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

Nature Cell Biology volume 17, pages 1049–1061 (2015)Cite this article

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An Author Correction to this article was published on 06 April 2021

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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.

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Figure 1: Rapamycin decreases the SASP through MTORC1.
Figure 2: SASP production is MTOR dependent.
Figure 3: Rapamycin suppresses NF-κB transcriptional activity.
Figure 4: Rapamycin suppresses IL1A signalling.
Figure 5: Rapamycin inhibits IL1A translation.
Figure 6: Rapamycin does not reverse cellular senescence.
Figure 7: Sustained effect of rapamycin on the SASP.
Figure 8: Rapamycin suppresses the tumour-promoting activity of senescent cells.

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References

  1. Vijg, J. & Campisi, J. Puzzles, promises and a cure for ageing. Nature 454, 1065–1071 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Kapahi, P. et al. With TOR, less is more: a key role for the conserved nutrient-sensing TOR pathway in aging. Cell Metab. 11, 453–465 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Stanfel, M. N., Shamieh, L. S., Kaeberlein, M. & Kennedy, B. K. The TOR pathway comes of age. Biochim. Biophys. Acta 1790, 1067–1074 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Harrison, D. E. et al. Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature 460, 392–395 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Campisi, J. & d’Adda di Fagagna, F. Cellular senescence: when bad things happen to good cells. Nat. Rev. Mol. Cell Biol. 8, 729–740 (2007).

    Article  CAS  PubMed  Google Scholar 

  6. Acosta, J. C. et al. Chemokine signaling via the CXCR2 receptor reinforces senescence. Cell 133, 1006–1018 (2008).

    Article  CAS  PubMed  Google Scholar 

  7. Bavik, C. et al. The gene expression program of prostate fibroblast senescence modulates neoplastic epithelial cell proliferation through paracrine mechanisms. Cancer Res. 66, 794–802 (2006).

    Article  CAS  PubMed  Google Scholar 

  8. Coppe, J. P. et al. A human-like senescence-associated secretory phenotype is conserved in mouse cells dependent on physiological oxygen. PLoS ONE 5, e9188 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Coppe, J. P. et al. Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol. 6, 2853–2868 (2008).

    CAS  PubMed  Google Scholar 

  10. Kuilman, T. et al. Oncogene-induced senescence relayed by an interleukin-dependent inflammatory network. Cell 133, 1019–1031 (2008).

    Article  CAS  PubMed  Google Scholar 

  11. Orjalo, A. V., Bhaumik, D., Gengler, B. K., Scott, G. K. & Campisi, J. Cell surface-bound IL-1α is an upstream regulator of the senescence-associated IL-6/IL-8 cytokine network. Proc. Natl Acad. Sci. USA 106, 17031–17036 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Baker, D. J. et al. Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature 479, 232–236 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Krtolica, A., Parrinello, S., Lockett, S., Desprez, P. & Campisi, J. Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging. Proc. Natl Acad. Sci. USA 98, 12072–12077 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Liu, D. & Hornsby, P. J. Senescent human fibroblasts increase the early growth of xenograft tumors via matrix metalloproteinase secretion. Cancer Res. 67, 3117–3126 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Freund, A., Patil, C. K. & Campisi, J. p38MAPK is a novel DNA damage response-independent regulator of the senescence-associated secretory phenotype. EMBO J. 30, 1536–1548 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Vasto, S. et al. Inflammation, ageing and cancer. Mech. Ageing Dev. 130, 40–45 (2009).

    Article  CAS  PubMed  Google Scholar 

  17. de Visser, K. E., Eichten, A. & Coussens, L. M. Paradoxical roles of the immune system during cancer development. Nat. Rev. Cancer 6, 24–37 (2006).

    Article  CAS  PubMed  Google Scholar 

  18. Glass, C. K., Saijo, K., Winner, B., Marchetto, M. C. & Gage, F. H. Mechanisms underlying inflammation in neurodegeneration. Cell 140, 918–934 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Nathan, C. & Ding, A. Nonresolving inflammation. Cell 140, 871–882 (2010).

    Article  CAS  PubMed  Google Scholar 

  20. Grivennikov, S. I., Greten, F. R. & Karin, M. Immunity, inflammation, and cancer. Cell 140, 883–899 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Franceschi, C. et al. Inflammaging and anti-inflammaging: a systemic perspective on aging and longevity emerged from studies in humans. Mech. Ageing Dev. 128, 92–105 (2007).

