MTOR regulates the pro-tumorigenic senescence-associated secretory phenotype by promoting IL1A translation

Abstract

The TOR (target of rapamycin) kinase limits longevity by poorly understood mechanisms. Rapamycin suppresses the mammalian TORC1 complex, which regulates translation, and extends lifespan in diverse species, including mice. We show that rapamycin selectively blunts the pro-inflammatory phenotype of senescent cells. Cellular senescence suppresses cancer by preventing cell proliferation. However, as senescent cells accumulate with age, the senescence-associated secretory phenotype (SASP) can disrupt tissues and contribute to age-related pathologies, including cancer. MTOR inhibition suppressed the secretion of inflammatory cytokines by senescent cells. Rapamycin reduced IL6 and other cytokine mRNA levels, but selectively suppressed translation of the membrane-bound cytokine IL1A. Reduced IL1A diminished NF-κB transcriptional activity, which controls much of the SASP; exogenous IL1A restored IL6 secretion to rapamycin-treated cells. Importantly, rapamycin suppressed the ability of senescent fibroblasts to stimulate prostate tumour growth in mice. Thus, rapamycin might ameliorate age-related pathologies, including late-life cancer, by suppressing senescence-associated inflammation.

Figure 1

Rapamycin decreases the SASP through MTORC1. (a) HCA2 fibroblasts, non-senescent (NS) or induced to senesce by ionizing radiation (IR; 10 Gy), were treated with the indicated concentrations of rapamycin (Rapa; or the highest concentration of DMSO as a control) immediately after ionizing radiation exposure. Conditioned media were collected 7 days later and analysed by ELISA for IL6. The level of IL6 secretion by non-senescent cells was arbitrarily set at 1. (b) HCA2 cells were induced to senesce by lentiviral-mediated overexpression of RAS or MKK6EE, 2 mM sodium butyrate (NaBu) for 3 days, replicative exhaustion (Rep), or 250 nM doxorubicin for 24 h (Doxo). The cells were given 12.5 nM rapamycin or DMSO (control) after treatment. Conditioned media were collected 7 days later and analysed by ELISA for IL6. (c) Conditioned media from non-senescent (NS) or senescent (ionizing radiation; Sen (IR)) cells treated with rapamycin or DMSO for 6 days were analysed by antibody arrays. The average signal from DMSO-treated cells was used as the baseline. Colour intensities represent log₂-fold changes from the baseline. Signals higher than the baseline are shown in yellow; signals lower than the baseline are shown in blue. Shown is the average of three independent experiments. (d) Effects of two different rapamycin treatment regimens on IL6 secretion by senescent (ionizing radiation; Sen (IR)) HCA2 cells, one starting immediately after irradiation (d0) and continuing for 13 days, the other starting 7 days after irradiation and continuing for an additional 6 days. (e,f) Senescent (ionizing radiation; Sen (IR)) HCA2 cells were treated with DMSO or rapamycin and immunostained for the DNA-SCAR marker 53BP1. The number of 53BP1 foci was determined using CellProfiler. Shown is the percentage of cells with >2 53BP1 foci (e), and the average number of foci per cell (f). NS cells are shown for comparison. For all panels except c, shown is one representative of two independent experiments, each with triplicate samples. For raw data, see Supplementary Table 4.

Figure 2

SASP production is MTOR dependent. (a) HCA2 cells were infected with lentiviruses expressing GFP shRNA (control) or one of three different shRNAs targeting raptor. Cells were then irradiated; 7 days later, conditioned media were collected and analysed by ELISA for IL6. (b) HCA2 cells were infected with lentiviruses expressing GFP shRNA (control) or one of three different shRNAs targeting MTOR. Cells were then irradiated; 7 days later, conditioned media were collected and analysed by ELISA for IL6. (c) IL8 was quantified by ELISA in conditioned media from PSC27 cells in which S6K was depleted by siRNA or 4EBP1 overexpressed by transfection with a 4EBP1-encoding plasmid. Cells treated with rapamycin (Rapa) are shown for comparison. (d) After inducing senescence by ionizing radiation, protein extracts from PSC27 cells transfected with S6K siRNA or trans-4EBP1, or treated with rapamycin were analysed by western blotting for the indicated SASP factors. Unprocessed original scans of blots are shown in Supplementary Fig. 9. (e) Extracts from non-senescent (NS) or senescent (ionizing radiation; Sen (IR)) HCA2 cells, treated or not with rapamycin, were assayed for the indicated proteins by western blotting. β-actin served as the loading control. Unprocessed original scans of blots are shown in Supplementary Fig. 9. For a–c, shown is one representative of two independent experiments, each with triplicate samples. For d and e shown is one representative of two independent biological replicates; each replicate required multiple blots, which were probed at the same time. For raw data, see Supplementary Table 4.

