The Senescence-Associated Secretory Phenotype: The Dark Side of Tumor Suppression Jean-Philippe Copp ´ e, 1 Pierre-Yves Desprez, 2,3 Ana Krtolica, 1 and Judith Campisi 1,2 1 Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 2 Buck Institute for Age Research, Novato, California 94945 3 California Pacific Medical Center, Cancer Research Institute, San Francisco, California 94107; email: [email protected]Annu. Rev. Pathol. Mech. Dis. 2010. 5:99–118 First published online as a Review in Advance on September 28, 2009 The Annual Review of Pathology: Mechanisms of Disease is online at pathmechdis.annualreviews.org This article’s doi: 10.1146/annurev-pathol-121808-102144 Copyright c 2010 by Annual Reviews. All rights reserved 1553-4006/10/0228-0099$20.00 Key Words aging, cancer, inflammation, proliferation, invasion Abstract Cellular senescence is a tumor-suppressive mechanism that perma- nently arrests cells at risk for malignant transformation. However, ac- cumulating evidence shows that senescent cells can have deleterious effects on the tissue microenvironment. The most significant of these effects is the acquisition of a senescence-associated secretory phenotype (SASP) that turns senescent fibroblasts into proinflammatory cells that have the ability to promote tumor progression. 99 Annu. Rev. Pathol. Mech. Dis. 2010.5:99-118. Downloaded from www.annualreviews.org by University of Sussex on 10/10/12. For personal use only.
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The Senescence-AssociatedSecretory Phenotype:The Dark Side of TumorSuppressionJean-Philippe Coppe,1 Pierre-Yves Desprez,2,3
Ana Krtolica,1 and Judith Campisi1,2
1Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 947202Buck Institute for Age Research, Novato, California 949453California Pacific Medical Center, Cancer Research Institute, San Francisco,California 94107; email: [email protected]
Annu. Rev. Pathol. Mech. Dis. 2010. 5:99–118
First published online as a Review in Advance onSeptember 28, 2009
The Annual Review of Pathology: Mechanisms ofDisease is online at pathmechdis.annualreviews.org
This article’s doi:10.1146/annurev-pathol-121808-102144
AbstractCellular senescence is a tumor-suppressive mechanism that perma-nently arrests cells at risk for malignant transformation. However, ac-cumulating evidence shows that senescent cells can have deleteriouseffects on the tissue microenvironment. The most significant of theseeffects is the acquisition of a senescence-associated secretory phenotype(SASP) that turns senescent fibroblasts into proinflammatory cells thathave the ability to promote tumor progression.
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INTRODUCTION
The tissue microenvironment is defined by thephenotypes of the cells in the immediate areaand by the physical and chemical propertiesof the soluble and insoluble factors surround-ing cells within a given tissue. These proper-ties include temperature and oxygen tension,as well as various molecules that may be pro-duced locally—for example, growth factors andcytokines. Further, cells within tissues form adynamic network that contributes to their mi-croenvironment. At the same time, the tissuemicroenvironment regulates cell behavior. Thisreciprocal relationship determines tissue func-tion and repair and is also central to a numberof pathologies, including cancer.
A permissive microenvironment supportsand promotes tumor growth and cancer cellaggressiveness (1–4). Alterations in the cellu-lar and molecular composition of the connec-tive tissues surrounding carcinomas allow tu-mors to evade detection by the immune sys-tem as well as to proliferate inappropriately,invade the surrounding tissue structure, andeventually metastasize. The synergy betweenan altered microenvironment and the geneticalterations acquired by tumor cells allows thesecells to evade preventive mechanisms and be-come fully malignant. Cellular senescence isnow recognized as a potent tumor-suppressivemechanism that arrests the growth of cells atrisk for malignant transformation (5–12). How-ever, recent studies show that senescent cellsdevelop altered secretory activities that may in-duce changes in the tissue microenvironment,relaxing its control over cell behavior and pro-moting tumorigenesis (13–18).
How can the senescence response be bothtumor suppressive and procarcinogenic? It isimportant to consider that a biological processsuch as cellular senescence can be both bene-ficial and deleterious. The idea that processescan have such dual effects is consistent witha major evolutionary theory of aging termedantagonistic pleiotropy (19). The senescence-associated secretory phenotype (SASP) repre-sents one of the darkest sides of the senescence
response and is the focus of this review. We par-ticularly emphasize the potential effects of theSASP (20) on cell behavior in the context oftumor progression.
CELLULAR SENESCENCE
Cellular senescence occurs in culture and invivo as a response to excessive extracellular orintracellular stress. The senescence programlocks the cells into a cell-cycle arrest that pre-vents the spread of damage to the next cellgeneration and precludes potential malignanttransformation (19). Senescent cells have beenshown to accumulate over the life span of ro-dents, nonhuman primates, and humans (21).These cells are found primarily in renewabletissues and in tissues that experience prolongedinflammation.
