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 neuroimmune system is involved in development, normal functioning, aging, and injury of the central nervous system Microglia, first described a century ago, are the main neuroimmune cells and have three essential functions: a sentinel function involved in constant sensing of changes in their environment, a housekeeping function that promotes neuronal well-being and normal operation, and a defense function necessary for responding to such changes and providing neuroprotection Microglia use a defined armamentarium of genes to perform these tasks In response to specific stimuli, or with neuroinflammation, microglia also have the capacity to damage and kill neurons Injury to neurons in Alzheimer’s, Parkinson’s, Huntington’s, and prion diseases, as well as in amyotrophic lateral sclerosis, frontotemporal dementia, and chronic traumatic encephalopathy, results from disruption of the sentinel or housekeeping functions and dysregulation of the defense function and neuroinflammation Pathways associated with such injury include several sensing and housekeeping pathways, such as the Trem2, Cx3cr1 and progranulin pathways, which act as immune checkpoints to keep the microglial inflammatory response under control, and the scavenger receptor pathways, which promote clearance of injurious stimuli Peripheral interference from systemic inflammation or the gut microbiome can also alter progression of such injury Initiation or exacerbation of neurodegeneration results from an imbalance between these microglial functions; correcting such imbalance may be a potential mode for therapy

Microglia in neurodegeneration.

MicroRNAs (miRNAs) are endogenous ∼23 nt RNAs that play important gene-regulatory roles in animals and plants by pairing to the mRNAs of protein-coding genes to direct their posttranscriptional repression. This review outlines the current understanding of miRNA target recognition in animals and discusses the widespread impact of miRNAs on both the expression and evolution of protein-coding genes.

MicroRNAs: Target Recognition and Regulatory Functions

Amyotrophic lateral sclerosis

Stress granules, P-bodies and cancer

Deep analysis of next-generation sequencing data unveils numerous small non-coding RNAs with distinct functions. Recently, fragments derived from tRNA, named as tRNA-derived small RNA (tsRNA), have attracted broad attention. There are mainly two types of tsRNAs, including tRNA-derived stress-induced RNA (tiRNA) and tRNA-derived fragment (tRF), which differ in the cleavage position of the precursor or mature tRNA transcript. Emerging evidence has shown that tsRNAs are not merely tRNA degradation debris but have been recognized to play regulatory roles in many specific physiological and pathological processes. In this review, we summarize the biogeneses of various tsRNAs, present the emerging concepts regarding functions and mechanisms of action of tsRNAs, highlight the potential application of tsRNAs in human diseases, and put forward the current problems and future research directions.

tRNA-Derived Small RNA: A Novel Regulatory Small Non-Coding RNA

As a member of the vertebrate-specific secreted ribonucleases, angiogenin (ANG) was first isolated and identified solely by its ability to induce new blood vessel formation, and now, it has been recognized to play important roles in various physiological and pathological processes through regulating cell proliferation, survival, migration, invasion, and/or differentiation. ANG exhibits very weak ribonucleolytic activity that is critical for its biological functions, and exerts its functions through activating different signaling transduction pathways in different target cells. A series of recent studies have indicated that ANG contributes to cellular nucleic acid metabolism. Here, we comprehensively review the results of studies regarding the structure, mechanism, and function of ANG over the past three decades. Moreover, current problems and future research directions of ANG are discussed. The understanding of the function and mechanism of ANG in a wide context will help to better delineate its roles in diseases, especially in cancer and neurodegenerative diseases.

Three decades of research on angiogenin: a review and perspective.

