Oncogene-induced senescence mitochondrial metabolism and bioenergetics drive the secretory phenotype: further characterization and comparison with other senescence-inducing stimuli

Abstract

Cellular senescence is characterized by proliferation arrest and a senescence-associated secretory phenotype (SASP), that plays a role in aging and the progression of various age-related diseases. Although various metabolic alterations have been reported, no consensus exists regarding mitochondrial bioenergetics. Here we compared mitochondrial metabolism of human fibroblasts after inducing senescence with different stimuli: the oxidant hydrogen peroxide (H₂O₂), the genotoxic doxorubicin, serial passage, or expression of the H-RAS^G12V oncogene (RAS). In senescence induced by H₂O₂, doxorubicin or serial passage a decrease in respiratory control ratio (RCR) and coupling efficiency was noted, in relation to control cells. On the contrary, oncogene-induced senescent cells had an overall increase in respiration rates, RCR, spare respiratory capacity and coupling efficiency. In oncogene-induced senescence (OIS) the increase in respiration rates was accompanied by an increase in fatty acid catabolism, AMPK activation, and a persistent DNA damage response (DDR), that were not present in senescent cells induced by either H₂O₂ or doxorubicin. Inhibition of AMPK reduced mitochondrial oxygen consumption and secretion of proinflammatory cytokines in OIS. Assessment of enzymes involved in acetyl-CoA metabolism in OIS showed a 3- to 7.5-fold increase in pyruvate dehydrogenase complex (PDH), a 40% inhibition of mitochondrial aconitase, increased phosphorylation and activation of ATP-citrate lyase (ACLY), and inhibition of acetyl-CoA carboxylase (ACC). There was also a significant increase in expression and nuclear levels of the deacetylase sirtuin 6 (SIRT6). These changes can influence the sub-cellular distribution of acetyl-CoA and modulate protein acetylation reactions in the cytoplasm and nuclei. In fact, ACLY inhibition reduced histone 3 acetylation (H3K9Ac) in OIS and secretion of SASP components. In summary, our data show marked heterogeneity in mitochondrial energy metabolism of senescent cells, depending on the inducing stimulus, reveal new metabolic features of oncogene-induced senescent cells and identify AMPK and ACLY as potential targets for SASP modulation.

Keywords: Bioenergetics; Fatty acid oxidation; Mitochondria; RAS oncogene; Senescence; TCA cycle.

Graphical abstract

Fig. 1

Senescence induction and fatty acid oxidation in human fibroblasts exposed to different stimuli. Early passage IMR-90 human fibroblasts were incubated with the different senescence inducing stimuli. Measurements were made 14 days after treatment. A) Fibroblasts were exposed to H₂O₂ in complete media (600 μM) for 2 h, (two exposures separated by a five day interval). (B) Fibroblasts were exposed to doxorubicin 0.2 μM for 24 h (Doxo) or the vehicle DMSO (Control) in complete media. (C) Fibroblasts were transduced with lentiviral particles containing a plasmid coding for the H-RAS^G12V oncogene (RAS) or a control plasmid (Control), and selected with puromycin. (A, B, C) SA-β-Gal activity was determined. (D, E, F) Oxygen consumption rate (OCR) was measured in a Seahorse XFe24 in media containing an oleate:BSA conjugate (0.1 mM:0.2% m/v). The CPT1 inhibitor etomoxir (Eto, 100 μM) and complex III inhibitor Antimycin A (AA, 2.5 μM) were sequentially added to the culture. Fatty acid oxidation was determined as the fraction of the OCR sensitive to etomoxir, calculated as the ratio between etomoxir sensitive OCR and basal OCR. Results are the mean ± SEM. Significant differences between senescent cells and the control condition were determined with Student's t-test. ∗P < 0.05 (n = 3–5).

Fig. 2

Mitochondrial bioenergetics in senescent human fibroblasts induced with different stimuli. Early passage IMR-90 human fibroblasts were incubated with the different senescence inducing stimuli, and after 14 days oxygen consumption rate (OCR) was measured in the Seahorse XFe24 in media containing glucose 5 mM, pyruvate 1 mM, glutamine 2 mM. The ATP synthase inhibitor oligomycin (Oligo, 0.5 μM), the uncoupler FCCP (1 μM) and complex III inhibitor antimycin A (AA, 1 μM) were sequentially added to the culture. Respiratory parameters were determined as described in the Methods section. (A) Fibroblasts were exposed to H₂O₂ in complete media (600 μM) for 2 h, (two exposures separated by a five day interval) (n = 4–5). (B) Fibroblasts were exposed to doxorubicin 0.2 μM for 24 h (Doxo) or the vehicle DMSO (Control) in complete media (n = 6). (C) Fibroblasts were infected with lentiviral particles containing a plasmid coding for the H-RAS^G12V oncogene (RAS) or a control plasmid (Control). Cells were selected with puromycin (n = 6–7). Results are the mean ± SD. Significant differences between senescent cells and the control condition were determined with Student's t-test. ∗P < 0.05, ∗∗P < 0.001, ∗∗∗P < 0.0001.