A plethora of stresses can provoke cellu-lar senescence (22, 23). These stresses includetelomeric dysfunction (telomere uncapping) re-sulting from repeated cell division (termedreplicative senescence), mitochondrial deteri-oration, oxidative stress, severe or irrepara-ble DNA damage and chromatin disruption(genotoxic stress), and the expression of certainoncogenes (oncogene-induced senescence) (seeFigure 1) (24–31). Stresses that cause cellularsenescence can be induced by external or inter-nal chemical and physical insults encounteredduring the course of the life span, during thera-peutic interventions (for example, X-irradiationor chemotherapy), or as a consequence of en-dogenous processes such as oxidative respira-tion and mitogenic signals. External mitogenicsignals, for example growth-related oncogenealpha (GROα) secretion by tumor cells in closeproximity to normal cells (32) or circulating an-giotensin II (33, 34), have also been shown toinduce cellular senescence. All somatic cells thathave the ability to divide can undergo senes-cence. Regardless of the disparate mechanismsof senescence-inducing stresses, the senescenceprogram is activated once a cell has sensed acritical level of damage or dysfunction. So far,the senescence growth arrest has been shown
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to depend on the activities of the major tumor-suppressor pathways controlled by p16INK4a andpRB (retinoblastoma protein), as well as by p53.Some of the molecules involved in pathwaysupstream and downstream of the senescence-associated phenotype have been used as mark-ers to detect senescent cells in culture andin vivo.
THE SECRETORY PHENOTYPEOF SENESCENT CELLS
The senescent phenotype is not limited to an ar-rest of cell proliferation. In fact, a senescent cellis a potentially persisting cell that is metabol-ically active and has undergone widespreadchanges in protein expression and secretion,ultimately developing the SASP. This pheno-type has also been termed the senescence-messaging secretome (35). We recently pro-vided a large-scale characterization of the SASP,using antibody arrays to quantitatively mea-sure factors secreted by human fibroblasts andepithelial cells (18), as well as mouse fibrob-lasts ( J.P. Coppe & J. Campisi, unpublisheddata). The potential existence of the SASP wasalready suggested by large-scale comparativegene (mRNA) expression studies performed onfibroblasts from different-aged donors and dif-ferent tissues of origin (36–46). Among the cellsthat have been shown to senesce and secrete bi-ologically active molecules are liver stellate cells(47), endothelial cells (36, 48–51), and epithelialcells of the retinal pigment, mammary gland,colon, lung, pancreas, and prostate (8, 18, 36,41, 52–56).
Senescence-associated changes in gene ex-pression are specific and mostly conservedwithin individual cell types. Most differencesbetween the molecular signatures of presenes-cent and senescent cells entail cell-cycle- andmetabolism-related genes, as well as genes en-coding the secretory proteins that constitutethe SASP. The SASP includes several familiesof soluble and insoluble factors (see Table 1).These factors can affect surrounding cells by ac-tivating various cell-surface receptors and cor-responding signal transduction pathways that
Short/dysfunctionaltelomeres
NontelomericDNA damage
Oncogenes/oncogenicmutations
Chromatininstability
Strong mitogenic/stress signals
Irreversiblecell-cycle
arrest
Overexpressed cell-cycle inhibitors(p16, p21)
DNA damage = SASP No DNA damage = No SASP
Figure 1Multiple types of stimuli can provoke cellular senescence and a senescence-associated secretory phenotype (SASP). When irreversible cell-cycle arrest istriggered by severe DNA damage (i.e., dysfunctional telomeres or oncogenicstress), the SASP occurs in senescent cells. However, when a senescent-likephenotype is triggered in cells that overexpress cell-cycle inhibitors such as p16or p21, cells undergo a growth arrest with many characteristics of senescentcells, but not a SASP.
may lead to multiple pathologies, includingcancer. SASP factors can be globally dividedinto the following major categories: soluble sig-naling factors (interleukins, chemokines, andgrowth factors), secreted proteases, and se-creted insoluble proteins/extracellular matrix(ECM) components. SASP proteases can havethree major effects: (a) shedding of membrane-associated proteins, resulting in soluble versionsof membrane-bound receptors, (b) cleavage/degradation of signaling molecules, and/or(c) degradation or processing of the ECM.These activities provide potent mechanisms bywhich senescent cells can modify the tissue mi-croenvironment. In the following sections, wediscuss these SASP subsets and some of theirknown paracrine effects on nearby cells, withan emphasis on their ability to facilitate cancerprogression.
Soluble Signaling Factors as MajorComponents of the Senescence-Associated Secretory Phenotype
Senescent cells secrete interleukins, inflamma-tory cytokines, and growth factors that can af-fect surrounding cells.
