Cellular Senescence: Defining a Path Forward.

Cellular senescence is a cell state implicated in various physiological processes and a wide spectrum of age-related diseases. Recently, interest in therapeutically targeting senescence to improve healthy aging and age-related disease, otherwise known as senotherapy, has been growing rapidly. Thus, the accurate detection of senescent cells, especially in vivo, is essential. Here, we present a consensus from the International Cell Senescence Association (ICSA), defining and discussing key cellular and molecular features of senescence and offering recommendations on how to use them as biomarkers. We also present a resource tool to facilitate the identification of genes linked with senescence, SeneQuest (available at http://Senequest.net). Lastly, we propose an algorithm to accurately assess and quantify senescence, both in cultured cells and in vivo.

Cellular senescence in ageing: from mechanisms to therapeutic opportunities.

Cellular senescence, first described in vitro in 1961, has become a focus for biotech companies that target it to ameliorate a variety of human conditions. Eminently characterized by a permanent proliferation arrest, cellular senescence occurs in response to endogenous and exogenous stresses, including telomere dysfunction, oncogene activation and persistent DNA damage. Cellular senescence can also be a controlled programme occurring in diverse biological processes, including embryonic development. Senescent cell extrinsic activities, broadly related to the activation of a senescence-associated secretory phenotype, amplify the impact of cell-intrinsic proliferative arrest and contribute to impaired tissue regeneration, chronic age-associated diseases and organismal ageing. This Review discusses the mechanisms and modulators of cellular senescence establishment and induction of a senescence-associated secretory phenotype, and provides an overview of cellular senescence as an emerging opportunity to intervene through senolytic and senomorphic therapies in ageing and ageing-associated diseases.

Non-cell-autonomous tumor suppression by p53.

The p53 tumor suppressor can restrict malignant transformation by triggering cell-autonomous programs of cell-cycle arrest or apoptosis. p53 also promotes cellular senescence, a tumor-suppressive program that involves stable cell-cycle arrest and secretion of factors that modify the tissue microenvironment. In the presence of chronic liver damage, we show that ablation of a p53-dependent senescence program in hepatic stellate cells increases liver fibrosis and cirrhosis associated with reduced survival and enhances the transformation of adjacent epithelial cells into hepatocellular carcinoma. p53-expressing senescent stellate cells release factors that skew macrophage polarization toward a tumor-inhibiting M1-state capable of attacking senescent cells in culture, whereas proliferating p53-deficient stellate cells secrete factors that stimulate polarization of macrophages into a tumor-promoting M2-state and enhance the proliferation of premalignant cells. Hence, p53 can act non-cell autonomously to suppress tumorigenesis by promoting an antitumor microenvironment, in part, through secreted factors that modulate macrophage function.

Senescence is a developmental mechanism that contributes to embryonic growth and patterning.

Senescence is a form of cell-cycle arrest linked to tumor suppression and aging. However, it remains controversial and has not been documented in nonpathologic states. Here we describe senescence as a normal developmental mechanism found throughout the embryo, including the apical ectodermal ridge (AER) and the neural roof plate, two signaling centers in embryonic patterning. Embryonic senescent cells are nonproliferative and share features with oncogene-induced senescence (OIS), including expression of p21, p15, and mediators of the senescence-associated secretory phenotype (SASP). Interestingly, mice deficient in p21 have defects in embryonic senescence, AER maintenance, and patterning. Surprisingly, the underlying mesenchyme was identified as a source for senescence instruction in the AER, whereas the ultimate fate of these senescent cells is apoptosis and macrophage-mediated clearance. We propose that senescence is a normal programmed mechanism that plays instructive roles in development, and that OIS is an evolutionarily adapted reactivation of a developmental process.

The intricate nature of senescence in development and cell plasticity.

