Comparative Transcriptomic Analysis of Temozolomide Resistant Primary GBM Stem-Like Cells and Recurrent GBM Identifies Up-Regulation of the Carbonic Anhydrase CA2 Gene as Resistance Factor

Abstract

About 95% of patients with Glioblastoma (GBM) show tumor relapse, leaving them with limited therapeutic options as recurrent tumors are most often resistant to the first line chemotherapy standard Temozolomide (TMZ). To identify molecular pathways involved in TMZ resistance, primary GBM Stem-like Cells (GSCs) were isolated, characterized, and selected for TMZ resistance in vitro. Subsequently, RNA sequencing analysis was performed and revealed a total of 49 differentially expressed genes (|log2-fold change| > 0.5 and adjusted p-value < 0.1) in TMZ resistant stem-like cells compared to their matched DMSO control cells. Among up-regulated genes, we identified carbonic anhydrase 2 (CA2) as a candidate gene correlated with glioma malignancy and patient survival. Notably, we describe consistent up-regulation of CA2 not only in TMZ resistant GSCs on mRNA and protein level, but also in patient-matched clinical samples of first manifest and recurrent tumors. Co-treatment with the carbonic anhydrase inhibitor Acetazolamid (ACZ) sensitized cells to TMZ induced cell death. Cumulatively, our findings illustrate the potential of CA2 as a chemosensitizing target in recurrent GBM and provide a rationale for a therapy associated inhibition of CA2 to overcome TMZ induced chemoresistance.

Glioma stem cells and their roles within the hypoxic tumor microenvironment

“…CAXII inhibitors also reduced the growth of TMZ-resistant GSCs by inhibiting P-glycoprotein-mediated drug efflux 109 . TMZ resistance in GSCs was further shown to be CAII mediated, with the broader CA inhibitor acetazolamide showing efficacy in combination with TMZ in xenograft models 110 , 111 . Furthermore, CAIX is suggested as a target in GBM for CAR T-cells that could be effective against GSCs 112 .…”

The Expression of Carbonic Anhydrases II, IX and XII in Brain Tumors

“…Recently, Hannen and colleagues reported that CA II is upregulated in TMZ resistant stem-like cells compared to control cells [ 81 ]. The same phenomenon was observed in clinical specimens of first manifest and recurrent tumors.…”

Peptidylarginine Deiminase Isozyme-Specific PAD2, PAD3 and PAD4 Inhibitors Differentially Modulate Extracellular Vesicle Signatures and Cell Invasion in Two Glioblastoma Multiforme Cell Lines

“…PAD activation has indeed been linked to hypoxia in the CNS [53][54][55] and deiminated KEGG (Kyoto Encyclopedia of Genes and Genomes) protein pathways for HIF-1 regulation have been identified to be enriched in animal models of hypoxia-and cancer-resistance [56,57]. PADs also modulate neuronal stem cell growth and death [58], which is of importance considering that GBM tumour invasion, progression and chemo-resistance of recurrent GBM are partly linked to stem-ness [59,60]. Therefore, effects of PAD inhibitors on PAD mediated pathways including EV-mediated export of a range of cancer and hypoxia related microRNAs are also of considerable interest.…”