    Article  CAS  PubMed  Google Scholar 

  22. McElhaney, J. E. & Effros, R. B. Immunosenescence: what does it mean to health outcomes in older adults? Curr. Opin. Immunol. 21, 418–424 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Coppé, J. P., Desprez, P. Y., Krtolica, A. & Campisi, J. The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu. Rev. Pathol. 5, 99–118 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Freund, A., Orjalo, A., Desprez, P. Y. & Campisi, J. Inflammatory networks during cellular senescence: causes and consequences. Trends Mol. Med. 16, 238–248 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. d’Adda di Fagagna, F. Living on a break: cellular senescence as a DNA-damage response. Nat. Rev. Cancer 8, 512–522 (2008).

    Article  PubMed  CAS  Google Scholar 

  26. Beausejour, C. M. et al. Reversal of human cellular senescence: roles of the p53 and p16 pathways. EMBO J. 22, 4212–4222 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kortlever, R. M., Higgins, P. J. & Bernards, R. Plasminogen activator inhibitor-1 is a critical downstream target of p53 in the induction of replicative senescence. Nat. Cell Biol. 8, 877–884 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Wajapeyee, N., Serra, R. W., Zhu, X., Mahalingam, M. & Green, M. R. Oncogenic BRAF induces senescence and apoptosis through pathways mediated by the secreted protein IGFBP7. Cell 132, 363–374 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Binet, R. et al. WNT16B is a new marker of cellular senescence that regulates p53 activity and the phosphoinositide 3-kinase/AKT pathway. Cancer Res. 69, 9183–9191 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Chang, B. D. et al. Molecular determinants of terminal growth arrest induced in tumor cells by a chemotherapeutic agent. Proc. Natl Acad. Sci. USA 99, 389–394 (2002).

    Article  CAS  PubMed  Google Scholar 

  31. Coppe, J. P., Kauser, K., Campisi, J. & Beausejour, C. M. Secretion of vascular endothelial growth factor by primary human fibroblasts at senescence. J. Biol. Chem. 281, 29568–29574 (2006).

    Article  CAS  PubMed  Google Scholar 

  32. Krizhanovsky, V. et al. Senescence of activated stellate cells limits liver fibrosis. Cell 134, 657–667 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Novakova, Z. et al. Cytokine expression and signaling in drug-induced cellular senescence. Oncogene 29, 273–284 (2010).

    Article  CAS  PubMed  Google Scholar 

  34. Parrinello, S., Coppe, J. P., Krtolica, A. & Campisi, J. Stromal–epithelial interactions in aging and cancer: senescent fibroblasts alter epithelial cell differentiation. J. Cell Sci. 118, 485–496 (2005).

    Article  CAS  PubMed  Google Scholar 

  35. Dimri, G. P. et al. A novel biomarker identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl Acad. Sci. USA 92, 9363–9367 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Jeyapalan, J. C., Ferreira, M., Sedivy, J. M. & Herbig, U. Accumulation of senescent cells in mitotic tissue of aging primates. Mech. Ageing Dev. 128, 36–44 (2007).

    Article  CAS  PubMed  Google Scholar 

  37. Sedelnikova, O. A. et al. Senescing human cells and ageing mice accumulate DNA lesions with unrepairable double-strand breaks. Nat. Cell Biol. 6, 168–170 (2004).

    Article  CAS  PubMed  Google Scholar 

  38. Castro, P., Giri, D., Lamb, D. & Ittmann, M. Cellular senescence in the pathogenesis of benign prostatic hyperplasia. Prostate 55, 30–38 (2003).

    Article  CAS  PubMed  Google Scholar 

  39. Collado, M. et al. Tumor biology: senescent in premalignant tumours. Nature 436, 642 (2005).

    Article  CAS  PubMed  Google Scholar 

  40. Ding, G. et al. Tubular cell senescence and expression of TGF-β1 and p21(WAF1/CIP1) in tubulointerstitial fibrosis of aging rats. Exp. Mol. Path. 70, 43–53 (2001).

    Article  CAS  Google Scholar 

  41. Erusalimsky, J. D. & Kurz, D. J. Cellular senescence in vivo: its relevance in ageing and cardiovascular disease. Exp. Gerontol. 40, 634–642 (2005).

