Mediators of the anti-tumoral activity of TAT-Cx43266-283 in human glioblastoma stem cells

Resumen

Glioblastomas (GBMs), the most aggressive primary brain tumours with a median survival of 16 months, have high oncogenic c-Src activity. Glioblastoma stem cells (GSCs) rely on the acti- vity of this oncoprotein for survival, proliferation, stemness and invasion, and are responsible for GBM recurrence and hence the lethality of this tumour. Remarkably, a cell-penetrating peptide based on the physiologically inhibitory interaction of Cx43 and c-Src, TAT-Cx43266-283, specifically inhibits c-Src activity and consequently exerts potent anti-tumoral effects in human glioblastoma cells in vitro and in vivo, without affecting the survival or phenotype of healthy brain cells. In this PhD Thesis, we explored the impact of TAT-Cx43266-283 on pathways fundamental to GSC survival, especially under challenging conditions. As detailed in Chapter 1, we showed that TAT-Cx43266-283, through c-Src inhibition, decrea- sed glucose uptake and reduced oxidative phosphorylation without a compensatory increase in glycolysis in human GSCs. TAT-Cx43266-283 showed no effect on the metabolism of healthy brain cells, including rat neurons, human and rat astrocytes, and human neural stem cells. TAT-Cx43266- 283 impaired metabolic plasticity leading to reduced GSC growth and survival under different nu- trient environments. Importantly, mice glioma models in which GSCs were intracranially implan- ted with TAT-Cx43266-283 showed decreased levels of relevant metabolic targets for cancer therapy, such as hexokinase-2 (HK-2) and glucose transporter-3 (GLUT-3). Together, these results suggest that TAT-Cx43266-283 impairs metabolic plasticity specifically in GSCs in vitro and in vivo. Subsequently, as explained in Chapter 2, we found that TAT-Cx43266-283 blocks autophagic flux in GSCs, especially in nutrient-starvation induced dormant GSCs, and that this process parti- cipates in the anti-tumoral effect of the peptide. Remarkably, dasatinib, a c-Src inhibitor, could not replicate TAT-Cx43266-283 effect on dormant GSCs, revealing for the first time the involvement of pathways other than c-Src in TAT-Cx43266-283 effect. Finally, as presented in Chapter 3, we took advantage of state-of-the-art mass spectrometry proteomics to explore TAT-Cx43266-283 impact at the proteome level. This revealed an important number of pathways and proteins that may be acting as mediators of TAT-Cx43266-283 effect in GSCs. We combined the knowledge generated by this proteomic approach with publicly available GBM patient data to further our insights and identified several potential key proteins in the effect of TAT-Cx43266-283. In summary, the results presented in this PhD Thesis show how TAT-Cx43266-283, by inhibi- ting metabolic plasticity and autophagy, exerts potent, cell-specific and nutrient-context-indepen- dent anti-tumoral effects. In addition, our results open new avenues to continue the research effort into this promising molecule, in hopes of contributing to improve the outcome of GBM patients.