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

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Life at the Leading Edge

Regulation of SIRT1 in cellular functions: role of polyphenols.

The ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway (ALP) are the two most important mechanisms that normally repair or remove abnormal proteins. Alterations in the function of these systems to degrade misfolded and aggregated proteins are being increasingly recognized as playing a pivotal role in the pathogenesis of many neurodegenerative disorders such as Parkinson's disease. Dysfunction of the UPS has been already strongly implicated in the pathogenesis of this disease and, more recently, growing interest has been shown in identifying the role of ALP in neurodegeneration. Mutations of α-synuclein and the increase of intracellular concentrations of non-mutant α-synuclein have been associated with Parkinson's disease phenotype. The demonstration that α-synuclein is degraded by both proteasome and autophagy indicates a possible linkage between the dysfunction of the UPS or ALP and the occurrence of this disorder. The fact that mutant α-synucleins inhibit ALP functioning by tightly binding to the receptor on the lysosomal membrane for autophagy pathway further supports the assumption that impairment of the ALP may be related to the development of Parkinson's disease. In this review, we summarize the recent findings related to this topic and discuss the unique role of the ALP in this neurogenerative disorder and the putative therapeutic potential through ALP enhancement.

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The role of autophagy-lysosome pathway in neurodegeneration associated with Parkinson's disease.

It has been two decades since the yeast Ypt1 and Sec4 proteins and the mammalian Rab (Ras-related proteins in brain) GTPases were first identified as evolutionarily conserved, essential regulators of membrane trafficking ([Salminen and Novick, 1987][1]; [Schmitt et al., 1986][2]; [Touchot et al.,

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Rab GTPases at a glance.

Macroautophagy, the process by which cytosolic components and organelles are engulfed and degraded by a double-membrane structure, could be viewed as a specialized, multistep membrane transport process. As such, it intersects with the exocytic and endocytic membrane trafficking pathways. A number of Rab GTPases which regulate secretory and endocytic membrane traffic have been shown to play either critical or accessory roles in autophagy. The biogenesis of the pre-autophagosomal isolation membrane (or phagophore) is dependent on the functionality of Rab1. A non-canonical, Atg5/Atg7-independent mode of autophagosome generation from the trans-Golgi or endosome requires Rab9. Other Rabs, such as Rab5, Rab24, Rab33, and Rab7 have all been shown to be required, or involved at various stages of autophagosomal genesis and maturation. Another small GTPase, RalB, was very recently demonstrated to induce isolation membrane formation and maturation via its engagement of the exocyst complex, a known Rab effector. We summarize here what is now known about the involvement of Rabs in autophagy, and discuss plausible mechanisms with future perspectives.

Involvement of members of the Rab family and related small GTPases in autophagosome formation and maturation.

SIRT1 and neuronal diseases.

https://onlinelibrary.wiley.com/doi/pdf/10.1042/BC20100150

Rabs and other small GTPases in ciliary transport.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/j.febslet.2009.11.051

Rab35--a vesicular traffic-regulating small GTPase with actin modulating roles.

Rab GTPases are key regulators of eukaryotic membrane traffic, and their functions and activities are limited to particular intracellular transport steps and their membrane localization is by and large restricted. Some Rabs do participate in more than one transport steps, but broadly speaking, there is a clear demarcation between exocytic and endocytic Rabs. One Rab protein, Rab10, however, appears to be anomalous in this regard and has a diverse array of functions and subcellular localizations. Rab10 has been implicated in a myriad of activities ranging from polarized exocytosis and endosomal sorting in polarized cells, insulin-dependent Glut4 transport in adipocytes, axonal growth in neurons, and endo-phagocytic processes in macrophages. It's reported subcellular localizations include the endoplasmic reticulum (ER), Golgi/TGN, the endosomes/phagosomes and the primary cilia. In this review, we summarize and discuss the multitude of known roles of Rab10 in cellular membrane transport and the molecular players and mechanisms associated with these roles.

Christelle En Lin Chua

Papers

Involvement of members of the Rab family and related small GTPases in autophagosome formation and maturation.

SIRT1 and neuronal diseases.

Rabs and other small GTPases in ciliary transport.

Rab35--a vesicular traffic-regulating small GTPase with actin modulating roles.

Rab 10-a traffic controller in multiple cellular pathways and locations.