Angiogenin (ANG) promotes cell growth and survival. Under growth conditions, ANG undergoes nuclear translocation and accumulates in the nucleolus where it stimulates rRNA transcription. When cells are stressed, ANG mediates the production of tRNA-derived stress-induced small RNA (tiRNA), which reprograms protein translation into a survival mechanism. The ribonucleolytic activity of ANG is essential for both processes but how this activity is regulated is unknown. We report here that ribonuclease/angiogenin inhibitor 1 (RNH1) controls both the localization and activity of ANG. Under growth conditions, ANG is located in the nucleus and is not associated with RNH1 so that the ribonucleolytic activity is retained to ensure rRNA transcription. Cytoplasmic ANG is associated with and inhibited by RNH1 so that random cleavage of cellular RNA is prevented. Under stress conditions, ANG is localized to the cytoplasm and is concentrated in stress granules where it is not associated with RNH1 and thus remains enzymatically active for tiRNA production. By contrast, nuclear ANG is associated with RNH1 in stressed cells to ensure that the enzymatic activity is inhibited and no unnecessary rRNA is produced to save anabolic energy. Knockdown of RNH1 abolished stress-induced relocalization of ANG and decreased cell growth and survival.

/pdf/ribonuclease-angiogenin-inhibitor-1-regulates-stress-induced-h2lbyh4lg2.pdf

Ribonuclease/angiogenin inhibitor 1 regulates stress-induced subcellular localization of angiogenin to control growth and survival

N6-methyladenosine (m6 A) mRNA methylation has emerged as an important player in many biological processes by regulating gene expression. However, its roles in intestinal stem cell (ISC) homeostasis remain largely unknown. Here, we report that YTHDF1, an m6 A reader, is highly expressed in ISCs and its expression is upregulated by Wnt signaling at the translational level. Whereas YTHDF1 is dispensable for normal intestinal development in mice, genetic ablation of Ythdf1 dramatically blocks Wnt-driven regeneration and tumorigenesis with reduced ISC stemness. Mechanistically, YTHDF1 facilitates the translation of Wnt signaling effectors including TCF7L2/TCF4, while this process is enhanced during Wnt activation to augment β-catenin activity. Targeting YTHDF1 in ISCs of established tumors leads to tumor shrinkage and prolonged survival. Collectively, our studies unveil YTHDF1 as an amplifier of Wnt/β-catenin signaling at the translational level, which is required for the maintenance of ISCs during regeneration and tumorigenesis.

https://www.embopress.org/doi/pdf/10.15252/embr.201949229

YTHDF1-mediated translation amplifies Wnt-driven intestinal stemness

Metastasis of colorectal cancer (CRC) is the leading cause of CRC-associated mortality. Angiogenin (ANG), a member of the ribonuclease A superfamily, not only activates endothelial cells to induce tumor angiogenesis, but also targets tumor cells to promote cell survival, proliferation and/or migration. However, its clinical significance and underlying mechanism in CRC metastasis are still largely unknown. Here, we reported that ANG was upregulated in CRC tissues and associated with metastasis in CRC patients. We then revealed that ANG enhanced CRC growth and metastasis in both in vitro and in vivo systems. Intriguingly, we characterized a bunch of tRNA-derived stress-induced small RNAs (tiRNAs), produced through ANG cleavage, that was enriched in both CRC tumor tissues and highly metastatic cells, and functioned in ANG-promoted CRC metastasis. Moreover, higher level of a 5'-tiRNA from mature tRNA-Val (5'-tiRNA-Val) was observed in CRC patients and was correlated with tumor metastasis. Taken together, we propose that a novel ANG-tiRNAs-cell migration and invasion regulatory axis promotes CRC metastasis, which might be of potential target for CRC diagnosis and treatment.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/ijc.32245

Jinghao Sheng

Papers

tRNA-Derived Small RNA: A Novel Regulatory Small Non-Coding RNA

Three decades of research on angiogenin: a review and perspective.

Ribonuclease/angiogenin inhibitor 1 regulates stress-induced subcellular localization of angiogenin to control growth and survival

YTHDF1-mediated translation amplifies Wnt-driven intestinal stemness

Angiogenin promotes colorectal cancer metastasis via tiRNA production.