CENPE Inhibition Leads to Mitotic Catastrophe and DNA Damage in Medulloblastoma Cells

Abstract

Medulloblastoma (MB) is the most frequent brain tumor in children. The standard treatment consists in surgery, followed by radiotherapy and chemotherapy. These therapies are only partially effective since many patients still die and those who survive suffer from neurological and endocrine disorders. Therefore, more effective therapies are needed. Primary microcephaly (MCPH) is a rare disorder caused by mutations in 25 different genes. Centromere-associated protein E (CENPE) heterozygous mutations cause the MCPH13 syndrome. As for other MCPH genes, CENPE is required for normal proliferation and survival of neural progenitors. Since there is evidence that MB shares many molecular features with neural progenitors, we hypothesized that CENPE could be an effective target for MB treatment. In ONS-76 and DAOY cells, CENPE knockdown induced mitotic defects and apoptosis. Moreover, CENPE depletion induced endogenous DNA damage accumulation, activating TP53 or TP73 as well as cell death signaling pathways. To consolidate CENPE as a target for MB treatment, we tested GSK923295, an allosteric inhibitor already in clinical trial for other cancer types. GSK923295, induced effects similar to CENPE depletion with higher penetrance, at low nM levels, suggesting that CENPE's inhibition could be a therapeutic strategy for MB treatment.

Keywords: 53BP1; CENPE; DNA damage; childhood brain tumor; microcephaly; mitotic catastrophe; γH2AX.

Figure 1

The expression of CENPE correlates with MB patients’ prognosis. (A) Kaplan-Meier survival curves of MB patients. The two groups were defined on the basis of CENPE expression high (red) or low (blue) based on median value. p-value = 0.0318, Log-rank test statistics (B) Graph showing expression level by RSEM count of CENPE divided by molecular subgroups (C) Kaplan-Meier survival curves of MB patients divide by molecular subgroups and finally subdivided in two groups on the basis of CENPE expression high (red) or low (blue) based on median value. WNT p-value = 0.1385 SHH p-value = 0.0091 Group3 p-value = 0.2815 Group4 p-value = 0.1363, Log-rank test statistics. *, p < 0.05; **, p < 0.01; n.s., not significant.

Figure 1

The expression of CENPE correlates with MB patients’ prognosis. (A) Kaplan-Meier survival curves of MB patients. The two groups were defined on the basis of CENPE expression high (red) or low (blue) based on median value. p-value = 0.0318, Log-rank test statistics (B) Graph showing expression level by RSEM count of CENPE divided by molecular subgroups (C) Kaplan-Meier survival curves of MB patients divide by molecular subgroups and finally subdivided in two groups on the basis of CENPE expression high (red) or low (blue) based on median value. WNT p-value = 0.1385 SHH p-value = 0.0091 Group3 p-value = 0.2815 Group4 p-value = 0.1363, Log-rank test statistics. *, p < 0.05; **, p < 0.01; n.s., not significant.

Figure 2

Medulloblastoma cells are sensitive to CENPE knockdown. (A) Western blot analysis of total lysate from DAOY and ONS-76 cell lines, 48 h after treatment with non-targeting (siCtrl) or CENPE-specific (siCENPE) siRNA. The level of CENPE was analyzed and the internal loading control was tubulin (TUB). The original images are shown in the Figures S1 and S2. (B) Representative image of DAOY cells processed for immunofluorescence 48 h after transfection with siCtrl or siCENPE and stained with anti-CENPE antibody, anti-Tubulin antibody and DAPI. (C) Representative images of live imaging performed on DAOY 30 h after transfection. Time lapses were recorded overnight with an interval of 5 min. Magnification: 40× (D) Quantification of the time spent in metaphase by DAOY and ONS-76 cells analyzed as described in panel C. (E) Quantification of the percentage of cells showing metaphases lasting more than 30 min, in cells analyzed as described in panel C. Statistical test = Chi², considering absolute number of cells in experiments. (F,G) DAOY and ONS-76 proliferation assay: 50,000 cells were transfected with non-targeting (siCtrl) or CENPE-specific (siCENPE) siRNA. Growth curves were obtained by assessing cells’ number in each well at 48, 72 and 96 h after transfection. Statistical test = two-tailed Student t-test. All quantifications were based on at least 3 independent biological replicates. At least 60 cell divisions were recorded for any biological replicate. Error bars, SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Scale bars, 5 µm.