Figure 3

Rapamycin suppresses NF-κB transcriptional activity. (a) Transcripts for SASP factors secreted by DMSO- or rapamycin (Rapa)-treated senescent (ionizing radiation; Sen (IR)) HCA2 cells were quantified by qPCR 8 days after ionizing radiation exposure. Cells were incubated in serum-free media without rapamycin for the last 24 h. For the heatmap, the average signal from non-senescent (NS), senescent (ionizing radiation; Sen (IR)) DMSO-and rapamycin-treated HCA2 cells was used as the baseline (for numbers, see Supplementary Table 1). Colour intensities represent log₂-fold changes from the baseline. Signals higher than the baseline are shown in yellow; signals lower than the baseline are shown in blue. (b) Transcripts for SASP factors from DMSO- or rapamycin-treated senescent (ionizing radiation; Sen (IR)) HCA2 cells were quantified by qPCR 2 days after ionizing radiation exposure. For the heatmap, the average signal from non-senescent, senescent DMSO-and rapamycin-treated cells was used as the baseline (for numbers, see Supplementary Table 2). Colour intensities represent log₂-fold change from the baseline. Signals higher than the baseline are shown in yellow; signals lower than the baseline are shown in blue. (c) Cell extracts were prepared from non-senescent (NS) and senescent (ionizing radiation; Sen (IR)) HCA2 cells expressing an NF-κB–luciferase reporter construct. Cells were treated with DMSO (control) or rapamycin for 7 days, and analysed for luciferase activity as described previously^,. Non-senescent luciferase activity was set at 1. For a and b, shown are the average of three independent experiments. For c, shown is one representative of two independent experiments, each with triplicate samples. For raw data, see Supplementary Table 4.

Figure 4

Rapamycin suppresses IL1A signalling. (a) HCA2 cells were infected with lentiviruses expressing shRNAs against GFP (control) or raptor. Senescent (ionizing radiation; Sen (IR)) cells, treated with rapamycin (Rapa) or DMSO for 10 days after ionizing radiation exposure, were analysed by flow cytometry for cell-surface IL1A using a FITC-tagged antibody. The fluorescence signal was divided by the forward scatter signals to account for cell size variations; 10,000 flow cytometry events were recorded. Shown is the result of one of two independent experiments. (b) HCA2 cells infected with lentiviruses expressing GFP shRNA or IL1A shRNA were irradiated and treated with DMSO (D) or rapamycin (R); 7 days later conditioned media were collected and analysed by ELISA for IL6. (c) Proteins were extracted from DMSO- and rapamycin-treated senescent cells and analysed by western blotting for IRAK1, IκBα, phospho-S6 and S6 at the indicated intervals after ionizing radiation exposure. Recombinant (r) IL1A protein was added to one senescent (ionizing radiation) sample treated with rapamycin (right lane). Unprocessed original scans of blots are shown in Supplementary Fig. 9. Shown is one of two independent biological replicates; each replicate required multiple blots, which were probed at the same time. (d) Non-senescent (NS) and senescent (ionizing radiation; Sen (IR)) HCA2 cells were treated with DMSO or rapamycin for 6 d, after which rIL1A in serum-free medium was added for 24 h. Conditioned media were collected and analysed by ELISA for IL6. For b and d, shown is one representative of two independent experiments, each with triplicate samples. For raw data, see Supplementary Table 4.

Figure 5

Rapamycin inhibits IL1A translation. (a) Senescent (ionizing radiation; Sen (IR)) HCA2 cells were treated for 7 days with rapamycin (Rapa) or DMSO followed by 1 day in serum-free media, after which cells were collected and mRNA was collected for polysome profiling as described in the Methods. qPCR was performed on each fraction for IL1A, IL6, CCL13, TUBA1A, IL1B, IL8, TIMP1, IL3 and IL5 mRNA (one representative experiment is shown). Fractions 1–7: free RNA; 8–12: 40–60S; 13–20: polysome. (b) Polysome profiles used to determine the translated fractions; representative polysome traces are shown. NS, non-senescent. (c) Using the UCSC genome browser sequence, analysis of the IL1A gene was performed to determine potential start sites of transcription. (d) Potential transcription start sites (TSSs) and their 5’UTRs are shown from various sources, including the determination based on 5’RACE (see c). Transcription start site sequences start at the capital letter and pyrimidines are shown in red. (e) IL1A mRNA minimum free energy structure was determined using the default parameter obtained from the University of Vienna RNAfold web service (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). Scale represents the organizational entropy. The presence of a highly stable structure upstream of the start codon is detected. 5’RACE was performed once. For a and b, shown is one representative of two independent experiments, each with triplicate cell culture samples. For raw data, see Supplementary Table 4.

