The link between glioblastoma malignancy and calcium signalling

Glioblastoma (GBM), as one of the most malignant brain tumours, is mostly incurable. For a patient diagnosed with GBM, it is not a question whether the tumour will progress, but when and how fast the patient will die. In the past decades, medical doctors combined different treatments, such as surgical resection, radiation and chemotherapy, to save their lives. Unfortunately, the efficacy of these cures is limited, and patients usually die within 12-18 months. Researchers are trying to put immunotherapy, targeted therapy, and gene therapy into use. So that revealing the molecular changes in GBM and figuring out biomarkers could promote the development of new GBM treatments. Ca2+ signalling is crucial in cellular processes, especially in cancer: cell proliferation, cell migration, metastasis and apoptosis. Mitochondrial Ca2+ uniporter (MCU), locating in the inner mitochondrial membrane, can regulate mitochondrial Ca2+ uptake and shape the amplitude and spatio-temporal patterns of Ca2+ signals . According to previous research, MCU is highly expressed in GBM stem cells (GSC), embryonic stem cell lines (ESC) and GBM cell lines, compared with that in normal brain tissues [5]. The aims during my PhD: • To establish a real-time method to measure Ca2+ concentration. • To analyse Ca2+ signal and MCU expression level in GBM cells. • To link these characteristics with GBM hallmarks: proliferation and migration. I also tested GBM migration in a 3D neural scaffold in collaboration, and we compared the motility inhibition effects of inhibitors based on different molecular mechanisms. The main results of my thesis: 1) By co-staining with Fluo-4 AM and Fura Red AM, we measured real-time Ca2+ oscillations in U87 GBM cell line, GSC cells and human astrocytes. The resting Ca2+ levels are similar, but Ca2+ signal amplitude in GBM cells is much higher than in human astrocytes. 2) MCU expression level in GBM cells is much higher than in human astrocytes. MCU knockdown in U87 GBM cells decreased Ca2+ signal amplitudes, cell proliferation and migration. MCU overexpression in human astrocytes increased Ca2+ signal level but triggered cell death, suggesting that human astrocytes cannot sustain a high Ca2+ level. 3) MCU silencing in U87 GBM cells decreased proliferation and migration. Cell proliferation was slowed down by arresting cells in the G1 phase. 4) We cultured healthy cortical cells on 3D Interconnected Graphene–Carbon Nanotube and establish an in vitro model. By culturing GBM cells in the 3D neural model, we investigated GBM cells infiltration and revealed its potential to screen anticancer drugs. 5) We employed three known inhibitors of small GTPases: ML141, R-ketorolac and EHT 1864. These three inhibitors can reduce the infiltration propensity of GBM cells by targeting two prototypical small Rho GTPase (Rac1 and Cdc42). The binding poses of these drugs on the target proteins provide a rationale at the atomic-level of detail of their non-competitive inhibition mechanism.