Glioblastomas (GBM) are some bad prognosis brain tumors despite a conventional treatment associating surgical resection and subsequent radio-chemotherapy. Among these heterogeneous tumors, a subpopulation of chemo- and radioresistant GBM stem-like cells appears to be involved in the systematic GBM recurrence. Moreover, recent studies showed that differentiated tumor cells may have the ability to dedifferentiate and acquire a stem-like phenotype, a phenomenon also called plasticity, in response to microenvironment stresses such as hypoxia. We hypothesized that GBM cells could be subjected to a similar dedifferentiation process after ionizing radiations (IRs), then supporting the GBM rapid recurrence after radiotherapy. In the present study we demonstrated that subtoxic IR exposure of differentiated GBM cells isolated from patient resections potentiated the long-term reacquisition of stem-associated properties such as the ability to generate primary and secondary neurospheres, the expression of stemness markers and an increased tumorigenicity. We also identified during this process an upregulation of the anti-apoptotic protein survivin and we showed that its specific downregulation led to the blockade of the IR-induced plasticity. Altogether, these results demonstrated that irradiation could regulate GBM cell dedifferentiation via a survivin-dependent pathway. Targeting the mechanisms associated with IR-induced plasticity will likely contribute to the development of some innovating pharmacological strategies for an improved radiosensitization of these aggressive brain cancers.

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 αvβ3 and αvβ5 integrins in glioblastoma cell radioresistance and overall to decipher the downstream biological pathways. We first demonstrated that silencing αvβ3 and αvβ5 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 αvβ3 or αvβ5 integrin. The second pathway involved in cell radiosensitivity consists in RhoB activation by ionising radiation through ILK. Furthermore, we demonstrated that the αvβ3/αvβ5 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 αvβ3 and/or αvβ5 integrins via ILK and RhoB. Our results are clues that downstream effectors of αvβ3 and αvβ5 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.

The presence of hypoxic areas in glioblastoma is an important determinant in tumor response to therapy and, in particular, to radiotherapy. Here we have explored the involvement of integrins, up to now known as regulators of angiogenesis and invasion, in the regulation of tumor hypoxia driven from the tumor cell. We first show that hypoxia induces the recruitment of α_vβ₃ and α_vβ₅ integrins to the cellular membrane of U87 and SF763 glioblastoma cells, thereby activating the focal adhesion kinase (FAK). We then show that inhibiting α_vβ₃ or α_vβ₅ integrins in hypoxic cells with a specific inhibitor or with siRNA decreases the hypoxia-inducible factor 1α (HIF-1α) intracellular level. This integrin-dependent regulation of HIF-1α is mediated through the regulation of FAK, which in turn activates the small GTPase RhoB, leading to the inhibition of GSK3-β. Furthermore, silencing this pathway in glioma cells of established xenografts dramatically reduces glioma hypoxia, associated with a significant decrease in vessel density. Our present results unravel a new mechanism of hypoxia regulation by establishing the existence of an α_vβ₃/α_vβ₅ integrin–dependent loop of hypoxia autoregulation in glioma. Targeting this hypoxia loop may be crucial to optimizing radiotherapy efficiency. [Cancer Res 2009;69(8):3308–16]

Interest of integrins targeting in glioblastoma according to tumor heterogeneity and cancer stem cell paradigm: an update Interest of integrins targeting in glioblastoma according to tumor heterogeneity and cancer stem cell paradigm: an update

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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-1α in hypoxic conditions was mediated by the Akt/glycogen synthase kinase-3β 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. (Cancer Res 2006; 66(1): 482-9)