    Article  CAS  PubMed  Google Scholar 

  42. Martin, J. A. & Buckwalter, J. A. The role of chondrocyte senescence in the pathogenesis of osteoarthritis and in limiting cartilage repair. J. Bone Joint Surg. Am. 85, 106–110 (2003).

    Article  PubMed  Google Scholar 

  43. Matthews, C. et al. Vascular smooth muscle cells undergo telomere-based senescence in human atherosclerosis: effects of telomerase and oxidative stress. Circ. Res. 99, 156–164 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. McGlynn, L. M. et al. Cellular senescence in pretransplant renal biopsies predicts postoperative organ function. Aging Cell 8, 45–51 (2009).

    Article  CAS  PubMed  Google Scholar 

  45. Paradis, V. et al. Replicative senescence in normal liver, chronic hepatitis C, and hepatocellular carcinomas. Hum. Pathol. 32, 327–332 (2001).

    Article  CAS  PubMed  Google Scholar 

  46. Roberts, S., Evans, E. H., Kletsas, D., Jaffray, D. C. & Eisenstein, S. M. Senescence in human intervertebral discs. Eur. Spine J. 15, 312–316 (2006).

    Article  PubMed Central  Google Scholar 

  47. Davalos, A. R., Coppe, J. P., Campisi, J. & Desprez, P. Y. Senescent cells as a source of inflammatory factors for tumor progression. Cancer Metastasis Rev. 29, 273–283 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Schmitt, C. A. Cellular senescence and cancer treatment. Biochim. Biophys. Acta 1775, 5–20 (2007).

    CAS  PubMed  Google Scholar 

  49. Schmitt, C. A. et al. A senescence program controlled by p53 and p16INK4a contributes to the outcome of cancer therapy. Cell 109, 335–346 (2002).

    Article  CAS  PubMed  Google Scholar 

  50. te Poele, R. H., Okorokov, A. L., Jardine, L., Cummings, J. & Joel, S. P. DNA damage is able to induce senescence in tumor cells in vitro and in vivo. Cancer Res. 62, 1876–1883 (2002).

    CAS  PubMed  Google Scholar 

  51. Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D. & Lowe, S. W. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 88, 593–602 (1997).

    Article  CAS  PubMed  Google Scholar 

  52. Ogryzko, V. V., Hirai, T. H., Russanova, V. R., Barbie, D. A. & Howard, B. H. Human fibroblast commitment to a senescence-like state in response to histone deacetylase inhibitors is cell cycle dependent. Mol. Cell. Biol. 16, 5210–5218 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Rodier, F. et al. Persistent DNA damage signaling triggers senescence-associated inflammatory cytokine secretion. Nat. Cell Biol. 11, 973–979 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Rodier, F. et al. DNA-SCARS: distinct nuclear structures that sustain damage-induced senescence growth arrest and inflammatory cytokine secretion. J. Cell Sci. 124, 68–81 (2011).

    Article  CAS  PubMed  Google Scholar 

  55. Choo, A. Y., Yoon, S. O., Kim, S. G., Roux, P. P. & Blenis, J. Rapamycin differentially inhibits S6Ks and 4E-BP1 to mediate cell-type-specific repression of mRNA translation. Proc. Natl Acad. Sci. USA 105, 17414–17419 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Apsel, B. et al. Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases. Nat. Chem. Biol. 4, 691–699 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Parsyan, A. et al. mRNA helicases: the tacticians of translational control. Nat. Rev. Mol. Cell Biol. 12, 235–245 (2011).

    Article  CAS  PubMed  Google Scholar 

  58. Feoktistova, K., Tuvshintogs, E., Do, A. & Fraser, C. S. Human eIF4E promotes mRNA restructuring by stimulating eIF4A helicase activity. Proc. Natl Acad. Sci. USA 110, 13339–13344 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Demidenko, Z. N. et al. Rapamycin decelerates cellular senescence. Cell Cycle 8, 1888–1895 (2009).

    Article  CAS  PubMed  Google Scholar 

  60. Sun, Y. et al. Treatment-induced damage to the tumor microenvironment promotes prostate cancer therapy resistance through WNT16B. Nat. Med. 18, 1359–1368 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Chang, B. D. et al. A senescence-like phenotype distinguishes tumor cells that undergo terminal proliferation arrest after exposure to anticancer agents. Cancer Res. 59, 3761–3767 (1999).

