Birth and death of cellular senescence Wang, Boshi
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10.33612/diss.131223530
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Wang, B. (2020). Birth and death of cellular senescence. University of Groningen. https://doi.org/10.33612/diss.131223530
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CHAPTER VI
General summary and discussion
Boshi Wang
European Research Institute for the Biology of Ageing (ERIBA), University Medical Center Groningen (UMCG), 9713AV Groningen, The Netherlands
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This thesis focuses on better understanding the underlying mechanisms for senescence induction (Chapter 2 and Chapter 3) and clearance (Chapter 4 and Chapter 5). In Chapter 2, we reviewed how anticancer therapies induce cellular senescence in normal cells / tissues, and the subsequent side effects deriving from these senescent cells. Radio- and chemotherapy were among the first developed anti-cancer therapies and are yet arguably the most commonly used. Because of their mechanism of action, based on inflicting severe DNA damage, they can induce senescence and Senescence-Associated Secretory Phenotypes (SASP) of both cancer and normal cells. Paradoxically, SASP factors produced by senescent cells residing in the tumor microenvironment were shown to have pro-tumorigenic effects. Moreover, pro-inflammatory SASP factors, mainly activated through the NF-κB pathway, can also contribute to side effects of anti-cancer interventions, including fatigue, cardiac dysfunction and cancer relapse. Novel senolytic drugs targeting SASP-producing senescent cells in combination with cancer therapy seem to be very promising and are under investigation. However, for the studies available for CDK4/6 inhibitors (CDK4/6i; palbociclib and abemaciclib) induced senescent cancer cells, whether SASP is established in these cells is under debate. Moreover, little is known about the characteristics of CDK4/6i-induced senescent normal cells. In Chapter 3, my study went deeper to dissect whether senescence-associate phenotypes are induced by CDK4/6i. First, we have shown that CDK4/6i indeed could induce senescence in normal fibroblasts, and we were also the first to show the systemic senescence induction by CDK4/6i using a senescence reporter mouse model. Second, the CDK4/6i-induced stable growth arrest was dependent on p53 transcriptional activity via the repression of the DNA methyltransferase DNMT1, even in absence of a chronic DNA damage response (DDR). Third, we found that the CDK4/6i-induced senescent program engaged only a partial SASP enriched in p53-associated genes but without pro-inflammatory and detrimental NF-κB-dependent factors in human cells, mouse tissues and plasma of metastatic breast cancer patients. Fourth, we demonstrated that CDK4/6i-induced p16+ senescent cells did not exert pro-tumorigenic and detrimental effects in culture and in vivo.
Fifth, our data suggested that short-term treatment of CDK4/6i on chemotherapy-induced senescent cells was sufficient to partially reduce an already established detrimental κB-dependent SASP factors and improve the healthspan of treated mice via p53 activation and NF-κB repression. Another important message from this chapter is that not all senescent cells are detrimental, and it is dependent on the context of stimuli. In Chapter 4, we confirmed that depletion of p16+ senescent cells contributed to the improvements of healthspan in mice using
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our senescence reporter mouse model, and the platform was established for further exploration of biomarkers. In Chapter 5, we analyzed whether common extrinsic and intrinsic factors can influence immune-mediated clearance of senescent cells. We confirmed that functional immune systems are essential to achieve efficient clearance, but this might be also gender-dependent. Moreover, we further explored another question raised from Chapter 3 that if distinct pro-inflammatory SASP in CDK4/6i-induced senescent cells reduces the immune clearance of these cells. The results showed that CDK4/6i-induced senescent cells with reduced pro-inflammatory SASP were cleared even faster than the chemotherapy-induced cells, implying that the cell surface markers might be more relevant variables to mediate senescence immune-mediated clearance.
Based on the data obtained during my PhD, I now have formulated new questions to address in the coming years:
(1) How do pharmaceutical CDK4/6 inhibitors induce irreversible growth arrest?
