Background
Glioblastoma (GBM) represents the most common primary malignant brain tumor in adults, with a median survival rate of less than two years. GBM presents with therapeutic resistance and high rates of recurrence, largely attributed to the presence of glioblastoma stem cells (GSCs). GSCs are frequently located in specific niches, including a hypoxic niche and a normoxic, perivascular niche. Targeting GSCs is a potential therapeutic avenue for treating GBM, but the details of GSC function in hypoxic conditions remains poorly understood. Further, most gene expression findings are limited to epigenetic or transcriptional regulation assays, overlooking the importance of the translatome. This study expands on conclusions made using steady-state mRNA levels by including ribosome profiling to evaluate translational regulation mechanisms. 

Methods
We selected two patient-derived GSC lines (GSC 215, GSC 316) and cultured them in both hypoxic (5% O2) and normoxic (21% O2) conditions. RNA-seq libraries were prepared in parallel with RIBO-seq libraries for the lines in both conditions, with biological replicates for each treatment group, after which Illumina sequencing was performed. Raw sequencing reads were assessed for quality. Single-end reads were aligned and counted using command line versions of STAR, BEDTools, and SAMTools. Differential changes in ribosome-protected fragments (∆RPF) and changes in transcript levels (∆RNA) were calculated using DESeq2. GO term analyses for transcripts was performed with Reactome using a hypergeometric statistical test and BH FDR correction.

Results
Comparing ∆RNA and ∆RPF levels between hypoxic and normoxic GSCs reveals 9 functional clusters of genes. These categories indicate genes which are post-transcriptionally controlled in 3 manners: translationally suppressed (28.19%), translationally upregulated (32.87%), or concurrently translated (38.93%). Altogether, more than 60% of mRNA show marked regulation on the translational level.

Significant enrichment of genes in the NOTCH signaling pathway (p = 1.09e-02), WNT signaling pathway (p = 1.37e-04), and RHO GTPase cycle (p = 2.37e-07) are present among the transcripts that experience translational upregulation in hypoxic conditions.

Conclusion
This study may lead to the identification of potential therapeutic targets as evidenced by their function as regulatory hubs in cellular networks responsible for hypoxic adaptation. These observations are largely undetected by RNA-seq measurements.


Evaluating the Role of Translation Regulation in Glioblastoma Stem Cells for Hypoxic Adaptation

Basic Science | Hematology / Oncology / Pathology

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Evaluating the Role of Translation Regulation in Glioblastoma Stem Cells for Hypoxic Adaptation

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