    CAS  PubMed  Google Scholar 

  62. Sheiban, I. et al. Next-generation drug-eluting stents in coronary artery disease: focus on everolimus-eluting stent (Xience V). Vasc. Health Risk Manage. 4, 31–38 (2008).

    Article  CAS  Google Scholar 

  63. Ciuffreda, L., Di Sanza, C., Incani, U. C. & Milella, M. The mTOR pathway: a new target in cancer therapy. Curr. Cancer Drug Targets 10, 484–495 (2010).

    Article  CAS  PubMed  Google Scholar 

  64. Meric-Bernstam, F. & Gonzalez-Angulo, A. M. Targeting the mTOR signaling network for cancer therapy. J. Clin. Oncol. 27, 2278–2287 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Xue, W. et al. Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas. Nature 445, 656–660 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Finkel, T., Serrano, M. & Blasco, M. A. The common biology of cancer and ageing. Nature 448, 767–774 (2007).

    Article  CAS  PubMed  Google Scholar 

  67. Huang, S., Bjornsti, M. A. & Houghton, P. J. Rapamycins: mechanism of action and cellular resistance. Cancer Biol. Ther. 2, 222–232 (2003).

    Article  CAS  PubMed  Google Scholar 

  68. Weichhart, T. et al. The TSC-mTOR signaling pathway regulates the innate inflammatory response. Immunity 29, 565–577 (2008).

    Article  CAS  PubMed  Google Scholar 

  69. Hayward, S. W. et al. Establishment and characterization of an immortalized but non-transformed human prostate epithelial cell line: BPH-1. In Vitro Cell. Dev. Biol. Anim. 31, 14–24 (1995).

    Article  CAS  PubMed  Google Scholar 

  70. Bae, V. L. et al. Metastatic sublines of an SV40 large T antigen immortalized human prostate epithelial cell line. Prostate 34, 275–282 (1998).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank A. Rogers, B. Kennedy, G. Lithgow, S. Melov and members of the Campisi laboratory (Buck Institute) for comments and discussions, I. Coleman for assistance with figure preparation and data analysis, and R. Strong (U Texas Health Science Center) for providing the rapamycin encapsulated chow. This work was supported by grants from the National Institutes of Health (NIH) AG045288 to R.-M.L., Hillblom Medical Foundation (CP), NIH T32 training grant AG000266-16 to K.A.W.-E., NIH grants CA143858, CA155679 and CA071468 to C.C.B., NIH grants AG032113 and AG025901 to P.K., DOD-PCRP grant PC111703 and National Natural Science Foundation of China 81472709 to Y.S., NIH grants CA164188, CA165573 and CA097186 and the Prostate Cancer Foundation to P.S.N., and NIH grants AG09909 and AG017242 to J.C. and AG041122 to J.C. and Y.I.

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Author notes

  1. Pankaj Kapahi and Peter S. Nelson: These authors contributed equally to this work.

Authors and Affiliations

  1. Buck Institute for Research on Aging, Novato, California 94945, USA

    Remi-Martin Laberge, Arturo V. Orjalo, Christopher K. Patil, Adam Freund, Lili Zhou, Samuel C. Curran, Albert R. Davalos, Kathleen A. Wilson-Edell, Su Liu, Chandani Limbad, Marco Demaria, Patrick Li, Pierre-Yves Desprez, Christopher C. Benz, Pankaj Kapahi & Judith Campisi

  2. Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA

    Yu Sun & Peter S. Nelson

  3. Key Lab of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

    Yu Sun

  4. Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, USA

    Gene B. Hubbard & Yuji Ikeno

  5. Department of Pathology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, USA

    Gene B. Hubbard & Yuji Ikeno

  6. Research Service, San Antonio, Texas 78229, USA

    Yuji Ikeno

  7. GRECC, Audie Murphy VA Hospital (STVHCS), San Antonio, Texas 78229, USA

    Yuji Ikeno

  8. Department of Psychiatry, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, USA

    Martin Javors

  9. California Pacific Medical Center, Research Institute, San Francisco, California 94107, USA

    Pierre-Yves Desprez

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  1. Remi-Martin Laberge
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Contributions

R.-M.L., Y.S., A.V.O., C.K.P., A.F., L.Z., S.C.C., A.R.D., K.A.W.-E., S.L., C.L., M.D. and P.L. acquired and interpreted the data. M.J. analysed rapamycin concentrations. G.B.H. and Y.I. provided pathological assessment. R.-M.L., Y.S., C.C.B., P.-Y.D., P.K., P.S.N. and J.C. designed experiments and interpreted data. R.-M.L., Y.S., P.-Y.D., P.K., P.S.N. and J.C. wrote the paper.

