REPOSITORY > RESULTS

Abstract

Proteases are involved in different processes of cancer progression by cleaving peptide bonds and thus modifying proteins. One of the processes is the invasive spread of the brain tumour glioblastoma (GBM). Besides single GBM cell invasion into surrounding brain parenchyma, GBM is characterized by significant heterogeneity. In this thesis, the GBM invasion is addressed in relation to invasion-associated proteases and the heterogeneous nature of GBM. Four GBM subtypes have been identified on the basis of clinical behaviour and patient’s survival. GBM cell heterogeneity is a consequence of both different cancer cells and different stromal cells, such as mesenchymal stem cells (MSCs).
It was shown that GBM cell lines U87 and U373 differ in their invasive properties, with the U87 cell line being more invasive and expressing higher levels of proteases. It was proven that the effects of tumour-infiltrating MSCs on GBM cell invasion is GBM cell type-specific. MSCs increased the invasion of U373 cells and inhibited the invasion of U87 cells. MSCs enhanced invasion of U373 cells via overexpression of proteases, such as cathepsin B, calpain1, uPA/uPAR, MMP-2, -9 and -14, and increased activities of some of these proteases, whereas selective protease inhibitors had no effect on U87 cell invasion upon MSC co-culturing. New selective cathepsin B inhibitors were tested and GBM cell type-specific inhibition of cell migration in MSC and GBM cell co-cultures was demonstrated, implicating the importance of addressing the GBM heterogeneity when testing novel anti-GBM drugs. By monitoring GBM cell invasion upon their co-injection with MSCs into the brain of zebrafish embryo, GBM cell type-dependent responses to MSCs were confirmed in vivo. It was demonstrated that zebrafish embryos are an excellent model for studying GBM heterogeneity.
The research described in this thesis was focused on the expression of the lysosomal cysteine cathepsins B, K and X, which were found to be upregulated in GBM tissue. High cathepsin X mRNA levels were associated with poor GBM patient survival. Moreover, it was found that cathepsins K and X were heterogeneously expressed and that GBM subtype–dependent expression had predictive value with respect to overall survival, indicating the importance of taking into account GBM heterogeneity when exploring novel biomarkers.
Therapy-resistant GBM stem cells (GSCs) reside in specific tumour tissue niches and contribute to the heterogeneity of GBM. It was demonstrated that cathepsin X, K and B are localized in GSC niches both in paraffin and cryostat GBM tissue sections. In vitro, it was demonstrated that SDF-1α induces chemotactic responses of CXCR4- and CXCR7-positive GSCs supporting the concept that this cytokine recruits GSCs to niches. Moreover, it was shown that cathepsin K can cleave SDF-1α at the N-terminal site and thus inhibits its chemotactic effects on CXCR4/7-positive GSCs. This implies that cathepsin activity may facilitate the invasion of GSCs out of their niches. The clinical relevance of this finding is supported by correlations that were observed between mRNA expression of cathepsins and GSC niche markers in GBM tissues. In summary, previously unknown roles of proteases in GBM pathobiology, such as mediation of GBM-MSC interactions and regulation of GSC invasion from their niches were demonstrated.