Chemotherapy-mediated p53-dependent DNA damage response in clear cell renal cell carcinoma: role of the mTORC1/2 and hypoxia-inducible factor pathways

Abstract

The DNA-damaging agent camptothecin (CPT) and its analogs demonstrate clinical utility for the treatment of advanced solid tumors, and CPT-based nanopharmaceuticals are currently in clinical trials for advanced kidney cancer; however, little is known regarding the effects of CPT on hypoxia-inducible factor-2α (HIF-2α) accumulation and activity in clear cell renal cell carcinoma (ccRCC). Here we assessed the effects of CPT on the HIF/p53 pathway. CPT demonstrated striking inhibition of both HIF-1α and HIF-2α accumulation in von Hippel-Lindau (VHL)-defective ccRCC cells, but surprisingly failed to inhibit protein levels of HIF-2α-dependent target genes (VEGF, PAI-1, ET-1, cyclin D1). Instead, CPT induced DNA damage-dependent apoptosis that was augmented in the presence of pVHL. Further analysis revealed CPT regulated endothelin-1 (ET-1) in a p53-dependent manner: CPT increased ET-1 mRNA abundance in VHL-defective ccRCC cell lines that was significantly augmented in their VHL-expressing counterparts that displayed increased phosphorylation and accumulation of p53; p53 siRNA suppressed CPT-induced increase in ET-1 mRNA, as did an inhibitor of ataxia telangiectasia mutated (ATM) signaling, suggesting a role for ATM-dependent phosphorylation of p53 in the induction of ET-1. Finally, we demonstrate that p53 phosphorylation and accumulation is partially dependent on mTOR activity in ccRCC. Consistent with this result, pharmacological inhibition of mTORC1/2 kinase inhibited CPT-mediated ET-1 upregulation, and p53-dependent responses in ccRCC. Collectively, these data provide mechanistic insight into the action of CPT in ccRCC, identify ET-1 as a p53-regulated gene and demonstrate a requirement of mTOR for p53-mediated responses in this tumor type.

Figure 1

Effect of CPT and apigenin on HIF-1α, HIF-2α and HIF-α target genes in RCC4 and 786-O cells. (a and b) 786-O or RCC4 cells were treated with CPT or apigenin at the concentrations indicated or vehicle control (DMSO). Panels, whole-cell lysates were assayed by western blot for HIF-1α, HIF-2α and cyclin D1 proteins. Actin and/or tubulin were used as loading controls. Graphs, conditioned media were harvested after 24 h and secreted protein levels of VEGF were determined by ELISA and normalized to cell number. (c) RCC4 cells were treated with 2 μM CPT or DMSO vehicle control for the times indicated. Conditioned media were harvested and secreted protein levels of VEGF, PAI-1 and ET-1 were determined by ELISA and normalized to cell number. Mean±S.E. of duplicate values of one representative experiment is shown. *P<0.05, **P<0.01, t-test compared with control

Figure 2

CPT inhibits HIF-1α and HIF-2α protein synthesis. (a) RCC4/VHL cells were incubated with 500 μM desferrioxamine (DFX) in the absence or presence of 2 μM CPT for 24 h as indicated. Whole-cell lysates were assayed by western blot for HIF-2α. Actin was used as a loading control. (b) The 786-O cells were incubated with 2 μM CPT or vehicle control (DMSO) for 24 h and analyzed for mRNA expression of HIF-2α by real-time quantitative PCR relative to GAPDH. (c and d) RCC4 cells were incubated with or without 2 μM CPT for 6 h in the absence or presence of 10 μM MG-132 or 10 μM cycloheximide (CHX) for the final 3 h as indicated. Whole-cell lysates were assayed by western blot for HIF-1α and HIF-2α. Actin was used as a loading control

Figure 3

CPT-mediated increase in p53 levels and apoptosis is augmented in VHL-competent cells. (a) The 786-O and RCC4 cells and their VHL-expressing counterparts were treated with increasing concentrations of CPT for 24 h. Whole-cell lysates were assayed by western blot for p53 and phosphorylated-S15-p53 proteins and cleaved PARP. Actin was used as a loading control. (b) The 786-O and 786-O/VHL cells were treated with 100 nM CPT or vehicle for 24 h, fixed, stained with propidium iodide and the percentage (%) of cells in subG1 was assessed by flow cytometry. Mean±S.E. of three replicates is shown

Figure 4

DNA-damaging agents increase ET-1 mRNA abundance in a p53-dependent manner. (a) The 786-O and 786-O/VHL cells were treated with increasing concentrations of CPT for 24 h and analyzed for mRNA expression of ET-1 by real-time quantitative PCR relative to GAPDH. (b) Cells as indicated were treated with 2 μM CPT (+) or vehicle DMSO (−) for 24 h and analyzed for mRNA expression of ET-1 by real-time quantitative PCR relative to GAPDH. (c) The 786-O/VHL cells were treated with vehicle DMSO, 2 μM CPT, 100 μM etoposide (ETO) or 10 μM nutlin-3a (N3) and analyzed for mRNA expression of ET-1 by real-time quantitative PCR relative to GAPDH. (d) The 786-O and RCC4 cells and their VHL-expressing counterparts were treated with vehicle control, 10 μM or 50 μM nutlin-3a (N3) for 2 or 24 h. Whole-cell lysates were assayed by western blot for phosphorylated-S15-p53, p53 and cleaved PARP proteins. Actin was used as a loading control. In the graphs, mean±S.E. of duplicate values of one representative experiment is shown. * P<0.05 as compared with control

