Regulation of DNA double strand break repair by EGF and VEGF signalling reveals Akt to be a critical therapeutic target in glioblastoma

Abstract

Glioblastoma (GBM) is currently incurable. Its radioresistance has been attributed to a subpopulation of cells termed 'GBM stem-like cells' characterised by multipotentiality and tumorigenicity. The discrepancy between pre-clinical and clinical effects of molecular targeted agents on radiosensitivity indicates that 2D in vitro models of GBM do not recapitulate the clinical scenario. In a 3D model developed in our laboratory, EGFR inhibitors failed to enhance radiosensitivity recapitulating their lack of efficacy in the clinic, contrasting with their radiosensitising activity in 2D cultures. Conversely, inhibition of VEGF signalling caused significant radiosensitisation of 3D cultures but had no effect in 2D conditions. The critical role of the DNA damage response in mediating these effects is illustrated by the consistent correlation between radiosensitivity, unrepaired double-strand breaks (γH2AX foci), mitotic catastrophe and micronuclei in both 2D and 3D models. Further investigation revealed unrepaired DSB to be associated with delayed resolution of phosphorylated DNA-PKcs nuclear foci and reduced formation of Rad51 foci. Hence in 2D conditions, EGFR signalling appeared to promote efficient non-homologous end-joining (NHEJ) repair, while in 3D conditions this process was dependent on VEGF signalling. Motivated by previous reports that radiation induced EGFR signalling promotes NHEJ via Akt mediated phosphorylation of DNA-PKcs, we investigated the role of Akt signalling in 2D and 3D systems. Specific inhibition of Akt using the small molecule inhibitor MK-2206 or Akti knockdown increased radiation sensitivity in both 2D and 3D models to a similar extent as EGFR or VEGF inhibition respectively. In keeping with this, phosphorylation of Akt was EGFR dependent in 2D GSC cultures but VEGF dependent in the 3D model. MK-2206 induced radiosensitivity was correlated with increased unrepaired DSBs and extended mouse survival in an U87MGLuc2 orthotopic model compared to radiation only. Our data identify Akt as a promising therapeutic target in combination with radiation for GBM.