PLoS BIOLOGY Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor Jean-Philippe Coppe 1 , Christopher K. Patil 1[ , Francis Rodier 1,2[ , Yu Sun 3 , Denise P. Mun oz 1,2 , Joshua Goldstein 1¤ , Peter S. Nelson 3 , Pierre-Yves Desprez 1,4 , Judith Campisi 1,2* 1 Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America, 2 Buck Institute for Age Research, Novato, California, United States of America, 3 Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 4 California Pacific Medical Center Research Institute, San Francisco, California, United States of America Cellular senescence suppresses cancer by arresting cell proliferation, essentially permanently, in response to oncogenic stimuli, including genotoxic stress. We modified the use of antibody arrays to provide a quantitative assessment of factors secreted by senescent cells. We show that human cells induced to senesce by genotoxic stress secrete myriad factors associated with inflammation and malignancy. This senescence-associated secretory phenotype (SASP) developed slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. Remarkably similar SASPs developed in normal fibroblasts, normal epithelial cells, and epithelial tumor cells after genotoxic stress in culture, and in epithelial tumor cells in vivo after treatment of prostate cancer patients with DNA- damaging chemotherapy. In cultured premalignant epithelial cells, SASPs induced an epithelial–mesenchyme transition and invasiveness, hallmarks of malignancy, by a paracrine mechanism that depended largely on the SASP factors interleukin (IL)-6 and IL-8. Strikingly, two manipulations markedly amplified, and accelerated development of, the SASPs: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated the promalignant paracrine activities of the SASPs. Our findings define a central feature of genotoxic stress-induced senescence. Moreover, they suggest a cell-nonautonomous mechanism by which p53 can restrain, and oncogenic RAS can promote, the development of age-related cancer by altering the tissue microenvironment. Citation: Coppe JP, Patil CK, Rodier F, Sun Y, Mun oz DP, et al. (2008) Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol 6(12): e301. doi:10.1371/journal.pbio.0060301 Introduction Cancer is a multistep disease in which cells acquire increasingly malignant phenotypes. These phenotypes are acquired in part by somatic mutations, which derange normal controls over cell proliferation (growth), survival, invasion, and other processes important for malignant tumorigenesis [1]. In addition, there is increasing evidence that the tissue microenvironment is an important determinant of whether and how malignancies develop [2,3]. Normal tissue environ- ments tend to suppress malignant phenotypes, whereas abnormal tissue environments such at those caused by inﬂammation can promote cancer progression. Cancer development is restrained by a variety of tumor suppressor genes. Some of these genes permanently arrest the growth of cells at risk for neoplastic transformation, a process termed cellular senescence [4–6]. Two tumor suppressor pathways, controlled by the p53 and p16INK4a/pRB proteins, regulate senescence responses. Both pathways integrate multiple aspects of cellular physiology and direct cell fate towards survival, death, proliferation, or growth arrest, depending on the context [7,8]. Several lines of evidence indicate that cellular senescence is a potent tumor-suppressive mechanism [4,9,10]. Many poten- tially oncogenic stimuli (e.g., dysfunctional telomeres, DNA PLoS Biology | www.plosbiology.org damage, and certain oncogenes) induce senescence [6,11]. Moreover, mutations that dampen the p53 or p16INK4a/pRB pathways confer resistance to senescence and greatly increase cancer risk [12,13]. Most cancers harbor mutations in one or both of these pathways [14,15]. Lastly, in mice and humans, a senescence response to strong mitogenic signals, such as those delivered by certain oncogenes, prevents premalignant lesions from progressing to malignant cancers [16–19]. Academic Editor: Julian Downward, Cancer Research UK, United Kingdom Received June 27, 2008; Accepted October 22, 2008; Published December 2, 2008 Copyright: O 2008 Coppe et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abbreviations: CM, conditioned medium; DDR, DNA damage response; ELISA, enzyme-linked immunosorbent assay; EMT, epithelial–mesenchymal transition; GSE, genetic suppressor element; IL, interleukin; MIT, mitoxantrone; PRE, presenescent; PrEC, normal human prostate epithelial cell; REP, replicative exhaustion; SASP, senescence-associated secretory phenotype; SEN, senescent; shRNA, short hairpin RNA; XRA, X-irradiation * To whom correspondence should be addressed. E-mail: jcampisi@lbl.gov [ These authors contributed equally to this work. ¤ Current address: Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America December 2008 | Volume 6 | Issue 12 | e301

Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor

https://www.cell.com/trends/cell-biology/pdf/S0962-8924(18)30020-5.pdf

Hallmarks of Cellular Senescence.

The recognition of microbial nucleic acids is a major mechanism by which the immune system detects pathogens. Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that activates innate immune responses through production of the second messenger cGAMP, which activates the adaptor STING. The cGAS-STING pathway not only mediates protective immune defense against infection by a large variety of DNA-containing pathogens but also detects tumor-derived DNA and generates intrinsic antitumor immunity. However, aberrant activation of the cGAS pathway by self DNA can also lead to autoimmune and inflammatory disease. Thus, the cGAS pathway must be properly regulated. Here we review the recent advances in understanding of the cGAS-STING pathway, focusing on the regulatory mechanisms and roles of this pathway in heath and disease.