Fig. 3

Time course of basal respiration of oncogene-induced senescent human fibroblasts. Early passage IMR-90 human fibroblasts were transduced with lentiviral particles carrying a Control or H-RAS^G12V (RAS) containing plasmid, and selected with puromycin. (A) SA-β-Gal activity was measured at different time points (n = 3). (B) Oxygen consumption rate (OCR) was measured continuously using a Resipher instrument. Measurements were made in complete media, in a CO₂ containing stove, from the day the selection started. Oxygen consumption rate of Control and RAS cells from day 1 (0 h) to day 17 (410 h) after selection. Wells including only media were measured to control for cell-independent OCR. Media was changed every 2–3 days (arrows). (C) Basal oxygen consumption rate of senescent and non-senescent cells from day 14–17 (345–410 h) after the addition of puromycin (D) Basal respiration at 400 h was normalized by protein content in the wells. (E) Basal respiration normalized by protein content 14 days after the addition of puromycin, measured using the Seahorse XFe24 or Resipher instruments was compared. Results are the mean ± SD. Significant differences between conditions were determined with Student's t-test. ∗P < 0.05, ∗∗P < 0.001, ∗∗∗P < 0.0001 (n = 4).

Fig. 4

Time course of mitochondrial bioenergetics in oncogene-induced senescent human fibroblasts. (A–D) Early passage BJ human fibroblasts carrying ERRAS fusion protein were cultured. Measurements were made at different time points after the addition of 4OHT (200 nM). (A) Representative western Blots (WB) of RAS, phosphorylated ATM (p-ATM) (Ser1981), ATM, p53, p16 and tubulin (loading control). (B) SA-β-Gal staining (n = 4). (C) Representative WB of phosphorylated AMPK (p-AMPK) (Thr172), AMPK, phosphorylated ACC (p-ACC) (Ser79), ACC, and tubulin as loading control. (D) Oxygen consumption rate (OCR) was measured and respiratory parameters determined as described in Fig. 2. Results are the mean ± SD (n = 4–5). (E–F) IMR-90 fibroblasts were exposed to H₂O₂ in complete media (600 μM) for 2 h, (two exposures separated by a five day interval). Measurements were made at different time points after the last addition of the oxidant. (E) SA-β-Gal activity (n = 3). (F) OCR was measured and respiratory parameters determined as described in Fig. 2 (n = 3–4). Results are the mean ± SD. Significant differences between senescent cells and the control condition were determined with ANOVA and Dunnett post-hoc for multiple comparisons (B and D), or Student's t-test (E and F). ∗P < 0.05, ∗∗P < 0.001, ∗∗∗P < 0.0001.

Fig. 5

RAS levels and the senescent phenotype. Early passage BJERRAS fibroblasts were incubated 4OHT (200 nM) (Sen + RAS) or the vehicle methanol (Control) for 20 days. A third group was exposed to 4OHT (200 nM) for 11 days, and on day 12 when the cells had acquired a senescent phenotype, the cultures were switched to vehicle and incubated in these conditions for 8 more days (Sen - RAS). (A) Scheme showing the culture conditions of the three groups: Control, Sen + RAS, Sen - RAS. (B) Representative western blots showing the levels of RAS, phosphorylated ERK (p-ERK) (Thr202/Tyr204), ERK, p21, p16, p65 and tubulin as loading control. Graphs show the quantification of the different proteins normalized to the control condition. Results are the mean ± SD (n = 3). (C) IL-8 secretion assessed by ELISA, normalized considering cell protein content (μg) (n = 8) and relative to the control. Significant differences between senescent cells with and without RAS and the control condition were determined with one-way ANOVA, and Tukey post-hoc for multiple comparisons ∗P < 0.05, ∗∗P < 0.001, ∗∗∗P < 0.0001.

