Serine/threonine protein phosphatase 6 modulates the radiation sensitivity of glioblastoma

Abstract

Increasing the sensitivity of glioblastoma cells to radiation is a promising approach to improve survival in patients with glioblastoma multiforme (GBM). This study aims to determine if serine/threonine phosphatase (protein phosphatase 6 (PP6)) is a molecular target for GBM radiosensitization treatment. The GBM orthotopic xenograft mice model was used in this study. Our data demonstrated that the protein level of PP6 catalytic subunit (PP6c) was upregulated in the GBM tissue from about 50% patients compared with the surrounding tissue or control tissue. Both the in vitro survival fraction of GBM cells and the patient survival time were highly correlated or inversely correlated with PP6c expression (R(2)=0.755 and -0.707, respectively). We also found that siRNA knockdown of PP6c reduced DNA-dependent protein kinase (DNA-PK) activity in three different GBM cell lines, increasing their sensitivity to radiation. In the orthotopic mice model, the overexpression of PP6c in GBM U87 cells attenuated the effect of radiation treatment, and reduced the survival time of mice compared with the control mice, while the PP6c knocking-down improved the effect of radiation treatment, and increased the survival time of mice. These findings demonstrate that PP6 regulates the sensitivity of GBM cells to radiation, and suggest small molecules disrupting or inhibiting PP6 association with DNA-PK is a potential radiosensitizer for GBM.

Figure 1

The expression of PP6 catalytic subunit (PP6c) is increased in glioblastoma tissues from patients and its protein levels inversely correlate with patient survival time. (a) The 380 sections from 38 glioblastoma multiforme (GBM) patients (10 slides each patient) were analyzed. The figure rows show representative pictures of non-tumor epilepsy brain section (the 1st row), para-tumor tissue (the 2nd row) and GBM sections (3rd–6th rows). The left panels show PP6c staining (green), the middle panels show 4′,6-diamidino-2-phenylindole (DAPI) staining (blue) and the right panels show merged images. The quantification analysis of PP6c fluorescence density (FD) was carried out. The patient samples were classified into four groups ranging from low to high expression (+, FD <20% ++, 20% ≤FD<40% +++, 40% ≤FD<60% ++++, FD ≥60%); the scale bar indicates 2 nm. (b) The expression microarray data of GBM tissue downloaded from the public database of the Cancer Genomic Center (TCGA, NIH) was statistically analyzed by Student's t-test. The bar represents log₂ copy number units of normal brain tissue or GBM tissue (median±S.D.). (c) The dose–effect relationship between PP6c protein levels and the survival time (months) of GBM patients was analyzed by the Mann–Whitney U-test. The coefficient of determination (R²) in the linear regression model is indicated

Figure 2

PP6 catalytic subunit (PP6c) protein levels correlate with radiation resistance of glioblastoma cells. (a) The dose–response relationship between PP6 protein levels and the radiosensitivity of glioblastoma cells was analyzed by the Mann–Whitney U-test. The coefficient of determination (R²) in the linear regression model is indicated. (b) T98G, A172 and U251 cells were left untreated or irradiated with 5 Gy. At 1 h after irradiation, cells were lysed and subjected to immunoblot analysis. The protein levels of DNA-PK catalytic subunit (DNA-PKcs) and PP6c in these cells were detected. (c) The protein levels of PP6c were quantified by densitometry analysis using the Image J software and the reading was normalized to internal controls of Ku86 and β-tubulin. The error bars represent the S.D. of the means of three independent experiments. The statistical significance of the differences of the PP6c amount between the different cell lines was determined by Student's t-test (^*P<0.001; ^#P<0.05). (d) The representative images from clonogenic assay. (e) In all, 100 T98G, A172 or U251 cells treated with the indicated doses of radiation were seeded in a 100 mm dish. Colony numbers were normalized to the untreated controls of each cell line. The error bars represent the S.D. of the means of three independent experiments