Figure 6

Rapamycin does not reverse cellular senescence. (a) Senescent (ionizing radiation; Sen (IR)) HCA2 cells were treated with DMSO or rapamycin (Rapa) for 6 days, and the percentage of cells expressing SA-β-gal was determined by light microscopy (right panel) and counting (left panel). (b) Cells were treated with rapamycin or DMSO for 28 days following ionizing radiation exposure, washed, and incubated in drug-free media for 14 days, at which point cell numbers were determined. (c) HCA2 cells, non-senescent (NS) or made senescent by ionizing radiation exposure and treated with DMSO or rapamycin for 6 days, were pulsed with BrdU for 24 h and the fraction that incorporated BrdU was determined by fluorescence microscopy. (d) Clonogenic assays comparing the effects of chronic treatment with rapamycin or PP242 (500 nM) of non-senescent (NS) and senescent (ionizing radiation; Sen (IR)) HCA2 cells. Cells were plated and drugs were added at the indicated times before or after ionizing radiation exposure and cells were cultured for 10–14 days (one representative experiment shown). (e) HCA2 cells were infected with a control lentivirus (L3P) or lentivirus carrying oncogenic RAS. Cell were grown in drug for three days (acute) and released or continuously treated (chronic) for 10–14 days after which clonogenic staining was performed. (f) HCA2 cells were co-infected with RAS and lentiviruses carrying shRNAs to deplete the indicated proteins. Transcripts for IL6 were quantified by qPCR. (g,h) HCA2 cells were infected with lentiviruses carrying shRNAs to deplete the indicated proteins. Clonogenic assays were performed on the infected cells induced to senesce by oncogenic RAS (g) or ionizing radiation (h) (one representative experiment is shown). (i) HCA2 cells were infected with a lentivirus carrying shRNAs against GFP (control) or raptor, induced or not (NS) to senesce by 10 Gy ionizing radiation (IR) or 250 nM doxorubicin (Doxo) for 24 h and treated with DMSO or rapamycin. Cells were cultured for 10–14 days and clonogenic staining was performed using crystal violet (one representative experiment is shown). (j) Cells were treated as in i. Seven days after senescence induction, conditioned medium was collected and analysed for IL6 by ELISA. For a–c, f and j shown is one representative of two independent experiments, each with triplicate cell culture samples. For d, e, g–i, shown is one representative clonogenic assay experiment replicated once. For raw data, see Supplementary Table 4.

Figure 7

Sustained effect of rapamycin on the SASP. (a) HCA2 cells were treated with rapamycin (Rapa) for 1 day immediately after ionizing radiation exposure, and conditioned medium was collected 6 days later and analysed for IL6 secretion by ELISA; shown is one representative of two independent experiments, each with triplicate cell culture samples. (b) The amount of rapamycin was measured in non-senescent (NS) and senescent (ionizing radiation; Sen (IR)) HCA2 cells 1, 3, 7, 15 and 31 days after acute treatment (single 1-day dose immediately after ionizing radiation exposure). Rapamycin was measured by high-performance liquid chromatography at the Biological Psychiatry Laboratories Services of the University of Texas Health Sciences Center San Antonio. Six samples were collected and pooled in groups of two for each condition. (c) Western blot analysis of phospho-S6 and S6 using proteins extracted 1, 3, 7 and 15 days after acute treatment (as in a) of HCA2 cells with rapamycin (one representative or two representative experiments is shown). Unprocessed original scans of blots are shown in Supplementary Fig. 9. (d) IL6 secretion by X-irradiated (IR) senescent HCA2 cells at day 7, 13, 19 and 25 after rapamycin treatment, relative to non-senescent (NS) secretion; shown is one representative of two independent experiments, each with triplicate cell culture samples. (e) SA-β-gal activity in non-senescent and senescent cells treated with DMSO and rapamycin was determined by light microscopy 7 or 25 days after treatment (one representative of two representative independent experiments is shown). For raw data, see Supplementary Table 4.

Figure 8

Rapamycin suppresses the tumour-promoting activity of senescent cells. (a,b) Percentage of BPH1, M12 and PC3 prostate cancer cell migration (a) and invasion (b) was determined on co-culture with conditioned media from irradiated PSC27 fibroblasts that were treated with rapamycin (Rapa) or vehicle (control). The human cervical cancer line HeLa was used as a positive control in both experiments. (c) The indicated prostate epithelial cells were incubated with conditioned media from PSC27 human prostate fibroblasts, non-senescent (NS) or induced to senesce by irradiation (Sen (IR)), untreated or treated with rapamycin. Epithelial cell numbers were determined 3 days later. (d) PC3 prostate cancer cells were implanted subcutaneously with non-senescent (PSC27-NS) or senescent (ionizing radiation; PSC27-Sen (IR)) prostate fibroblasts that had been pretreated for 8 days in culture with DMSO or rapamycin. Tumour volumes were determined as described in the Methods (mean ± s.e.m., n = 8 animals). (e) Prostate cancer cell viability was determined by MTT assays following exposure to mitoxantrone for 72 h at twice the IC₅₀ dose for each cell line in the presence of conditioned media from non-senescent (PSC27-NS) or senescent (PSC27-Sen (IR)) PSC27 fibroblasts that were co-treated with rapamycin or vehicle. (f) In vivo effect of rapamycin on chemotherapy resistance was determined by injecting PC3 cancer cells with or without PSC27 prostate fibroblasts into SCID mice followed by treatment with mitoxantrone or vehicle and co-administration of rapamycin or vehicle. Tumour volumes were determined after an 8-week treatment period (mean ± s.e.m., n = 10 animals). For a–c and e shown is one representative of three independent experiments, each with triplicate cell culture samples. For d and f, a standard t-test served to determine P values. For raw data, see Supplementary Table 4.