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Table 1 The senescence-associated secretory phenotype (SASP). Factors significantlyaltered between presenescent and senescent states are listed
SASP factorsaSecretory profile of
senescent cellsbChanges in the SASP due to the lossof p53 and/or gain of oncogenic RAS
senescent cellsbChanges in the SASP due to the lossof p53 and/or gain of oncogenic RAS
IGFBP-2, -3, -4, -6, -7 ↑ ↑ or ×Proteases and regulatorsMMP-1, -3, -10, -12, -13, -14 ↑ ↑ or ×TIMP-1 ↓ or × ×TIMP-2 ↑ ×PAI-1, -2; tPA; uPA ↑ ×Cathepsin B ↑ ×Soluble or shed receptors or ligandsICAM-1, -3 ↑ ×OPG ↑ ↑sTNFRI ↑ ×TRAIL-R3, Fas, sTNFRII ↑ ×Fas ↑ ×uPAR ↑ ↑SGP130 ↑ ↑EGF-R ↑ ×Nonprotein soluble factorsPGE2 ↑ –Nitric oxide ↑ –Reactive oxygen species Altered –Insoluble factors (ECM)Fibronectin ↑ –Collagens Altered –Laminin Altered –
aFactors are arranged by family.bThe secretory changes that occur at senescence are indicated by upward arrows (increase), crosses (no change),and downward arrows (decrease). Loss of p53 or gain of oncogenic RAS increases (upward arrows) or decreases(downward arrows) the secretion of several SASP factors.cAbbreviations: bFGF, basic fibroblast growth factor; ECM, extracellular matrix; EGF, endothelial growth factor;GRO, growth-related oncogene; HGF, hepatocyte growth factor; ICAM, intercellular adhesion molecule;IGFBP, insulin-like growth factor–binding protein; MCP, membrane cofactor protein; MMP, matrixmetalloproteinase; NGF, nerve growth factor; OPG, osteoprotegerin; PAI, plasminogen activator inhibitor;PGE2, prostaglandin E2; PIGF, placental growth factor; SCF, stem cell factor; SDF, stromal cell–derived factor;sTNFR, soluble tumor necrosis factor receptor; t-PA, tissue-type plasminogen activator; TIMP, tissue inhibitor ofmetalloproteinases; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand; u-PA, urokinase-typeplasminogen activator; uPAR, u-PA receptor; VEGF, vascular endothelial growth factor.
IL-6. The most prominent cytokine of theSASP is interleukin-6 (IL-6), a pleiotropicproinflammatory cytokine (see Figure 2).IL-6 has been shown to be associated withDNA damage– and oncogenic stress–inducedsenescence of mouse and human keratinocytes,melanocytes, monocytes, fibroblasts, and
epithelial cells (16, 18, 57, 58). Further, IL-6secretion appears to be directly controlledby persistent DNA-damage signaling (ATMand CHK2), independent of the p53 pathway(59). Through IL-6 expression, senescentcells can directly affect neighboring cells thatexpress the IL-6R (gp80) and gp130 signaling
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IL-6 IL-8 p16
PRE
REP SEN
IR SEN
RAS SEN
Figure 2Human fibroblasts, either presenescent (PRE) or senescent (SEN), wereimmunostained for the inflammatory cytokines interleukin (IL)-6 and IL-8, aswell as the senescence marker p16. Cells were made senescent either byreplicative exhaustion (REP) or ionizing radiation (IR) or by expression ofoncogenic RAS (RAS).
complex at their surface, such as epithelialand endothelial cells of various functions andorigins.
IL-1. Another interleukin signaling pathwaydemonstrated to be upregulated by senescentcells is that of IL-1 (60, 61). Both IL-1α and-β are overexpressed and secreted by senes-cent endothelial cells (62), fibroblasts (63, 64),and chemotherapy-induced senescent epithe-lial cells (53). These cytokines can affect neigh-boring cells through the cell-surface receptors(IL-1 receptor/Toll-like receptor superfamily),which act primarily to trigger the nuclear factorkappa B and activating protein 1 pathways (65).
Chemokines (CXCL and CCL). Most senes-cent cells overexpress IL-8 (CXCL-8) (seeFigure 2), along with GROα and GROβ
(CXCL-1 and -2; the murine CXCL-1 is namedKC) (58, 66, 67). CCL family members thatare generally upregulated in senescent cells in-clude MCP-2, -4, and -1 (CCL-8, -13, and-2); HCC-4 (CCL-16); eotaxin-3 (CCL-26);
and macrophage inflammatory protein (MIP)-3α and -1α (CCL-20, -3). MCP-3 (CCL-7) isoverexpressed by senescent liver stellate cellsand by prostate and skin fibroblasts. Fibroblastsinduced to senesce by oncogenic RAS secretehigh levels of MCP-3 as well as I-309 (CCL-1).In addition, both fibroblasts induced to senesceby RAS and stellate cells induced to senesce byliver damage secrete high levels of another twomembers of the CXCL family, GCP-2 (CXCL-6) and ENA-78 (CXCL-5). Overexpression ofPF-4 (CXCL-4) and SDF-1 (CXCL-12) wasobserved in senescent prostate fibroblasts (46,68). Recently, it was shown that cells undergo-ing oncogene-induced senescence secrete mul-tiple CXCR-2 (IL-8RB)-binding chemokines(15). It was proposed that senescent cells ac-tivate a self-amplifying secretory network inwhich CXCR-2-binding chemokines reinforcegrowth arrest.