Cellular senescence, a stable form of cell cycle arrest, accompanied by pronounced secretory activity, has functional roles in both physiological and pathological conditions. Although senescence has been linked for a long time with cancer and ageing, recent studies have revealed a functional role of senescence in development, regeneration and reprogramming. Notably, the transient presence of senescent cells may be beneficial, in contrast to the potential deleterious effects of persistent senescence in aged or chronically damaged tissues. We will discuss how senescence contributes to embryonic development, cell plasticity and tissue regeneration, as a highly coordinated and programmed cellular state.

Molecular pathways of senescence regulate placental structure and function.

The placenta is an autonomous organ that maintains fetal growth and development. Its multinucleated syncytiotrophoblast layer, providing fetal nourishment during gestation, exhibits characteristics of cellular senescence. We show that in human placentas from pregnancies with intrauterine growth restriction, these characteristics are decreased. To elucidate the functions of pathways regulating senescence in syncytiotrophoblast, we used dynamic contrast-enhanced MRI in mice with attenuated senescence programs. This approach revealed an altered dynamics in placentas of p53-/- , Cdkn2a-/- , and Cdkn2a-/- ;p53-/- mice, accompanied by histopathological changes in placental labyrinths. Human primary syncytiotrophoblast upregulated senescence markers and molecular pathways associated with cell-cycle inhibition and senescence-associated secretory phenotype. The pathways and components of the secretory phenotype were compromised in mouse placentas with attenuated senescence and in human placentas from pregnancies with intrauterine growth restriction. We propose that molecular mediators of senescence regulate placental structure and function, through both cell-autonomous and non-autonomous mechanisms.

Senescence of activated stellate cells limits liver fibrosis.

Cellular senescence acts as a potent mechanism of tumor suppression; however, its functional contribution to noncancer pathologies has not been examined. Here we show that senescent cells accumulate in murine livers treated to produce fibrosis, a precursor pathology to cirrhosis. The senescent cells are derived primarily from activated hepatic stellate cells, which initially proliferate in response to liver damage and produce the extracellular matrix deposited in the fibrotic scar. In mice lacking key senescence regulators, stellate cells continue to proliferate, leading to excessive liver fibrosis. Furthermore, senescent activated stellate cells exhibit gene expression profile consistent with cell-cycle exit, reduced secretion of extracellular matrix components, enhanced secretion of extracellular matrix-degrading enzymes, and enhanced immune surveillance. Accordingly natural killer cells preferentially kill senescent activated stellate cells in vitro and in vivo, thereby facilitating the resolution of fibrosis. Therefore, the senescence program limits the fibrogenic response to acute tissue damage.

Senescent cells communicate via intercellular protein transfer.

Mammalian cells mostly rely on extracellular molecules to transfer signals to other cells. However, in stress conditions, more robust mechanisms might be necessary to facilitate cell-cell communications. Cellular senescence, a stress response associated with permanent exit from the cell cycle and the development of an immunogenic phenotype, limits both tumorigenesis and tissue damage. Paradoxically, the long-term presence of senescent cells can promote tissue damage and aging within their microenvironment. Soluble factors secreted from senescent cells mediate some of these cell-nonautonomous effects. However, it is unknown whether senescent cells impact neighboring cells by other mechanisms. Here we show that senescent cells directly transfer proteins to neighboring cells and that this process facilitates immune surveillance of senescent cells by natural killer (NK) cells. We found that transfer of proteins to NK and T cells is increased in the murine preneoplastic pancreas, a site where senescent cells are present in vivo. Proteomic analysis and functional studies of the transferred proteins revealed that the transfer is strictly dependent on cell-cell contact and CDC42-regulated actin polymerization and is mediated at least partially by cytoplasmic bridges. These findings reveal a novel mode of intercellular communication by which senescent cells regulate their immune surveillance and might impact tumorigenesis and tissue aging.

Senolytic elimination of Cox2-expressing senescent cells inhibits the growth of premalignant pancreatic lesions.

