Hallmarks of Cellular Senescence
Cellular senescence is a permanent state of cell cycle arrest that promotes tissue remodeling during development and after injury, but can also contribute to the decline of the regenerative potential and function of tissues, to inflammation, and to tumorigenesis in aged organisms. Therefore, the identification, characterization, and pharmacological elimination of senescent cells have gained attention in the field of aging research. However, the nonspecificity of current senescence markers and the existence of different senescence programs strongly limit these tasks. Here, we describe the molecular regulators of senescence phenotypes and how they are used for identifying senescent cells in vitro and in vivo. We also highlight the importance that these levels of regulations have in the development of therapeutic targets.
The Complexity of the Senescence Phenotype
The functional decline of an organism throughout its life affects multiple organs and is accompanied by the appearance of several diseases. This general decline of functional capabilities is known as aging (see Glossary) and is fairly conserved among species [1].
A main feature of aged organisms is the accumulation of cellular senescence [1], a state of permanent cell cycle arrest in response to different damaging stimuli [2] (Box 1). Excessive and aberrant accumulation of senescent cells in tissues can negatively affect regenerative capacities and create a proinflammatorymilieu favorable forthe onset and progression of various age-related diseases, including cancer[3,4]. However, senescent cells have several beneficialfunctions forthe organism. Duetothe activation of an irreversible proliferation arrest, cellular senescence is seen as a strong safeguard against tumorigenesis [3]. Moreover, senescent cells can act via both cell and non-cell autonomous mechanisms as positive regulators of tissue remodeling and repair during development and adulthood[5,6].Deleteriousfunctions of senescent cells are potentially powerful targets for antiaging approaches [7], but the existence of beneficial senescence programs complicates the development of interventions without incurring toxicities.
The senescence phenotype is often characterized by the activation of a chronic DNA damage response (DDR), the engagement of various cyclin-dependent kinase inhibitors (CDKi), enhanced secretion of proinflammatory and tissue-remodeling factors, induction of antiapoptotic genes, altered metabolic rates, and endoplasmic reticulum (ER) stress (Figure 1, Key Figure). As a consequence of these signaling pathways, senescent cells show structural aberrations, from enlarged and more flattened morphology, altered composition of the plasma membrane (PM), accumulation of lysosomes and mitochondria, and nuclear changes (Figure 2).
The understanding of how these different hallmarks are regulated and how they are present in non-senescence states is essential to choosing the right methods to measure them. However, there are two important problems for the identification, isolation, and characterization of senescent cells. First, many of the senescence-associated molecular and morphological features are present in other cellular states and conditions. Second, the phenotype of senescent cells is highly heterogeneous and dynamic, possibly a consequence of various distinct senescence programs. These limitations have to be taken into account carefully for the generation of therapies targeting senescent cells. Here, we describe the main hallmarks of senescent cells, the methods used to measure them, and the limitations for their use as markers. Finally, we discuss how these senescenceassociated hallmarks are currently being exploited for antisenescence interventions.
Box 1. Types of Senescence In vitro senescence can be induced by different stimuli [2]. Whether all these ‘types of senescence’ actually occur in in vivo is not yet known. Here, we describe the main models of senescence used in research. At least two other biological events, namely wound healing and development, are known to rely on senescence [3]. However, these types have been less described and, thus, are beyond the scope of this review.
Replicative senescence: this refers to the decrease in proliferation potential observed after multiple cell divisions that ultimately leads to total arrest [160]. The shortening of telomeres as a consequence of multiple cell divisions in nontransformed cells has been blamed for this type of senescence [3].
DNA damage-induced senescence: irreparable DNA damage can induce either senescence or apoptosis, depending on the magnitude of the damage [2]. In vitro, multiple DNA-damaging agents are used to induce this type of senescence, including radiation (ionizing and UV) or multiple drugs (see ‘chemotherapy-induced senescence’) [2].
Oncogene-induced senescence (OIS): the activation of oncogenes, such as Ras or BRAF, or the inactivation of tumor suppressors, such as PTEN, can lead to OIS [2,3]. Oxidative stress-induced senescence: either oxidizing products of the cell metabolism or known oxidative agents (e.g., H2O2) can cause senescence [49]. Although oxidizing agents exert their effect partly through DNA damage, other cellular components and processes are also affected.
Chemotherapy-induced senescence: multiple anticancer drugs are able to induce senescence. Some (such as bleomycin or doxorubicin) induce DNA damage, while others can act through different mechanisms, such as inhibition of CDKs (e.g., abemaciclib and palbociclib) [163].
Mitochondrial dysfunction-associated senescence (MiDAS): it was recently reported that induction of mitochondrial dysfunction also leads to senescence [56]. The phenotype, particularly the SASP, appears to be characteristic of this type of senescence [56].
