Hypoxia sustains glioblastoma radioresistance through ERKs/DNA-PKcs/HIF-1α functional interplay

The molecular mechanisms by which glioblastoma multiforme (GBM) refracts and becomes resistant to radiotherapy treatment remains largely unknown. This radioresistance is partly due to the presence of hypoxic regions, which are frequently found in GBM tumors. We investigated the radiosensitizing effects of MEK/ERK inhibition on GBM cell lines under hypoxic conditions. Four human GBM cell lines, T98G, U87MG, U138MG and U251MG were treated with the MEK/ERK inhibitor U0126, the HIF-1 alpha inhibitor FM19G11 or gamma-irradiation either alone or in combination under hypoxic conditions. Immunoblot analysis of specific proteins was performed in order to define their anti-oncogenic or radiosensitizing roles in the different experimental conditions. MEK/ERK inhibition by U0126 reverted the transformed phenotype and significantly enhanced the radiosensitivity of T98G, U87MG, U138MG cells but not of the U251MG cell line under hypoxic conditions. U0126 and ERK silencing by siRNA reduced the levels of DNA protein kinase catalytic subunit (DNA-PKcs), Ku70 and K80 proteins and clearly reduced HIF-1 alpha activity and protein expression. Furthermore, DNA-PKcs siRNA-mediated silencing counteracted HIF-1 alpha activity and downregulated protein expression suggesting that ERKs, DNA-PKcs and HIF-1 alpha cooperate in radioprotection of GBM cells. Of note, HIF-1 alpha inhibition under hypoxic conditions drastically radiosensitized all cell lines used. MEK/ERK signal transduction pathway, through the sustained expression of DNA-PKcs, positively regulates HIF-1 alpha protein expression and activity, preserving GBM radioresistance in hypoxic condition.