OBJECTIVE: Cellular senescence limits tumourigenesis by blocking the proliferation of premalignant cells. Additionally, however, senescent cells can exert paracrine effects influencing tumour growth. Senescent cells are present in premalignant pancreatic intraepithelial neoplasia (PanIN) lesions, yet their effects on the disease are poorly characterised. It is currently unknown whether senolytic drugs, aimed at eliminating senescent cells from lesions, could be beneficial in blocking tumour development. DESIGN: To uncover the functions of senescent cells and their potential contribution to early pancreatic tumourigenesis, we isolated and characterised senescent cells from PanINs formed in a Kras-driven mouse model, and tested the consequences of their targeted elimination through senolytic treatment. RESULTS: We found that senescent PanIN cells exert a tumour-promoting effect through expression of a proinflammatory signature that includes high Cox2 levels. Senolytic treatment with the Bcl2-family inhibitor ABT-737 eliminated Cox2-expressing senescent cells, and an intermittent short-duration treatment course dramatically reduced PanIN development and progression to pancreatic ductal adenocarcinoma. CONCLUSIONS: These findings reveal that senescent PanIN cells support tumour growth and progression, and provide a first indication that elimination of senescent cells may be effective as preventive therapy for the progression of precancerous lesions.

p21 maintains senescent cell viability under persistent DNA damage response by restraining JNK and caspase signaling.

Cellular senescence is a permanent state of cell cycle arrest that protects the organism from tumorigenesis and regulates tissue integrity upon damage and during tissue remodeling. However, accumulation of senescent cells in tissues during aging contributes to age-related pathologies. A deeper understanding of the mechanisms regulating the viability of senescent cells is therefore required. Here, we show that the CDK inhibitor p21 (CDKN1A) maintains the viability of DNA damage-induced senescent cells. Upon p21 knockdown, senescent cells acquired multiple DNA lesions that activated ataxia telangiectasia mutated (ATM) and nuclear factor (NF)-κB kinase, leading to decreased cell survival. NF-κB activation induced TNF-α secretion and JNK activation to mediate death of senescent cells in a caspase- and JNK-dependent manner. Notably, p21 knockout in mice eliminated liver senescent stellate cells and alleviated liver fibrosis and collagen production. These findings define a novel pathway that regulates senescent cell viability and fibrosis.

Cell fusion induced by ERVWE1 or measles virus causes cellular senescence.

Cellular senescence limits proliferation of potentially detrimental cells, preventing tumorigenesis and restricting tissue damage. However, the function of senescence in nonpathological conditions is unknown. We found that the human placental syncytiotrophoblast exhibited the phenotype and expressed molecular markers of cellular senescence. During embryonic development, ERVWE1-mediated cell fusion results in formation of the syncytiotrophoblast, which serves as the maternal/fetal interface at the placenta. Expression of ERVWE1 caused cell fusion in normal and cancer cells, leading to formation of hyperploid syncytia exhibiting features of cellular senescence. Infection by the measles virus, which leads to cell fusion, also induced cellular senescence in normal and cancer cells. The fused cells activated the main molecular pathways of senescence, the p53- and p16-pRb-dependent pathways; the senescence-associated secretory phenotype; and immune surveillance-related proteins. Thus, fusion-induced senescence might be needed for proper syncytiotrophoblast function during embryonic development, and reuse of this senescence program later in life protects against pathological expression of endogenous fusogens and fusogenic viral infections.

Tissue-specific and reversible RNA interference in transgenic mice.

Genetically engineered mice provide powerful tools for understanding mammalian gene function. These models traditionally rely on gene overexpression from transgenes or targeted, irreversible gene mutation. By adapting the tetracycline (tet)-responsive system previously used for gene overexpression, we have developed a simple transgenic system to reversibly control endogenous gene expression using RNA interference (RNAi) in mice. Transgenic mice harboring a tet-responsive RNA polymerase II promoter driving a microRNA-based short hairpin RNA targeting the tumor suppressor Trp53 reversibly express short hairpin RNA when crossed with existing mouse strains expressing general or tissue-specific 'tet-on' or 'tet-off' transactivators. Reversible Trp53 knockdown can be achieved in several tissues, and restoring Trp53 expression in lymphomas whose development is promoted by Trp53 knockdown leads to tumor regression. By leaving the target gene unaltered, this approach permits tissue-specific, reversible regulation of endogenous gene expression in vivo, with potential broad application in basic biology and drug target validation.

