Temozolomide suppresses MYC via activation of TAp63 to inhibit progression of human glioblastoma

Abstract

Glioblastoma multiforme (GBM) is a highly invasive and chemoradioresistant brain malignancy. Temozolomide (TMZ), a DNA-alkylating agent, is effective against GBM and has become the standard first-line drug. However, the mechanism by which TMZ regulates the progression of GBM remains elusive. Here, we demonstrate that TMZ targets TAp63, a p53 family member, inducing its expression to suppress the progression of human GBM. High levels of TAp63 expression in GBM tissues after TMZ treatment was an indicator of favourable prognosis. In human GBM cells, TMZ-induced TAp63 directly repressed MYC transcription. Activation of this TAp63-MYC pathway by TMZ inhibited human GBM progression both in vitro and in vivo. Furthermore, downregulation of MYC mRNA levels in recurrent GBMs after TMZ treatment correlated with better patient survival. Therefore, our results suggest that the TAp63-mediated transcriptional repression of MYC is a novel pathway regulating TMZ efficacy in GBM.

Figure 1. High TAp63 expression correlates with favourable prognoses in human GBM.

(a) TAp63 mRNA expression detected by quantitative RT-PCR (qRT-PCR) in 69 newly diagnosed malignant glioma samples (59 GBM; 7 anaplastic astrocytoma; 3 anaplastic oligoastrocytoma). TAp63 expression was normalised to ACTB mRNA, and designated high (n = 37) or low (n = 32) based on the normal human brain expression (dashed red line). (b) Overall survival of subjects with newly diagnosed malignant gliomas according to relative TAp63 expression levels before chemotherapy (n = 69; high, n = 37; low, n = 32). P value by log-rank test. (c) p63 immunohistochemical staining in human GBM. Scale bar, 50 μm. (d) Recurrence-free survival of 20 TMZ-treated GBM subjects according to p63 immunohistochemical staining in newly diagnosed samples. P values by log-rank test.

Figure 2. TMZ-induced TAp63 suppresses MYC expression in human GBM cells.

(a) TAp63 and MYC mRNA levels in GBM cells with increasing TMZ concentrations, 24 h. (b) Immunoblot of TAp63 and MYC from GBM cells treated with increasing concentrations of TMZ, 24 h. (c) RT-PCR analyses of relative MYC expression in U87MG and YH-13 cells following siTAp63 transfection, normalised to ACTB mRNA. (d) Analysis of MYC expression in YH-13 cells transfected with siControl or siTAp63 by western blotting. (e) RT-PCR analyses showing MYC suppression after TAp63α overexpression in GBM cell lines. (f) Positions of PCR primer sets R1, R2, R3 and R4 for the chromatin immunoprecipitation (ChIP) assays. (g) Identification of the TAp63-binding region in the MYC promoter by ChIP assays. YH-13 cells were transfected with or without indicated siRNAs. Genomic DNA was amplified by PCR using the indicated primers. (h) Semi-quantitative PCR and (i) quantitative PCR of ChIP assays showing endogenous TAp63 recruitment onto the MYC promoter after 24 h TMZ treatment, 150 μM. *P < 0.005 (two-tailed t-test). (j) Luciferase activity of MYC reporters after lentiviral TAp63α or GFP infection of U87MS cells. Data shown as the fold change in the luciferase activity compared with control cells.

Figure 3. TAp63-MYC pathway is critical for glioblastoma sphere-forming ability and invasion in vitro.

(a) Sphere formation assay of YH-13 cells, showing induction of sphere-forming activity after knockdown of TAp63 and MYC. (b) The graph indicates the differences in the sphere numbers per microscopic field at 400× magnification. The values represent the mean ± SD of triplicate samples from a single representative experiment (n = 9). **P < 0.0005, ***P < 0.00005 (two-tailed t-test). (c) Cell viability assay of TAp63 and MYC knockdown in YH-13 cells. (e) The graph indicates the percentage of YH-13 cells invading the Matrigel relative to control migration, following TAp63 and MYC knockdown. *P < 0.05, **P < 0.0005 (two-tailed t-test).

Figure 4. TMZ inhibits GBM progression via the TAp63-MYC pathway.

(a) Cell viability assay showing suppression of proliferation in cells treated with TMZ, 75 μM. (b) Cellular invasion was suppressed by day 5, at which point the treated and control cultures were adjusted to 5 × 10⁴ cells/500 μl and subjected to Boyden chamber invasion assays. *P < 0.05 (two-tailed t-test). (c) Percentage of U87MG cells invading the Matrigel relative to control migration, following TAp63 knockdown and TMZ treatment. Corresponding mRNA analysis. *P < 0.05 (two-tailed t-test). (d) Effect of TAp63 knockdown on the U87MG cells treated with TMZ treatment at 14 days after subcutaneous transplantation in nude mice. The end volumes were compared to the volumes at implantation. Tumour growth was measured with callipers and calculated by the formula: volume = length (A) × width (B) × width (B) × 0.5, where A and B are the long and short axes respectively. TMZ in PBS was administered intraperitoneally at 15 mg/kg once a week. Data are representative of five independent experiments (n = 4). *P < 0.05 by two-tailed t-test. Photographs in (d) are representative of n = 4 mice. Bar, 10 mm. (e) MYC downregulation in recurrent tumours after TMZ plus radiotherapy (n = 20; solid line: mean difference; dashed lines: 95% confidence interval. P value by paired t-test. (f) Overall survival according to MYC downregulation (≥1.5-fold decrease in primary/recurrent (p/r) MYC mRNA), post-TMZ treatment. MYC decrease: n = 11, mean p/r = 2.65 ± 0.31 s.d.; no change: n = 9, mean p/r = 0.9 ± 0.13 s.d. P value by log-rank test.