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Cited by 124Telomeres, Telomerase, and SenescenceCancer-related Molecular PathwaysGenetics, Aging, and Longevity in Model Organisms

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Small-molecule MDM2 antagonists attenuate the senescence-associated secretory phenotype

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Wiley, Christopher D., Schaum, Nicholas, Alimirah, Fatouma, Lopez-Dominguez, Jose Alberto, Orjalo, Arturo V., Scott, Gary, Desprez, Pierre-Yves, Benz, Christopher, Davalos, Albert R., Campisi, Judith·2018·Scientific Reports
DOI 10.1038/s41598-018-20000-4PMC PMC5799282
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Abstract

Processes that have been linked to aging and cancer include an inflammatory milieu driven by senescent cells. Senescent cells lose the ability to divide, essentially irreversibly, and secrete numerous proteases, cytokines and growth factors, termed the senescence-associated secretory phenotype (SASP). Senescent cells that lack p53 tumor suppressor function show an exaggerated SASP, suggesting the SASP is negatively controlled by p53. Here, we show that increased p53 activity caused by small molecule inhibitors of MDM2, which promotes p53 degradation, reduces inflammatory cytokine production by senescent cells. Upon treatment with the MDM2 inhibitors nutlin-3a or MI-63, human cells acquired a senescence-like growth arrest, but the arrest was reversible. Importantly, the inhibitors reduced expression of the signature SASP factors IL-6 and IL-1α by cells made senescent by genotoxic stimuli, and suppressed the ability of senescent fibroblasts to stimulate breast cancer cell aggressiveness. Our findings suggest that MDM2 inhibitors could reduce cancer progression in part by reducing the pro-inflammatory environment created by senescent cells.