Physiological and pathological consequences of cellular senescence.

Cellular senescence, a permanent state of cell cycle arrest accompanied by a complex phenotype, is an essential mechanism that limits tumorigenesis and tissue damage. In physiological conditions, senescent cells can be removed by the immune system, facilitating tumor suppression and wound healing. However, as we age, senescent cells accumulate in tissues, either because an aging immune system fails to remove them, the rate of senescent cell formation is elevated, or both. If senescent cells persist in tissues, they have the potential to paradoxically promote pathological conditions. Cellular senescence is associated with an enhanced pro-survival phenotype, which most likely promotes persistence of senescent cells in vivo. This phenotype may have evolved to favor facilitation of a short-term wound healing, followed by the elimination of senescent cells by the immune system. In this review, we provide a perspective on the triggers, mechanisms and physiological as well as pathological consequences of senescent cells.

Senescent cells: SASPected drivers of age-related pathologies.

The progression of physiological ageing is driven by intracellular aberrations including telomere attrition, genomic instability, epigenetic alterations and loss of proteostasis. These in turn damage cells and compromise their functionality. Cellular senescence, a stable irreversible cell-cycle arrest, is elicited in damaged cells and prevents their propagation in the organism. Under normal conditions, senescent cells recruit the immune system which facilitates their removal from tissues. Nevertheless, during ageing, tissue-residing senescent cells tend to accumulate, and might negatively impact their microenvironment via profound secretory phenotype with pro-inflammatory characteristics, termed senescence-associated secretory phenotype (SASP). Indeed, senescent cells are mostly abundant at sites of age-related pathologies, including degenerative disorders and malignancies. Interestingly, studies on progeroid mice indicate that selective elimination of senescent cells can delay age-related deterioration. This suggests that chronic inflammation induced by senescent cells might be a main driver of these pathologies. Importantly, senescent cells accumulate as a result of deficient immune surveillance, and their removal is increased upon the use of immune stimulatory agents. Insights into mechanisms of senescence surveillance could be combined with current approaches for cancer immunotherapy to propose new preventive and therapeutic strategies for age-related diseases.

Identification of senescent, TREM2-expressing microglia in aging and Alzheimer's disease model mouse brain.

Alzheimer's disease (AD) and dementia in general are age-related diseases with multiple contributing factors, including brain inflammation. Microglia, and specifically those expressing the AD risk gene TREM2, are considered important players in AD, but their exact contribution to pathology remains unclear. In this study, using high-throughput mass cytometry in the 5×FAD mouse model of amyloidosis, we identified senescent microglia that express high levels of TREM2 but also exhibit a distinct signature from TREM2-dependent disease-associated microglia (DAM). This senescent microglial protein signature was found in various mouse models that show cognitive decline, including aging, amyloidosis and tauopathy. TREM2-null mice had fewer microglia with a senescent signature. Treating 5×FAD mice with the senolytic BCL2 family inhibitor ABT-737 reduced senescent microglia, but not the DAM population, and this was accompanied by improved cognition and reduced brain inflammation. Our results suggest a dual and opposite involvement of TREM2 in microglial states, which must be considered when contemplating TREM2 as a therapeutic target in AD.

Immunosurveillance of senescent cells: the bright side of the senescence program.

