AKT induces senescence in human cells via mTORC1 and p53 in the absence of DNA damage: implications for targeting mTOR during malignancy

Abstract

The phosphatidylinositol 3-kinase (PI3K)/AKT and RAS oncogenic signalling modules are frequently mutated in sporadic human cancer. Although each of these pathways has been shown to play critical roles in driving tumour growth and proliferation, their activation in normal human cells can also promote cell senescence. Although the mechanisms mediating RAS-induced senescence have been well characterised, those controlling PI3K/AKT-induced senescence are poorly understood. Here we show that PI3K/AKT pathway activation in response to phosphatase and tensin homolog (PTEN) knockdown, mutant PI3K, catalytic, α polypeptide (PIK3CA) or activated AKT expression, promotes accumulation of p53 and p21, increases cell size and induces senescence-associated β-galactosidase activity. We demonstrate that AKT-induced senescence is p53-dependent and is characterised by mTORC1-dependent regulation of p53 translation and stabilisation of p53 protein following nucleolar localisation and inactivation of MDM2. The underlying mechanisms of RAS and AKT-induced senescence appear to be distinct, demonstrating that different mediators of senescence may be deregulated during transformation by specific oncogenes. Unlike RAS, AKT promotes rapid proliferative arrest in the absence of a hyperproliferative phase or DNA damage, indicating that inactivation of the senescence response is critical at the early stages of PI3K/AKT-driven tumourigenesis. Furthermore, our data imply that chronic activation of AKT signalling provides selective pressure for the loss of p53 function, consistent with observations that PTEN or PIK3CA mutations are significantly associated with p53 mutation in a number of human tumour types. Importantly, the demonstration that mTORC1 is an essential mediator of AKT-induced senescence raises the possibility that targeting mTORC1 in tumours with activated PI3K/AKT signalling may exert unexpected detrimental effects due to inactivation of a senescence brake on potential cancer-initiating cells.

Figure 1

PTEN depletion or PIK3CA mutant expression induces senescence in BJ-T cells. (a) BJ-T cells were transduced with pGIPZ-NS-shRNA or pGIPZ-PTEN-shRNA lentivirus or pBABE or pBABE-PIK3CA^E545K retrovirus. Lysates of these cells were collected and immunoblotted with the indicated antibodies. Actin is used as a loading control. (b) BJ-T cells as above at day 10 post-transduction were trypsinised and analysed on a Coulter counter to obtain cell size measurements (cell volume in fL) (n=4, bars represent mean±s.e.m., *P<0.05, **P<0.01). (c) Cells as above were fixed and incubated with SAβGAL staining solution overnight. Cells were washed, stained with DAPI and imaged for both SAβGAL and the number of nuclei. Percentage of SAβGAL-positive cells was determined for a minimum of 10 low-power magnification fields (n=4–5, bars represent mean±s.e.m., *P<0.05).

Figure 2

Activated AKT isoforms induce markers of senescence and proliferation arrest in BJ-T cells. (a) BJ-T cells were transduced with pBABE, pBABE-myr-AKT isoforms or pBABE-H-RAS^V12. At day 10 post-transduction, cells were harvested and lysates immunoblotted with the indicated antibodies to demonstrate construct expression, activation of downstream signalling pathways and accumulation of senescence markers. Actin is used as a loading control. Black arrowheads indicate nonspecific bands from the 12CA5 anti-HA antibody. (b) Cells prepared as in (a) were fixed, incubated with SAβGAL staining solution overnight, and then stained with DAPI to visualise non-senescent cells and quantitate total cell number. The percentage of senescent cells was quantitated manually (n=4, bars represent mean±s.e.m., **P<0.05, ***P<0.001). (c) BJ-T cells as above were trypsinised and analysed on a Coulter counter to determine cell size measurements (fL) (n=6, bars represent mean±s.e.m., *P<0.01, **P<0.05, ***P<0.001). (d) To visualise SAHFs, BJ-T cells expressing myr-AKT1 of H-RAS^V12 at day 10 post-transduction were stained with DAPI and imaged using confocal microscopy. Scale bar represents 10 μm. (e) Proliferation of BJ-T cells expressing myr-AKT1 or H-RAS^V12. At 4 days post-transduction, cells were plated at equal cell number and harvested daily for cell counting on a Coulter counter over a period of 5 days (n=5, error bars represent mean±s.e.m.).

Figure 3

myr-AKT1 fails to induce markers of DNA damage. (a) BJ-T cells transduced with pBABE, pBABE-myr-AKT1 or pBABE-H-RAS^V12 were immunostained with phospho-Ser139-γH2A.X. Cells were imaged and the percentage that exhibited nuclear phospho-Ser139-γH2A.X foci was quantitated for a minimum of 200 cells per experiment (n=3, bars represent mean±s.e.m., ***P<0.001). (b) Lysates of BJ-T cells expressing pBABE, pBABE-myr-AKT1 or H-RAS^V12 were immunoblotted with phospho-p53(Ser15) and p53 antibodies. Lysate of γ-irradiated A549 cells were included as a positive control.