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Correspondence to Judith Campisi.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 4 Rapamycin suppresses the SASP in multiple cell strains and lines.

(A) IL-6 secretion by IR-induced senescent cells, normalized to replicatively senescent IMR-90 normal fetal lung human fibroblasts. Shown are WI-38 normal fetal lung fibroblasts, MCF10-A and 184A1 immortal human breast epithelial cells and PSC27 normal adult prostate fibroblasts, treated with either DMSO (blue bars) or rapamycin (red bars). (B) We treated non-senescent (NS) HCA2 cells with rapamycin for 6 days, collected CM and analyzed IL-6 by ELISA. IL-6 secretion by irradiated senescent cells (Sen (IR)) is shown for comparison. (C) Immunofluorescence staining for 53BP1 was compared between NS and senescent (Sen IR) cells treated with DMSO or rapamycin (one representative image is shown). (D) We determined the number of foci in NS or senescent (Sen IR) cells treated with DMSO or rapamycin. With the exception of 184A1 experiment which was performed once, for panels A, B and D, shown is one representative of two or more independent experiments, each with triplicate samples. Raw data could be found in Supplementary Table 4.

Supplementary Figure 5 The SASP is MTOR dependent.

(A) HCA2 cells were infected with lentiviruses expressing shRNAs against GFP (shGFP; control) or one of three different shRNAs against raptor. Raptor transcript levels were measured and are shown relative the level in cells expressing shGFP. (B) HCA2 cells were infected with lentiviruses expressing shGFP (control) or one of three different shRNA against MTOR. The relative transcript level of MTOR was measured. (C) Normal HCA2 human foreskin fibroblasts, non-senescent (NS) or induced to senesce by IR, were treated with DMSO (control) or the indicated concentrations of the MTOR kinase inhibitor PP242. 7 days later, CM was collected and analyzed for IL-6 by ELISA. Values were normalized to the senescent cell level. For all panels, shown is one representative of two independent experiments, each with triplicate samples. Raw data could be found in Supplementary Table 4.

Supplementary Figure 6 Rapamycin reduces SASP transcript levels and NF-kB activity in senescent prostate fibroblasts.

(A) Transcript levels of indicated SASP factors were quantified by qRT-PCR in NS and Sen (IR) PSC27 fibroblasts with or without treatment with rapamycin for 7 days. (B) NF-κB activity was measured after treatment with rapamycin for 7 days using a reporter assay. For all panels, shown is one representative of three independent experiments, each with triplicate samples. Raw data could be found in Supplementary Table 4.

Supplementary Figure 7 FACS analysis of cell surface IL-1α.

(A) Flow cytometry for cell surface IL-1α was performed using NS or senescent (IR) HCA2 cells expressing shRNAs against GFP or raptor and treated with DMSO or rapamycin and a FITC-tagged antibody. Slope of a trend-line of fluorescence over forward scattering (FSC) was determined to discriminate fluorescence intensity from the effect of cell size (shown is the result of one of two independent experiments, 10,000 cells were counter before gating). (B) HCA2 cells were infected with a lentivirus expressing shRNA against IL-1 α and the relative IL-1 α mRNA level was measured. Shown is one representative of two independent experiments, each with triplicate samples. Raw data could be found in Supplementary Table 4.

Supplementary Figure 8 Translation status of various transcripts after rapamycin treatment.

(A) After IR, senescent (Sen (IR)) HCA2 cells were treated for 1 day with rapamycin or DMSO followed by 1 day in serum-free media, after which cells were harvested and mRNA collected for polysome profiling. qPCR was performed on each fraction for IL1A, IL1B, IL6, IL8, IL3, IL5, TIMP1, CCL13 and TUBA1A mRNA (one representative experiment is shown). Fractions 1–7: Free RNA; 8–12: 40-60S; 13–20: polysomes. (B) NS or senescent (Sen (IR)) HCA2 cells were cultured for 7 days and then treated with DMSO or rapamycin for 4 h, after which cells were harvested and mRNA collected for polysome profiling. qPCR was performed on each fraction for IL1A, TUBA1A, IL6, and EEF2 mRNA (one representative experiment is shown). Fractions 1–4: Free RNA; 5–6: 40-60S; 7–11: polysomes. Right panel: polysome profiles used to determine the translated fractions. All polysome profile data are based on at least two independent replicates; representative polysome traces are shown. Raw data could be found in Supplementary Table 4.

