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

Dahan P, Martinez Gala J, Delmas C, Monferran S, Malric L, Zentkowski D, Lubrano V, Toulas C, Cohen-Jonathan Moyal E, Lemarie A - Cell Death Dis (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: INSERM UMR 1037, Centre de Recherches en Cancérologie de Toulouse (CRCT), Université Toulouse III Paul Sabatier, Toulouse, France.

ABSTRACT
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.

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Requirement of the IR-induced survivin overexpression for the dedifferentiationprocess in GBM cells. (a–d) Differentiated GBM cells werepre-treated for 24 h with either a survivin inhibitor (YM-155 7 nM)or an AKT inhibitor (MK-2206 250 nM) and then irradiated or not at3 Gy before being placed 2 days after in either FCS or SCM medium,complemented or not with fresh YM-155 or MK-2206 inhibitors. (a) Westernblot analysis of the effect of YM-155 and MK-2206 treatment on Survivin expressionin GBM cells (I cell line) irradiated or not and kept in SCM medium for 1additional week. Efficiency of MK-2206 toward AKT was also checked as a control bythe blotting of phospho-AKT1 (pAKT1). (b and c) At the end of thededifferentiation protocol, the effects of YM-155 and MK-2206 were measured on theNS generation potential in response to IR by NS counting in phase-contrastmicroscopy (original magnification: × 4, scale bar:17 μm) (b) and subsequent quantification in theindicated cell lines (c). Results are expressed as the means±S.E.M.of three independent experiments. ∗∗P<0.01,∗∗∗P<0.001 compared with the 3-GySCM condition. (d) The involvement of Survivin in the IR-induced GBMreprogramming was checked by western blotting by analyzing the expression of thestem markers Nestin, Sox2 and Olig2 at the end of the dedifferentiation protocolin the presence or absence of YM-155 and MK-2206. Concerning western blottings,equal gel loading and transfer efficiency were checked with an anti-actin, AKT1 orβ2-microglobulin (β2M) antibody, and results wererepresentative of at least three independent experiments on the indicated cellline and reproduced in all the cell lines
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fig8: Requirement of the IR-induced survivin overexpression for the dedifferentiationprocess in GBM cells. (a–d) Differentiated GBM cells werepre-treated for 24 h with either a survivin inhibitor (YM-155 7 nM)or an AKT inhibitor (MK-2206 250 nM) and then irradiated or not at3 Gy before being placed 2 days after in either FCS or SCM medium,complemented or not with fresh YM-155 or MK-2206 inhibitors. (a) Westernblot analysis of the effect of YM-155 and MK-2206 treatment on Survivin expressionin GBM cells (I cell line) irradiated or not and kept in SCM medium for 1additional week. Efficiency of MK-2206 toward AKT was also checked as a control bythe blotting of phospho-AKT1 (pAKT1). (b and c) At the end of thededifferentiation protocol, the effects of YM-155 and MK-2206 were measured on theNS generation potential in response to IR by NS counting in phase-contrastmicroscopy (original magnification: × 4, scale bar:17 μm) (b) and subsequent quantification in theindicated cell lines (c). Results are expressed as the means±S.E.M.of three independent experiments. ∗∗P<0.01,∗∗∗P<0.001 compared with the 3-GySCM condition. (d) The involvement of Survivin in the IR-induced GBMreprogramming was checked by western blotting by analyzing the expression of thestem markers Nestin, Sox2 and Olig2 at the end of the dedifferentiation protocolin the presence or absence of YM-155 and MK-2206. Concerning western blottings,equal gel loading and transfer efficiency were checked with an anti-actin, AKT1 orβ2-microglobulin (β2M) antibody, and results wererepresentative of at least three independent experiments on the indicated cellline and reproduced in all the cell lines

Mentions: We then investigated whether this survivin overexpression during the IR-induceddedifferentiation was a consequence of this reprogramming or could be an essentialstep supporting IR-induced GBM plasticity to a stem-like phenotype. To this end,we treated GBM cells during the dedifferentiation process with 7 nM YM-155,a selective inhibitor of survivin used in anti-cancer clinicaltrials.50 As an additionalcontrol, we used MK-2206 (250 nM), a selective AKT inhibitor,51 as we and others52 showed that AKT can control the expression of survivinin GBM cells (Figure 8a). We first checked theefficiency of these inhibitors used at non-toxic concentrations (data not shown)on survivin expression by western blotting during the IR-promoteddedifferentiation (Figure 8a). Next, we observed theireffect at the end of the dedifferentiation protocol and showed that bothinhibitors induced a potent blockade of the 3-Gy-induced NS generation in SCMmedium (Figures 8b and c). Moreover, YM-155 andMK-2206 markedly inhibited at the protein level the overexpression of the stemmarkers Olig2, Sox2 and Nestin in response to IR in SCM medium (Figure 8d). Altogether, these results strongly suggest that theIR-induced reprogramming in GBM cells was associated and supported by theupregulation of the anti-apoptotic protein survivin.