Figure 2

Medulloblastoma cells are sensitive to CENPE knockdown. (A) Western blot analysis of total lysate from DAOY and ONS-76 cell lines, 48 h after treatment with non-targeting (siCtrl) or CENPE-specific (siCENPE) siRNA. The level of CENPE was analyzed and the internal loading control was tubulin (TUB). The original images are shown in the Figures S1 and S2. (B) Representative image of DAOY cells processed for immunofluorescence 48 h after transfection with siCtrl or siCENPE and stained with anti-CENPE antibody, anti-Tubulin antibody and DAPI. (C) Representative images of live imaging performed on DAOY 30 h after transfection. Time lapses were recorded overnight with an interval of 5 min. Magnification: 40× (D) Quantification of the time spent in metaphase by DAOY and ONS-76 cells analyzed as described in panel C. (E) Quantification of the percentage of cells showing metaphases lasting more than 30 min, in cells analyzed as described in panel C. Statistical test = Chi², considering absolute number of cells in experiments. (F,G) DAOY and ONS-76 proliferation assay: 50,000 cells were transfected with non-targeting (siCtrl) or CENPE-specific (siCENPE) siRNA. Growth curves were obtained by assessing cells’ number in each well at 48, 72 and 96 h after transfection. Statistical test = two-tailed Student t-test. All quantifications were based on at least 3 independent biological replicates. At least 60 cell divisions were recorded for any biological replicate. Error bars, SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Scale bars, 5 µm.

Figure 3

CENPE knockdown induces apoptosis and DNA damage in MB cells. (A) DAOY and ONS-76 cell death, measured by CellTox™ Green Cytotoxicity assay, 48 h after treatment with siCtrl or siCENPE. Values are plotted as Relative Light Unit (RLU). (B) Western blot analysis of total lysate from DAOY and ONS-75 cell lines, 48 h after treatment with siCtrl or siCENPE. The levels of CENPE, cleaved Caspase 3 (cCASP3) and γH2AX were analyzed and the internal loading control was vinculin (VINC). The original images are shown in the Figures S1 and S2. (C,D) Quantification of the relative density of cCASP3 (C) and γH2AX in DAOY and ONS-76 treated cells. (E) Representative image of DAOY cells processed for immunofluorescence 48 h after transfection with siCtrl or siCENPE and stained with anti-53BP1 antibody and DAPI. (F) Quantification of 53BP1 foci per nucleus in cells treated as in (E). All quantifications were based on at least three independent biological replicates. 53BP1 foci were counted in >300 cells per condition. Error bars, SEM. *, p < 0.05, **, p < 0.01; two-tailed Student t-test for blots and CellTox™ assay. ***, p < 0.001 Mann–Withney U test for 53BP1 foci. Scale bars, 5 µm. A.U., arbitrary units.

Figure 4

CENPE inhibition reduces cell proliferation and induces cell death. (A,B) Dose response curve of DAOY and ONS-76 cells, respectively, 24, 48 and 72 h after treatment with GSK923295. (C,D) DAOY and ONS-76 cell death, measured by CellTox™ Green Cytotoxicity assay, 24 h after treatment with DMSO or GSK923295. Values are plotted as Relative Light Unit (RLU). (E,F) DAOY and ONS-76 proliferation assay: 50,000 cells were treated with DMSO or 25 nM GSK923295. Growth curves were obtained by assessing cells’ number in each well 24, 48 and 72 h after treatment. All quantifications were based on at least three independent biological replicates. Error bars, SEM; *, p < 0.05; ***, p < 0.001; two-tailed Student t-test.

Figure 4

CENPE inhibition reduces cell proliferation and induces cell death. (A,B) Dose response curve of DAOY and ONS-76 cells, respectively, 24, 48 and 72 h after treatment with GSK923295. (C,D) DAOY and ONS-76 cell death, measured by CellTox™ Green Cytotoxicity assay, 24 h after treatment with DMSO or GSK923295. Values are plotted as Relative Light Unit (RLU). (E,F) DAOY and ONS-76 proliferation assay: 50,000 cells were treated with DMSO or 25 nM GSK923295. Growth curves were obtained by assessing cells’ number in each well 24, 48 and 72 h after treatment. All quantifications were based on at least three independent biological replicates. Error bars, SEM; *, p < 0.05; ***, p < 0.001; two-tailed Student t-test.

Figure 5

CENPE inhibition abolished the in vitro clonogenic potential of SHH MB cells. (A,B) Low magnification images of plates of DAOY and ONS-76 cells, treated with DMSO or GSK923295 25 nM for 7 days, stained with crystal violet. Scale bars 25 mm (C,D) Example of DAOY and ONS-76 treated with DMSO or GSK923295 25 nM for 7 days in phase contrast. Scale bar 20 μm.

