or mesenchymal-like (M) (Figure S1A, B). To verify whether the expression of miR-128 can drive molecular subclass speciation, we screened both subpopulations of GSCs, and found that expression of miR-128 varied coherently between them, being the lowest in M GSCs (Figure 1A, S1C–E).…”

“…Mycoplasma testing was routinely performed by PCR. Mesenchymal and proneural subclass classification was performed by gene expression profiling as previously described (Mineo et al, 2016). Stable GSCs with either overexpression or knock-down of miR-128 in M and P GSCs were created by lentiviral infection with pCDH-miR-128 and anti-miR-ZIP128 respectively (Godlewski et al, 2008; Peruzzi et al, 2013).…”

“…For GSC co-implantation experiments, total 5 × 10 4 cells were implanted with a ratio of 1:500 in both combinations when M GSCs stably overexpressing miR-128 mixed with either P GSC stably overexpressing NC or miR-ZIP128. Animals were sacrificed either at earlier time points to determine the tumor growth or at terminal stage to determine the survival, as per protocol and brain tissue was immediately processed as described (Bronisz et al, 2014; Mineo et al, 2016). Brain sections were imaged using a confocal microscope Zeiss LSM710 and in vivo magnetic resonance imaging (MRI) was performed using a 7.0T Bruker BioSpec USR (Bruker BioSpin Corp.) in the Small Animal Imaging Laboratory (SAIL) at the Brigham and Women’s Hospital as described (Mineo et al, 2016).…”

IMPs: an RNA-binding protein family that provides a link between stem cell maintenance in normal development and cancer

“…Thus, interaction of IMP2 with the long ncRNA (lncRNA) MyoD was found to prevent translation of its associated transcripts, N-Ras and C-myc (Gong et al 2015). A study conducted on mesenchymal GBM showed that IMP2 interacts with the hypoxia-sensitive lncRNA HIF1a-AS2 and facilitates its association with its target transcripts, such as HMGA1 (Mineo et al 2016). IMPs also appear to have the potential to destabilize lncRNAs, as shown by the ability of IMP1 to recruit the CCR4-NOT deadenylase complex to degrade the liver-specific lncRNA HULC (Hammerle et al 2013).…”

Long Noncoding RNA NEAT1, Regulated by the EGFR Pathway, Contributes to Glioblastoma Progression Through the WNT/β-Catenin Pathway by Scaffolding EZH2

“…Bromodomain protein BRD4 promotes the expression of HOTAIR to regulate the cell cycle of glioma cells (6). In a hypoxic microenvironment, HIF1A-AS2 facilitates the maintenance of mesenchymal glioblastoma stem-like cells (7). A lncRNA transcribed from the 5-prime end of the HOXA transcript HOXA11-AS contributes to the malignant progression of glioblastoma (8).…”