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).

Apoptosis Resistance in Senescent cells

Taken from: Cellular Senescence: From Growth Arrest to Immunogenic Conversion

In order to develop senotherapeutic drugs (targeting cellular senescence),  it is important to understand the molecular mechanisms governing the pro-survival phenotype of senescent cells. 

Senescent cells are frequently referred to as ‘apoptosis resistant’.  This apparent resistance to an apoptotic stimulus in vitro was originally reported by Wang (1995) who observed that late passage (58 population doubling) WI38 fibroblasts were resistant to death caused by serum withdrawal compared to WI38 cultures at less than 15 or approximately 38 population doublings.  All of these human cell populations were dramatically more resistant to death by growth factor deprivation than Swiss 3T3 fibroblasts.  This death resistant phenotype was linked to maintenance of Bcl2 protein levels in senescent WI38 cells.  Subsequent studies extended the resistance phenotype to treatment with both UV light (120mJ) and staurosporin (35nM) and linked it to reduced expression of caspase 3 (Marcotte et al. 2004).  Subsequent work (Ryu et al. 2007) using human dermal fibroblasts confirmed resistance to staurosporin-induced cell death and demonstrated significant resistance to thapsigargin (up to 700nM).  The enhanced survival of senescent dermal fibroblasts under these conditions was attributed to a failure to down regulate Bcl2 under conditions of cellular stress. 

It has been proposed that resistance to apoptotic cell death is a feature of the senescent phenotype that may promote their persistence in vivo, thereby favoring immune clearance over cell death. However, key questions around this phenotypic aspect remain and may be summarized as (i) what are the primary molecular players driving apoptosis resistance in senescent human dermal and lung fibroblasts? (ii) is this phenomenon a general one across tissues and between species?

It is possible that the pro-survival response observed in fibroblasts normally facilitates DNA repair, but is maintained when persistent DNA damage activates the senescent program.  For example, when low levels of DSBs are present, ATM and ATR can result in ERK/NFkB pro-survival signaling (Khalil et al. 2010, Hawkins et al. 2011, Janssens and Tschopp, 2006) that has been associated with the induction of senescent cells by various triggers.  Paradoxically ATM-deficient human fibroblasts are significantly more resistant to cell death triggered by exposure to doxorubicin or low dose ionizing radiation than wild type controls (Park et al. 2012).  However, the population doublings levels of the wild type and mutant cultures were not reported.   If significantly different, this has the potential to confound studies of this type (since normal fibroblast cultures are mixtures of senescent and proliferating cells, the proportions of which alter as the culture is passaged).

In addition to activating cell cycle arrest in response to DNA damage, the p53/p21 pathway can also initiate a pro-survival response.  In some studies, p21 has been shown to play a role in cell survival through its cytoplasmic localization, rather than its nuclear localization associated with cell cycle arrest (Gartel and Tyner, 2002, Piccolo and Crispi 2012, Kreis et al. 2014).  Interestingly, p21 has been reported to be a negative regulator of p53-mediated apoptosis (Gartel and Tyner, 2002), a known response reported in senescent fibroblasts (Seluanov et al. 2001).  p21 has also been reported to promote cell survival in response to oxidative stress by integrating the DDR with endoplasmic reticulum (ER) stress signaling (Vitiello et al. 2009).  However, the up-regulation of p21 may also be required for cells to enter and maintain quiescence (Perucca et al. 2009), suggesting a pro-survival response may occur independent of DNA damage, but dependent upon growth state. 

Autophagy is another feature of senescent cells which can also be initiated by DNA damage and promote cell survival (Rodriguez-Rocha et al. 2011, Singh et al. 2012).  Autophagy promotes cell survival by the degradation of damaged cellular components (Codogno and Meijer, 2005), probably as a result of elevated ROS (Scherz-Shouval and Elazar, 2011) in the case of cell senescence.  Interestingly, there is crosstalk between autophagy and apoptosis pathways (Zhou et al. 2011, Xu et al. 2013, Lindqvist and Vaux, 2014), with particular emphasis on the anti-apoptotic Bcl2 protein family.   