Figure 5

The p53-dependent regulation of ET-1 in RCC4 cells. (a) RCC4 cells were transfected with 10 nM siRNA to p53 or nonsilencing control (NSC) duplex for 24 h before addition of 2 μM CPT (+) or DMSO vehicle control (−) for a further 24 h. Panels, whole-cell lysates were assayed by western blot for p53 protein. Actin was used as a loading control. Graph, mRNA expression of ET-1 by real-time quantitative PCR relative to GAPDH. Mean±S.E. of duplicate values of one representative experiment is shown. (b) RCC4 cells were transfected with 10 nM siRNA to p53, HIF-1α (H1), or HIF-2α (H2) or NSC duplex for 24 h. Panels, whole-cell lysates were assayed by western blot for p53, HIF-1α and HIF-2α proteins. Tubulin was used as a loading control. Graph, conditioned media were harvested and secreted protein levels of ET-1 were determined by ELISA and normalized to cell number. Mean±S.E. of duplicate values of one representative experiment is shown. *P<0.05, t test compared with control

Figure 6

DNA damage response is suppressed in HIF-α-expressing cells. (a) RCC4 and RCC4/VHL cells were treated with 2 μM CPT, 100 μM etoposide (ETO) or 400 nM of the mTORC1/2 kinase inhibitor pp242 for 24 h. Whole-cell lysates were assayed by western blot for phosphorylated p53 (S15), CHK1 (S317, S345), γH2AX (S139), total p53 and CHK1 proteins. Actin was used as a loading control. (b) The 786-O and 786-O/VHL cells were transfected with siRNA to ATR or nonsilencing control (NSC) for 24 h before addition of 2 μM CPT for a further 24 h. mRNA expression of ET-1 was assessed by real-time quantitative PCR relative to GAPDH. Mean±S.E. of duplicate values of one representative experiment is shown. (c) RCC4/VHL cells were treated with 10 μM ATM inhibitor (ATMi), 400 nM pp242 or vehicle control (DMSO) in the absence (−) or presence (+) of 2 μM CPT for 24 h. Whole-cell lysates were assayed by western blot for phosphorylated p53 (S15), and p53 proteins. Short and long exposure times are shown for P-S15-p53. Actin was used as a loading control. (d) RCC4/VHL and 786-O/VHL cells were preincubated with 10 μM ATMi for 1 h before addition of 2 μM CPT for a further 24 h and mRNA expression of ET-1 was assessed by real-time quantitative PCR relative to GAPDH. Mean±S.E. of duplicate values of one representative experiment is shown

Figure 7

Attenuation of mTORC1/2 kinase ablates CPT-induced p53- and p53-dependent responses in ccRCC. (a) The 786-O and 786-O/VHL cells were treated with 400 nM pp242, 2 μM CPT, 10 μM nutlin-3a (N3) or vehicle control (DMSO) alone for 24 h, or preincubated with 400 nM pp242 for 1 h before addition of 2 μM CPT or 10 μM N3 for a further 24 h. Whole-cell lysates were assayed by western blot for HIF-2α, mTOR, phosphorylated p53 (S15), mTOR (S2448) and p70S6K (T389) proteins. Actin was used as a loading control. Short and long exposure times are shown for P-S15-p53. (b) RCC4 and RCC4/VHL cells were treated with 400 nM pp242 or vehicle control (DMSO) alone or preincubated with 400 nM pp242 for 1 h before addition of either 1 μM or 2 μM CPT for 24 h. Whole-cell lysates were assayed by western blot for mTOR, p53, phosphorylated p53 (S15), mTOR (S2448) and p70S6K (T389) proteins. Actin was used as a loading control. (c) RCC4 and RCC4/VHL cells were preincubated with 400 nM pp242 for 1 h before addition of 2 μM CPT for 24 h and mRNA expression of ET-1 and PAI-1 were assessed by real-time quantitative PCR relative to GAPDH. Mean±S.E. of duplicate values of one representative experiment is shown. (d) RCC4 cells were pretreated with 400 nM pp242 for 1 h before addition of 2 μM CPT for 24 h. Conditioned media were harvested and assessed for secreted ET-1 protein levels by ELISA and normalized to total protein levels. Mean±S.E. of duplicate values of one representative experiment is shown

Figure 8

mTORC1/2 kinase inhibition reverses CPT-induced cell death in ccRCC. (a) RCC4/VHL and 786-O/VHL cells were incubated with (open bars) or without (solid bars) 400 nM pp242 for 1 h before addition of increasing concentrations of CPT as indicated for 24 or 48 h. Cell viability was determined by reduction of MTT at A 595 nm. Mean±S.E. of triplicate values is shown. (b) RCC4/VHL and 786-O/VHL cells were incubated with 400 nM pp242 or 500 μM DFX for 1 h before addition of 2 μM CPT as indicated for 48 h. Whole-cell lysates were assayed for cleaved caspases 3, 7 and 9 and cleaved PARP. Actin was used as a loading control