Fig. 6

AMPK activation drives the increase in cell respiration and the SASP in OIS. (A) Early passage BJERRAS fibroblasts were incubated as described in Fig. 5. Representative western blots showing the levels of phosphorylated ACC (p-ACC) (Ser79), ACC, and tubulin as loading control. The graph shows the p-ACC/ACC ratio relative to the control condition. Results are the mean ± SD (n = 3). (B) Oxygen consumption rate (OCR) was measured and respiratory parameters determined as described in Fig. 2. (C) IMR-90 human fibroblasts were transduced with lentiviral particles containing H-RAS^G12V oncogene (RAS) or a control plasmid (Ctl). Senescent cells were incubated with CC (10 μM) for 6 h and western blots preformed with antibodies against p-ACC (Ser79), ACC, and tubulin as loading control. (D) Oxygen consumption rate (OCR) was measured and respiratory parameters determined for IMR-90 fibroblasts incubated with CC (10 μM) or vehicle (n = 6–7). (E) Cytokine secretion was assessed by ELISA, and normalized considering cell protein content (n = 3) for IMR-90 fibroblasts incubated with CC (10 μM) or oligomycin (1 μM) for 6 h (n = 6–7). Results are the mean ± SD. Significant differences between senescent cells with and without RAS and the control condition were determined with one-way ANOVA, and Tukey or Dunnett's post-hoc for multiple comparisons ∗P < 0.05, ∗∗P < 0.001, ∗∗∗P < 0.0001.

Fig. 7

Mitochondrial metabolic features of oncogene-induced senescent human fibroblasts. Early passage IMR-90 human fibroblasts were transduced with lentiviral particles containing a plasmid coding for the H-RAS^G12V oncogene (RAS) or a control plasmid (Control), and selected with puromycin. Measurements were made 14 days after transduction. (A) Data from metabolomic experiments reported in Ref. [13] (n = 6). (B) Representative WB for Pyruvate dehydrogenase complex subunits. (C and D) Graphs show the quantification of the subunits E1α, and E2 proteins relative to tubulin and normalized to the control condition. Results are the mean ± SD (n = 3). (E) Aconitase activity was determined by spectrophotometric techniques (n = 3). (F) Intracellular oxidant levels measured assessing chloromethyl-2′,7′-dichlorofluorescein (CM-DCF) fluorescence intensity by flow cytometry analysis (n = 7), the geometric mean fluorescence intensity relative to the control condition is shown. Results are the mean ± SD. Significant differences between senescent cells and the control condition were determined with Student's t-test. ∗P < 0.05, ∗∗P < 0.001.

Fig. 8

ACLY-dependent histone lysine acetylation in oncogene-induced senescent cells. (A–D) Early passage IMR-90 human fibroblasts expressing the H-RAS^G12V oncogene (RAS) or a control plasmid (Control). (A) Representative WB for ACLY and phosphorylated ACLY (p-ACLY) (Ser455) and tubulin as loading control. (B–D) Quantification of ACLY and p-ACLY relative to tubulin and the ratio between the phosphorylated and total enzyme, normalized to the control condition. (E) WB of BJERRAS fibroblasts at different time points after the addition of 4OHT (200 nM) or the vehicle showing an increase in RAS, p-ACLY, ACLY, p-ACC, ACC and GAPDH as loading control. (F–I) OIS IMR-90 fibroblasts treated with the ACLY inhibitor NDI-091143 (NDI, 40 μM) for 18 h or the vehicle: (F) Histone acetylation measured by confocal microscopy after immunocytochemistry using anti-H3K9Ac antibody (green) and staining the nuclei with Dapi (blue). (G) Quantification of nuclear H3K9Ac (n > 300). (H and I) Secretion of IL-8 and IL-6 measured by ELISA (n = 4). (J) SIRT6 levles obtained by confocal microscopy after immunocytochemistry with an anti-SIRT6 antibody (green), and nuclei stained with Dapi (white). (J) Quantification of nuclear SIRT6 (n = 8–25). (K) Expression levels of SIRT6 determined by RT-qPCR (n = 3–4). Significant differences between conditions were determined with Student's t-test or one-way ANOVA and Tukey post-hoc for multiple comparisons ∗P < 0.05, ∗∗P < 0.001, ∗∗∗P $\le$ 0.0001.

Fig. 9

Mitochondrial energy metabolism and metabolic fates of acetyl-CoA in OIS cells. Glucose and fatty-acid oxidation increase in OIS resulting in the formation of acetyl-CoA. Acetyl-CoA condenses with oxaloacetate forming citrate, that can be oxidized in the TCA cycle or exported to the cytoplasm, and regenerate acetyl-CoA. Since lipid synthesis is inhibited in these cells, acetyl-CoA can be rerouted and participate in protein lysine acetylation, promoting histone acetylation and SASP gene expression. AMPK and ACLY activation play key roles regulating metabolic reactions and the SASP. Enzymes and pathways where evidence of an increase or decrease in activity was found are shown with a red arrow or a blue arrow, respectively. ACC: acetyl-CoA carboxylase; ACLY: ATP-citrate lyase; ACO: mitochondrial aconitase; AMPK: AMP activated protein kinase; PDH: pyruvate dehydrogenase complex. The figure was created with BioRender.com.