Resumen de Anti-Tumour effects of the c-src inhibitor peptide, tat-cx43266-283, in human glioma stem cells
Glioma stem cells (GSCs) constitute one of the challenges in treating glioblastoma, the most severe and frequent brain tumour. They have self-renewal capacity, multilineage differentiation properties, high oncogenic potential, resistance to conventional therapies and aggressive infiltration into the brain parenchyma. All these features make difficult the whole tumour resection and facilitate tumour recurrence. GSCs express very low levels of connexin43 (Cx43), the main gap-junction channel-forming protein in astrocytes. One important channel-independent function of Cx43 is to inhibit the oncogene c-Src, involved in cell proliferation, migration and survival. In fact, restoring Cx43 inhibits the strong activity of this oncogene in glioma cells, including GSCs. Interestingly, a cell-penetrating peptide based on the region of Cx43 that inhibits c-Src, TAT-Cx43266-283, reverses GSC phenotype and reduces neurosphere formation. In this thesis, we analysed the anti-tumour effects of TAT-Cx43266-283 on human G166 GSCs and more importantly, we set up a protocol for the culture of primary patient-derived GSCs that allowed us to broaden our study to these cells and to gain insight into the anti-tumour mechanism of TAT-Cx43266-283. First, we showed that TAT-Cx43266-283 inhibits G166 and patient-derived GSCs proliferation. To corroborate the proposed mechanism for Cx43-c-Src interaction previously described in rat C6 glioma cells and astrocytes, we used a biotinylated TAT-Cx43266-283 cell-penetrating peptide as a bait to identify its interacting partners. This confirmed that TAT-Cx43266-283 serves as a docking platform that favours the proximity of active c-Src with its endogenous inhibitors, PTEN and Csk, in GSCs. In addition to c-Src inhibition, TAT-Cx43266-283 upregulated PTEN contributing to the reduction of GSC proliferation by the downregulation of AKT activity in human GSCs. Since the inhibition of GSC proliferation by TAT-Cx43266-283 is lost when PTEN is silenced, it could be proposed that this phosphatase is required for the anti-proliferative effect of TAT-Cx43266-283 on GSCs.
Because TAT-Cx43266-283 targets c-Src and PTEN, two regulators of the focal adhesion kinase (FAK), which in turn controls migration and invasion, we addressed the effects of TAT-Cx43266-283 on FAK activity. FAK active levels decreased after 15 h (Y576 and Y577 FAK) and 24 h (Y397 FAK) of treatment with TAT-Cx43266-283 on patient-derived GSCs. The inhibition of this c-Src downstream motility cascade was patent when individual G166 and patient-derived GSC trajectories were tracked because they showed a significant decrease in their lengths. Invasive properties through Matrigel-inserts were also reduced in G166 and patient-derived GSCs when these cells were exposed to TAT-Cx43266-283.
Furthermore, we performed a time-lapse microscopy study on fresh tumour explants from the same surgical specimens used to obtain GSCs. Importantly, these movies showed a strong decrease in cell proliferation, migration and survival of those tumour explants treated with TAT-Cx43266-283.
Finally, another region of Cx43 (amino acids 274-291) was used to confirm the specificity of TAT-Cx43266-283. We showed that TAT-Cx43274-291 did not affect significantly migration or proliferation in G166 and patient-derived GSCs, thus verifying TAT-Cx43266-283 specificity.
This PhD thesis advances the understanding of the molecular mechanisms triggered by Cx43-Src interaction that includes the PTEN-FAK axis. As a consequence of this signalling pathway, this study shows the anti-tumour effects of TAT-Cx43266-283 on the reduction of cell proliferation, migration and survival in two highly relevant glioma models, such as patient-derived GSCs and freshly removed surgical specimens of malignant gliomas.