Supplementary Figure 9 MTORC1 inhibition does not reverse the senescence growth arrest.

(A) Effect of rapamycin on the number of senescent cells with detectable senescence-associated β-gal (SA-β-gal) activity. (B) Proliferative potential of PSC27 fibroblasts was measured under the indicated culture conditions. (C) PSC27 cells, NS or made senescent by IR 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) SA-β-gal expression was determined for the cell populations described in Fig. 1 (one representative experiment is shown). (E) Clonogenic assays were performed on HCA2 cells to compare the effects of rapamycin and DMSO on NS cells or cells irradiated at 6, 8 or 10 Gy (one representative experiment is shown). (F) BJ and HCA2 human fibroblasts were irradiated at 5 or 10 Gy and clonogenic assays performed in the presence of DMSO or rapamycin (one representative experiment is shown). For panels A, B and C, shown is one representative of three independent experiments, each with triplicate cell culture samples. For panel D, E and F, shown is one clonogenic assay experiment replicated once. Raw data could be found in Supplementary Table 4.

Supplementary Figure 10 Model for SASP suppression by rapamycin.

(1) Senescence signals activate IL-1α transcription; (2) IL-1α translation is MTOR-dependent and sensitive to rapamycin; (3) IL-1α at the plasma membrane binds the IL1R; (4) IL1R occupancy activates IL1R signaling; (5) IL1R signaling releases IκB, allowing NF-κB translocation to the nucleus, where it activates the transcription of genes encoding SASP factors; (6) SASP factors are transcribed, translated and secreted.

Supplementary Figure 11 Rapamycin suppresses tumour cell growth.

(A) Experimental timeline corresponding to Fig. 8a–c, e. (B) Experimental timeline corresponding to Fig. 8d. (C) PC3 prostate tumour cells were implanted subcutaneously with or without PSC27 prostate fibroblasts, and tumour sizes were measured every 2 weeks. PC3 cells were either exposed to DMSO or rapamycin ex vivo before implantation. PSC27 cells were exposed to ionizing radiation (IR), rapamycin (Rapa) or both ex vivo before implantation (n = 8 per type of treatment). (D) Timeline corresponding to the cell culture experiment presented in Fig. 8f. (E) Timeline corresponding to the in vivo experiment presented in Fig. 8f. Scheduled timing of mitoxantrone given as 3 doses 2 weeks apart, and rapamycin given every 2 days, to SCID mice over the course of an 8 week regimen. The mice were engrafted with PC3 cells alone, or combined with either PSC27-NS (control fibroblasts, without pre-treatment) or PSC27-Sen (IR) (fibroblasts pretreated with IR in culture). At the end of the treatment period, tumours were excised and volumes were determined, with 8-10 mice used per treatment arm. (F) PC3 prostate tumour cells were implanted subcutaneously with or without PSC27 prostate fibroblasts. After 2 weeks of tumour growth, mice were treated with vehicle (control), rapamycin (Rapa) and/or mitoxantrone (MIT). Tumour sizes were measured every 2 weeks (n = 10 per type of treatment). (G) (1) Tumour cells (orange) are surrounded by stromal cells (grey); (2) Treatment with DNA-damaging chemotherapy induces senescence in the stroma (blue cell). The SASP (red arrows) from these senescent cells fuels the proliferation of the remaining tumour cells; (3) Rapamycin reduces the intensity of the SASPs induced by chemotherapy, and tumour cell proliferation is decreased.

Supplementary Table 1 shRNA list.
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Supplementary Table 2 Antibodies used for western blotting.
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Supplementary Table 3 Primers and probes list.
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Laberge, RM., Sun, Y., Orjalo, A. et al. MTOR regulates the pro-tumorigenic senescence-associated secretory phenotype by promoting IL1A translation. Nat Cell Biol 17, 1049–1061 (2015). https://doi.org/10.1038/ncb3195

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