ROCK inhibition reduces the sensitivity of mutant p53 glioblastoma to genotoxic stress through a Rac1-driven ROS production.

Abstract

Resistance to radio and chemotherapy in Glioblastoma (GBM) is correlated with its malignancy, invasiveness, and aggressiveness. The Rho GTPase pathway plays important roles in these processes, but its involvement in the GBM response to genotoxic treatments remains unsolved. Inhibition of this signaling pathway has emerged as a promising approach for the treatment of CNS injuries and diseases, proving to be a strong candidate for therapeutic approaches. To this end, Rho-associated kinases (ROCK), classic downstream effectors of small Rho GTPases, were targeted for pharmacological inhibition using Y-27632 in GBM cells, expressing the wild-type or mutated p53 gene, and exposed to genotoxic stress by gamma ionizing radiation (IR) or cisplatin (PT). The use of the ROCK inhibitor (ROCKi) had opposite effects in these cells: in cells expressing wild-type p53, ROCKi reduced survival and DNA repair capacity (reduction of γH2AX foci and accumulation of strand breaks) after stress promoted by IR or PT; in cells expressing the mutant p53 protein, both treatments promoted longer survival and more efficient DNA repair, responses further enhanced by ROCKi. The target DNA repair mechanisms of ROCK inhibition were, respectively, an attenuation of NHEJ and NER pathways in wild-type p53 cells, and a stimulation of HR and NER pathways in mutant p53 cells. These effects were accompanied by the formation of reactive oxygen species (ROS) induced by genotoxic stress only in mutant p53 cells but potentiated by ROCKi and reversed by p53 knockdown. The p53-dependent actions of ROCKi were completely abolished by N-acetyl-L-cysteine (NAC) treatment or Rac1 knockdown, as Rac-GTP levels were specifically elevated by ROCK inhibition only in mutant p53 cells. Combining IR or PT and ROCKi treatments broadens our understanding of the sensitivity and resistance of, respectively, GBM expressing wild-type or mutant p53 to genotoxic agents. Our proposal may be a determining factor to improve the efficiency and assertiveness of CNS antitumor therapies based on ROCK inhibitors. SIGNIFICANCE: The use of ROCK inhibitors in association with radio or chemotherapy modulates GBM resistance and sensitivity depending on the p53 activity, suggesting the potential value of this protein as therapeutic target for tumor pre-sensitization strategies.