Targeting Cellular Senescence: One Drug for Many Diseases?

Cellular senescence is an altered cell state associated with permanent cell cycle arrest and an immunogenic, pro-inflammatory secretome that can contribute to the development and progression of age-related diseases.  Because cellular senescence can occur in different cell types (i.e. pancreatic beta cells, vascular smooth muscle cells, astrocytes) that undertake different biological functions, then their appearance can manifest differently and we refer to these manifestations as different diseases.  These include, diabetes, cardiovascular disease, COPD and cancer.  The mechanisms by which senescent cells can cause disease include:

        (1)  Loss of cellular regenerative capacity.  
        (2) Loss of normal cell function.  
        (3) Persistent pro-inflammatory tissue damage.
        (4) Altering the behaviour of neighbouring cells.
        (5) Protease-mediated degradation of extracellular structural proteins.

Although scientists researching cell senescence have long suspected that senescent cells play an important role in ageing and age-related disease, convincing evidence had not been provided until 2011 when Scientists from the Mayo Clinic in the US published their findings on the elimination of senescent cells in mice.  The elimination of senescent cells using transgenic (genetically engineered) mice delayed the onset of disease, thereby increasing healthspan.  However, likely owing to the use of an accelerated ageing mouse model, no life extension was observed in this instance.  However, a follow-up study by the same group using naturally aged mice lead to delayed tumorigenesis and reduced age-related decline leading to significant increase (up to 35%) in lifespan.  Studies like these thus provide a convincing rationale for developing therapeutic approaches for targeting senescent cells, so-called “senotherapeutics”.  These may include:

  1.        Specifically inducing cell death in senescent cells (i.e. small-molecule compounds).
  2.        Inhibiting the senescent secretome (i.e. inhibitors of inflammation).
  3.        Preventing senescence induction (i.e. telomerase activators, geroprotectors).
  4.        Boost immune response towards senescent cells (i.e. immunotherapy).

A few recent studies have published findings regarding the elimination of senescent cells by small-molecule compounds.  Wang et al (2016) identified the compound ABT-263 as a potent inducer of cell death in senescent cells leading to rejuvenation of aged tissue stem cells.  In another study, Yosef et al (2016) identified ABT-737 which through the elimination of senescent cells from the epidermis of the skin of mice lead to increased hair-follicle stem cell proliferation.  Both ABT-263 and ABT-737 inhibit proteins (BCL-2 family) known to play a role in cell survival.

Studies focused on the elimination of senescent cells are only beginning to emerge and will no doubt gain momentum as they show tremendous potential for improving health and wellbeing.  One intriguing notion that may arise from this research is concerned with the question of whether it may one day be possible to treat many diseases with a single drug.  If senescent cells play a role in the development of many different diseases, then a drug that can eliminate senescent cells in all cell types could act as both a preventative and a treatment for many diseases.

One of the obstacles preventing research into senotherapeutics from advancing and ultimately becoming translational to help increase healthspan of individuals within the general public, is funding.  However, this has not discouraged some researchers who were determined enough to acquire funding through the help of crowdfunding.  The Major Mouse Testing Programme (MMTP) raised over $50,000 towards research focused on eliminating senescent cells and is still ongoing (Click Here). 

A new start-up company, CellAge (click here) is also interested in targeting senescent cells and has recently announced a crowdfunding campaign (click here) to raise funds for their ongoing research.  CellAge aims are to “Increase human healthspan and reduce the incidence of age-related diseases by helping the human body eliminate senescent cells.  Our breakthrough technology concept harvests the promise of synthetic biology and recent findings in ageing research to deliver novel products and therapies to enable people to live healthier longer lives”

So if you are interested in stimulating research in this field for the benefit of all, then please make a donation (link here).

NKG2D ligands mediate immunosurveillance of senescent cells


Cellular senescence is a stress response mechanism that limits tumorigenesis and tissue damage. Induction of cellular senescence commonly coincides with an immunogenic phenotype that promotes self-elimination by components of the immune system, thereby facilitating tumor suppression and limiting excess fibrosis during wound repair. The mechanisms by which senescent cells regulate their immune surveillance are not completely understood. Here we show that ligands of an activating Natural Killer (NK) cell receptor (NKG2D), MICA and ULBP2 are consistently up-regulated following induction of replicative senescence, oncogene-induced senescence and DNA damage - induced senescence. MICA and ULBP2 proteins are necessary for efficient NK-mediated cytotoxicity towards senescent fibroblasts. The mechanisms regulating the initial expression of NKG2D ligands in senescent cells are dependent on a DNA damage response, whilst continuous expression of these ligands is regulated by the ERK signaling pathway. In liver fibrosis, the accumulation of senescent activated stellate cells is increased in mice lacking NKG2D receptor leading to increased fibrosis.  Overall, our results provide new insights into the mechanisms regulating the expression of immune ligands in senescent cells and reveal the importance of NKG2D receptor-ligand interaction in protecting against liver fibrosis.

