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

The mechanistic target of rapamycin (mTOR) coordinates eukaryotic cell growth and metabolism with environmental inputs, including nutrients and growth factors. Extensive research over the past two decades has established a central role for mTOR in regulating many fundamental cell processes, from protein synthesis to autophagy, and deregulated mTOR signaling is implicated in the progression of cancer and diabetes, as well as the aging process. Here, we review recent advances in our understanding of mTOR function, regulation, and importance in mammalian physiology. We also highlight how the mTOR signaling network contributes to human disease and discuss the current and future prospects for therapeutically targeting mTOR in the clinic.

mTOR Signaling in Growth, Metabolism, and Disease

Placebo

Ageing results from complex genetically and epigenetically programmed processes that are elicited in part by noxious or stressful events that cause programmed cell death Here, we report that administration of spermidine, a natural polyamine whose intracellular concentration declines during human ageing, markedly extended the lifespan of yeast, flies and worms, and human immune cells In addition, spermidine administration potently inhibited oxidative stress in ageing mice In ageing yeast, spermidine treatment triggered epigenetic deacetylation of histone H3 through inhibition of histone acetyltransferases (HAT), suppressing oxidative stress and necrosis Conversely, depletion of endogenous polyamines led to hyperacetylation, generation of reactive oxygen species, early necrotic death and decreased lifespan The altered acetylation status of the chromatin led to significant upregulation of various autophagy-related transcripts, triggering autophagy in yeast, flies, worms and human cells Finally, we found that enhanced autophagy is crucial for polyamine-induced suppression of necrosis and enhanced longevity

Induction of autophagy by spermidine promotes longevity

Metagenomics is expanding our knowledge of the gene content, functional significance, and genetic variability in natural microbial communities. Still, there exists limited information concerning the regulation and dynamics of genes in the environment. We report here global analysis of expressed genes in a naturally occurring microbial community. We first adapted RNA amplification technologies to produce large amounts of cDNA from small quantities of total microbial community RNA. The fidelity of the RNA amplification procedure was validated with Prochlorococcus cultures and then applied to a microbial assemblage collected in the oligotrophic Pacific Ocean. Microbial community cDNAs were analyzed by pyrosequencing and compared with microbial community genomic DNA sequences determined from the same sample. Pyrosequencing-based estimates of microbial community gene expression compared favorably to independent assessments of individual gene expression using quantitative PCR. Genes associated with key metabolic pathways in open ocean microbial species—including genes involved in photosynthesis, carbon fixation, and nitrogen acquisition—and a number of genes encoding hypothetical proteins were highly represented in the cDNA pool. Genes present in the variable regions of Prochlorococcus genomes were among the most highly expressed, suggesting these encode proteins central to cellular processes in specific genotypes. Although many transcripts detected were highly similar to genes previously detected in ocean metagenomic surveys, a significant fraction (≈50%) were unique. Thus, microbial community transcriptomic analyses revealed not only indigenous gene- and taxon-specific expression patterns but also gene categories undetected in previous DNA-based metagenomic surveys.

https://www.pnas.org/content/pnas/105/10/3805.full.pdf

Microbial community gene expression in ocean surface waters

Oxidative stress in skin plays a major role in the aging process. This is true for intrinsic aging and even more for extrinsic aging. Although the results are quite different in dermis and epidermis, extrinsic aging is driven to a large extent by oxidative stress caused by UV irradiation. In this review the overall effects of oxidative stress are discussed as well as the sources of ROS including the mitochondrial ETC, peroxisomal and ER localized proteins, the Fenton reaction, and such enzymes as cyclooxygenases, lipoxygenases, xanthine oxidases, and NADPH oxidases. Furthermore, the defense mechanisms against oxidative stress ranging from enzymes like superoxide dismutases, catalases, peroxiredoxins, and GSH peroxidases to organic compounds such as L-ascorbate, α-tocopherol, beta-carotene, uric acid, CoQ10, and glutathione are described in more detail. In addition the oxidative stress induced modifications caused to proteins, lipids and DNA are discussed. Finally age-related changes of the skin are also a topic of this review. They include a disruption of the epidermal calcium gradient in old skin with an accompanying change in the composition of the cornified envelope. This modified cornified envelope also leads to an altered anti-oxidative capacity and a reduced barrier function of the epidermis.