In Chapter 3, we investigated the mechanisms underlying the CDK4/6i-induced irreversible growth arrest and found that DNMT1 and p53 were the important mediators involved in this process. The first problem is that we could not identify how DNMT1 regulate the p53 transcriptional activity. Since the DNA methylation in the promoter or enhancer region silence the gene expression in many cases, and indeed DNMT1 mediated the methylation in the promoter region of p21 (Choi et al., 2013; Laget et al., 2014), which is the most powerful p53 direct target (Fischer, 2017). Our working hypothesis is currently the following: in proliferating cells, high level of DNMT1 maintains promoter methylation of p53 downstream target genes, which are usually negative regulators of cell cycle (Fischer, 2017). In contrast, in CDK4/6i-induced senescent cells, DNMT1 expression is reduced via decreased phosphorylation of Rb (Chapter 3; Goel et al., 2017), so that the promoters of p53 downstream genes could be accessible for the binding and positive regulation by p53. This hypothesis needs to be tested via checking the methylated status for p53 target gene promoters under the treatments of CDK4/6i. An alternative and/or complementary hypothesis is that DNMT1 physically interacts with p53 and blocks its transcriptional activities in proliferating cells. There are already some evidences that DNMT1 can bind to p53 (Esteve et al., 2005), but there is no evidence of this interaction leading to p53 inactivation. This hypothesis can be tested by co-immunoprecipitation of p53 and DNMT1 in both proliferating and senescent (CDK4/6i) cells. A second important question on this project is that we still do not know what downstream targets of p53 are important for
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the cell cycle arrest arising as consequence of CDK4/6 inhibition. RNA-sequencing analysis of CDK4/6i-treated control or p53 knock-out cells can help to answer this question.
(2) Do CDK4/6 inhibitors induce SASP in cancer cells?
There are three selective CDK4/6 inhibitors approved by the U.S. FDA for treating ER+ HER2-
advanced breast cancer patients, namely palbociclib, ribociclib and abemaciclib (Klein et al., 2018). Some other CDK4/6 inhibitors, such as trilaciclib (Deng et al., 2017; He et al., 2017) and SHR6390 (Long et al., 2019; Wang et al., 2017), are also under preclinical characterization. Palbociclib and ribociclib were approved earlier and their structures are very similar, and abemaciclib was the last approved and the only one used as monotherapy (Hafner et al., 2019). Due to their similar mechanism of action to the senescence marker p16, these drugs were considered potential inducers of cellular senescence. However, we still lack information of which senescence-associated phenotypes cancer cells treated with CDK4/6i exhibit. For example, we have limited data on the SASP.
It first has been shown that long-term (8 days) treatment with 1 μM palbociclib on melanoma-derived cells induced cellular senescence, and the induction of SASP factors (IL6, IL8 and CXCL1) was observed during the 8 days treatment (Yoshida et al., 2016). Unfortunately, the irreversibility of the growth arrest, which is the fundamental feature of cellular senescence (Gorgoulis et al., 2019), was not investigated in this study. A study of palbociclib on liposarcoma cells also found that SASP factors (CXCL1, GMCSF, IL6 and IL8) were induced by the CDK4/6 inhibitor palbociclib (Kovatcheva et al., 2017). Conversely, in gastric cancer cells it was proposed that a SASPless senescence program could be induced by CDK4/6 inhibition, but experimental evidence for this hypothesis is lacking (Valenzuela et al., 2017). Later, a study focused on NK cell-mediated cytotoxicity showed that 8 days palbociclib treatment (500 nM) alone was unable to induce senescence in lung cancer cells (e.g. A549) and SASP induction was also not observed. Nevertheless, combinatorial treatments of MEK inhibitor (trametinib) and palbociclib clearly induced senescence and SASP. Given that trametinib treatment alone already activate a very strong profile of SASP factors that is comparable to the combinatorial treatment (Ruscetti et al., 2018), whether palbociclib contributed to the SASP activation in the combination setting need to be further investigated. Of note, a study recently showed that IMR90 human fibroblasts induced into senescence by palbociclib treatments (10 μM for 7 days) did not induce SASP when the factors (IL1B and IL6) were checked (Hari et al., 2019). Interestingly, the study of another CDK4/6 inhibitor abemaciclib on anti-tumor immunity effects also discovered that SASP factors (IL6, IL1A and
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IL1B) were not induced in drug-treated breast cancer cells and xenografts, even though up-regulation of SA-β-galactosidase activity was observed (Goel et al., 2017). Again, as also stated in the study, that only a senescence-like phenotype was induced since irreversibility of growth arrest was also not achieved. However, the other study of abemaciclib on immunity supported the above notion that SASP factors such as IL1A, IL6, IL8 and PAI1 were not detected (Schaer et al., 2018).
Possible explanations for the lack of clarity of whether SASP is engaged in CDK4/6i-induced senescence are: 1) SASP is a collection of multiple factors (Coppé et al., 2008), so probably several selected factors could not represent the entire phenotype and profile; 2) there are important structural, and potentially functional, differences among the three CDK4/6 inhibitors (Hafner et al., 2019); 3) the definition of senescence, and the markers to identify senescent cells, remain very miscellaneous in cancer studies, is very heterogenous; 4) cancer cell lines are highly unstable and harbor a vast array of mutations which can hit essential genes for senescence.
(3) What are the roles of p53-driven SASP factors in mediating the immune clearance of senescent cells?
In Chapter 3, we identified that CDK4/6i-induced senescent cells only activated a partial SASP, mostly driven by p53. In Chapter 5, we found that CDK4/6i-induced senescent cells were cleared even faster in vivo compared to the chemotherapy-induced senescent cells, which activate a full set of SASP. Taken together, it is highly possible that: 1) p53-driven SASP is playing a fundamental role in attracting immune cells; 2) the NF-κB-driven SASP interferes with immune activity; 3) treatment with CDK4/6i promotes expression of ligands for immune cell recognition (Chapter 5; Goel et al., 2017). Experiments on studying the effect of removing p53 or activating NF-κB in CDK4/6i-treated cells, and the accurate study of expression of membrane proteins on CDK4/6i-induced senescent cells will help to address these points.
(4) What is the phenotype of beneficial and well-tolerated senescent cells?
Senescent cells that transiently appeared during wound healing (Demaria et al., 2014), in injury-caused fibrosis (Krizhanovsky et al., 2008) and in developmental stages (Storer et al., 2013) are regarded as beneficial. Wound-associated senescent cells are able to promote skin regeneration through inducing myofibroblast differentiation by secreting the SASP factors PDGF-AA. Further isolation of these senescent cells unraveled that the major cells types entering senescence during healing were fibroblasts and endothelial cells, but the upstream senescence
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inducer(s) is still not known (Demaria et al., 2014). Isolation of the pre-senescent cells at the very early stage of the wounding and performing a transcriptomic analysis of those cells would be informative to check the intermediate effectors between wounding and senescence induction, and subsequently providing more insights about what defines beneficial senescent cells. However, instead of p16, which is a late senescence marker, the early response p53-p21 pathway can serve for the purpose of isolating pre-senescent cells during wound healing. Moreover, we have shown that CDK4/6 inhibitors (abemaciclib) induced senescent cells were not detrimental, but whether they are beneficial or neutral is not known. An interesting future direction could be to expand the analysis on the capacity of abemaciclib to reduce the detrimental effects of high SASP senescent cells (Chapter 3).
(5) What contribute to the difference of immune clearance in females and males?
In Chapter 5, we showed that the clearance of endogenous senescent cells induced by systemic administration of chemotherapy (doxorubicin) or UVB irradiation was faster in females compared to male mice. Moreover, we subcutaneously transplanted exogenous senescent cells of the same origin to female and male mice, and we also observed faster clearance in female mice. However, what factors contribute to the difference still need to be dissected, as two important variables need to be considered: 1) senescence-extrinsic environmental differences; 2) senescence-intrinsic epi-genetic differences.
Undoubtedly, immune environments, both the innate and adaptive immune responses are differential in different genders: 1) the activities of macrophages and neutrophils are higher in females than males; 2) females also have higher counts of CD4+ T cells and higher proportion
of activated CD4+ and CD8+ T cells than males (Klein and Flanagan, 2016). This is
evolutionarily conserved (Klein and Flanagan, 2016) and can result in different outcomes for the clearance of senescent cells. Importantly, both sex hormones and sex chromosome genes can influence immune environments and partially explain the differences observed between different genders (Klein and Flanagan, 2016). Further experiments modulating the hormone levels and key sex chromosome gene expression in males and monitor the senescent cell clearance would be informative to dissect these mechanisms.
The senescent cells derived from different gender possibly also hold different features, since they have partial differential genomic backgrounds (sex chromosomes). Little is known whether senescent cells induced by the same stimulus but with different gender origin have the same phenotypes or not. Subcutaneous transplantation of senescent cells with different gender background into the same mouse (male or female) will help to provide more insights. However,
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the individual variation among the same gender still needs to be taken into account. RNA-sequencing analysis and comparison of female and male senescent cells can also contribute to answer this question.
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