Sylvie Monferran

αvβ3/αvβ5 Integrins-FAK-RhoB: A Novel Pathway for Hypoxia Regulation in 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 alpha(v)beta(3) and alpha(v)beta(5) integrins to the cellular membrane of U87 and SF763 glioblastoma cells, thereby activating the focal adhesion kinase (FAK). We then show that inhibiting alpha(v)beta(3) or alpha(v)beta(5) integrins in hypoxic cells with a specific inhibitor or with siRNA decreases the hypoxia-inducible factor 1alpha (HIF-1alpha) intracellular level. This integrin-dependent regulation of HIF-1alpha is mediated through the regulation of FAK, which in turn activates the small GTPase RhoB, leading to the inhibition of GSK3-beta. 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 alpha(v)beta(3)/alpha(v)beta(5) integrin-dependent loop of hypoxia autoregulation in glioma. Targeting this hypoxia loop may be crucial to optimizing radiotherapy efficiency.

αvβ3 and αvβ5 integrins control glioma cell response to ionising radiation through ILK and RhoB

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.

Alphavbeta3 and alphavbeta5 integrins control glioma cell response to ionising radiation through ILK and RhoB.

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.

Activation of RhoB by Hypoxia Controls Hypoxia-Inducible Factor-1α Stabilization through Glycogen Synthase Kinase-3 in U87 Glioblastoma Cells

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.

Ionizing radiations sustain glioblastoma cell dedifferentiation to a stem-like phenotype through survivin: possible involvement in radioresistance

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.

Radiation-induced mitotic cell death and glioblastoma radioresistance: A new regulating pathway controlled by integrin-linked kinase, hypoxia-inducible factor 1alpha and survivin in U87 cells

We have previously shown that integrin-linked kinase (ILK) regulates U87 glioblastoma cell radioresistance by modulating the main radiation-induced cell death mechanism in solid tumours, the mitotic cell death. To decipher the biological pathways involved in these mechanisms, we constructed a U87 glioblastoma cell model expressing an inducible shRNA directed against ILK (U87shILK). We then demonstrated that silencing ILK enhanced radiation-induced centrosome overduplication, leading to radiation-induced mitotic cell death. In this model, ionising radiations induce hypoxia-inducible factor 1 alpha (HIF-1α) stabilisation which is inhibited by silencing ILK. Moreover, silencing HIF-1α in U87 cells reduced the surviving fraction after 2 Gy irradiation by increasing cell sensitivity to radiation-induced mitotic cell death and centrosome amplification. Because it is known that HIF-1α controls survivin expression, we then looked at the ILK silencing effect on survivin expression. We show that survivin expression is decreased in U87shILK cells. Furthermore, treating U87 cells with the specific survivin suppressor YM155 significantly increased the percentage of giant multinucleated cells, centrosomal overduplication and thus U87 cell radiosensitivity. In consequence, we decipher here a new pathway of glioma radioresistance via the regulation of radiation-induced centrosome duplication and therefore mitotic cell death by ILK, HIF-1α and survivin. This work identifies new targets in glioblastoma with the intention of radiosensitising these highly radioresistant tumours.

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

Glioblastomas are malignant brain tumors with dismal prognosis despite standard treatment with surgery and radio/chemotherapy. These tumors are defined by an important cellular heterogeneity and notably contain a particular subpopulation of Glioblastoma-initiating cells, which recapitulate the heterogeneity of the original Glioblastoma. In order to classify these heterogeneous tumors, genomic profiling has also been undertaken to classify these heterogeneous tumors into several subtypes. Current research focuses on developing therapies, which could take into account this cellular and genomic heterogeneity. Among these targets, integrins are the subject of numerous studies since these extracellular matrix transmembrane receptors notably controls tumor invasion and progression. Moreover, some of these integrins are considered as membrane markers for the Glioblastoma-initiating cells subpopulation. We reviewed here integrin expression according to glioblastoma molecular subtypes and cell heterogeneity. We discussed their roles in glioblastoma invasion, angiogenesis, therapeutic resistance, stemness and microenvironment modulations, and provide an overview of clinical trials investigating integrins in glioblastomas. This review highlights that specific integrins could be identified as selective glioblastoma cells markers and that their targeting represents new diagnostic and/or therapeutic strategies.

PARP-1-dependent RND1 transcription induced by topoisomerase I cleavage complexes confers cellular resistance to camptothecin

RHO GTPases regulate essential functions such as the organization of the actin cytoskeleton. The classic members cycle between an active GTP-bound and an inactive GDP-bound conformation whereas atypical members are predominantly GTP-bound. Besides their well-established role, the classic RHO GTPases RHOB and RAC1, are rapidly induced and/or activated by genotoxic stress and contribute to the DNA damage response. Here we used camptothecin, a selective topoisomerase I (TOP1) inhibitor that stabilizes TOP1 cleavage complexes (TOP1cc), to search for other potential early DNA damage-inducible RHO GTPase genes. We identified that an atypical RHO GTPase, RND1, is rapidly induced by camptothecin. RND1 induction is closely associated with the presence of TOP1cc induced by camptothecin or by DNA lesions that elevate TOP1cc levels such as UV and hydrogen peroxide. We further demonstrated that camptothecin increases RND1 gene transcription and mRNA stability. Camptothecin also increases poly(ADP-ribose) polymerase 1 (PARP-1) activity, whose inhibition reduces RND1 transcription. In addition, overexpression of RND1 increases PARP-1, suggesting a cross-talk between PARP-1 and RND1. Finally, RND1 protects cells against camptothecin-induced apoptosis, and hence favors cellular resistance to camptothecin. Together, these findings highlight RND1 as an atypical RHO GTPase early induced by TOP1cc, and show that the TOP1cc-PARP-1-RND1 pathway protects cells against apoptosis induced by camptothecin.

αvβ3 and αvβ5 integrins control glioma cell response to ionising radiation through ILK and RhoB: Integrins and Glioma Radioresistance

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.