Elizabeth Cohen‐Jonathan‐Moyal

Integrins are extracellular matrix receptors involved in tumour invasion and angiogenesis. Although there is evidence that inhibiting integrins might enhance the efficiency of radiotherapy, little is known about the exact mechanisms involved in the integrin-dependent modulation of tumor radiosensitivity. The purpose of this study was to investigate the role of alphavbeta3 and alphavbeta5 integrins in glioblastoma cell radioresistance and overall to decipher the downstream biological pathways. We first demonstrated that silencing alphavbeta3 and alphavbeta5 integrins with specific siRNAs significantly reduced the survival after irradiation of 2 glioblastoma cell lines: U87 and SF763. We then showed that integrin activity and integrin signalling pathways controlled the glioma cell radiosensitivity. This regulation of glioma cell response to ionising radiation was mediated through the integrin-linked kinase, ILK, and the small GTPase, RhoB, by two mechanisms. The first one, independent of ILK, consists in the regulation of the intracellular level of RhoB by alphavbeta3 or alphavbeta5 integrin. The second pathway involved in cell radiosensitivity consists in RhoB activation by ionising radiation through ILK. Furthermore, we demonstrated that the alphavbeta3/alphavbeta5 integrins/ILK/RhoB pathway controlled the glioma cells radiosensitivity by regulating radiation-induced mitotic cell death. This work identifies a new biological pathway controlling glioblastoma cells radioresistance, activated from the membrane through alphavbeta3 and/or alphavbeta5 integrins via ILK and RhoB. Our results are clues that downstream effectors of alphavbeta3 and alphavbeta5 integrins as ILK and RhoB might also be promising candidate targets for improving the efficiency of radiotherapy and thus the clinical outcome of patients with glioblastoma.

Recurrence of GBM is thought to be due to GBMSCs, which are particularly chemo-radioresistant and characterized by a high capacity to invade normal brain. Evidence is emerging that modulation of m6A RNA methylation plays an important role in tumor progression. However, the impact of this mRNA modification in GBM is poorly studied. We used patient-derived GBMSCs to demonstrate that high expression of the RNA demethylase, ALKBH5, increases radioresistance by regulating homologous recombination (HR). In cells downregulated for ALKBH5, we observed a decrease in GBMSC survival after irradiation likely due to a defect in DNA-damage repair. Indeed, we observed a decrease in the expression of several genes involved in the HR, including CHK1 and RAD51, as well as a persistence of γ-H2AX staining after IR. We also demonstrated in this study that ALKBH5 contributes to the aggressiveness of GBM by favoring the invasion of GBMSCs. Indeed, GBMSCs deficient for ALKBH5 exhibited a significant reduced invasion capability relative to control cells. Our data suggest that ALKBH5 is an attractive therapeutic target to overcome radioresistance and invasiveness of GBMSCs.

Hypoxia is a crucial factor in tumor aggressiveness and resistance to treatment, particularly in glioma. Our previous results have shown that inhibiting the small GTPase RhoB increased oxygenation of U87 human glioblastoma xenografts, in part, by regulating angiogenesis. We investigated here whether RhoB might also control a signaling pathway that would permit glioma cells to adapt to hypoxia. We first showed that silencing RhoB with siRNA induced degradation and inhibition of the transcriptional activity of the hypoxia-inducible factor by the proteasome in U87 hypoxic cells. This RhoB-dependent degradation of hypoxia-inducible factor-1alpha in hypoxic conditions was mediated by the Akt/glycogen synthase kinase-3beta pathway. While investigating how hypoxia could activate this signaling pathway, using the GST-Rhotekin RBD pulldown assay, we showed the early activation of RhoB by reactive oxygen species under hypoxic conditions and, subsequently, its participation in the ensuing cellular adaptation to hypoxia. Overall, therefore, our results have not only highlighted a new signaling pathway for hypoxia controlled by the small GTPase RhoB, but they also strongly implicate RhoB as a potentially important therapeutic target for decreasing tumor hypoxia.