/pdf/oxidative-stress-in-aging-human-skin-3ohc7c9zri.pdf

Oxidative Stress in Aging Human Skin

Anterior specification of embryonic ectoderm: the role of the Xenopus cement gland-specific gene XAG-2

In late summer, pollen grains originating from Compositae weeds (e.g., mugwort, ragweed) are a major source of allergens worldwide. Here, we report the isolation of a cDNA clone coding for Art v 1, the major allergen of mugwort pollen. Sequence analysis showed that Art v 1 is a secreted allergen with an N-terminal cysteine-rich domain homologous to plant defensins and a C-terminal proline-rich region containing several (Ser/Ala)(Pro)2-4 repeats. Structural analysis showed that some of the proline residues in the C-terminal domain of Art v 1 are posttranslationally modified by hydroxylation and O-glycosylation. The O-glycans are composed of 3 galactoses and 9-16 arabinoses linked to a hydroxyproline and represent a new type of plant O-glycan. A 3-D structural model of Art v 1 was generated showing a characteristic "head and tail" structure. Evaluation of the antibody binding properties of natural and recombinant Art v 1 produced in Escherichia coli revealed the involvement of the defensin fold and posttranslational modifications in the formation of epitopes recognized by IgE antibodies from allergic patients. However, posttranslational modifications did not influence T-cell recognition. Thus, recombinant nonglycosylated Art v 1 is a good starting template for engineering hypoallergenic vaccines for weed-pollen therapy.

Art v 1, the major allergen of mugwort pollen, is a modular glycoprotein with a defensin-like and a hydroxyproline-rich domain

Expression profiling of aging in the human skin.

The clinical presentation of non-pollen related allergy to hazelnut can be severe and systemic. So far, only a limited number of non-pollen related hazelnut allergens have been identified and characterized. The aim of this study was to identify and clone new hazelnut allergens. A lambda ZAP cDNA library of hazelnut was constructed. The library was screened with serum of six hazelnut allergic patients displaying different IgE-binding patterns on hazelnut immunoblot. Rapid amplification of cDNA ends (RACE) protocols were applied to obtain full-length clones. Expression experiments were carried out in Eschericchia coli. Expression was monitored by SDS-PAGE, protein staining and immunoblotting. A hazelnut cDNA library was constructed. IgE screening resulted in the cloning of two isoforms of a novel putative hazelnut allergen. The clones were identified as oleosins, with theoretical molecular masses of 16.7 and 14.7 kDa and pI of 10.5 and 10.0, respectively. The isoforms demonstrated only 37% amino acid sequence identity but contained the typical hydrophobic stretch in the middle of the protein (53% identity) with the characteristic oleosin proline knot region (11/12 amino acids identical). Expression in E. coli of the longer isoform resulted in a clear band on SDS-PAGE. The expressed protein was recognized on an immunodot blot by IgE from serum that was used for screening the cDNA library. Hazelnut contains multiple isoforms of oleosin. IgE binding of a hazelnut-allergic patient to a recombinant version suggest that hazelnut oleosin is an allergen, as has been described for peanut and sesame.

Klaus Richter

Papers

Oxidative Stress in Aging Human Skin

Anterior specification of embryonic ectoderm: the role of the Xenopus cement gland-specific gene XAG-2

Art v 1, the major allergen of mugwort pollen, is a modular glycoprotein with a defensin-like and a hydroxyproline-rich domain

Expression profiling of aging in the human skin.

Cloning of oleosin, a putative new hazelnut allergen, using a hazelnut cDNA library