Figure 3

Ionizing radiation (IR)-induced DNA-dependent protein kinase (DNA-PK) activity is correlated with radiation resistance in glioblastoma cells. (a) T98G, A172 and U251 cells were treated with Nu7026 (10 μM) 30 min before irradiation (5 Gy) or without radiation. At 1 h post-irradiation, the nuclear fractions from these cells were isolated, and then subjected to a DNA-PK kinase assay using a specific peptide as substrate (^*P<0.001; ^#,**P<0.05). (b) The dose–response relationship between DNA-PK activity and the survival fraction of glioblastoma cells was analyzed by the Mann–Whitney U-test. The coefficient of determination (R²) in the linear regression model is indicated. (c) T98G, A172 and U251 cells were treated with Nu7026 (10 μM) for 30 min before camptothecin (CPT) treatment. At 3 h post-irradiation, whole-cell lysates were subjected to immunoblotting for total or phospho-specific RPA32 detection using RPA32 antibody (top row). β-Actin was used as a loading control. (d) Phosphorylated and unphosphorylated RPA32 were quantified by the Image J software, and the readings were normalized to the internal control of β-tubulin. The error bars represent the S.D. of the means of three independent experiments. The statistical significance of the differences between the ratio of phosphorylated to unphosphorylated RPA32 in each cell line was determined by Student's t-test (^*P<0.001; ^#,**P<0.05)

Figure 4

Small interfering RNA (siRNA) knockdown of PP6 catalytic subunit (PP6c) reduces DNA-dependent protein kinase (DNA-PK) kinase activity in three different glioblastoma cell line and increase the sensitivity of glioblastoma cells to radiation. (a) T98G, A172 and U251 cells were transfected with PP6c-specific siRNA or non-specific siRNA. At 48 h after transfection, the cells were treated with 5 Gy radiation or sham radiation, and subjected to the DNA-PK kinase assay using a specific peptide as substrate.^, The data were normalized to the number of non-irradiated and non-PP6c siRNA-transfected T98G cells. The error bars represent the S.D. of the means of three independent experiments. The statistical significance of the differences between the DNA-PK activity in the irradiated PP6c siRNA-treated or control siRNA-treated cells was determined by Student's t-test (^{*, #, **}P<0.001). The western blotting showed the siRNA knocking-down efficiency of PP6c in T98G, A172 and U251 cells. (b) The T98G cells infected by the lentivirus containing PP6c siRNAs or control siRNA were treated with camptothecin (CPT)- and/or DNA-PK-specific inhibitor Nu7026, the Ser 4/8 phosphorylation of RPA32 were detected by western blot and the bottom panel shows PP6c knocking-down efficiency in stable T98G cells. (c) The U251 cells infected by the lentivirus containing PP6c coding sequence or control siRNA were treated with CPT- and/or DNA-PK-specific inhibitor Nu7026, the Ser 4/8 phosphorylation of RPA32 were detected by western blot and the bottom panel shows PP6c protein level in stable U251 cells. (d) T98G, A172 and U251 cells were transfected with PP6c-specific siRNA or nonspecific siRNA and then treated with the indicated dose of radiation. All colony numbers were normalized to the untreated control of each cell line. The error bars represent the S.D. of the means of three independent experiments

Figure 5

Small interfering (siRNA) knockdown of PP6 catalytic subunit (PP6c) improves the radiation therapeutic efficacy on the mice bearing orthotopic xenografted glioblastoma. (a) The mice were euthanized at the time of reaching a moribund condition, and all survival times in each group were analyzed using Kaplan–Meier method. This experiment was independently repeated two times, six mice in each group at the start time point. The analyzed mice with radiation treatment in empty virus group (control), PP6c knocking-down or PP6c overexpression group were 10, 11, 12, respectively, while the mice without radiation treatment in the control group, PP6c knocking-down or PP6c overexpression group were 11, 11, 12, respectively. The radiation enhancements of survival time in each group were calculated by the formula: the survival time with radiation divided by the survival time without radiation. The significance of radiation enhancement difference between control siRNA group and PP6c siRNA group were analyzed by the χ² method (^*P<0.001). (b) The representative tumor pictures from the PP6c overexpression, the PP6c knocking-down or the control group were captured by magnetic resonance imaging (MRI) imaging at the 9th and 21st day with (+IR (ionizing radiation)) or without radiation treatment (−IR), and the tumor volumes were calculated by the formula: ½ × L × S² (L: maximum length; S: minimum length). (c) The representative PP6c immunofluorescence staining (red) in glioblastoma sections from the PP6c overexpression, the PP6c knocking-down or the control group