Epigenetically induced senescence: inhibitors of DNA methylases (e.g., 5-aza-20 -deoxycytidine) or histone deacetylases (e.g., suberoylanilide hydroxamic acid and sodium butyrate) are also known to cause senescence [163].
Paracrine senescence: senescence induced by the SASP produced by a primary senescent cell [70].
Signaling pathways as Hallmarks of Senescence
DNA Damage Response
In the presence of DNA damage, cells activate a robust response, the DDR. Double-strand DNA breaks (DSBs) are powerful activators of DDR, and can lead to cellular senescence when unresolved. DSBs promote the recruitment and binding of ATM kinase to the DNA damage site [8,9]. This recruitment drives phosphorylation of the histone H2AX, which facilitates the assembly of specific DNA repair complexes (Figure 1) [10]. Histone methylation can also contribute to the assembly of damage response components; a complex of kap-1, HP1, and the H3K9 methyltransferase suv39h1 is loaded directly onto the chromatin at DSBs, leading to the methylation of H3K9. H3K9me3 functions as a binding site and activates the acetyltransferase Tip60, which subsequently acetylates and activates ATM [11]. Therefore, H3K9 methylation is required for ATM-mediated DNA damage signaling during early stages of the DDR, but H3K9 methylation has to be later reversed to promote the repair process. DDR provokes the degradation of G9a/GLP methyltransferase, which causes a global reduction in H3K9 dimethylation, including that of IL-6 and IL-8 promoters, two components of the senescence-associated secretory phenotype (SASP; discussed below) [12]. Many substrates are phosphorylated by ATM, including the two essential kinases CHK1 and CHK2, which propagate the signal by further phosphorylating their substrates [13,14].
The persistence of DDR induces the phosphorylation of p53 at multiple serine residues, which enhances the ability of p53 to induce the transcription of many genes [15]. Inductions of g-H2AX nuclear foci or phosphorylated p53 are commonly used as markers of senescence. However, the DDR is activated by a variety of DNA-damaging stimuli that do not lead cells into a senescent state. Moreover, not all senescence programs are a consequence of DDRs.
Cyclin-Dependent Kinase Inhibitors and Cell Cycle Arrest
CDKs phosphorylate and regulate multiple proteins involved in cell cycle progression (Figure 1). Main drivers of the cell cycle arrest in senescence are the CDKis encoded in the CDKN2A (p16INK4a, hereafter p16), CDKN2B (p15INK4b, hereafter p15)m and CDKN1A (p21CIP, hereafter p21) loci. P16 comprises a 136-kb protein that directly interacts and inhibits CDK4/6. P16 is often used as a unique and specific marker for senescence (Box 2), and its transcriptional activation has been used extensively to report the presence of senescent cells in vivo [6,16,17].
Experimental evidence suggests that the main inducers of P16 levels are epigenetic changes, but other regulators, from promoter accessibility to protein stability, have been described. The methyl-transferase DNMT3b is responsible for the de novo methylation of the p16 promoter [18], while DNMT1 maintains existing methylation. Inhibitors of DNMT1 cause demethylation of the p16 promoter and a senescence-like phenotype [19–21]. However, methylation levels do not always correlate with p16 gene expression [22]. The Polycomb group repressive complexes 1 and 2 (PRC1 and PRC2) are also responsible for the deposition of repressive histone modifications at the CDKN2A locus [23], and can be recruited to the p16 promoter by the antisense long noncoding RNA for p16, ANRIL [24]. Other epigenetic marks, such as the repressive histone variant macroH2A1, are enriched in the inactive, but depleted in the active, p16 locus [25]. Transcription factors, such as Sp1, Ets, AP1 (particularly JunB subunit), and PPARg [26–29], bind to the p16 promoter and trigger its transcription, while repressor mechanisms, such as the INK4A transcription silence element (ITSE), YB1, ID1, and AP-1 (c-Jun subunit), balance the activation of p16 [23,27,30,31].
The RNA-binding proteins hnRNP A1 and A2 promote the stability of p16 transcripts [32], while the ribonuclear protein AUF1 binds p16 mRNA and promotes its degradation [33]. Interestingly, there are also hints that p16 can suppress the expression of AUF1 [34]. Translation of the p16 mRNA can be modulated through a region on its 50 untranslated region (UTR) end, which contains an internal ribosome entry site (IRES) [35], and the affinity of p16 for CDK4 can be modulated by Ser140 phosphorylation and Arg138 methylation [36]. Finally, p16 protein is degraded by N terminus polyubiquitinylation and, ultimately, the proteasome and this elimination is favored upon conditions of subconfluence [37,38].
