Caroline Delmas

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.

Preclinical evidence that SSR128129E--a novel small-molecule multi-fibroblast growth factor receptor blocker--radiosensitises human glioblastoma.

Resistance of glioblastoma to radiotherapy is mainly due to tumour cell radioresistance, which is partially controlled by growth factors such as fibroblast growth factor (FGF). Because we have previously demonstrated the role of FGF-2 in tumour cell radioresistance, we investigate here whether inhibiting FGF-2 pathways by targeting fibroblast growth factor receptor (FGFR) may represent a new strategy to optimise the efficiency of radiotherapy in glioblastoma. Treating radioresistant U87 and SF763 glioblastoma cells with the FGFR inhibitor, SSR12819E, radiosensitises these cells while the survival after irradiation of the more radiosensitive U251 and SF767 cells was not affected. SSR128129E administration to U87 cells increases the radiation-induced mitotic cell death. It also decreased cell membrane availability of the FGFR-1 mainly expressed in these cells, increased this receptors ubiquitylation, inhibited radiation-induced RhoB activation and modulated the level of hypoxia inducible factor, HIF-1α, a master regulator of hypoxia, thus suggesting a role of FGFR in the regulation of hypoxia pathways. Moreover, treating orthotopically U87 xenografted mice with SSR128129E before two subsequent local 2.5Gy irradiations significantly increased the animals neurological sign free survival (NSFS) compared to the other groups of treatment. These results strongly suggest that targeting FGFR with the FGFR blocker SSR128129E might represent an interesting strategy to improve the efficiency of radiotherapy in glioblastoma.

FGFR1 Induces Glioblastoma Radioresistance through the PLCγ/Hif1α Pathway.

FGF2 signaling in glioblastoma induces resistance to radiotherapy, so targeting FGF2/FGFR pathways might offer a rational strategy for tumor radiosensitization. To investigate this possibility, we evaluated a specific role for FGFR1 in glioblastoma radioresistance as modeled by U87 and LN18 glioblastomas in mouse xenograft models. Silencing FGFR1 decreased radioresistance in a manner associated with radiation-induced centrosome overduplication and mitotic cell death. Inhibiting PLCγ (PLCG1), a downstream effector signaling molecule for FGFR1, was sufficient to produce similar effects, arguing that PLCγ is an essential mediator of FGFR1-induced radioresistance. FGFR1 silencing also reduced expression of HIF1α, which in addition to its roles in hypoxic responses exerts an independent effect on radioresistance. Finally, FGFR1 silencing delayed the growth of irradiated tumor xenografts, in a manner that was associated with reduced HIF1α levels but not blood vessel alterations. Taken together, our results offer a preclinical proof of concept that FGFR1 targeting can degrade radioresistance in glioblastoma, a widespread problem in this tumor, prompting clinical investigations of the use of FGFR1 inhibitors for radiosensitization. Cancer Res; 76(10); 3036-44. ©2016 AACR.

Alpha-6 integrin promotes radioresistance of glioblastoma by modulating DNA damage response and the transcription factor Zeb1

Radiotherapy is the cornerstone of glioblastoma (GBM) standard treatment. However, radioresistance of cancer cells leads to an inevitable recurrence. In the present study, we showed that blocking α6-integrin in cells derived from GBM biopsy specimens cultured as neurospheres, sensitized cells to radiation. In cells downregulated for α6-integrin expression, we observed a decrease in cell survival after irradiation and an increase in radio-induced cell death. We also demonstrated that inhibition of α6-integrin expression affects DNA damage checkpoint and repair. Indeed, we observed a persistence of γ-H2AX staining after IR and the abrogation of the DNA damage-induced G2/M checkpoint, likely through the downregulation of the checkpoint kinase CHK1 and its downstream target Cdc25c. We also showed that α6-integrin contributes to GBM radioresistance by controlling the expression of the transcriptional network ZEB1/OLIG2/SOX2. Finally, the clinical data from TCGA and Rembrandt databases demonstrate that GBM patients with high levels of the five genes signature, including α6-integrin and its targets, CHK1, ZEB1, OLIG2 and SOX2, have a significantly shorter overall survival. Our study suggest that α6-integrin is an attractive therapeutic target to overcome radioresistance of GBM cancer cells.

Abstract B85: In vivo radiosensitizer effect of the HDAC inhibitor S78454 on orthotopic human glioblastoma.

Glioblastoma is an aggressive primary brain tumor with poor outcome, despite a treatment associating surgery and radiotherapy combined with temozolomide. Recent data have shown an in vitro synergy of the HDAC inhibitor S78454 (also called PCI-24781) with ionizing radiation, via inhibition of homologous recombinational (HR) repair of DNA double-strand breaks. We propose in this study to investigate the in vivo radiosensitizer effect of S78454 on human glioblastoma model. For this, we generated orthotopic U87 human glioblastoma xenografts in nude mice. Intra-peritoneal (i.p.). S 78454 treatment was performed daily starting 10 days after the engraftment. Irradiation was performed in one daily 2 Gy localized fraction during 4 days followed by a second similar 4 days treatment performed after 3 days off (for a total dose of 16 Gy administrated on 11 days). Survival experiments were conducted in mice bearing xenografts in the four different groups of treatment (vehicle, S 78454 alone, radiation alone, S 78454 + radiation). Mice bearing orthotopic xenografts were sacrificed at the onset of neurological signs. Survival curves were then performed. We first determined the S78454 dosing schedule to obtain a pharmacodynamic effect of the compound in the xenograft. Inhibition of RAD 51 expression, histone H3 and tubulin acetylation in the tumor were obtained 24 hours after daily i.p. treatment with 50mg/kg S78454. These results allowed us to define the schedule treatment combining S78454 with radiotherapy : mice were treated with S78454 50 mg/kg/d i.p., daily from Days 10 to14 and then from Days 17 to 21; one 2 Gy fraction radiotherapy was performed daily from Days 11 to14 and Days 18 to 21. In these conditions of treatment, S78454 was well tolerated Survival of the mice treated with S78454 alone was not significantly increased while the survival of the mice treated with irradiation alone was significantly increased compared to the control group (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B85.