IGF pathway. The insulin-like growth fac-tor (IGF)/IGF receptor network may also con-tribute to the effect senescent cells exert ontheir microenvironment. Senescent endothe-lial, epithelial, and fibroblast cells express highlevels of almost all the IGF-binding proteins(IGFBPs), including IGFBP-2, -3, -4, -5, and-6 (18, 69, 70) and their regulators, IGFBP-rP1 and -rP2 [also known as connective tis-sue growth factor (CTGF)] (44, 71). Recently,activation of the BRAF oncogene in primaryfibroblasts was shown to lead to the secre-tion of IGFBP-7, which acts through au-tocrine/paracrine pathways to induce senes-cence and apoptosis in neighboring cells (72).
Other soluble factors. There are additionalsoluble factors associated with the SASP. Forexample, inflammatory cytokines such as thecolony-stimulating factors (CSFs, includingGM-CSF and G-CSF) are secreted at highlevels by senescent fibroblasts (18). In addi-tion, osteoprotegerin, a secreted decoy recep-tor for tumor necrosis factor alpha, is presentat high levels in the extracellular milieu ofsenescent fibroblasts. Other molecules upreg-ulated at senescence include prostaglandin E2
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(PGE2) (57, 73) and Cox-2, the enzyme re-sponsible for the production of PGE2 and otherprostaglandins.
Extracellular Proteasesas an Important Subsetof the Senescence-AssociatedSecretory Phenotype
In addition to secreting soluble signaling cy-tokines and growth factors, senescent cells alsosecrete proteases such as matrix metallopro-teinases (MMPs).
MMP family. The MMP family membersthat are consistently upregulated in humanand mouse fibroblasts undergoing replicativeor stress-induced senescence are stromelysin-1 and -2 (MMP-3 and -10, respectively) andcollagenase-1 (MMP-1) (74–78). In some in-stances, the MMP-1 and -3 produced by senes-cent cells (79) can also regulate the activity ofthe soluble factors present in the SASP. For ex-ample, these MMPs can cleave MCP-1, -2, and-4 and IL-8 (80). A variety of other CXCL/CCL family members that constitute the SASPcan also be cleaved by MMP-9, -2, or -7. TheseCXCL and CCL cytokines can originate fromneighboring cells, such as leukocytes or tumorcells (81).
Serine proteases and their inhibitors.Another family of proteases involved in car-cinogenesis and present in the SASP comprisesserine proteases and regulators of the plasmino-gen activation pathway. Members of this familyinclude urokinase- or tissue-type plasminogenactivators (uPA or tPA, respectively), the uPAreceptor (uPAR), and inhibitors of these serineproteases (PAI-1 and -2) (82). Indeed, a >50-fold increase in plasminogen activator activityhas been reported in senescent endothelialcells and lung and skin fibroblasts (83, 84).PAI-1 is also upregulated in fibroblasts andendothelial cells from aged donors (85–87).Like the CXCR-2 cytokines, PAI-1 also seemsto reinforce the senescence growth arrest(88).
Extracellular Insoluble Molecules
Fibronectin is a large multidomain glycopro-tein found in connective tissue, on cell sur-faces, and in plasma and other body fluids. Itinteracts with a variety of macromolecules, in-cluding cell-surface receptors, components ofthe cytoskeleton, and other ECM molecules.Through its interactions with cell-surface re-ceptors, primarily integrins, fibronectin can af-fect cell adhesion, survival, growth, and migra-tion. Fibronectin production is upregulated inpremature aging Werner syndrome fibroblasts(89). Moreover, cells undergoing senescence inculture and in vivo (90) increase fibronectinexpression.
Nonprotein Secretions
As a result of senescence-induced changes incellular metabolism, senescent cells may ex-ert influences on tissue microenvironments dueto the secretion of molecules other than pro-teins. These molecules include reactive oxygenspecies and transported ions. For example,senescent cells have been shown to releasenitric oxide and reactive oxygen species dueto alterations in inducible nitric oxide syn-thase, endothelial nitric oxide synthase, andsuperoxide-dismutase activities (91–95). Thesereactive molecules are known modulators ofcellular phenotype, such as the differentiationof monocytes. In addition, these moleculescan enhance cancer cell aggressiveness and canpromote aging and age-related degeneration(96, 97).