It has long been recognized that cytokines and their binding proteins can act to modulate cell survival (Lotem and Sachs, 1999).  Given the altered secretory phenotype of some senescent cells, it would be unsurprising if this did not contribute to altered death dynamics, but the mechanisms by which this could occur are potentially highly complex.   For example Interleukin-6 (secreted by senescent cells) has been shown to promote cell survival in transformed cells (Biroccio et al. 2013), and its secretion by cancer-associated fibroblasts protects luminal breast cancer cells from tamoxifen treatment (Sun et al. 2014).  Whilst inhibition of insulin-like growth factor-1 (IGF-1) has been shown to induce apoptosis in senescent fibroblasts (Luo et al. 2014), the alteration of IGF-1 binding proteins are just as likely to influence cell survival.  For example, insulin-like growth factor binding protein 3 (IGFBP-3) is both transcriptionally up-regulated and secreted in elevated amounts by senescent human fibroblasts (Hampel et al. 2005).  IGFBP-3 triggers enhance apoptotic cell death in tumor cells when internalized and translocated to the nucleus, where it targets intracellular regulators of apoptosis (Hampel et al. 2005). Endocytotic uptake of IGFBP-3 in senescent human fibroblasts did not occur.  This has the potential to render them apoptosis resistant and capable of promoting apoptosis in cells nearby.  It could be speculated that in a microenvironment characterized by high cell turnover, both senescent and precancerous cells could be in close proximity. Elevated local IGFBP-3 generated by senescent cells could thus act as a paracrine tumour suppression mechanism.  This idea remains untested.

It seems doubtful that global apoptosis resistance is a general feature of senescent cells.  For example, early work by one of us (RGAF) failed to show any elevation in spontaneous apoptosis rates in HUVECs cultured to senescence (although baseline apoptosis rates as measured by TUNEL were significantly higher than those seen in fibroblasts) (Kalashnik et al. 2000).  Later studies (Hoffman et al. 2001) demonstrated that late passage HUVECs were more sensitive to apoptosis induced by oxidized LDL or TNFα compared to early passage cells.  Jeon and Boo (2013) have recently shown that up-regulation of the Fas receptor at both the mRNA and protein level in senescent HUVECs probably underlies their enhanced potential to undergo programmed cell death.  Perhaps most compellingly, Hample et al. (2004) demonstrated in parallel culture experiments that whilst senescent human dermal fibroblasts were more resistant to cell death induced by exposure to ceramide than early passage cells, senescent HUVECs were significantly more apoptosis prone.

It is interesting that minimal changes in baseline apoptosis rates could be detected in senescent HUVEC populations despite their increased sensitivity to Fas or ceramide-induced killing.  However Wang et al. (2004) reported an analogous phenomenon in senescent human keratinocytes.  This study demonstrated that spontaneous apoptosis rates did not alter in cultures of senescent human keratinocytes (duplicating an earlier report by Norsgaard et al. 1996).  Nonetheless, levels of Fas and related apoptotic effectors (e.g. FLICE) increased whilst Bcl2 declined significantly (as measured by ELISA).  The authors showed that antibody-mediated Fas activation or medium exhaustion increased the apoptotic fraction from 3-5% to 30% in senescent keratinocytes, whilst leaving apoptosis levels unchanged in early passage cultures.

Interestingly, Crescenzi et al. (2011) have recently shown that induction of premature senescence in human cancer cell lines also induces Fas expression, and concomitant susceptibility to Fas-induced apoptosis.  Fibroblasts rendered senescent by serial passage are also susceptible to Fas-mediated killing (Tepper et al. 2000).  Thus it is possible that at senescence, human cell types differ in their resistance to apoptosis induced by stressors, but show a common susceptibility to Fas/TNFα mediated killing.  If immunogenic conversion were a key hallmark of senescence, then this would seem plausible.  It does however require significant additional experimental study.

As with the secretory response, it should not be assumed that an “apoptosis resistant” phenotype is conserved across species.  For example Mayogora et al. (2004) demonstrated that cultures of cardiac fibroblasts from Sprague-Dawley rats were more resistant to apoptosis induced by serum withdrawal or staurosporin, than dermal fibroblast cultures initiated from the same animals.  Dermal fibroblasts from this species apparently lacked Bcl2 protein as measured by Western blot (although it remained readily detectable in cardiac fibroblasts).  This is a clear species difference and suggests that researchers working in other systems should not assume that the features observed in human cells are duplicated across the animal kingdom.

