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.