SPECIFICITY OF THESENESCENCE-ASSOCIATEDSECRETORY PHENOTYPE
Despite the fact that a significant number of fac-tors increase their secretion upon senescence,the SASP is not a general or nonspecific upreg-ulator of secretion. The levels of expression ofmany secreted factors do not change when cellssenesce. Interestingly, among these unchangedsecreted molecules are anti-inflammatory
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soluble factors such as IL-2, -4, -10, -11, and-12 (18). Fractalkine (CX3CL-1), GCP-2,GITR, PDGF-BB, and LIGHT (all essentialto leukocyte differentiation or proliferation)also remain unchanged when fibroblasts areinduced to senesce by X-irradiation, RASoverexpression, or replicative exhaustion.Intriguingly, no factor was significantlydownregulated in different senescent states(18).
Despite a specific, conserved core of up-regulated and unchanged secreted molecules,different senescent states appear to displaysome unique features. Whereas cells inducedto senesce by replicative exhaustion, telomeredisruption, X-irradiation, or chromatin disrup-tion seem to express closely related SASPs (18,59), fibroblasts induced to senesce by oncogenicRAS oversecrete more GM-CSF, IL-6, -7, -8,-1β, -13, and GROα than do cells induced tosenesce by other means. Moreover, such cellssecrete high levels of factors such as ENA-78,I-309, G-CSF, and interferon (IFN)-γ that arenot secreted by other senescent cells. By con-trast, cells induced to senesce by overexpres-sion of the p16INK4a tumor-suppressor proteindo not express a SASP despite other hallmarksof senescence ( J.P. Coppe, F. Rodier & J. Camp-isi, unpublished data). Cells that senesce withdysfunctional p53 develop a SASP that resem-bles the SASP caused by oncogenic RAS (18).Thus, although a core of SASP factors is a fea-ture of all senescent cells (with the exception ofp16INK4a-induced senescence), there are varia-tions in the quantity and quality of the SASPthat depend on the cell type and senescenceinducer.
Another feature of the SASP is its dynamicdevelopment over time (18). In culture, cellsdevelop a full SASP >5 days after senescenceinduction, and the cells’ growth arrests within24 h of damage. Not all SASP factors begin to besecreted at the same time. This gradual pheno-typic transition is a feature conserved betweencell types and senescence inducers. Genetic al-terations, such as loss of p53 or gain of onco-genic RAS, lead to a more rapid acquisition of
the SASP, suggesting that the SASP is a specificprogram triggered by genotoxic stress.
Finally, mouse senescent fibroblasts also dis-play a SASP. Under standard cell-culture con-ditions, which include 20% oxygen, mousecells undergo an arrest that has been termedsenescence but that does not include a SASP.By contrast, under physiological 3% oxygen,the mouse SASP closely resembles the hu-man SASP. These findings suggest that thesenescent cell secretome is specific and evolu-tionarily conserved ( J.P. Coppe & J. Campisi,unpublished data).
Intracellular Signaling, Transcription,and Chromatin: Locking in theSenescence-AssociatedSecretory Phenotype
Overall, the gene (mRNA) expression profilesof senescent cells determined by microarraysresemble the profiles of secreted proteins de-termined by antibody arrays (18; J.P. Coppe &J. Campisi, unpublished data). This finding sug-gests that the secretory phenotype of senescentcells is at least partly regulated at the transcrip-tional level. However, because the changes ingene expression that occur at senescence areso widespread, the transcriptional control maywell be at the level of chromatin organization,rather than due to changes in specific transcrip-tion factors. In fact, dramatic chromatin alter-ations are known to occur at senescence (98–101). Further support for the idea that geneexpression specific to the senescence programmay be partially attributed to larger changesin chromatin conformation is suggested by thephysical clustering of genes that constitute theSASP ( J.P. Coppe & J. Campisi, unpublisheddata).
Our data suggest that a large propor-tion of the SASP of senescent fibroblastsis irreversible once established (18). Indeed,
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senescent human fibroblasts that express lowlevels of p16INK4a can revert and resume pro-liferation upon p53 inactivation (102). Thesereverted cells, however, retain the SASP (18).This may imply that, once senescence is es-tablished, unknown mechanisms—potentiallyrelated to chromatin alterations—permanentlylock the SASP in an irreversible open chro-matin confirmation, analogous to the way thep16INK4a/pRB pathway is proposed to lockgrowth-promoting genes into a heterochro-matic state (103). Another implication of thesefindings is that the senescence-associated cell-cycle arrest and the SASP can be uncoupled (seeFigure 1). Further, the SASP is a more per-manent characteristic of senescence than is thegrowth-arrested state.
