Molecular mechanisms of therapy resistance and recurrence in glioblastoma multiforme

Glioblastoma multiforme (GBM) WHO grade 4 is the most malignant and frequent primary brain tumor with a five-year overall survival rate of 9.8%. Virtually every time, the standard treatment with surgery and radio-/chemotherapy applying temozolomide (TMZ) fails, and the tumor progresses, develops res...

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Bibliographic Details
Main Author: Schäfer, Agnes
Contributors: Bartsch, Jörg-Walter (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2023
Online Access:PDF Full Text
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Summary:Glioblastoma multiforme (GBM) WHO grade 4 is the most malignant and frequent primary brain tumor with a five-year overall survival rate of 9.8%. Virtually every time, the standard treatment with surgery and radio-/chemotherapy applying temozolomide (TMZ) fails, and the tumor progresses, develops resistance mechanisms, and recurs. Thus, it is crucial to further expand the research field for a proper understanding of those mechanisms enabling deeper classifications, the assembly of combinatorial biomarkers, and the development of new therapeutic approaches to overcome therapy resistances. The first publication thematizes TMZ-resistance mechanisms based on the tumor’s changed metabolism and pH-value regulation. Carbonic anhydrase 2 (CA2) was previously identified as highly upregulated in TMZ-resistant glioblastoma stem-like cells (GSCs) as well as recurrent GBM tissue samples. Here, the metalloenzyme was functionally analyzed. Increased oxygen consumption and extracellular acidification rates were detected in CA2-overexpressing GBM cell lines induced via CRISPR/Cas9. This observation underlines the known function of the proton exchanger to enhance aerobic glycolysis (Warburg effect) and proton efflux, thereby leading to extracellular acidification and a pro-invasive and pro-inflammatory microenvironment in cancer. The pan-CA inhibitor acetazolamide (ACZ) repressed the oxygen consumption and extracellular acidification rates more efficiently than the more selective CA2 inhibitor brinzolamide (BRZ) in CA2-overexpressing GBM cells, indicating the crucial role of additional CA isozymes mediating tumor pH regulation and contributing to the Warburg effect. On the contrary, BRZ reduced infiltrative rates and augmented TMZ-induced autophagy causing cell death more efficiently than ACZ in CA2-overexpressing cells, CA2-highly expressing GSCs and TMZ-resistant GSCs. Those data provide evidence for a BRZ-induced pH shift to a physiological level, optimizing the conditions for an efficient TMZ treatment. In the second publication, signaling pathways contributing to tumor progression and resistance were examined for the influence of the disintegrin and metalloproteinase 8 (ADAM8). Next to its ability to cleave extracellular matrix proteins thereby shaping the tumor microenvironment, ADAM8 is characterized as a multi-domain enzyme and can bind to integrins thereby modulating various signaling pathways. Here, the mechanism of ADAM8 regulating miRNA expression profiles through intra- and extracellular signaling was investigated. Several dysregulated miRNAs were identified, comparing GBM cells with endogenous ADAM8 expression or a CRISPR/Cas9-induced ADAM8 knockout. In GBM, miR-181a-5p is described as a tumor-suppressor. Here, this miRNA was identified being upregulated in ADAM8 knockout cells. Mechanistically, ADAM8 was found to mediate miR-181a-5p repression via STAT3 and MAPK signaling. MiR-181a-5p mimic transfection suppressed GBM cell proliferation partially through the post-transcriptional inhibition of MEK1/2, ERK1/2, and CREB-1, indirectly targeting MMP9. Breaking down the ADAM8-mediated signaling involving miR-181a-5p, an ADAM8/STAT3/miR-181a-5p/osteopontin and ADAM8/ERK1/2/CREB-1/miR-181a-5p axis with a negative feedback loop targeting MMP9 was examined. Upon loss of ADAM8 expression, enriched miR-181a-5p levels were detected in extracellular vesicles. Thus, the ADAM8-mediated repression of miR-181a-5p could prevent surrounding cells from MMP9 repression, stimulating invasion and proliferation. Clinically, miR-181a-5p was detected in serum-EVs and downregulated in GBM tissue samples compared to ADAM8, which correlated with MMP9 mRNA expression. The versatile influences of ADAM8 on progression- and resistance-driving signaling pathways, also via miRNA regulation, justifies further experimental approaches targeting ADAM8 in GBM. Lastly, the miRNA expression patterns of three patient-derived GSCs compared to their differentiated astrocytic states were investigated. Thirty-one significantly changed miRNAs were identified, including ten highly dysregulated miRNAs involved in GBM progression and stem cell fate. Among them, miR-425-5p was highly expressed in GSCs, and miR-425-5p mimic transfection reduced the PTEN and GFAP protein expression in patient-derived GBM cell lines, suggesting to induce the GSC phenotype partially. A miRNA-target relationship KEGG enrichment analysis revealed a section of the dynamic miRNA/signaling network deciding about stem cell fate. Thus, we further classified the cellular subtype GSCs revealing potential targets to stimulate GSC differentiation, which could modulate the sensitivity towards GBM treatment strategies.