mTOR kinase leads to PTEN-loss-induced cellular senescence by phosphorylating p53

Abstract

Loss of PTEN, the major negative regulator of the PI3K/AKT pathway induces a cellular senescence as a failsafe mechanism to defend against tumorigenesis, which is called PTEN-loss-induced cellular senescence (PICS). Although many studies have indicated that the mTOR pathway plays a critical role in cellular senescence, the exact functions of mTORC1 and mTORC2 in PICS are not well understood. In this study, we show that mTOR acts as a critical relay molecule downstream of PI3K/AKT and upstream of p53 in PICS. We found that PTEN depletion induces cellular senescence via p53-p21 signaling without triggering DNA damage response. mTOR kinase, a major component of mTORC1 and mTORC2, directly binds p53 and phosphorylates it at serine 15. mTORC1 and mTORC2 compete with MDM2 and increase the stability of p53 to induce cellular senescence via accumulation of the cell cycle inhibitor, p21. In embryonic fibroblasts of PTEN-knockout mice, PTEN deficiency also induces mTORC1 and mTORC2 to bind to p53 instead of MDM2, leading to cellular senescence. These results collectively demonstrate for the first time that mTOR plays a critical role in switching cells from proliferation signaling to senescence signaling via a direct link between the growth-promoting activity of AKT and the growth-suppressing activity of p53.

Fig. 1

No DNA damage response is involved in the activation of p53-p21 signaling in PICS. a–c MCF7 cells were transfected with Con Si or PTEN Si. After 4 days, cellular morphology and SA-β-Gal positivity were assessed, and the percentage of senescent cells was quantified (a). Western blot (WB) analyses were performed at the indicated times after siRNA transfection (b, c). d Immunocytochemical staining was conducted 1 day after PTEN Si transfection. e–g MCF7 cells were transfected with a vector encoding HA-tagged Myr-AKT and experiments were performed as described for panels 1a–c. h Analysis of intracellular ROS levels. H₂O₂-treated MCF7 cells were used as the positive control. i Cells were harvested at the indicated times after Si transfection and then subjected to WB analysis. Cells exposed to 6 Gy IR were used as the positive control (PC) (b, c). Error bars indicate the ±SD; n = 3; ***P < 0.001

Fig. 2

mTORC1 and mTORC2 are activated during PICS. a, b MCF7 cells were transfected with Con Si or PTEN Si for 6 h, and then treated with the indicated inhibitors. After 4 days, SA-β-Gal positivity was assessed (left panel) and the percentage of senescent cells was quantified (right graph) (a). WB analyses were performed 2 days later (b). Rapamycin (Rapa), LY294002 (LY), PP2, PD098059 (PD), SB203580 (SB), and SP600125 (SP) inhibit mTORC1, PI3K, Src, Erk, p38, and JNK, respectively. c WB analyses were performed at the indicated times after siRNA transfection. d MCF cells were transfected with either AKT Si or PTEN Si. After 2 days of transfection, WB analyses were performed. The values represent the mean ± SD; n = 3; *P < 0.05; ***P < 0.001

Fig. 3

mTORC1 and mTORC2 are critical for PICS, but S6K and 4EBP1 are not. a MCF7 cells were transfected with either SIN1 Si or PTEN Si. The cells were harvested at 2 days after transfection for immunoblotting. b MCF cells were transfected with Con Si or PTEN Si. After transfection of siRNA, MCF7 cells were incubated with or without Rapa or Torin1. WB analysis and quantification of SA-β-Gal activity were performed after 2 and 4 days, respectively. c, d All experiments were performed as described in Fig. 3a except using S6K1/2 Si (c) 4EBP Si (d). WB and qRT-PCR were performed 2 days, and SA-β-Gal activity assays were conducted 4 days after transfection. The values represent the mean ± SD; n = 3; ^#P > 0.05; **P < 0.01; ***P < 0.001

Fig. 4

mTORC1 and mTORC2 physically associate with p53 during PICS. a MCF7 cells were transfected with vectors encoding Flag-tagged wild-type (WT) mTOR or Flag-tagged kinase-dead (KD) mTOR. The next day, the cells were transfected with the indicated siRNAs. Two days later, WB analyses were conducted. b, d MCF7 cells were transfected with Con Si, PTEN Si (b, c), or Myr-AKT (d). After 2 days, the transfected cells were lysed, immunoprecipitated with α-p53, α-Raptor, and α-Rictor antibodies, and immunoblotted with the indicated antibodies. e In vitro competition assay. f MCF7 cells were transfected with Con Si or mTOR Si. After 6 h, half of each group was transfected with Con Si, and the other half was transfected with PTEN Si. After 2 days, transfected cells were lysed and immunoprecipitated with anti-p53 antibody

Fig. 5

mTORC1 and mTORC2 directly phosphorylate p53 at S15 in PICS. a MCF7 cells exogenously expressing flag-tagged WT, Δ40, and Δ133 p53 along with Myc-tagged mTOR were transfected with Con Si or PTEN Si. Two days after siRNA transfection, the cells were immunoprecipitated with anti-Flag antibody. b Cells were transfected with Con Si or PTEN Si, harvested at the indicated days, and subjected to immunoblotting. c In vitro kinase assay. MCF7 cells were transfected with Con Si or PTEN Si. After 2 days, cell lysates were immunoprecipitated with α-Raptor and α-Rictor antibodies and used for in vitro kinase assay. d MCF7 cells exogenously expressing Flag-tagged WT p53 or S15A mutant p53, in combination with Myc-tagged mTOR, were transfected with Con Si or PTEN Si. Two days after siRNA transfection, the cells were immunoprecipitated with anti-Flag antibody. SA-β-Gal activity assays were conducted 4 days after siRNA transfection. The values represent the mean ± SD; n = 3; ^#P > 0.05; ***P < 0.001

Fig. 6

The binding of mTORC1 and mTORC2 to p53 in PTEN-deficient mouse embryonic fibroblasts. a–e CrePTEN^fl/fl MEFs were cultured with or without 0.5 μmol/L 4-OHT for 3 days and subjected to immunoblotting (a). Relative cell number (upper graph) and Annexin V positivity (lower graph) were determined at 6 and 4 days, respectively, after 4-OHT treatment (b). Morphology and SA-β-Gal positivity were observed at 7 days after 4-OHT treatment (c). MEFs were preincubated with Rapa or Torin1 for 1 h, and then cultured with or without 0.5 μmol/L 4-OHT for 7 days. SA-β-Gal activity was determined (d). IP was performed 3 days after 4-OHT treatment (e). f A proposed signaling cascade illustrating the roles of mTORC1 and mTORC2 in directly binding and phosphorylating p53 during PICS. The values represent the mean ± SD; n = 3; ^#P > 0.05; **P < 0.01; ***P < 0.001