Cell-Nonautonomous TumorSuppressors: The Guardiansof the Senescence-AssociatedSecretory Phenotype
The p16INK4a tumor suppressor is a posi-tive regulator of the pRB tumor-suppressorpathway. High levels of ectopic expression ofp16INK4a induce senescence. Induction of en-dogenous p16INK4a is associated with tumorprevention and the general age-associated de-cline in stem cell and tissue function (104–109).Although p16INK4a is a very efficient inducerof cell-cycle arrest (including the senescence-associated arrest), p16INK4a does not seem toplay a major role in the development of theSASP ( J.P. Coppe, F. Rodier & J. Campisi, un-published data). Cells induced to senesce by ec-topic p16INK4a expression secrete significantlylower levels of SASP factors compared to cellsinduced to senesce by most other senescence in-ducers. Senescence induced by p16INK4a has po-tential therapeutic applications. As an example,p16INK4a gene therapy for rheumatoid arthritiswas demonstrated to efficiently stop the diseaseevolution and decrease the inflammatory state(110). In cancer, such an approach could takeadvantage of both the cell-autonomous prop-erties (inhibition of cell growth) and the cell-nonautonomous properties (senescence arrest
without a SASP) of p16INK4a. That is, p16INK4a
induction/delivery could actively suppress cellproliferation without triggering the proinflam-matory SASP.
The p53 tumor suppressor can also pro-mote aging (111, 112) and senescence (7, 11,12) in mice (see Figure 3). Along with mu-tations in p16INK4a, mutations leading to p53inactivation occur very frequently in cancercells; that is, p53 is well known to act asa cell-autonomous tumor suppressor by con-trolling apoptosis and cell-cycle arrest, bothin culture and in vivo. However, p53 mu-tations have also been found in the stromalvicinity of carcinomas, and this p53-deficientstroma was shown to promote tumorigenesis(32). These data suggest that p53 may havebeneficial cell-nonautonomous effects in pre-venting cancer development (32, 113). Strik-ingly, we found that p53 actively restrains theSASP, suggesting a potential mechanism bywhich p53 may suppress tumorigenesis—thatis, by restraining the development of a pro-tumorigenic/proinflammatory tissue microen-vironment (18). Thus, loss of p53 activity bysenescing or damaged fibroblasts greatly en-hances the SASP and the stimulatory effects ofthe SASP on malignant epithelial cells (as dis-cussed further below) (18).
EFFECTS ON CELL BEHAVIOR
Factors secreted by senescent cells can pro-mote tumor development in vivo and malignantphenotypes such as proliferation and invasive-ness in cell-culture models. These effects havebeen observed in a number of tissues, includingbreast (13, 18, 77, 78, 114), skin (115), prostate(18, 116), pancreas (117), and oropharyngialmucosa (14). The effects of the complex SASPare, of course, dependent on the tissue context.Thus, different models show different effects ofthe SASP. In the following sections, we discussin greater detail the various behavioral changescells can undergo when residing in the prox-imity of senescent cells and how the senescenttissue microenvironment can facilitate tumorinitiation and progression.
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Damage sensorsand transducers
Transient arrest andfaithful repair p53
Senescencearrest
Cancer,aging, pathologies SASP
Alarm signals andtissue repair
Chronic SASPand persistentinflammation
Early(?)
Later(?)
Intrinsic or extrinsicgenotoxic stress
Figure 3The DNA damage signaling pathway leads to the activation of the p53 tumor suppressor. Activated p53triggers cell fate decisions, such as senescence or apoptosis. Depending on the cell context, p53 can suppresscancer through transient cell-cycle arrest and activation of the DNA-repair machinery. Additionally, p53restrains the senescence-associated secretory phenotype (SASP). Regulation of the SASP by p53 suggests acell-nonautonomous function of this tumor suppressor. In the short term, the SASP may promote tissuerepair. In the long term, it may promote chronic inflammation, which in turn can drive cancer and aging.
The Senescence-AssociatedSecretory Phenotype PromotesCell Proliferation
One of the most protumorigenic effects of theSASP is to promote the proliferation of epithe-lial cells.
Breast cancer. In the case of breast epithe-lial cells, senescent human fibroblasts can stim-ulate the growth of premalignant and malig-nant mammary epithelial cells (13, 18, 77). Thisstimulation may be due in large measure to se-cretion of GROα, which is a prominent SASP
component ( J.P. Coppe & J. Campisi, unpub-lished data). Irradiated stromal cells, which arepresumed to be senescent, have been shownto perturb mammary epithelial microenviron-ment and to fuel inappropriate epithelial cellgrowth in the mammary gland (114, 118). Fur-thermore, MMPs secreted by senescent fibro-blasts have been shown to be responsible forthe higher tumorigenicity of breast epithelialcell xenografts in mice, most likely by allowingmitogenic and chemotactic signals greater ac-cess to breast cancer cells (78, 114). In additionto secreted soluble factors, there is evidencethat the matrix laid down by senescent cells can
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also stimulate mammary epithelial cell growth(13).