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.

The senescent phenotype and promiscuous gene expression

Senescent cells are often associated with changes in gene expression that appear to occur independent of the regulated gene expression linked to aspects of the senescent phenotype such as cell cycle arrest, the secretory response and apoptosis resistance. This phenomenon has been termed promiscuous gene expression (pGE) (Burton and Krizhanovsky, 2014) and can be more specifically defined as gene expression that is uncoupled from tissue or developmental regulation. 

pGE can be observed in microarray analysis by comparing the gene expression profiles of different senescent cell types and lines. Zhang et al (2003) has demonstrated that the up-regulation of genes in senescent fibroblasts was associated with gene clustering (150 of the 376 gene up-regulated), whereas the down-regulation of genes (313) was not. 48.1% of the up-regulated genes were designated as membrane-associated proteins, 10.5% related to apoptosis and 15.8% to transport, whereas 17.9% of the down-regulated genes are involved in cell cycle regulation. Gene expression changes in senescent human mammary epithelial cells (HMECs) were shown to be drastically different than that of the fibroblasts, despite both undergoing senescence induced by telomere attrition. Only five genes up-regulated and seven genes down-regulated in HMECs showed similar regulation in fibroblasts. However, like senescent fibroblasts, HMECs also demonstrated gene clustering associated with up-regulated genes only. Zhang et al postulated at the time, that if senescence is a response to DNA damage, then the observed differences in gene expression between senescent fibroblasts and HMECs imply that the effects of DNA damage must vary according to cell type and line. This study also suggested that processes occurring during senescence may lead to localized alteration in chromatin and the consequent up-regulation of groups of genes within “opened” domains. 

Shelton et al (1999) also demonstrated that senescence-mediated gene expression between different cell lineages varies greatly. BJ fibroblasts, HUVECs and retinal pigment epithelial cells (RPE340) that underwent replicative senescence demonstrated substantial variation in gene expression. A genomic comparison of three different senescent fibroblasts strains also demonstrated significant differences in gene expression, but also shared trends were apparent. If indeed pGE is uncoupled from tissue or developmental regulation, then stochastic processes that alter chromatin structure could be at play and the different response between cell types and cell strains could reflect differences in cell-specific chromatin architecture important for cell-specific gene expression. Elevated levels of oxidative stress, a feature of senescent cells could be one such stochastic process. 

Bahar et al demonstrated that although gene expression levels varied among cardiomyocytes taken from hearts of young mice, the heterogeneity is elevated with age (Bahar et al. 2006). This increased stochastic gene expression with age was suggested to be the result of genomic damage, as mouse embryonic fibroblasts treated with hydrogen peroxide in culture resulted in significant cell-cell variation in gene expression in conjunction with these cells showing morphological signs of cellular senescence (Bahar et al. 2006). 

So how could DNA damage induced by oxidative stress result in stochastic changes in gene expression? When cells sustain DNA damage, chromatin undergoes remodeling to facilitate DNA repair (Price and D’Andrea, 2013, House et al. 2014). This remodeling or “opening” of tightly packed DNA could allow transcription factors access to previously inaccessible genes. Therefore, persistent DNA damage and consequently continuous chromatin remodeling may facilitate pGE. While the induction of DNA damage is likely a stochastic process, the sites of DNA damage may not be completely random, as certain areas of the genome may be more or less prone to genomic insults (Ma et al. 2012). The clustering phenomenon reported by Zhang et al may be the result of these DNA damage prone sites (Zhang et al. 2003). If this were indeed the case, while there may be substantial differences in gene expression at a cell-cell comparison, an overall comparison between cell cultures would likely demonstrate consistent gene alterations resulting from an average expression of all cells within a culture. 

In addition to oxidative stress, a number of other possible mechanisms may exist for generating pGE. Senescent fibroblasts are known to undergo methylation changes (Cruickshanks et al. 2013) and these alterations may lead to epigenetic alterations that promote stochastic changes in gene expression. Alternatively, it has been suggested that DNA damage may modulate gene expression by altering the binding capacity of transcription factors (Rose et al. 2012). 

