Involvement of TBK1 and PREP signalling pathways in Glioblastoma Multiforme progression: novel therapeutic perspectives

Gliomas are common malignant brain tumours, among which glioblastoma multiforme (GBM) has the worst prognosis. Different studies revealed that inflammation plays a key role in GBM pathogenesis. TANK-Binding Kinase 1 (TBK1) is a serine/threonine protein kinase; it is a member of the IκB kinase (IKK) family which is involved in NF-κB pathway activation. Evidence have proven high levels of TBK1 in GBM patients, suggesting that its inhibition could represent a valid strategy to counteract cancer progression by modulating inflammatory process. Moreover, in the last years great attention was focused on the role of prolyl-endopeptidase (PREP), a serine protease involved in the release of pro-angiogenic and anti-fibrogenic molecules. Various angiogenic factors such as vascular endothelial growth factor (VEGF) and angiopoietins (Ang) are up regulated in GBM that generate highly permeable and functionally immature blood vessels, contributing to tumor growth. Thus, the modulation of angiogenesis process through PREP inhibition could represent another possible strategy to counteract GBM growth. Therefore, based on these findings, the aim of the present thesis was to investigate the key roles of TBK1 and PREP signalling pathways in GBM, and the beneficial effects of BX795, TBK1 inhibitor, and KYP-2047, PREP inhibitor, using an in vitro, ex vivo and in vivo model of GBM. In vitro data demonstrated that BX795, TBK1 inhibitor, at the concentrations of 1 µM and 10 µM was able to reduce GBM cell viability, exerting a cytotoxic effect in GBM cell lines. The key role of TBK1 in the pathogenesis of GBM was demonstrated by an in vitro and ex vivo model, showing that the treatment with BX795 was able to significantly reduce TBK1, as well as NIK, IKKα, TNF-α, and SOX3 expression, counteracting inflammatory process. Additionally, BX795 significantly modulated angiogenesis by a downregulation of VEGF and IRF3 levels. In vivo results demonstrated that KYP-2047, PREP inhibitor, at doses of 2.5 mg/kg and 5 mg/kg was able to reduce tumor growth by a significant reduction of PREP level. Moreover, KYP-2047 significantly reduced VEGF, angiopoietin, and endothelial-nitric oxide synthase (eNOS) expression, counteracting angiogenesis. In conclusion, these findings have proven that TBK1 and PREP are involved in GBM pathogenesis, demonstrating that BX795, thanks to its anti-inflammatory effect, as well as KYP-2047, thanks to its angiogenic effect, could represent alternatives therapeutics strategies for GBM treatment, whose mortality rate is increasing.