Prostate cancer. Fibroblasts from the humanprostate gland that undergo senescence in cul-ture have been shown to create a local tissueenvironment that favors prostate epithelial cellhyperproliferation, in part owing to amphireg-ulin secretion (46). Furthermore, CTGF (orIGFBP-rP2) is upregulated in senescent fibro-blasts (44), and this protein was shown to reg-ulate prostate tumor progression in xenograftsand to be expressed by the cancer-associated re-active stroma (119). Whereas upregulation ofCXCR-4 is observed in most cancer cells, onlysenescent stromal cells of the prostate displayhigh levels of expression of its ligand SDF-1α (CXCL-12). Thus, SDF-1α secretion bysenescent fibroblasts may therefore play a se-lective role in fueling prostate cancer. It hasrecently been determined that senescence in-duced by irradiation in prostate cancer patientsis associated with a significantly increased re-lease of exosome-like microvesicles (120). Thisnovel secretory phenotype depends on the acti-vation of p53. Finally, the propensity of prostatecancer patients to relapse after chemotherapymay be due to the accumulation of senescenttumor cells with inflammatory characteristics(18).
Other carcinomas. In the skin, unidentifiedfactors secreted by human fibroblasts wereshown to be capable of inducing clonal ex-pansion of keratinocytes (121). In addition,senescent endometrial fibroblasts promotedanchorage-independent epithelial cell growth,primarily because of IL-1 oversecretion (64). Inthe orobucal cavity, tobacco-driven senescenceof supportive stromal cells was shown to stim-ulate the hyperplastic growth of epithelial cellsand was associated with the loss of tight junc-tions and epithelial integrity (14).
Melanoma. Melanocytic nevi (moles) are of-ten composed of senescent melanocytes thatare induced owing to oncogenic mutations inBRAF (V600E mutations) (9). Only rare cell
variants in nevi can evolve into melanoma.Malignant melanocytes express high levels ofthe CXCR-2 receptor (122) and can be stim-ulated to grow by its ligands GROα (123)and IL-8 (124). Given that both GROα andIL-8 form part of the core SASP, the senes-cent microenvironment may therefore stimu-late the proliferation of rare premalignant cellsin nevi, thereby leading to the development ofmelanoma.
Other tumor-associated cells. During an-giogenesis, endothelial cells can undergo pro-liferation, which is stimulated by vascular en-dothelial growth factor (VEGF), IL-8, I-309,and eotaxin (125–127). The proangiogenic ef-fects of the SASP were shown in vivo in mousexenografts of breast cancer cells. The blood ves-sel density was significantly higher when the tu-mors developed in the presence of senescent butnot presenescent fibroblasts (127). RAS-driventumors are also known to contain significantnumbers of senescent cells (8), and these tu-mors are also highly vascularized (128). Manyof the SASP factors can also affect leukocyteproliferation during the course of cancer devel-opment. For example, IL-7 directly promoteslymphocyte proliferation in peripheral tissues,and GM-CSF stimulates myeloid suppressorcells, which are known to have important im-munosuppressive functions that affect cancerprogression (129).
The Senescence-Associated SecretoryPhenotype Stimulates Cell Motility(Invasion, Migration, Metastasis)
Senescent cells secrete an array of factors thatcan create a gradient to promote cell migrationand invasion.
Epithelial cells. In pancreatic cancer, hepato-cyte growth factor (HGF), and to a lesser de-gree basic fibroblast growth factor (bFGF), pro-motes cancer cell invasion in culture and canpotentially drive cancer dissemination in vivo(117, 130). In breast cancer, high levels of IL-6 and -8 secreted by senescent fibroblasts are
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responsible for enhancing the invasiveness of apanel of cancer cell lines in cell-culture mod-els (18). Moreover, the secretion of MMP-2and -3 by senescent cells can also promotethe invasion of multiple epithelial cell types(77, 78, 114, 131, 132). Other proteases, suchas uPA and its regulator (PAI-1), are likewiseimplicated in cancer cell invasion. Senescentstromal cells may promote an epithelial-to-mesenchymal transition (18), which is an im-portant phenotypic switch that enables cancercells to migrate and invade. Through use of co-cultures of smokeless tobacco extract–exposedfibroblasts and human epidermal keratinocytes,factors secreted by extract-modified fibroblastsincreased the invasiveness of partially trans-formed epithelial cells in conjunction with aloss of E-cadherin, zonula occludens 1, and in-volucrin expression (14). Thus, senescent cellsand the SASP can induce phenotypes in nearbyhuman epithelial cells that are common duringcancer progression.
Endothelial cells. In cell-culture models, en-dothelial cells are induced to migrate by fac-tors secreted by senescent fibroblasts (127).This is in part due to VEGF secretion andchemokine gradients set up by senescent cells(133). Neoangiogenesis, which is dependent onendothelial cell motility and invasion, is en-hanced in xenograft models containing senes-cent fibroblasts (127). Further, it is knownthat IL-1, a SASP component, activates theendothelium and consequently increases theadhesive potential of cancer cells to vesselwalls (134). Thus, senescent cells may pro-mote extravasation of cancer cells to secondarymetastatic sites. However, the effects of senes-cent cells on angiogenesis may depend oncell type. For example, senescent keratinocytesoversecrete maspin, which displays paracrineantiangiogenic activity and acts as a domi-nant inhibitor of endothelial cell migration(135).