Cellular senescence, a state of irreversible cell cycle arrest, is a robust mechanism used to mediate tumor suppression and control the tissue damage response following short-term insults. In addition, the senescence associated-secretory phenotype (SASP), one of the most profound characteristics of the senescence program, facilitates the immunosurveillance of senescent cells. The SASP includes many chemokines, cytokines and adhesion molecules that can recruit and activate distinct immune cells from both the innate and adaptive immune system such as NK cells, monocytes/macrophages and T cells. Furthermore, senescent cells can upregulate specific immune ligands on their cell surface that can mediate the recognition of these cells by specific immune cell subsets and lead to activation of the immune cells. Consequently, the activated immune cells engage explicit regulatory mechanisms to eliminate senescent cells. For example, recent work from our laboratory showed that perforin-granzyme exocytosis mediates NK-cell killing of senescent cells. Here, we summarize the current advances in our knowledge of the mechanisms underlying specific immune-mediated elimination of senescent cells.

Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas.

Although cancer arises from a combination of mutations in oncogenes and tumour suppressor genes, the extent to which tumour suppressor gene loss is required for maintaining established tumours is poorly understood. p53 is an important tumour suppressor that acts to restrict proliferation in response to DNA damage or deregulation of mitogenic oncogenes, by leading to the induction of various cell cycle checkpoints, apoptosis or cellular senescence. Consequently, p53 mutations increase cell proliferation and survival, and in some settings promote genomic instability and resistance to certain chemotherapies. To determine the consequences of reactivating the p53 pathway in tumours, we used RNA interference (RNAi) to conditionally regulate endogenous p53 expression in a mosaic mouse model of liver carcinoma. We show that even brief reactivation of endogenous p53 in p53-deficient tumours can produce complete tumour regressions. The primary response to p53 was not apoptosis, but instead involved the induction of a cellular senescence program that was associated with differentiation and the upregulation of inflammatory cytokines. This program, although producing only cell cycle arrest in vitro, also triggered an innate immune response that targeted the tumour cells in vivo, thereby contributing to tumour clearance. Our study indicates that p53 loss can be required for the maintenance of aggressive carcinomas, and illustrates how the cellular senescence program can act together with the innate immune system to potently limit tumour growth.

Senescent cells in the brain and where to find them.

Cellular senescence is a process in which cells change their characteristic phenotype in response to stress and enter a state of prolonged cell cycle arrest accompanied by a distinct secretory phenotype. Cellular senescence has both beneficial and detrimental outcomes. With age, senescent cells progressively accumulate in tissues and might be the bridge connecting ageing to many age-related pathologies. In recent years, evidence emerged supporting the accumulation of brain senescent cells during neurological disorders and ageing. Here, we will discuss the different brain cell populations that exhibit a senescent phenotype. Subsequently, we will explore several senolytic strategies which have been developed to eliminate senescent cells. Finally, we will examine their potential to directly eliminate these senescent brain cells.

p21 facilitates chronic lung inflammation via epithelial and endothelial cells.

Cellular senescence is a stable state of cell cycle arrest that regulates tissue integrity and protects the organism from tumorigenesis. However, the accumulation of senescent cells during aging contributes to age-related pathologies. One such pathology is chronic lung inflammation. p21 (CDKN1A) regulates cellular senescence via inhibition of cyclin-dependent kinases (CDKs). However, its role in chronic lung inflammation and functional impact on chronic lung disease, where senescent cells accumulate, is less understood. To elucidate the role of p21 in chronic lung inflammation, we subjected p21 knockout (p21-/-) mice to repetitive inhalations of lipopolysaccharide (LPS), an exposure that leads to chronic bronchitis and accumulation of senescent cells. p21 knockout led to a reduced presence of senescent cells, alleviated the pathological manifestations of chronic lung inflammation, and improved the fitness of the mice. The expression profiling of the lung cells revealed that resident epithelial and endothelial cells, but not immune cells, play a significant role in mediating the p21-dependent inflammatory response following chronic LPS exposure. Our results implicate p21 as a critical regulator of chronic bronchitis and a driver of chronic airway inflammation and lung destruction.