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Figure 1 MDM2 inhibitors induce a senescence-like state. (A,B) HCA2 fibroblasts were treated using the indicated concentrations of MI-63 (A) or nutlin-3a (B). p53 and p21 levels were analyzed by western blotting. Actin levels served as a loading control. (C) IMR-90 fibroblasts were transduced with a p53 luciferase reporter and treated with MI-63 or nutlin-3a. Extracts were prepared and analyzed by luminometry. (D,E) HCA2 cells were treated with MI-63 or induced to senesce by 10 Gy IR. SA-β-gal staining is shown in (D) and percentage of positive cells is shown in (E). (F,G) HCA2 cells were treated with nutlin-3a or induced to senesce by 10 Gy IR. SA-β-gal staining is shown in (F) and percentage of positive cells is shown in (G). (H-I) HCA2 cells were irradiated (IR) or treated with MI-63 or nutlin-3a, and immunostained for γH2AX. Representative images are shown in (H) and percentage of cells with >3 γH2AX nuclear foci is shown in (I). Data are representative of two independent experiments. ***p < 0.001 by 1-way ANOVA.
Figure 1 MDM2 inhibitors induce a senescence-like state. (A,B) HCA2 fibroblasts were treated using the indicated concentrations of MI-63 (A) or nutlin-3a (B). p53 and p21 levels were analyzed by western blotting. Actin levels served as a loading control. (C) IMR-90 fibroblasts were transduced with a p53 luciferase reporter and treated with MI-63 or nutlin-3a. Extracts were prepared and analyzed by luminometry. (D,E) HCA2 cells were treated with MI-63 or induced to senesce by 10 Gy IR. SA-β-gal staining is shown in (D) and percentage of positive cells is shown in (E). (F,G) HCA2 cells were treated with nutlin-3a or induced to senesce by 10 Gy IR. SA-β-gal staining is shown in (F) and percentage of positive cells is shown in (G). (H-I) HCA2 cells were irradiated (IR) or treated with MI-63 or nutlin-3a, and immunostained for γH2AX. Representative images are shown in (H) and percentage of cells with >3 γH2AX nuclear foci is shown in (I). Data are representative of two independent experiments. ***p < 0.001 by 1-way ANOVA.CC BY · PubMed Central
Figure 2 Reversibility of MDM2 antagonist-induced senescence. (A,B) Proliferating HCA2 fibroblasts were treated with DMSO or nutlin-3a, and cell number measured over the ensuing 6 d, at which point nutlin-3a was either replaced or exchanged for DMSO (Release) for an additional 6 d. Representative images of the cell cultures are shown in (A) and cell numbers are shown in (B). (C–E) IMR-90 fibroblasts were treated as in (A,B), and proliferation measured by EdU incorporation (24 h labeling period). (C) Representative EdU labeling images. (D) Percentage of EdU-positive cells. (E) Percentage of cells that stained positive for SA-β-gal. (F,H) IMR-90 cells were cultured in 3% or 20% O2, and DMSO or nutlin-3a for 6. Cells were then reseeded in drug-free medium and cultured for additional 6 d, followed by analysis for (F) clonogenic growth, (G) EdU labeling (24 h), and (H) SA-β-gal. Data are representative of three independent experiments. ***p < 0.001 by 1-way ANOVA or t-test.
Figure 2 Reversibility of MDM2 antagonist-induced senescence. (A,B) Proliferating HCA2 fibroblasts were treated with DMSO or nutlin-3a, and cell number measured over the ensuing 6 d, at which point nutlin-3a was either replaced or exchanged for DMSO (Release) for an additional 6 d. Representative images of the cell cultures are shown in (A) and cell numbers are shown in (B). (C–E) IMR-90 fibroblasts were treated as in (A,B), and proliferation measured by EdU incorporation (24 h labeling period). (C) Representative EdU labeling images. (D) Percentage of EdU-positive cells. (E) Percentage of cells that stained positive for SA-β-gal. (F,H) IMR-90 cells were cultured in 3% or 20% O2, and DMSO or nutlin-3a for 6. Cells were then reseeded in drug-free medium and cultured for additional 6 d, followed by analysis for (F) clonogenic growth, (G) EdU labeling (24 h), and (H) SA-β-gal. Data are representative of three independent experiments. ***p < 0.001 by 1-way ANOVA or t-test.CC BY · PubMed Central
Figure 3 MDM2 antagonist attenuates the SASP. (A) IL-6 ELISA using conditioned media (CM) from HCA2 and IMR-90 fibroblasts that were induced to senesce by IR and treated with DMSO or nutlin-3a at two concentrations. (B) IL-6 in CM from IMR-90 cells (mock or IR) after treatment with DMSO, rapamycin (RAPA) or MI-63. (C,D) IMR-90 cells were treated for 24 h with the chemotherapeutic agents doxorubicin (DOXO) (C), taxol or cisplatin (D), and IL-6 secretion measured by ELISA in cells cultured with MI-63 (C) or nutlin-3a (D) for 24 h following removal of the drugs. (E) Heat map indicating gene expression of several SASP factors in IMR-90 fibroblasts (mock or IR) treated DMSO or nutlin-3a. (F,G) Relative expression of (F) IL-1α and (G) IL-1β in control or irradiated (IR) fibroblasts cultured with nutlin-3a or DMSO. (H) HCA2 cells were transduced with a lentiviral NF-κB luciferase reporter construct, and luciferase activity in DMSO- or nutlin-3a-treated (mock or IR) cells was measured. (I) HCA2 fibroblasts were transduced with lentiviruses expressing either control (shGFP) or p53-targeted shRNAs (shp53) or a p53 genetic suppressor element (GSE22) and treated with DMSO or nutlin-3a for 24 h following mock or X-irradiation (IR). IL-6 secretion was measured by ELISA. Data are representative of three independent experiments. *p < 0.05, ***p < 0.001 by 2-way ANOVA.
Figure 3 MDM2 antagonist attenuates the SASP. (A) IL-6 ELISA using conditioned media (CM) from HCA2 and IMR-90 fibroblasts that were induced to senesce by IR and treated with DMSO or nutlin-3a at two concentrations. (B) IL-6 in CM from IMR-90 cells (mock or IR) after treatment with DMSO, rapamycin (RAPA) or MI-63. (C,D) IMR-90 cells were treated for 24 h with the chemotherapeutic agents doxorubicin (DOXO) (C), taxol or cisplatin (D), and IL-6 secretion measured by ELISA in cells cultured with MI-63 (C) or nutlin-3a (D) for 24 h following removal of the drugs. (E) Heat map indicating gene expression of several SASP factors in IMR-90 fibroblasts (mock or IR) treated DMSO or nutlin-3a. (F,G) Relative expression of (F) IL-1α and (G) IL-1β in control or irradiated (IR) fibroblasts cultured with nutlin-3a or DMSO. (H) HCA2 cells were transduced with a lentiviral NF-κB luciferase reporter construct, and luciferase activity in DMSO- or nutlin-3a-treated (mock or IR) cells was measured. (I) HCA2 fibroblasts were transduced with lentiviruses expressing either control (shGFP) or p53-targeted shRNAs (shp53) or a p53 genetic suppressor element (GSE22) and treated with DMSO or nutlin-3a for 24 h following mock or X-irradiation (IR). IL-6 secretion was measured by ELISA. Data are representative of three independent experiments. *p < 0.05, ***p < 0.001 by 2-way ANOVA.CC BY · PubMed Central
Figure 4 Effect of MDM2 inhibitors on ability of senescent cells to stimulate cancer cell aggressiveness. (A,B) ZR75-1 human breast cancer cells were cultured in the presence of CM from DMSO- or MI-63-treated (mock or IR) HCA2 fibroblasts for 3 d, and immunostained for ZO-1, cytokeratin 18 or vimentin. (A) Representative images of the staining. (B) Quantification of staining (percentage of positive cells). (C) CM described in (A) was assayed for ability to stimulate ZR75-1 cancer cells to invade a basement membrane. Invasion stimulated by CM from DMSO-treated mock fibroblasts was given a value of one, and other conditions were normalized to this value. Data are means of 4 independent experiments (*p < 0.05, t-test). Error bars indicate standard deviation of the mean.
Figure 4 Effect of MDM2 inhibitors on ability of senescent cells to stimulate cancer cell aggressiveness. (A,B) ZR75-1 human breast cancer cells were cultured in the presence of CM from DMSO- or MI-63-treated (mock or IR) HCA2 fibroblasts for 3 d, and immunostained for ZO-1, cytokeratin 18 or vimentin. (A) Representative images of the staining. (B) Quantification of staining (percentage of positive cells). (C) CM described in (A) was assayed for ability to stimulate ZR75-1 cancer cells to invade a basement membrane. Invasion stimulated by CM from DMSO-treated mock fibroblasts was given a value of one, and other conditions were normalized to this value. Data are means of 4 independent experiments (*p < 0.05, t-test). Error bars indicate standard deviation of the mean.CC BY · PubMed Central