Figure 6

CENPE inhibition alters mitotic spindle assembly and induces mitotic catastrophe. (A) Representative images of DAOY cells, processed for immunofluorescence 24 h after treatment with DMSO or 25 nM GSK923295, immunostained with anti-CENPE and anti-Tubulin antibodies and counterstained with DAPI. (B) Representative frames of live-imaging experiments, performed on DAOY cells 24 h after treatment with DMSO or 25 nM GSK923295. Time lapses were recorded overnight with an interval of 5 min. (C) Quantification of the time spent in metaphase, in DAOY and ONS-76 cells analyzed as described in panel B. Statistical test = two-tailed Student t test. (D) Quantification of the percentage of metaphases lasting more than 30 min, in DAOY and ONS-76 cells analyzed as described in panel B. Statistical test = Chi², considering absolute number of cells in experiments (E) Quantification of the percentage of correct cell division (Division), cytokinesis failure (Failure) and mitotic catastrophe (Death) in DAOY and ONS-76 cells analyzed as described in panel B. Statistical test = Chi², considering absolute number of cells in experiments (F) Western blot analysis of total lysates from DAOY and ONS-75 cells, 24 h after treatment with DMSO or 25 nM GSK923295. The levels of CENPE, cleaved Caspase 3 (cCASP3) and γ H2AX were analyzed and the internal loading control was vinculin (VINC). The original images are shown in the Figures S1 and S2 (G,H) Quantification of the relative density of cCASP3 (G) and γH2AX (H) in DAOY and ONS-76 cells treated as described in panel A. Statistical test = two-tailed Student t-test. All quantifications were based on at least three independent biological replicates. At least 60 cell divisions were recorded for any biological replicate. Error bars, SEM; **, p < 0.01; ***, p < 0.001. Scale bars, 5 µm.

Figure 6

CENPE inhibition alters mitotic spindle assembly and induces mitotic catastrophe. (A) Representative images of DAOY cells, processed for immunofluorescence 24 h after treatment with DMSO or 25 nM GSK923295, immunostained with anti-CENPE and anti-Tubulin antibodies and counterstained with DAPI. (B) Representative frames of live-imaging experiments, performed on DAOY cells 24 h after treatment with DMSO or 25 nM GSK923295. Time lapses were recorded overnight with an interval of 5 min. (C) Quantification of the time spent in metaphase, in DAOY and ONS-76 cells analyzed as described in panel B. Statistical test = two-tailed Student t test. (D) Quantification of the percentage of metaphases lasting more than 30 min, in DAOY and ONS-76 cells analyzed as described in panel B. Statistical test = Chi², considering absolute number of cells in experiments (E) Quantification of the percentage of correct cell division (Division), cytokinesis failure (Failure) and mitotic catastrophe (Death) in DAOY and ONS-76 cells analyzed as described in panel B. Statistical test = Chi², considering absolute number of cells in experiments (F) Western blot analysis of total lysates from DAOY and ONS-75 cells, 24 h after treatment with DMSO or 25 nM GSK923295. The levels of CENPE, cleaved Caspase 3 (cCASP3) and γ H2AX were analyzed and the internal loading control was vinculin (VINC). The original images are shown in the Figures S1 and S2 (G,H) Quantification of the relative density of cCASP3 (G) and γH2AX (H) in DAOY and ONS-76 cells treated as described in panel A. Statistical test = two-tailed Student t-test. All quantifications were based on at least three independent biological replicates. At least 60 cell divisions were recorded for any biological replicate. Error bars, SEM; **, p < 0.01; ***, p < 0.001. Scale bars, 5 µm.

Figure 7

CENPE depletion or inhibition induces TP53 or TP73 pathway. (A,B) Western blot analysis and quantification of total lysate from ONS-76, 48 h after treatment with the indicated siRNAs. The levels of phospho-Ser15-TP53 (pTP53) and P21 were analyzed, the internal loading control was vinculin (VINC). (C,D) Western blot analysis and quantification, performed as in panel A, of total lysate from ONS-76, 24 h after treatment with 25 nM GSK923295. (E,F) Western blot analysis and quantification of total lysate from DAOY, 48 h after treatment with the indicated siRNAs. The levels of TP73 and P21 were analyzed, the internal loading control was vinculin (VINC). (G,H) Western blot analysis and quantification, performed as in panel E, of total lysate from DAOY, 24 h after treatment with 25 nM GSK923295. All quantifications were based on at least four independent biological replicates. Error bars, SEM; **, p < 0.01; ***, p < 0.001; two-tailed Student t-test. The original images are shown in the Figures S1 and S2.