Figure 4

myr-AKT1 expression induces upregulation of senescence-associated secretory factors. (a) RT–PCR analysis of IL-1α, IL-1β, IL-6, IL-8 and GAPDH expression in BJ-T cells expressing pBABE, myr-AKT1 or H-RAS^V12 at 10–11 days post-transduction (n=4–6, bars represent mean±s.e.m., *P<0.05, **P<0.01). (b) CBA analysis of secreted IL-1α, IL-1β, IL-6 and IL-8 in the conditioned media from BJ-T cells expressing pBABE, myr-AKT1 or H-RAS^V12 at 10 days post-transduction (n=3, bars represent mean±s.e.m.).

Figure 5

AKT-induced senescence is p53-dependent. (a) BJ-T cells were transduced with retrovirus encoding control or p53 shRNA. These cell lines were then transduced with MSCV or MSCV-myr-AKT. Lysates were harvested at day 10 post-transduction and immunoblotted with p53 and p21 antibodies. (b) Control or p53 shRNA expressing BJ-T cell lines transduced with MSCV or MSCV-myr-AKT. Cells were plated at equal cell number and harvested 3 days later. Cell numbers were used to determine population doubling time (n=4, bars represent mean±s.e.m., *P<0.05, **P<0.01). (c) Cells as in (b) were analysed on the Coulter counter to obtain cell size measurements (n=3, bars represent mean±s.e.m., **P<0.01, ***P<0.001). (d) Cells as in (b) were analysed for SAβGAL activity. Cells were imaged under brightfield for SAβGAL- and DAPI-stained nuclei, and the cellular autofluorescence was imaged to indicate cell morphology. The percentage of cells that stained positive for SAβGAL was determined (n=4, bars represent mean±s.e.m., ***P<0.001).

Figure 6

MDM2 activity is reduced with the expression of AKT. (a) BJ-T cells expressing pBABE or pBABE-myr-AKT1 were subjected to [³⁵S]Met/Cys pulse-chase analysis. Lysates were collected at various chase times and p53 immunoprecipitations (IPs) performed. Complexes were resolved by SDS–PAGE and gels exposed to phosphorimager storage screens. p53 band intensity was quantified using the ImageQuant software (GE Healthcare, Uppsala, Sweden). p53 half-life determination values were standardised to t=0 for the particular cell population. p53 half-life, which was determined using regression analysis, is indicated (n=2). (b) The amount of [³⁵S]Met/Cys-labelled p53 following a 30 min pulse period (t=0) was quantitated (n=3, bars represent mean values±s.e.m., *P<0.05). (c) BJ-T cells expressing pBABE or pBABE-myr-AKT1 were treated with MG132 (30 μ) for 3 h and subjected to rapid lysis in SDS–PAGE reducing buffer. Lysates were run on SDS–PAGE and immunoblotted with p53 antibodies. (d) Cells as in (a) were fixed, immunostained with MDM2 (red) and fibrillarin (green) antibodies and co-stained with DAPI (blue). Cells were imaged at the same laser attenuation. Scale bar represent 10 μm. The total nucleolar MDM2, as a percentage of total nuclear MDM2, was quantitated (n=10–11 fields, ***P<0.001).

Figure 7

AKT-induced senescence is dependent on mTORC1 activity. (a) BJ-T cells were transduced with pBABE or pBABE-myr-AKT1 and treated with either vehicle or 20 n rapamycin for 11 days. Cells were fixed, incubated with SAβGAL staining solution overnight, and then stained with DAPI to visualise non-senescent cells and quantitate total cell number (DAPI not shown). The percentage of senescent cells was quantitated manually (n=4–7, bars represent mean±s.e.m., ***P<0.001). (b) BJ-T cells as above were trypsinised and analysed on a Coulter counter to obtain cell size measurements (fL) (n=4–6, bars represent mean±s.e.m., ***P<0.001). (c) RNA was harvested from cells as in (a). RT–PCR analysis was performed for IL-1α, IL-1β and vimentin expression. Values are expressed relative to myr-AKT–Rapa (n=4, bars represent mean±s.e.m., ***P<0.001). (d) Cell lysates were prepared from BJ-T cells as in (a) and immunoblotted with the indicated antibodies. Actin is used as a loading control.

Figure 8

Proposed mechanism of AKT-induced senescence. (a) Physiological levels of AKT signalling lead to the activation of MDM2 at levels that maintain low p53 protein expression. (b) Chronic hyperactivation of AKT results in mTORC1-dependent increases in p53 translation and simultaneously stimulates MDM2 sequestration within the nucleolus, inhibiting p53 ubiquitination to further enhance p53 accumulation leading to cellular senescence.