Regulation and function of the cGAS-STING pathway of cytosolic DNA sensing

During virus infection, the adaptor proteins MAVS and STING transduce signals from the cytosolic nucleic acid sensors RIG-I and cGAS, respectively, to induce type I interferons (IFNs) and other antiviral molecules. Here we show that MAVS and STING harbor two conserved serine and threonine clusters that are phosphorylated by the kinases IKK and/or TBK1 in response to stimulation. Phosphorylated MAVS and STING then bind to a positively charged surface of interferon regulatory factor 3 (IRF3) and thereby recruit IRF3 for its phosphorylation and activation by TBK1. We further show that TRIF, an adaptor protein in Toll-like receptor signaling, activates IRF3 through a similar phosphorylation-dependent mechanism. These results reveal that phosphorylation of innate adaptor proteins is an essential and conserved mechanism that selectively recruits IRF3 to activate the type I IFN pathway.

https://science.sciencemag.org/content/sci/early/2015/01/28/science.aaa2630.full.pdf

Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation

Chromosomal instability is a hallmark of cancer that results from ongoing errors in chromosome segregation during mitosis. Although chromosomal instability is a major driver of tumour evolution, its role in metastasis has not been established. Here we show that chromosomal instability promotes metastasis by sustaining a tumour cell-autonomous response to cytosolic DNA. Errors in chromosome segregation create a preponderance of micronuclei whose rupture spills genomic DNA into the cytosol. This leads to the activation of the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) cytosolic DNA-sensing pathway and downstream noncanonical NF-κB signalling. Genetic suppression of chromosomal instability markedly delays metastasis even in highly aneuploid tumour models, whereas continuous chromosome segregation errors promote cellular invasion and metastasis in a STING-dependent manner. By subverting lethal epithelial responses to cytosolic DNA, chromosomally unstable tumour cells co-opt chronic activation of innate immune pathways to spread to distant organs.

/pdf/chromosomal-instability-drives-metastasis-through-a-1615fx6140.pdf

Chromosomal instability drives metastasis through a cytosolic DNA response.

Cellular senescence is a natural barrier to tumorigenesis and it contributes to the antitumor effects of several therapies, including radiation and chemotherapeutic drugs. Senescence also plays an important role in aging, fibrosis, and tissue repair. The DNA damage response is a key event leading to senescence, which is characterized by the senescence-associated secretory phenotype (SASP) that includes expression of inflammatory cytokines. Here we show that cGMP-AMP (cGAMP) synthase (cGAS), a cytosolic DNA sensor that activates innate immunity, is essential for senescence. Deletion of cGAS accelerated the spontaneous immortalization of mouse embryonic fibroblasts. cGAS deletion also abrogated SASP induced by spontaneous immortalization or DNA damaging agents, including radiation and etoposide. cGAS is localized in the cytoplasm of nondividing cells but enters the nucleus and associates with chromatin DNA during mitosis in proliferating cells. DNA damage leads to accumulation of damaged DNA in cytoplasmic foci that contain cGAS. In human lung adenocarcinoma patients, low expression of cGAS is correlated with poor survival. These results indicate that cGAS mediates cellular senescence and retards immortalization. This is distinct from, and complementary to, the role of cGAS in activating antitumor immunity.

https://www.pnas.org/content/pnas/114/23/E4612.full.pdf

cGAS is essential for cellular senescence

The cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) detects microbial and self-DNA in the cytosol to activate immune and inflammatory programs cGAS also associates with chromatin, especially after nuclear envelope breakdown when cells enter mitosis How cGAS is regulated during cell cycle transition is not clear Here, we found direct biochemical evidence that cGAS activity was selectively suppressed during mitosis in human cell lines and uncovered two parallel mechanisms underlying this suppression First, cGAS was hyperphosphorylated at the N terminus by mitotic kinases, including Aurora kinase B The N terminus of cGAS was critical for sensing nuclear chromatin but not mitochondrial DNA Chromatin sensing was blocked by hyperphosphorylation Second, oligomerization of chromatin-bound cGAS, which is required for its activation, was prevented Together, these mechanisms ensure that cGAS is inactive when associated with chromatin during mitosis, which may help to prevent autoimmune reaction

https://science.sciencemag.org/content/sci/early/2021/02/03/science.abc5386.full.pdf

Phosphorylation and chromatin tethering prevent cGAS activation during mitosis

The transcription factor interferon regulatory factor 5 (IRF5) is essential for the induction of inflammatory cytokines, but the mechanism by which IRF5 is activated is not well understood. Here we present evidence that the kinase IKKβ phosphorylates and activates IRF5 in response to stimulation in several inflammatory pathways, including those emanated from Toll-like receptors and retinoic acid-inducible gene I–like receptors. IKKβ phosphorylates mouse IRF5 at specific residues, including serine 445 (S446 in human IRF5 isoform 1), as evidenced by mass spectrometry analysis and detection with a phosphospecific antibody. Recombinant IKKβ phosphorylated IRF5 at Ser-445 in vitro, and a point mutation of this serine abolished IRF5 activation and cytokine production. Depletion or pharmacologic inhibition of IKKβ prevented IRF5 phosphorylation. These results indicate that IKKβ is an IRF5 kinase that instigates inflammation.

https://www.pnas.org/content/pnas/111/49/17438.full.pdf

Junyao Ren

Papers

Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation

cGAS is essential for cellular senescence

Phosphorylation and chromatin tethering prevent cGAS activation during mitosis

IKKβ is an IRF5 kinase that instigates inflammation