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Metadata

Authors
Wiley, C. D., Schaum, N., Alimirah, F., Lopez-Dominguez, J. A., Orjalo, A. V., Scott, G., Desprez, P.-Y., Benz, C., Davalos, A. R., & Campisi, J.
Affiliations
Buck Institute for Research on Aging (US)
Corresponding author
  • Davalos, Albert R.
  • Campisi, Judith
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2018-02-05
Volume
8
Issue
1
Pages
2410-2410
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-018-20000-4
PMC
PMC5799282
OpenAlex
W2802384001
Cited by
124
References
55
Citations by year
2026: 10 · 2025: 10 · 2024: 13 · 2023: 8 · 2022: 25 · 2021: 25 · 2020: 20 · 2019: 8 · 2018: 5
FWCI
9.97
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
Funders
  • National Institutes of Health (AG09909, AG017242)
  • National Institute on Aging (R56AG009909, R37AG009909, P01AG017242)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Telomeres, Telomerase, and Senescence
  • Health Sciences › Medicine › Oncology › Cancer-related Molecular Pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Aging › Genetics, Aging, and Longevity in Model Organisms
Keywords
MDM2 inhibitors, senescence-associated secretory phenotype, cellular senescence, p53, Nutlin-3a, interleukin-6, IL-1α, inflammation, breast cancer progression, genotoxic stress, aging
MeSH terms
Interleukin-6 Inhibitors, Cell Line, Enzyme Inhibitors, Epithelial Cells, Fibroblasts, Gamma Rays, Humans, Imidazoles, Indoles, Lung, Male, Piperazines, Spiro Compounds, Interleukin-6, Tumor Suppressor Protein p53, Cellular Senescence, Proto-Oncogene Proteins c-mdm2, Foreskin, Interleukin-1alpha, Cell Cycle Checkpoints

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