Interestingly, the reprogramming of fibroblasts into induced pluripotent stem cells (iPSCs) via the addition of OCT4, SOX2, KLF4 and MYC (OSKM) requires a long stochastic phase of gene activation associated with changes in histone modifications at somatic genes and activation of DNA repair and RNA processing (Buganim et al. 2013). This stochastic gene expression may be the result of “promiscuous binding” by OCT4, SOX2 and KLF4, where they occupy accessible chromatin and bind to promoters of genes that are active or repressed (Buganim et al. 2013). It is possible that pGE in senescent cells partly mimics stochastic gene activation associated with cellular reprogramming. However, whether pGE in senescent cells is associated with factors that can undergo “promiscuous binding” has yet to be determined. 

Whether pGE plays a functional role in cell senescence has yet to be determined. However, it can be speculated that pGE may function to generate an array of tissue-restricted proteins that can subsequently be processed into peptides by autophagic proteases for presentation on MHC molecules (Dengjel et al. 2005). Similar to the presentation of tumour-associated antigens (Reuschenbach et al. 2009), senescent cells may also present antigens that can be recognized by immune cells, thereby becoming antigen-presenting cells (APCs). Although the up-regulation of MHC molecules on senescent cells have yet to be fully evaluated, the up-regulation of MHC class I but not MHC class II in response to DNA damage in fibroblasts has been reported (Tang et al. 2014). It remains to be determined whether pGE is a component of immunogenic conversion.

Atypical senescent states: Experimental induction of cyclin-dependent kinase inhibitors (e.g. p16, p21)

For many researchers, irreversible cell cycle arrest is the canonical trait of senescent cells.   Such growth arrest can be induced experimentally by the up-regulation or over-expression of cyclin dependent kinase inhibitors (CDKi).  Thus valuable models are, at least potentially, available in which to study the physiological effect of growth arrest distinct from the DDR or any other upstream response.   Unfortunately there has been little characterization of the phenotype of cells rendered ‘senescent’ by this means.

Blagosklonny and co-workers (Korotchkina et al. 2009) used an isopropyl-thio-galactosidase (IPTG)-inducible p21 expression construct to induce a senescence-like state in an HT1080-derived cell line (HT-p21-9).   Characterisation of the phenotype of these cells does not appear to have been attempted beyond observing irreversible growth arrest and the presence of increased SA-β-Gal activity.  Given that HT1080 is a highly tumorigenic fibrosarcoma carrying an activated N-ras oncogene (Benedict et al. 1984), it probably represents a poor genetic background in which to assess whether markers of immunogenic conversion or resistance to cell death can be induced by CDKi overexpression alone.  However, the basic principle of using such a construct for that purpose is sound.

Tokarsky-Amiel et al (2013) showed that overexpression of p14ARF in the epidermis of the skin of mice (using a tetracyclin-inducible construct) resulted in mass apoptosis and cell cycle arrest.  As measured by SA-β-Gal activity, the p14ARF transgene drove senescence in up to 8% of the surviving cells in the epithelium by a p53-dependent mechanism (demonstrated by ablation of p53 through co-expression of a specific shRNA directed against it).  These senescent cells were viable within the epidermis for several weeks consistent with lack of clearance.  Unfortunately, minimal analysis of their phenotype was conducted (beyond assessment of the message levels for the senescence-associated genes Pai-1 and Dcr2).  Thus, the immune state of the p14ARF-senescent cells is currently unclear and the picture is complicated by the fact that senescent rodent cells do not display a senescent secretome under some conditions.  However, given that alopecia and follical stem cell dysfunction were observed in the animals, it is clear that cells rendered ‘senescent’ in this manner can exert phenotypic effects.  Thus, there is some evidence that cell cycle arrest alone may be sufficient to cause problems in highly mitotic tissues such as the epidermis, but large amounts of work remain to be done.  

