Article
Metabolic targeting of Carbonic Anhydrase 2 (CA-2) overcomes temozolomide resistance in GBM stem cells
Metabolisches Targeting der Carboanhydrase 2 (CA-2) überwindet Temozolomid-Resistenz in Glioblastom-Stammzellen
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Published: | May 25, 2022 |
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Objective: About 95 % of GBM patients show tumor relapse leaving them with limited therapeutic options as recurrent tumors are often resistant to temozolomide (TMZ). GBM-like stem cells (GSCs) are considered as the major obstacle in therapy resistance, so the characterization of their response to TMZ is informative to identify genes associated with TMZ resistance. In TMZ resistant GSCs, we identified Carbonic Anhydrase 2 (CA-2) as a major resistance factor responsible for lactate transport and maintenance of the “Warburg effect” in GBM cells.
Methods: Four independent patient-derived GSC lines and the GBM cell lines U251 and U87 were analyzed for expression levels of CA2 by qPCR. CA-2 expression in GBM sections was detected by IHC-staining with established markers (GFAP, Nestin). Stable CA2- expressing U251 and U87 cells were transfected with a CA-2 construct and analyzed for proliferation, cell migration, invasion, and cell viability assays using co-treatment of TMZ with CA-2 inhibitors. To assess the energy metabolism of these cells, “Seahorse” experiments were performed to measure metabolic parameters in these cells, such as oxygen consumption and glycolysis. Autophagy was investigated in GSCs treated with TMZ and brinzolamide by determining LC-3I to LC-3II ratio and p62 induction.
Results: CA-2 expression was detected in GBM tissue and is accumulated in GSCs of recurrent GBM. CA-2 is highly expressed in GSCs, but merely present in U87 and U251 GBM cell lines. Using acetazolamide as a pan-CA inhibitor, we showed that co-treatment of TMZ with100-400 mM Acetazolamide (ACZ) significantly sensitizes GSCs to TMZ (p<0.01). More effective in sensitizing GSCs to TMZ-mediated cell death was the more “specific” CA-2 inhibitor brinzolamide (100 and 400 mM). Stable CA-2 overexpressing U251 and U87 cells exerted a significantly (at least 2.3-fold, p<0.001) higher resistance to TMZ. In addition, CA-2 overexpressing cells show enhanced proliferation, enhanced cell viability and are metabolically more active as revealed by higher oxygen consumption rate and extracellular acidification rate. However, when treated with TMZ and brinzolamide, CA-2 overexpressing cells showed reduced viability and undergo autophagic cell death, as indicated by LC-3 conversion and p62 activation.
Conclusion: CA-2 has a high metabolic impact in GBM cells and GSCs. Our results provide a rationale for combination therapy targeting the lactate metabolism by inhibition of CA-2 to enhance the therapeutic efficacy of TMZ.