Article
Characterisation of carbonic anhydrase 2 (CA-2) as a factor conferring temozolomide resistance in GBM stem cells
Charakterisierung der Carbonic Anhydrase 2 (CA-2) als ein Faktor der Resistenzvermittlung in GBM-Stammzellen
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Published: | June 4, 2021 |
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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 that characterization of their response to TMZ is informative to identify genes associated with TMZ resistance. We have identified Carbonic Anhydrase 2 (CA-2) as a major factor responsible for TMZ resistance of GBM stem cells.
Methods: Four independent patient derived GSC lines and the GBM cell lines U251 and U87 were analysed 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.
Results: CA-2 expression is detected in GBM tissue and is accumulated in GSCs of recurrent GBM. Moreover, CA-2 is highly expressed in GSCs, but merely present in U87 and U251 cells. Using acetazolamide as pan-CA inhibitor, we showed that co-treatment of TMZ with100-400 μM Acetazolamide (ACZ) significantly sensitizes GSCs to TMZ (p<0.01). Similar effects were observed using a novel specific CA-2 inhibitor brinzolamide (100 μM) in GSCs. 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.
Conclusion: We described Carbonic Anhydrase 2 as a potential resistance factor against temozolomide treatment in GSCs and demonstrate here that CA-2 has a high metabolic impact on tumor cells when expressed in amounts that are comparable to that of GSCs. Our results form the basis for a rational combination therapy to enhance the therapeutic efficacy of TMZ.