CDKi overexpression systems clearly have the potential to be valuable tools.  However the extent to which these are physiologically reflective can legitimately be challenged.  This can be understood in two ways (i) the mechanism by which the growth arrest is induced has not been reported in vivo and (ii) cells do not become senescent en mass but gradually as a result of tissue turnover throughout life.  Thus, findings made with these systems could be considered ‘artefactual’

By way of addressing these concerns, it is worth remembering that for many years replicative senescence was dismissed as a ‘tissue culture artefact’ because senescent cells had not been observed in vivo (evidence for their existence in tissue remained severely limited until the late 1990s).  By the same token, elevation of CDKi alone in cells in vivo is not impossible.  Absence of evidence is never evidence of absence.   Similarly, many over-expression systems model systems can be said to be non-physiological.  However, valuable data is routinely gathered using them and in this instance could allow researchers to gage the maximum physiological impact that irreversible growth arrest can have on tissue function.  Thus, if these limits are recognized, such models are potentially utile, especially when combined with detailed analysis of phenotypes known to exist in other ‘senescent cells’ (e.g. apoptosis resistance, immune ligand presentation and the secretory response) 

Atypical senescent states: Endoplasmic Reticulum stress induced senescence

Endoplasmic reticulum (ER) stress may also promote a senescent-like response.  The accumulation of unfolded proteins in the ER triggers a stress-signaling pathway that can result in cell cycle arrest mediated by p27 (Han et al. 2013) and the p53/47 isoform (Bourougaa et al. 2010).  Furthermore, ER stress has also been shown to induce an inflammatory response via NFkB activation (Garg et al. 2012) and induce cytokines such as MCP-1, IL-6 and IL-8 (Schroder, 2008), which are capable of attracting and activating immune cells (Sagiv and Krizhanovsky, 2013). ER stress has also been shown to promote cell survival, another feature of cell senescence (Raciti et al. 2012).  Interestingly, a senescent state via activation of ER stress-dependent p21 signaling has been reported in proximal tubular epithelial cells, triggered by receptors for advanced glycation end-products (RAGE) (Liu et al. 2014).  Although, ER stress-induced senescence has the potential induce an immunogenic phenotype in the absence of DNA damage, a full evaluation of the phenotype is required to determine if this is so.

Atypical senescent states: Metabolic stress-induced senescence

Metabolic stress, defined here as a combination of aerobic glycolysis and mitochondria dysfunction can potentially trigger a senescent state.  All organisms that use aerobic glycolysis form reactive acyclic α-oxoaldehydes (e.g. methylglyoxal and glyoxal) spontaneously from triosephosphates and by a wide variety of other routes (Thornalley, 2009).  These dicarbonyl compounds are highly reactive and damage proteins through non-enzymatic modification producing a wide variety of covalent adducts (AGEs).  Elevated levels of methylglyoxal and glyoxal are known to be cytotoxic and although the mechanism of action remains imprecisely defined, it can be blocked by ROS scavengers, suggesting that oxidative stress mediates at least some of the deleterious effects (Shangari and O’Brian, 2004).

Cytosolic and mitochondrial protection from dicarbonly damage is primarily mediated through the action of the glyoxalase system that consists of two enzymes, glyoxalase I and II.  However, in cultures of WI38 fibroblasts a significant reduction in the activity of glyoxalase-I occurs with serial passage (Ahmed et al. 2010).  Treatment of cultures of ASF2 human adult dermal fibroblasts with micro or millimolar concentrations of glyoxal or methylglyoxal renders them senescent within 72 hours.  This was defined by the presence of typical senescent morphology, irreversible growth arrest and increased SA-β-Gal activity (Sejersen & Rattan, 2009).  Further studies (Larsen et al. 2012) extended these observations to immortalized human mesenchymal stem cells (MSCs) and demonstrated that treatment with physiologically reflective (Han et al. 2007) concentrations of glyoxal for 72 hours led to senescence without significant cell death (although massive cell death occurred at higher glyoxal concentrations).  Elevated levels of SA-β-Gal, p16 and DNA damage (as measured by COMET) accompanied the growth arrest.  Interestingly, a profound reduction in the ability of these senescent MSCs to differentiate into functional osteoblasts (as determined by alkaline phosphatase and mineralization assays) was also observed.   Given the imbalances in glucose metabolism that accompany mammalian ageing (and diabetes), the authors proposed that this type of metabolic stress might underlie age-related changes in bone function.   Unfortunately, no markers of immunogenic conversion have yet been measured in this system and whilst the presence of DNA damage could indicate the likelihood of a secretory response, this cannot be assumed.  Thus, the propensity of senescence human MSCs to be cleared by the immune system remains unknown and is of considerable physiological significance.

The main focus of ageing research is to prevent/combat age-related disease and disability, allowing everyone to live healthier lives for longer.