RNA Sequencing in Hypoxia-Adapted T98G Glioblastoma Cells Provides Supportive Evidence for IRE1 as a Potential Therapeutic Target

Abstract

Glioblastoma (GBM) is an aggressive brain cancer with a median survival time of 14.6 months after diagnosis. GBM cells have altered metabolism and exhibit the Warburg effect, preferentially producing lactate under aerobic conditions. After standard-of-care treatment for GBM, there is an almost 100% recurrence rate. Hypoxia-adapted, treatment-resistant GBM stem-like cells are thought to drive this high recurrence rate. We used human T98G GBM cells as a model to identify differential gene expression induced by hypoxia and to search for potential therapeutic targets of hypoxia adapted GBM cells. RNA sequencing (RNAseq) and bioinformatics were used to identify differentially expressed genes (DEGs) and cellular pathways affected by hypoxia. We also examined expression of lactate dehydrogenase (LDH) genes using qRT-PCR and zymography as LDH dysregulation is a feature of many cancers. We found 2630 DEGs significantly altered by hypoxia (p < 0.05), 1241 upregulated in hypoxia and 1389 upregulated in normoxia. Hypoxia DEGs were highest in pathways related to glycolysis, hypoxia response, cell adhesion and notably the endoplasmic reticulum, including the inositol-requiring enzyme 1 (IRE1)-mediated unfolded protein response (UPR). These results, paired with numerous published preclinical data, provide additional evidence that inhibition of the IRE1-mediated UPR may have therapeutic potential in treating GBM. We propose a possible drug repurposing strategy to simultaneously target IRE1 and the spleen tyrosine kinase (SYK) in patients with GBM.

Inhibition of epigenetic and cell cycle-related targets in glioblastoma cell lines reveals that onametostat reduces proliferation and viability in both normoxic and hypoxic conditions

“…S9 ). In literature, a recent study in the same cell line reported a substantial number of DEGs following the 72-h hypoxia treatment of passage number 3 cells, with upregulation of the Warburg effect-related genes such as inositol-requiring enzyme 1 ( IRE1 ) or lactate dehydrogenase A ( LDHA ) 56 . Neither IRE1 nor LDHA were identified as DEGs in our study, which may be explained by the shorter treatment times and higher cell passage number in our experiments.…”

Inhibition of epigenetic and cell cycle-related targets in glioblastoma cell lines: onametostat reduces proliferation and viability in both normoxic and hypoxic conditions

“…The moderate effect of hypoxic treatment on the gene transcription in the DMSO-treated T98-G cells confirmed this notion (Figure 4). In literature, a recent study in the same cell line reported a substantial number of DEGs following the 72-h hypoxia treatment of passage number 3 cells, with upregulation of the Warburg effect-related genes such as inositol-requiring enzyme 1 (IRE1) or lactate dehydrogenase A (LDHA) 30 . Neither IRE1 nor LDHA were identified as DEGs in our study, which may be explained by the shorter treatment times and higher cell passage number in our experiments.…”

The unfolded protein response machinery in glioblastoma genesis, chemoresistance and as a druggable target

“… 69 White et al. 70 found that IRE1 was among the highest hypoxia DEGs in the T98G cell line and had a key role in the neovascularization and invasiveness of GB. The modulation of TME is essential for GB progression.…”

“…The preceding led to reduced tumor growth, invasiveness and decreased angiogenesis, and blood perfusion with increased overall survival in xenograph mice. 69 White et al 70 found that IRE1 was among the highest hypoxia DEGs in the T98G cell line and had a key role in the neovascularization and invasiveness of GB. The modulation of TME is essential for GB progression.…”