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

Dahan P, Martinez Gala J, Delmas C, Monferran S, Malric L, Zentkowski D, Lubrano V, Toulas C, Cohen-Jonathan Moyal E, Lemarie A - Cell Death Dis (2014)

Requirement of the IR-induced survivin overexpression for the dedifferentiationprocess in GBM cells. (a–d) Differentiated GBM cells werepre-treated for 24 h with either a survivin inhibitor (YM-155 7 nM)or an AKT inhibitor (MK-2206 250 nM) and then irradiated or not at3 Gy before being placed 2 days after in either FCS or SCM medium,complemented or not with fresh YM-155 or MK-2206 inhibitors. (a) Westernblot analysis of the effect of YM-155 and MK-2206 treatment on Survivin expressionin GBM cells (I cell line) irradiated or not and kept in SCM medium for 1additional week. Efficiency of MK-2206 toward AKT was also checked as a control bythe blotting of phospho-AKT1 (pAKT1). (b and c) At the end of thededifferentiation protocol, the effects of YM-155 and MK-2206 were measured on theNS generation potential in response to IR by NS counting in phase-contrastmicroscopy (original magnification: × 4, scale bar:17 μm) (b) and subsequent quantification in theindicated cell lines (c). Results are expressed as the means±S.E.M.of three independent experiments. ∗∗P<0.01,∗∗∗P<0.001 compared with the 3-GySCM condition. (d) The involvement of Survivin in the IR-induced GBMreprogramming was checked by western blotting by analyzing the expression of thestem markers Nestin, Sox2 and Olig2 at the end of the dedifferentiation protocolin the presence or absence of YM-155 and MK-2206. Concerning western blottings,equal gel loading and transfer efficiency were checked with an anti-actin, AKT1 orβ2-microglobulin (β2M) antibody, and results wererepresentative of at least three independent experiments on the indicated cellline and reproduced in all the cell lines
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4260760&req=5

fig8: Requirement of the IR-induced survivin overexpression for the dedifferentiationprocess in GBM cells. (a–d) Differentiated GBM cells werepre-treated for 24 h with either a survivin inhibitor (YM-155 7 nM)or an AKT inhibitor (MK-2206 250 nM) and then irradiated or not at3 Gy before being placed 2 days after in either FCS or SCM medium,complemented or not with fresh YM-155 or MK-2206 inhibitors. (a) Westernblot analysis of the effect of YM-155 and MK-2206 treatment on Survivin expressionin GBM cells (I cell line) irradiated or not and kept in SCM medium for 1additional week. Efficiency of MK-2206 toward AKT was also checked as a control bythe blotting of phospho-AKT1 (pAKT1). (b and c) At the end of thededifferentiation protocol, the effects of YM-155 and MK-2206 were measured on theNS generation potential in response to IR by NS counting in phase-contrastmicroscopy (original magnification: × 4, scale bar:17 μm) (b) and subsequent quantification in theindicated cell lines (c). Results are expressed as the means±S.E.M.of three independent experiments. ∗∗P<0.01,∗∗∗P<0.001 compared with the 3-GySCM condition. (d) The involvement of Survivin in the IR-induced GBMreprogramming was checked by western blotting by analyzing the expression of thestem markers Nestin, Sox2 and Olig2 at the end of the dedifferentiation protocolin the presence or absence of YM-155 and MK-2206. Concerning western blottings,equal gel loading and transfer efficiency were checked with an anti-actin, AKT1 orβ2-microglobulin (β2M) antibody, and results wererepresentative of at least three independent experiments on the indicated cellline and reproduced in all the cell lines
Mentions: We then investigated whether this survivin overexpression during the IR-induceddedifferentiation was a consequence of this reprogramming or could be an essentialstep supporting IR-induced GBM plasticity to a stem-like phenotype. To this end,we treated GBM cells during the dedifferentiation process with 7 nM YM-155,a selective inhibitor of survivin used in anti-cancer clinicaltrials.50 As an additionalcontrol, we used MK-2206 (250 nM), a selective AKT inhibitor,51 as we and others52 showed that AKT can control the expression of survivinin GBM cells (Figure 8a). We first checked theefficiency of these inhibitors used at non-toxic concentrations (data not shown)on survivin expression by western blotting during the IR-promoteddedifferentiation (Figure 8a). Next, we observed theireffect at the end of the dedifferentiation protocol and showed that bothinhibitors induced a potent blockade of the 3-Gy-induced NS generation in SCMmedium (Figures 8b and c). Moreover, YM-155 andMK-2206 markedly inhibited at the protein level the overexpression of the stemmarkers Olig2, Sox2 and Nestin in response to IR in SCM medium (Figure 8d). Altogether, these results strongly suggest that theIR-induced reprogramming in GBM cells was associated and supported by theupregulation of the anti-apoptotic protein survivin.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: INSERM UMR 1037, Centre de Recherches en Cancérologie de Toulouse (CRCT), Université Toulouse III Paul Sabatier, Toulouse, France.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus