Cellular Senescence: the Sought or the Wanted?

Yu Sun, 1, 2, * Jean-Philippe Coppé 3 and Eric W.-F. Lam 4

1. Key Laboratory of Tissue Microenvironment and Tumor, Shanghai

Institutes for Biological Sciences, University of Chinese Academy of Sciences,

Chinese Academy of Sciences, Shanghai 200031, China.

2. Department of Medicine and VAPSHCS, University of Washington,

Seattle, WA 98195, USA

3. Department of Laboratory Medicine, Helen Diller Family Comprehensive

Cancer Center, University of California San Francisco, CA 94115, USA

4. Department of Surgery and Cancer, Imperial College London, London, UK

W12 0NN

* Corresponding author. Email: sunyu@sibs.ac.cn

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Abstract

Cellular senescence is a process that results in irreversible cell cycle arrest

and is thought to be an autonomous tumor suppressor mechanism. During

senescence, cells develop distinctive metabolic and signaling features,

together referred to as the senescence-associated secretory phenotype

(SASP). The SASP is implicated in a number of aging-related pathologies,

including various malignancies. Accumulating evidence supports that cellular

senescence acts as a double-edged sword in human cancer, while new

agents and innovative strategies to tackle senescent cells are in development

pipelines to erase their dark sides in future clinics. We focus on recent

discoveries in senescence research and the SASP biology, and highlight the

potential of SASP-suppression and senescent cell clearance in advancing

precision medicine.

Keywords

Cellular senescence, senescence-associated secretory phenotype, aging-

related pathology, therapy-induced senescence, cancer, senolytics

Highlights

Cellular senescence is a highly conserved stress response that restrains

the proliferation of cells at risk of oncogenic transformation.

Senescent cells spatially occupy tissue environmental niches and

elaborate numerous extracellular factors encoded by the SASP,

contributing to aging-related disorders particularly cancer.

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In the tumor microenvironment, senescent cells can drive events that

support malignant progression including but not limited to therapeutic

resistance, disease relapse and distant metastasis.

In cancer clinics, the abundance of senescent cells can serve as a

“molecular” marker that predicts adverse outcomes, while senescent cell

clearance significantly mitigates pathological exacerbation.

A new class of agents termed senolytics has been demonstrated effective

in extending healthspan, reducing frailty and improving stem cell function

in animal models of aging.

Cellular Senescence Can Act an Autonomous Tumor Suppressor

Mechanism

Cellular senescence was initially identified as an irreversible loss of

proliferative capacity after successive culture of human diploid fibroblasts

(HDFs) [1]. Later termed replicative senescence, this form of proliferative

exhaustion is largely a result of telomere attrition of cells in culture. Although

replicative senescence was the first identified form of senescence,

subsequent studies revealed that senescence can be induced by diverse

stimulators, including oxidative stress, genotoxic damage, certain cytokines,

chromatin disorganization, oncogene activation, proteasome inhibition and

various mitogens, and is distinct from other forms of cell cycle arrest [2-5]

(Table 1).

A hallmark feature of senescence is persistent cell cycle arrest that is

unresponsive to extrinsic or environmental growth factor induction [3] (Box 1).

This arrest, usually irreversible, is frequently observed in the case of

oncogene-induced senescence (OIS), in which cells stop dividing even when

the Ras/MAPK pathway is continuously active, acting as a checkpoint against

neoplastic transformation [6]. Critical to the senescent phenotype is activation

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of the p53-p21CIP1 and p16INK4A-retinoblastomaprotein (Rb) pathways; loss of

these pathways, as occurs in many human cancers, permits senescence

bypass and carcinogenesis [7]. Importantly, a senescent phenotype can also

be induced by certain anticancer agents, termed “therapy-induced

senescence” (TIS), which results in senescent cells that remain chronically

present and promote local as well as systemic inflammation [8]. More

importantly, TIS can amplify many of the side effects of cancer treatments,

including but not limited to bone marrow suppression, cardiac dysfunction,

cancer relapse and metastasis [8]. However, appropriate development of

senescence in cancer cells after chemotherapy promotes disease regression

and improved prognosis of various malignancies such as nasopharyngeal

carcinoma, ovarian cancer and leukemia [9-11]. Therefore, senescence can

be considered an essential cell-autonomous tumor suppressor mechanism,

preventing the accumulation of damaged cells and malignant transformation.

Multiple cellular and molecular changes can be observed in senescent cells,

including proliferation arrest, chromatin remodeling, upregulation of cell cycle

inhibitors such as p16INK4A and p21CIP1, engagement of a DNA damage

response (DDR) (see Glossary), increase of lysosomal activity, enhancement

of metabolic activities and more importantly, a time course cascade

development of the senescence-associated secretory phenotype (SASP)

[12-15] (Figure 1). The SASP mediates the paracrine activities of senescent

cells via secretion of a myriad of soluble factors including growth factors,

proteases, cytokines, chemokines, extracellular matrix (ECM) components

[16-18]. Notably, both the composition and intensity of the SASP are subject

to the influence of interior or exterior factors including the senescence trigger,

cell type, environmental context and time since senescence initiation, thus

there is actually no singular form of SASP [4, 15, 19]. Of note, the SASP is

physiologically beneficial or even essential for embryonic patterning, tissue

repair, wound healing, cell stemness and plasticity, hepatic fibrosis controlling

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and immune surveillance [20-26]. However, increasing lines of evidence

suggested that the SASP can also be deleterious and is responsible for

multiple aging-related pathologies including cancer [27]. As the burden of

senescent cells within tissues increases gradually with age, they can be found

in both benign and malignant tumor foci [28]. Recent studies revealed that

selectively targeting and effectively eliminating senescent cells under in vivo

conditions can significantly promote therapeutic outcome and elongate the

lifespan of experimental animals [8, 29].

Overall, senescence is both a physiologically fundamental and pathologically

relevant program, with its role depending on the specific situation.

Understanding the regulatory mechanisms particularly those key modulators

of the SASP might inform therapeutic targeting of the SASP to promote an

anticancer response which often involves generation of many senescent cells

[30, 31]. In this review, we highlight the new insights into cellular senescence,

dissect the complexity of the SASP regulation, and project future directions to

exploit the benefits and prevent the detrimental aspects of senescent cells.

Dynamic Regulation of the SASP in Senescent Cells

Multiple studies have examined the mechanisms underlying the opposing and

contradictory pathophysiological activities of the SASP in different settings. A

group of cytoplasmic and nuclear factors including p38MAPK, Jak2/Stat3, the

inflammasome, mTOR, HSP90, miRNA-146a/b, GATA4, macroH2A1 and ATM

have been identified to be functionally involved in the development of SASP

[30, 32-41]. Of note, most SASP regulators appear to converge on the NF-κB

complex and C/EBP family, two transcriptional machineries that cooperatively

regulate expression of diverse SASP components [42-44]. However, a recent

study using HDFs expressing HRasG12V and a mouse liver NRasG12V model has

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found that OIS occurs with a dynamic fluctuation of Notch1 activity, which

generates a TGF-β-rich secretome and dampens the SASP through

suppression of C/EBPβ, while Notch1 inhibition during senescence facilitates

upregulation of the SASP, thus identifying Notch1 as a unique secretome

regulator by dictating a functional balance between two distinct secretome

states [45].

Certain inflammatory cytokines, such as IL-6 and IL-1α, can form positive

feedback loops with NF-κB and C/EBPβ [43, 46]. Specifically, ectopic IL-1α

expression in HDFs triggers a SASP-like response that phenocopies cells

undergoing Ras-induced OIS, which is characterized by expression of IL-1β,

IL-6, IL-8 and CCL2, suggesting that IL-1α expression can partially reproduce

SASP activation [35]. Senescent hepatocytes in a mouse OIS model driven by

NRasG12V developing the SASP can even transmit the same phenotype to

neighboring cells, a complex program orchestrated by the inflammasome that

involves IL-1α and TGF-β signaling to cause p16INK4A/p21CIP1-mediated

paracrine senescence of recipient cells [35]. Other components of the SASP,

such as IGFBP-7, PAI-1 and CXCR2 ligands including IL-8 and Groα, can

functionally reinforce senescence in HDFs or mouse fibroblasts [42, 47-49].

Thus, multiple factors of senescent cells are able to reinforce the state of

senescence or associated secretion by amplifying or conveying the SASP

signaling via autocrine or paracrine pathways.

The DDR caused by environmental stress or replicative exhaustion frequently

triggers the SASP, a process that implicates ATM, NBS1 and Chk2, but not

cell cycle inhibitors p53 and Rb [50]. Distinct from transient DNA damage,

persistent DNA lesions can form DNA segments with chromatin alterations

reinforcing senescence (DNA-SCARS) and regulates diverse aspects of

senescent cells [51]. A recent study dissecting senescence mechanisms with

HDF and mouse embryonic fibroblasts (MEFs) has revealed that transcription

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factor GATA4 is stabilized by suppression of autophagy in DNA-damaged

cells, and functionally enhances cellular senescence and the SASP through

TRAF3IP2 and IL1α-mediated activation of NF-κB, thus establishing GATA4

as a critical switch activated by the DDR in modulating senescence and the

SASP [41].

Recent work has implicated cGMP-AMP (cGAMP) synthase (cGAS) (see

Glossary) in cellular senescence and formation of the SASP. In mammalian

cells, cGAS is a cytoplasmic DNA sensor activated by double-stranded DNA

species including microbial and self-DNA fragments, which results in

production of the second messenger cGAMP and subsequent activation of the

stimulator of interferon genes (STING, also termed MITA, MPYS or

TMEM173) (see Glossary) [52]. STING then recruits IκB kinase and TANK-

binding kinase 1 (TBK1) to engage IFN regulatory factor 3 (IRF3) and NF-κB,

respectively, leading to the production of type I interferons (e.g. interferon-β)

and inflammatory cytokines [53]. Activation of an oncogene such as HRasV12

causes DNA hyperreplication, induces replication errors and initiates DNA

damage response [54]. Upon genotoxic stress, cGAS senses genomic DNA

damage and is recruited to micronuclei to promote senescent phenotypes,

whereby cGAMP is detectable by liquid chromatography-mass spectrometry

(LC-MS) [55]. Importantly, a recent study using MEFs showed that cGAS

elimination can abrogate the SASP triggered by spontaneous immortalization

or DNA damaging agents such as radiation and etoposide, suggesting that

cGAS mediates cellular senescence and restrains immortalization, a distinct

function that is complementary to the role of cGAS in activating antitumor

immunity [56]. The data are validated by multiple lines of supporting evidence

that consolidate the critical role of cGAS-STING pathway in promoting

development of innate immunity, expression of the SASP factors, and

establishment of cellular senescence in primary human and murine fibroblasts

as well as transgenic mouse models [55, 57, 58]. Given the critical role of

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cGAS-STING signaling in cellular senescence and development of the SASP,

inhibitors of this pathway may provide benefits to control senescence and its

associated phenotypes induced by DNA damage events.

Release of soluble factors such as IL-6 and Timp-1 from DNA-damaged cells

can occur rapidly within 24 hours of doxorubicin-mediated genotoxic

chemotherapy of Burkitt’s lymphoma mouse model, representing an early

phase of cellular response termed acute stress-associated phenotype

(ASAP) (see Glossary) [59]. In contrast, the SASP develops gradually over a

course of 7 to 10 days in most cell types and culminates only after

appearance of typical senescence-associated markers [59, 60]. Besides being

temporally distinct from the SASP, a recent study using tissue-specific

knockout mice and human endothelial cells showed that the ASAP is

modulated by a distinct mechanism, as the ASAP occurs independently of

DDR kinase activity or mTOR signaling, further distinguishing it from a

canonical SASP [61].

In cultured human endothelial cells, the ASAP occurs in a context of

PI3K/Akt/mTOR signaling suppression, while transition toward the SASP can

be secured by activating this signaling axis, implying that the PI3K/Akt/mTOR

pathway activity is involved in controlling the SASP development [61]. A new

study has revealed that expression of zinc finger and SCAN domain

containing 4 (Zscan4), a transcription factor, is enhanced by an ATM-TRAF6-

TAK1 axis during the acute DDR, and forms a positive feedback loop that

potentiates a transition from the transient ASAP to the chronic SASP in DNA-

damaged human stromal cells [31]. As a critical kinase activated in the course

of ASAP, TAK1 subsequently activates p38MAPK and PI3K/Akt/mTOR within

the cytoplasm to sustain a persistent SASP signaling until the SASP intensity

is culminated via a continuous development in 7-10 days after DNA damage

events [31]. Thus, initiation and progression of the SASP in a stressed setting

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involves multiple signaling molecules, resulting in the development of a

heterogeneous SASP.

Pathological Implications of the SASP in Human Cancer

Senescent fibroblasts generated by various stimuli particularly those produced

from genotoxic stress including chemotherapy, radiation and replicative

exhaustion, promote the proliferation, migration and invasion of cancer cells in

vitro, as demonstrated by co-culture data from breast, pancreatic and

squamous cell carcinoma studies [18, 62, 63]. In addition, co-implantation of

human senescent fibroblasts with prostate or breast cancer cells dramatically

enhances tumorigenicity in xenograft mouse models [30, 64]. Notably, the

contribution of certain SASP components can differ according to the

experimental conditions, and some exert specific effects on cancer initiation

and progression. For instance, elimination of amphiregulin (AREG) by small

interfering RNA (siRNA) from the full SASP spectrum reduced the growth of

neoplastic human prostate epithelial cells enhanced by conditioned media

from senescent human fibroblasts [65]. Senescent cancer-associated

fibroblasts (CAFs) secrete elevated concentrations of IL-8 to promote

pancreatic cancer invasion and metastasis, a process demonstrated by both

in vitro transwell invasion assays and in vivo xenograft mouse models, and

correlated with poor survival of patients in pancreatic cancer clinics,

highlighting senescent fibroblasts as a pathologically relevant feature of

pancreatic malignancy [63]. Although the mechanisms underlying obesity-

associated cancer remains elusive, a pathologically critical role of the SASP in

promoting obesity-associated hepatocellular carcinoma (HCC) by DNA-

damaged hepatic stellate cells (HSCs) in the liver has recently been disclosed

in transgenic p21WAF1/CIP1 mice with increased levels of deoxycholic acid

(DCA), a gut bacterial metabolite that induces DNA damage and provokes

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SASP phenotype resulting in the generation of various inflammatory and

tumor-promoting factors in the liver [66]. Importantly, blocking DCA production

or removing gut bacteria effectively prevents HCC occurrence in obese mice,

with similar results observed in mice lacking an SASP inducer or depleted of

senescent HSCs, thereby providing novel insights into the potential

mechanism behind obesity-associated cancer persistence and indicating new

opportunities for future clinical control [66].

Through producing a large array of soluble factors, the SASP can exert a wide

range of pro-tumorigenic effects, including enhanced angiogenesis,

accelerated invasion and increased metastasis. Many components of the

SASP are chemoattractive in nature, which allows senescent cells to actively

recruit immune cells to a damaged tumor microenvironment (TME), a

phenomenon that may be beneficial but can also generate adverse effects by

causing persistent local tissue inflammation or migration of immature myeloid

progenitors [16]. Specifically, brief reactivation of p53 function in a p53-

deficient RAS-driven liver carcinoma mouse model induced senescence-

associated differentiation and upregulation of inflammatory cytokines, and

resulted in migration of innate immune cells to the vicinity of the senescent

tumor area, consequently inducing complete tumor regression via

engagement of the innate immune system [67]. Secretion of multiple

cytokines and chemokines by senescent hepatocytes in NRasG12V-expressing

mice render these cells subject to immune-mediated clearance or senescence

surveillance, which is mediated by an intact CD4+ T-cell-involved and

monocyte/macrophage-required adaptive immune response [25]. To the

contrary, compromised immune surveillance of pre-malignant senescent

hepatocytes increased the occurrence of murine HCCs, suggesting that

senescence surveillance promotes tumor suppression in vivo [25]. However,

contradictory experimental evidence from a mouse model generated to mimic

the aged skin microenvironment showed that the stromal SASP particularly IL-

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6 can induce expansion of suppressive myeloid cells such as CD11b+Ly6GHi

and FOXP3+ Treg cells, and enhance their ability to inhibit antitumor T-cell

responses, eventually creating an immunosuppressive microenvironment [68].

Similarly, activation of the Jak2/Stat3 pathway in Pten-null senescent prostate

tumor models establishes an immunosuppressive TME that promotes tumor

growth and chemoresistance [34]. This process in prostate tumor was

sustained by downregulation of the protein tyrosine phosphatase

PTPN11/SHP2 but was reversed by inhibition of Jak2/Stat3 activities upon

treatment with docetaxel, thus generating an effective antitumor immune

response [34]. Continued investigation is required to define the specific

factors that control the pro- and antitumor surveillance activities of senescent

cells.

Senescent cells can induce epithelial-to-mesenchymal transition (EMT) in

neoplastic cells, including those derived from breast cancer, mesothelioma,

lung cancer and prostate cancer, as evidenced by expression changes of

EMT markers such as cytokeratins, E-cadherin, N-cadherin and vimentin, or

EMT regulators including Twist, Snail and Zeb [15, 39, 69-72]. In addition,

chemotherapy or radiation-induced damage of stromal cells in the TME can

promote therapeutic resistance of prostate cancer cells in vitro and in vivo of

mouse xenografts via production of WNT16B [72]. Specifically, WNT16B

activates canonical Wnt signaling in prostate cancer cells, a process that can

be observed even months after the completion of clinical treatment [72]. In the

treatment-damaged TME of mice xenografted with prostate tumor, the

biological activity of WNT16B can be further enhanced by SFRP2, another

SASP factor released by senescent stromal cells, which acts as an agonist of

WNT16B and significantly enhances the capacity of WNT16B in driving

chemoresistance of surviving cancer cells [73].

The accumulation of senescent cells during natural aging results in persistent

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inflammation, a process that is associated with a tumor-permissive

environment. A recent study reported that SFRP2 secreted from senescent

dermal fibroblasts in aged mice drives therapeutic resistance of melanoma

cells injected via tail vein to targeted agents such as vemurafenib (PLX4720),

and promotes local invasion, enhanced angiogenesis and distant metastasis

[74]. In this case, SFRP2 activates a multistep signaling cascade in

melanoma cells and generates increased oxidative stress but decreased β-

catenin and microphthalmia-associated transcription factor (MITF), eventually

causing the loss of a key redox effector APE1 and attenuating the response of

melanoma cells to DNA damage induced by reactive oxygen species (ROS)

[74].

Besides the SASP, extracellular vesicles (EVs) represent another type of

mediators for the pro-tumorigenic effect of senescent cells. Under in vitro

conditions, telomere attrition- or radiation-induced senescence of human

prostate cancer cells was associated with a significantly increased release of

EVs, a process dependent on the activation of p53 [75]. Triggered by serial

passaging, ionizing irradiation, chemotherapeutic reagents or oncogene

overexpression, senescence-associated EV production is a general feature of

cellular senescence and has been observed in multiple cell types including

stromal fibroblasts, epithelial cells and cancer cells [75, 76]. Specifically, EV-

associated EphA2 released from senescent HDFs or human retinal pigment

epithelial cells binds ephrin-A1, a molecule highly expressed in cancer cells,

enhancing malignant proliferation via EphA2/ephrin-A-mediated reverse

signaling [18]. EV sorting of EphA2 is increased in senescent cells because of

its enhanced phosphorylation caused by oxidative inactivation of PTP1B

phosphatase, illustrating a novel mechanism of ROS-regulated cargo sorting

for EV production, which is critical for the tumor-promoting effect of the

senescent cell secretome [18]. A recent study showed that the plasma

concentrations of EV-associated EphA2 are significantly increased in

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pancreatic cancer patients, suggesting that EV-associated EphA2 may also

be involved in cancer development in humans [77]. How EV-associated DNA

secreted from senescent cells might impact their surrounding

microenvironment remains largely unknown, however, recent data showed

that EV-delivered DNAs from senescent HDFs can trigger a DNA damage

response in recipient cells, implying that cellular senescence may be

reinforced or transmitted in the local niche [76].

Senescent Cells and the SASP: to Target or to Clear, and How?

Increasing lines of data suggest that development of the SASP can be

orchestrated by multiple signaling pathways and/or network, including but not

limited to p38MAPK, cGAS-STING, TGF-β, JAK-STAT, PI3K-AKT-mTOR, as

well as transcriptional factors such as NF-kB and C/EBP-β [6]. Extensive

crosstalk among pathways and networks has been observed, while each

SASP signaling pathway may drive the transcription, translation, or protein

stability of numerous SASP factors [30, 46]. Targeting these individual

molecules can result in substantial attenuation or even abrogation of the

SASP, although the mechanistic action of each component is largely

dependent on the cellular or environmental context, such as the type of cells

and stimuli [6].

For this purpose, senomorphic therapy, which can suppress the SASP and

dampen sterile inflammation associated with aging to extend healthspan and

potentially lifespan, is recently proposed as one of the emerging

senotherapeutic approaches [78]. To date, several classes of senomorphics

have been identified, which delay the appearance of senescence markers or

suppress the development of the SASP without overt cytotoxicity. These

include chemical inhibitors of Iκ-BKinase (IKK)/NF-κB, free radical

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scavengers, JAK pathway inhibitors, and even the mTOR suppressant

rapamycin [30, 36, 79].

Transient induction of cellular senescence, followed by tissue remodeling and

senescent cell elimination by the immune system is beneficial, as it facilitates

removal of damaged cells from the affected tissue. However, chronic

senescence or inability to eliminate the senescent cells is frequently observed

in aged individuals or in pathological contexts, resulting in the accumulation of

senescent cells which do generate adverse effects. Increasing evidence

shows that both pro-senescence and anti-senescence therapies can be

beneficial to tissue homeostasis. For instance, in the case of cancer, pro-

senescence therapies can minimize the damage by limiting aberrant activities

such as hyperactive proliferation, and more specifically, preventing or

delaying events of carcinogenesis, while anti-senescence treatments may

help remove accumulated senescent cells and allow tissue regeneration [2].

As a matter of term use in this field of research, anti-senescence does not

mean that senescence is blocked or prevented, but means that when

senescence is engaged it is subsequently pushed into apoptosis. Recently, a

two-step anticancer strategy was raised, which proposed senescence-

inducing treatments followed by senotherapy, thus providing a novel option to

maximize therapeutic efficacy and improve clinical outcome [6].

Although incidence of senescence can improve long-term outcomes for

cancer, potentially harmful properties of senescent cells persisting in vivo

make their quantitative elimination an outstanding therapeutic priority.

Notably, the SASP-producing TIS cells display an unfolded protein response

(UPR), endoplasmic reticulum (ER) stress, and increased ubiquitination, and

are sensitive to glucose utilization blockage or induced autophagy, which

causes their selective elimination through caspase-12- and caspase-3-

mediated ER-related apoptosis [80]. Thus, the hypercatabolic nature of TIS

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cells might be exploitable by synthetic and lethal metabolic targeted therapy.

A recent study used a transgenic mouse strain expressing inducible

dimerizable FK506-binding-protein–caspase 8 (FKBP–Casp8) under the

control of a minimal fragment of human p16INK4a promoter (INK-ATTAC model),

and elegantly showed that senescent cells can be selectively depleted by the

systemic administration of AP20187 (AP), a dimerizer that activates FKBP-

Casp 8 [81]. Clearance of p16Ink4a-positive cells delayed carcinogenesis in the

lifespan, attenuated age-related degeneration of several organs without

manifest side effects and preserved the functionality of adipocytes, glomeruli

and cardio-protective KATP channels in ATTAC transgenic mice established in

two distinct genetic backgrounds [81]. As supporting evidence, several studies

demonstrated strong effects of senolysis in vitro or in old animals, such as

reduced morbidity and mortality from cardiovascular diseases, delayed

osteoporosis and sustained intervertebral disk proteoglycans, results that can

be achieved employing similar genetic strains or through pharmacological

approaches [82-85]. Of note, there is an increased activity of pro-survival

networks in senescent cells versus their normal counterparts, which is

consistent with their established resistance to apoptosis [85]. Small

interference RNA-mediated silencing of BCL-xL, ephrins (EFNB1 or 3),

PI3Kdelta, p21 or plasminogen-activated inhibitor-2 (PAI2), effectively induces

apoptosis of senescent cells, but not quiescent or proliferating cells [83].

Furthermore, combined treatment with Dasatinib (a RTK inhibitor used for

chronic myelogenous leukemia) and Quercetin (a flavonoid widely distributed

in nature) reduced the burden of senescent cells in chronologically aged,

radiation-exposed and progeroid Ercc1(-/Delta) mice, proving the technical

feasibility of selectively ablating senescent cells and the practical efficacy of

senolytics for mitigating frailty symptoms and extending healthspan [83] (Box

2).

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By screening a library of compounds, a recent study identified ABT263 (a

specific inhibitor of the anti-apoptotic proteins BCL-2 and BCL-xL) as a potent

senolytic agent that can effectively kill senescent cells in a cell type- and

species-independent manner by inducing apoptosis, while in vivo data of

irradiated mice demonstrated that selective clearance of senescent cells by a

pharmacological agent such as ABT263 is beneficial partially via rejuvenation

of aged tissue stem cells [85]. These data are corroborated by another study

that showed upregulation of the anti-apoptotic proteins BCL-W and BCL-xL in

senescent cells, while concurrent inhibition of BCL-W and BCL-xL by siRNAs

or the chemical inhibitor ABT-737 potently and specifically induced senescent

cell apoptosis [86]. Treatment of mice with ABT-737 efficiently cleared

senescent cells in the lung induced by DNA damage and those generated in

the epidermis by transgenic p14ARF-mediated activation of p53 [86]. In a p16-

3MR transgenic mouse model which carries a trimodal reporter protein (3MR)

under the control of p16, selective depletion of senescent cells by a small

molecule chemical UBX0101 attenuates the development of post-traumatic

osteoarthritis and establishes a pro-regenerative microenvironment, validating

senescent cells as a new therapeutic target to delay pathologies and increase

healthy lifespan [87].

Applying transgenic and pharmacological methods to remove senescent cells

in low-density lipoprotein receptor-deficient (LDLR(-/-)) mice that exhibit

atherosclerosis, another study proved that senescent cells are key drivers of

atheroma and selective clearance of these cells with senolytic agents

represent a new approach to treat atherosclerosis [88]. To restore tissue

homeostasis of a fast-aging mouse model based on the human premature-

aging syndrome trichothiodystrophy (TTD, mouse termed XpdTTD/TTD), a new

study using a FOXO4 peptide that disturbs the FOXO4-p53 interaction and

selectively causes p53 nuclear exclusion and intrinsic apoptosis of senescent

cells, demonstrated that therapeutic targeting of senescent cells is feasible

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even when loss of health has already occurred [89]. Oxidation resistance 1

(OXR1), an antioxidant protein that regulates the expression of multiple

antioxidant enzymes, has recently been found to be upregulated in senescent

HDFs but can be directly bound by Piperlongumine (PL), a natural product

that can selectively kill senescent cells but has low toxicity, an excellent

PK/PD profile, and oral bioavailability [84, 90-92]. Specifically, PL has the

ability to induce OXR1 degradation through the ubiquitin-proteasome system

in an senescent cell-specific manner, underscoring OXR1 as a novel senolytic

target that can be exploited for the development of new senolytic agents [92].

Natural products enriched in polyphenols, which have antioxidant and anti-

inflammatory effects, might also have the potential to be harnessed as "anti-

senescence foods" to achieve healthier aging by limiting senescence cell-

associated activities such as the SASP-induced inflammation [93].

Concluding Remarks

Accumulating evidence suggests that in contrast to the cell-autonomous

tumor-suppressive mechanism of senescence, the paracrine effects of

senescent cells themselves, particularly those mediated by the SASP are

responsible for aging-related pathologies, among which cancer has attracted

increasing attention. Optimizing the beneficial impact while minimizing the

deleterious effects of cellular senescence, remains a serious challenge for

multiple fields of scientific and clinical research, including cancer biology (see

Outstanding Questions).

As senescent cells tend to accumulate in aged individuals over time and are

also present in the residual tumor foci of patients in the post-treatment period,

the SASP-provoked pathological events contribute to disease relapse and

ectopic metastasis, and hallmark SASP factors might have potential for use

as biomarkers for real-time medical surveillance in future clinics (Figure 2).

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Moreover, it is reasonable to speculate that these events may also be

involved in the initial steps of oncogenic transformation of normal cells, as

evidenced by a recent study involving human adamantinomatous

craniopharyngioma (ACP), showing that the stem cell-associated SASP

causes cell transformation and tumor initiation in vivo in an age-dependent

manner [94]. Specifically, β-catenin+ and Sox2+ stem cell clusters undergo

senescence and initiate the SASP during embryonic pituitary development in

the Hesx1Cre/+;Ctnnb1lox(ex3)/+;R26YFP/+ mice, a human ACP model

which expresses oncogenic β-catenin in the embryonic pituitary precursors,

while mice with reduced senescence and the SASP display reduced tumor-

inducing potential, supporting the notion that childhood-onset or paediatric

ACP is a developmental malignancy [94, 95].

To date, numerous promising translational opportunities have emerged

through exploiting various agents, including antibodies and small molecule

compounds to effectively modulate the SASP thereby minimizing its

detrimental consequences, or to selectively eliminate senescent cells by

senolytics. To this end, targeting the SASP and clearing senescent cells

together represent a new wave of efforts to control or prevent pathological

exacerbation of cancer patients, especially those at a risk of developing

advanced malignancies. Considering that the SASP-induced initial epigenetic

and genetic alterations allow the appearance of tumor-initiating cells in normal

tissue, timely ablation of senescent cells in pre-malignant lesions using

senolytic compounds or SASP-neutralizing inhibitors may be a potential future

chemotherapeutic strategy.

The most promising senolytics appear to be inhibitors of pro-survival BCL

family proteins, probably because senescent cells physiologically need these

factors to circumvent apoptosis for long term survival [85, 86]. This class of

agents has undergone extensive investigation in patients with chronic

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leukemia, with a final FDA approval of a selective BCL-2 inhibitor, venetoclax

[5]. However, venetoclax is not a potent senolytic agent in vitro, whereas its

homolog navitoclax has recently been disclosed to be one of the strongest

senolytics. Navitoclax effectively inhibits BCL-2, BCL-xL and BCL-W,

suggesting senolysis requires suppression of a wider range of anti-apoptotic

effectors rather than just BCL-2 alone. It is rational to propose a broad-

spectrum of BCL protein inhibitors to be adapted as potential senolysis

treatments in patients but such molecules would need to exhibit acceptable

toxicity through new or optimized formulation, delivery or administration

schedule (Table 2).

Our evolving understanding of the implications of senescent cells in human

malignancies may unveil therapeutic opportunities for cancer, one of the most

life-threatening age-related pathologies. A future goal will be to develop

effective pharmacologic methods to remove accumulated senescent cells or

dampen the SASP intensity. Such therapeutic trials are simpler from a clinical

trial viewpoint and yet provide hope for future strategies to minimize the

adverse consequences generated by the presence of an overwhelming

burden of senescent cells in human lifespan.

Acknowledgments

The authors cordially apologize to colleagues whose work in senescence and

cancer could not be cited due to space limitation. We are grateful to members

of Sun laboratory for inspiring discussion and insightful comments on the

manuscript. This work is supported in part by grants from National Key

Research and Development Program of China (2016YFC1302400), National

Natural Science Foundation of China (NSFC) (81472709, 31671425), Key

Lab of Stem Cell Biology of Chinese Academy of Sciences, the National 1000

Young Talents Research Program of China and the U.S. Department of

19

Defense (DoD) Prostate Cancer Research Program (PCRP) (Idea

Development Award PC111703) to Y.S.; NIH TR000005 T1 Award, the Give

Breast Cancer The Boot program and the Friends for an Earlier Breast

Cancer Test program (to J-P.C.); and CRUK (A12011), Breast Cancer Now

(2012MayPR070; 2012NovPhD016), the Medical Research Council of the

United Kingdom (MR/N012097/1), Cancer Research UK Imperial Centre,

Imperial ECMC and NIHR Imperial BRC to E.W.-F.L.

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Figure legends

Figure 1. Phenotypical hallmarks of cellular senescence. Cells exposed to

various types of interior and/or exterior stresses can enter a state termed

cellular senescence. The major attributes that distinguish senescent cells from

their normal counterparts include a permanent cell cycle arrest, increased

expression of CDK inhibitors p16INK4a (CDKN2A) and p21CIP1 (CDKN1A), and

prominent changes in cell size and morphology. Appearance of senescence-

associated heterochromatin foci (SAHFs), increased lysosomal activity or

senescence-associated-β-galactosidase (SA-β-Gal) positivity, shortened

telomere length, emergence of DNA segments with chromatin alterations

reinforcing senescence (DNA-SCARS), enhanced DNA damage response

(DDR), loss of lamin B1, secretion of oxidized high mobility group box 1

(HMGB1) and elaborate expression of multiple SASP factors, are frequently

observed in senescent cells. Although these markers are not unique or

specific for senescent cells, novel and universal features of cellular

senescence are still being actively explored.

Figure 2. Pathological implications of in vivo cellular senescence and

the SASP induced by clinical treatments (e.g. chemotherapy), and

exemplifying liquid biopsies to monitor the SASP development. In clinical

oncology, anticancer regimens such as chemotherapy remain the mainstay of

therapeutic strategies. Although cancer cells are induced to undergo

apoptosis, stromal cell subpopulations in the host TME are relatively static

and typically enter cellular senescence in vivo. During and after the

therapeutic regimen, senescent cells developing the SASP in the local TME

constantly produce numerous soluble factors, substantially fueling the

25

repopulation of surviving cancer cells. A high frequency of tumor relapse and

metastasis can be observed even after initial regression of primary tumors,

while the presence of senescent cells and their non-autonomous attribute, the

SASP, represent a major force that confer acquired resistance which

contributes to increased cancer mortality (A-C). SASP, senescence-

associated secretory phenotype. TME, tumor microenvironment.

Box 1. Biological facets of cellular senescence

Cellular senescence is a stress-induced, essentially irreversible cell-cycle

arrest of previously division-competent cells. Although the term was initially

used to describe the finite proliferative capacity of normal human diploid

fibroblasts (HDFs) in culture, cells with senescence features were later

observed in vivo of humans, and their number in solid tissues tend to increase

with age [5]. While the canonical hallmarks of senescent cells have been

extensively reviewed [96, 97], there are a few typical properties shared by

most cell types or lineages. For example, senescent cells have enhanced

expression of the cyclin-dependent kinase inhibitors, such as p16 INK4a and

p21CIP1, activation of stress-perceiving signal pathways including those

involving p38MAPK and the nuclear factor кB (NF-κB) complex, staining

positivity for senescence-associated β-galactosidase (SA-β-Gal), enlargement

of nucleus and nucleolus, formation of the senescence-associated secretory

phenotype (SASP) (Figure 1). In most cases, growth arrest of formerly

replication competent cells is induced by persistent DNA damage response

(DDR) which eventually causes constitutive activation of the p53-p16 INK4a-

retinoblastoma (RB) pathway [96, 98]. In contrast to apoptotic or quiescent

cells, senescent cells are highly metabolically active, a feature that is

consistent with development of the SASP, which is characterized by

transcriptional activation and translational synthesis of a myriad of soluble

factors with the potential to exert various biological activities on adjacent cells

26

in the tissue microenvironment (TME) [14, 17, 99]. The SASP may explain

how a relatively small number of senescent cells can generate durable, local

and systemic effects in vivo, ultimately chronically promoting aging-related

disorders including multiple forms of malignancies.

Box 2. The Clinician’s Corner

The composition or spectrum of the SASP can vary according to several

factors such as the stimulatory signal, cell lineage, damage extent and tissue

context, factors together shaping the heterogeneity of the SASP. However, a

subset of secreted molecules including but not limited to IL-6, IL-8, GM-CSF,

Groα, CCL20 and MMPs are universally expressed as the core SASP factors

in the vast majority of stressful conditions and across most cell lines [17, 99],

thus holding the potential to be developed as gold-standards or hallmarks of

the SASP for routine diagnosis in clinical medicine.

Although the SASP is frequently triggered by anticancer treatments including

chemotherapy and radiation, a signal network that regulates the therapy-

induced secretome can also be activated by targeted agents particularly

BRAF, ALK or EGFR kinase inhibitors [31, 100], implying that the SASP has

wide implications in clinical settings and may contribute to disease

progression.

Because clearance of senescent cells delays the onset of multiple

complications and extends lifespan in experimental animals [81, 96], it is

rational to speculate that small molecule compounds or other pharmaceutical

formats with a senescent cell-targeting potency in vivo for human patients are

within reach, and such senolytics can be naturally developed for clinical

purposes.

Since senescent cells can benefit wound healing and tissue regeneration, it is

likely that acute senolysis could impair these health-essential activities in

some situations. Further, senescent cells physically occupy spatial niches in

the tissue thus restraining rapid expansion of somatic stem cells; therefore,

27

unduly depleting senescent cells may impose a proliferation burden on stem

cells to fill the local microenvironment post-senolysis. Thus, when and how to

apply senolytics are challenging issues for prospective therapeutic trials.

Outstanding Questions Box

Besides the reported beneficial effects, are there any unknown detrimental

consequence of senescent cells during physiological processes such as

embryonic development, tissue repair, and would healing?

How is the cGAS-cGAMP-STING pathway connected to the major signaling

network of the SASP that comprises p38MAPK, mTOR, GATA4, TAK1 and

many other factors reported to be essential for the SASP so far?

Should DDR, cytosolic DNA sensors or innate immunity be targeted to prevent

the SASP development in cancer patients, particularly those undergoing

chemotherapy and/or radiation?

Are there molecules/pathways specifically upregulated in senescent cells that

confer substantial resistance to apoptosis and can they be exploited as potent

targets for development of new senolytics, beyond those targeting the BCL

family?

Should SASP inhibitors be used to minimize therapeutic resistance during

anticancer treatment, and if so, when and how (administered separately or

combined with classic agents)? Are they safe for cancer patients?

Does it provide more benefit to retain TIS to favor tissue repair and support

tissue regeneration, or should senescent cells be thoroughly eliminated in the

post-treatment period to avoid subsequent deleterious effects? And again, is

28

this safe for patients?

Table 1. Comparison of cellular senescence with several other forms of proliferation arrest

Senescence Quiescence Exhaustion Terminal Differentiation

Definition

A cellular state characterized with arrested cell cycle, enlarged and flattened cell shape, enhanced β-galactosidase activity, but active metabolism and development of an immunogenic phenotype consisting of a pro-inflammatory secretome, usually termed the SASP.

A special phase of cell cycle, usually referred to as G0 phase, when a cell is not dividing but has metabolic activity similar to their proliferating counterparts and is secreting proteins that help maintain tissues.

A state of cell dysfunction arising during chronic infections and carcinogenesis, usually referred to T cells of poor effector function and transcriptomic expression profile distinct from effector or memory T cells.

A precursor cell (or stem cell) formerly capable of cell division, permanently leaves the cell cycle, dismantles the cell cycle machinery and often expresses a range of genes characteristic of the cell's final function (e.g. actin and myosin for a muscle cell).

Cell type Many if not most replication competent cells (fibroblasts, epithelial cells, endothelial cells, adipocytes, pericytes, neuroendocrine cells, immune cells, mesenchymal

Many if not all dividing cells (somatic stem and progenitor cells, lymphocytes, hepatocytes, renal/pulmonary epithelial cells, chondrocytes,

T lymphocytes, most somatic stem cells

Many cell types (fibroblasts, epithelial cells, small muscle cells, neurons, adipocytes, endothelial cells, urothelial cells, cardiomyocytes, osteocytes, etc)

29

stem cells, etc.) glia, etc.)

Growth arrest

Essentially permanent

Generally reversible

Defective in response to antigenic challenge (T cells),limited proliferative potential (somatic stem cells)

Changes by cell type

DNA content

2N or 4N 2N 2N Usually 2N (with some exceptions, e.g., megakaryocytes, striated muscle)

Effectors p16INK4a, p14/p19ARF,p21CIP1, p53 and RB

p18INK4c, p21CIP1, p27KIP1, p107, p130, and repressive E2Fs

Persistent antigen and/or inflammatory signals [101]

p18INK4c, p21CIP1, p27KIP1, p107, p130, repressive E2Fs

Markers Shortened, dysfunctional telomeres,p16INK4a

expression,SAHF

Low RNA content and lack of cell proliferation markers, as well as by label retention as an indication of low turnover [102]

Expression of PD1, TIM3, LAG3 (T cells)

Cell type-specific markers such as α-SMA and fibronectin for cardiomyocytes, as well as proteoglycan neural/glial antigen for neurons [103, 104]

persistent DDR,SASP,SA-β-gal positivity,Lamin B1 lossp38MAPK and

30

NF-кB activation

SAHF, senescence-associated heterochromatin foci; DDR, DNA damage response; SASP, senescence-associated secretory phenotype; SA-β-gal, senescence-associated β galactosidase; MAPK, mitogen-activated protein kinase. Table contents adapted from He and Sharpless (5) with permission from Cell, Copyright 2017.

Table 2. Small molecule compounds that hold potential as candidate senotherapies

Agent Target (s) Target class Development status RefsABT-263 (Navitoclax)

BCL-2/ BCL-XL

Pro-survival or anti-apoptotic factors

Preclinical animal models/Clinical trials (phase I/II (NCT00406809 for leukemia and lymphoma/NCT00445198 for lung cancer), phase I (NCT00743028 for leukemia and lymphoma/NCT00982566 for lymphoma and solid tumors), and phase II (NCT02591095 for ovarian cancer/NCT01557777 for leukemia))

[85]

ABT-737 BCL-w/ BCL-XL

Pro-survival or anti-apoptotic factors

Preclinical animal models/Ex vivo evaluation of ovarian tumor (NCT01440504)

[86]

Dasatinib Pan-receptor tyrosine kinases

Receptor tyrosine kinases

Clinical trials (Phase I/II (NCT00597038 for melanoma/NCT00550615 for lymphoma), Phase I (NCT00652574 for mesothelioma/NCT0174

31

4652 for advanced cancers), Phase II (NCT02744768 for leukemia/NCT00429949 for myeloma), Phase III (NCT02013648 for leukemia),

Phase IV (NCT03216070 for leukemia))

Metformin The IKK complex and/or NF-κB

The SASP Approved for type II diabetes/Clinical trials for cancer (Phase I/ II (NCT02949700 for head and neck squamous cell carcinoma), Phase II (NCT03137186 for prostate cancer/NCT03398824 for Fanconi Anemia/NCT02506777 for breast cancer)), clinical trials for aging (Phase IV (NCT02745886 for aging/NCT02432287 for aging))

Rapamycin Mechanistic target of rapamycin kinase (mTOR)

The SASP Approved for immunosuppression/Clinical trials for cancer (Phase I (NCT02724332 for liver cancer/NCT03014297 for neuroendorine tumors))

RAD001 Mechanistic target

The SASP Approved for immunosuppression, clinical trials for cancer

32

of rapamycin kinase (mTOR)

(Phase I/II (NCT00516165 for liver

cancer/) ， Phase II

(NCT00782626 for glioma and astrocytoma/NCT01051791 for head and neck squamous cell carcinoma/NCT01152840 for adenoid cystic cancer))

LY2228820

p38MAPK

The SASP Clinical trials for cancer (Phase I (NCT01393990 for advanced cancer), Phase I/II (NCT01663857 for ovarian cancer, NCT02364206 for glioblastoma)

LY3007113

p38MAPK

The SASP Clinical trials for cancer (Phase I (NCT01463631 for advanced cancer))

Quercetin Lipoprotein lipase (LPL) and potassium voltage-gated channel subfamily E regulatory subunit 2 (KCNE2)

Antioxidant enzymes

Phase II clinical trial (NCT02848131) for chronic kidney disease

[105]

Piperlongumine

Oxidation resistance 1 (OXR1)

Antioxidant protein

Preclinical animal models

[90]

33

Anakinra IL-1 receptor

The SASP Clinical trials for cancer (Phase I (NCT01624766 for advanced cancer/NCT01802970 for metastatic breast cancer/NCT02021422 for pancreatic cancer) , Phase II (NCT03233776 for myeloma))

[46]

5Z-7-Oxozeaenol

Transforming growth factor-β1-activated kinase-1 (TAK1)

The SASP Preclinical animal models [3

1]

Alvespimycin (17-DMAG)

Heat shock protein 90 (HSP90)

Chaperone subfamily

Preclinical animal models/Clinical trials for cancer (Phase I (NCT01126502 for leukemia and lymphoma/NCT00089362 for metastatic or unresectable solid tumors/NCT00088868 for advanced solid tumor or lymphoma)

[106]

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