The removal of senescent cells using therapeutic agents

As discussed in the previous blog, one of the strategies for overcoming the detrimental effects of senescent cells is to remove them as they appear through the use of therapeutic agents. At present, no drug-based system exists which can specifically identify senescent cells and remove them. However, there is currently great interest in the development of drugs which specifically target and remove cancer cells. The problem with current cancer treatments (such as drugs used in chemotherapy) is that they are non-specific and as such can cause damage and undesirable changes to non-cancerous cells, causing side-effects. The development of cell-specific drug targeting is greatly needed and such research could be adapted to target senescent cells. Cell-specific drug targeting requires a carrier molecule containing a targeting agent which specifically recognises and binds to a specific receptor or binding site on the surface membrane of target cells and a therapeutic agent which could trigger programmed cell death, apoptosis. The following are crucial factors in determining the success of drug-targeting systems (Beljaars et al, 2001, Petrak 2005).

(1) Cellular specificity: For a drug to exert its desired effect it needs to be in physical contact with its physiological target, such as a receptor.

(2) Rate of elimination of the drug-carrier conjugate: It is essential that the drug-carrier conjugate is not removed too rapidly from the circulation. If it is eliminated from systemic circulation more rapidly than it is delivered to the target site, the amount of conjugate at the target site might never be enough to provide the required concentration of free (unbound) drug.

(3) Rate of release of free drug at the non-target site: Depending on the amount of drug, the release of drug away from the target site could nullify any benefits that might potentially come from delivering the drug to the target site.

(4) Rate of delivery of drug-carrier conjugate to the target site: If the drug conjugate reaches the target site too slowly, the supply of free drug might never be sufficient to generate the concentration required to elicit the desired therapeutic effect at the site of action.

(5) Rate of release of free drug at target site: The capacity of the system selected for the release of free drug from the conjugate should be considered. It needs to be suitable for processing the entirety of the drug-carrier conjugate arriving at the target site, doing so at a rate that also ensures drug accumulation at this site.

(6) Rate of removal of free drug from the target site: Drugs that benefit most from target-selective delivery are those that are retained at the site while acting on their target of action.

(7) Rate of elimination of the drug-carrier conjugate and free drug from the body: For optimal targeting, elimination of the complete drug-carrier system should be minimal.

One promising area of research in the development of drug delivery systems incorporates the use of nanotechnology (http://nano.cancer.gov/). Such technology has been used to create dendrimers, spheroid or globular nanostructures which are highly branched (Alexis et al, 2008). The branched regions of these dendrimers can be used to attach molecules such as targeting and therapeutic agents (Gillies and Frechet 2005). To test this nano-delivery system, invesitgators at the University of Michigan attached a targeting agent, a therapeutic agent and an imaging agent to the surface of dendrimers (Majoros et al, 2006, Shi et al 2007). The investigators chose folic acid as the tumour-targeting agent (a molecule which binds to a high-affinity receptor found on many types of tumour cells), paclitaxel as the therapeutic agent (a drug which triggers programmed cell death, apoptosis) and the fluorescent dye known as fluorescein isothiocyanate as the imaging agent. This nano-dilivery system was then tested on two sets of cancer cells in vitro: one that expresses the folic acid receptor and one that does not. Only the cells containing the folic acid receptor took up the dendrimer, visualised by the presence of the imaging agent. The dendrimer construct was highly toxic to these cells but had no effect on cells without the folic acid receptor. When both of these cells were exposed to dendrimers containing the targeting and imaging agent but no paclitaxel, no detrimental effects were observed.

These promising initial results thus call for tests to be carried out on animals with tumours that overexpress folic acid receptors. It is research like this that could one day be adapted to specifically target senescent cells. For this to be the case, a target agent is required that specifically recognises senescent cells. For this to be achieved, a deeper understanding of the changes which occur when a cell becomes senescent is required. Ideally a universally expressed senescent membrane receptor would be ideal, but at present no such receptor is known. If it did, it would also make a useful biomarker for detecting senescent cells in tissues.