Leukocytes. Senescent fibroblasts may pro-mote leukocyte recruitment because theychronically release chemokines (136). In
p53-deficient RAS-driven tumors induced tosenesce through reestablishment of p53 func-tion (12), innate immune cells were shown tomigrate into the vicinity of the senescent tumorarea. CSF-1, CXCL-1, or MCP-1 and ICAM-1 transcripts were found to be higher in thesesenescent tumor masses, and they may be re-sponsible for the immune response. For exam-ple, neutrophils express CXCR-1, -2, and -4 tosense their microenvironment and to invade tis-sues; eosinophils use the broad-spectrum recep-tor CCR-3 to fulfill their function; monocytesuse CCR-1, -2, and -5, CXCR-4, and CX3CR1to extravasate and enter peripheral sites, wherethey differentiate; natural killer cells expressCCR -2 and -5, CXCR-4, CX3CR1, and XCR1;and immature myeloid dendritic cells displayCCR-1, -2, -5, and -6 and CXCR-4, which fa-cilitate their transport, migration, and function(136–139).
The Senescence-AssociatedSecretory Phenotype RegulatesCell Differentiation
The factors secreted by senescent cells can alterthe differentiation status of neighboring cells.
Epithelial cells. Senescent human and mousefibroblasts disrupt the differentiation of mam-mary epithelial cells and inhibit the expressionof differentiation markers (77, 114). This activ-ity is due in large measure to the secretion ofMMP-3 by the senescent cells. Furthermore,weakly tumorigenic pancreatic (117) and mam-mary (18) epithelial cells undergo morphologicchanges in culture that resemble an epithelial-to-mesenchymal transition in the presence of asenescent conditioned medium. The effect onmammary epithelial cells is attributable to IL-6 and -8 (18), as well as to HGF, uPAR, andMMPs (77), all of which can disrupt epithelialcell clusters and stimulate dedifferentiation inculture and in vivo (140–142).
Endothelial cells. Strikingly, no angiostaticfactors have been reported among SASP con-stituents (e.g., IFN-γ, TSP-1, MIG, PF4,
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IP-10, IL-4 and -12, and endostatin). This con-trasts with the largely proangiogenic profile ofthe SASP (which includes IL-8, MCP-1 and-2, GROs, PGE2, VEGF, EGF, CSFs, u-/tPA,MMPs, fibronectin, and laminin) (143). Fur-thermore, there may be an amplifying activa-tion loop because senescent stromal cells se-crete MCPs, CSFs, MIPs, GROs, and CXCLs,which in turn recruit inflammatory and immunecells that also secrete proangiogenic factors(VEGFs, IL-8, and MMPs). Thus, senescentcells are poised to support the differentiationof a new vasculature around and within a pro-gressing tumor.
The Senescence-Associated SecretoryPhenotype Affects LeukocyteInfiltration and Tumor Immunology
No anti-inflammatory factors (e.g., IFN-α,IFN-γ, IL-3, and IL-5) are significantly se-creted by senescent fibroblasts, and some ofthese factors are even downregulated uponsenescence (e.g., IL-2 and -12). Nonetheless,some reports show that massive amounts ofeither MCP-1 or IL-8, which are prominentcomponents of the SASP, lead to tumor de-struction (144, 145). Senescent fibroblasts mayinfluence the macrophage balance in the tumorenvironment. Molecules that are implicated inthe recruitment and differentiation of circulat-ing monocytes to tumor sites also happen tobe overexpressed by senescent fibroblasts (146).These molecules can lead to an inadequate im-mune response within the close proximity ofsenescent cells. Senescent fibroblasts may affectlymphocytic populations infiltrating the tumor.Specific T cell populations associated with tu-mor progression (i.e., Th2 and regulatory Tcells) respond to inflammatory cytokines thatare commonly present in the fibroblast SASP.Other cells of the specific and innate immunesystem, such as natural killer cells, neutrophils,eosinophils, dendritic cells, and B cells, are alsosubject to regulation by cytokines that are pro-duced by senescent fibroblasts.
CONCLUSIONS AND FUTUREDIRECTIONS
Most insoluble components of the ECMare enzymatic targets of secreted proteases.Therefore, the senescence-associated changesin proteolytic activities could affect the phys-ical properties of the tissue structure aroundcells. In particular, the accumulation of senes-cent cells could lessen the supportive role of theECM, globally diminishing tissue tension andelasticity. In addition, the relaxed tissue struc-ture and higher levels of MMPs may help tumorcells migrate and invade through the ECM, thusenabling metastasis. The panel of proteases se-creted by senescent cells extensively overlapswith those found in malignant tumors. Further,senescence-induced alterations in the secretionof interleukins, chemokines, growth factors,proteases, and associated processing activitiestend to establish the SASP as protumorigenic.
Overall, senescence is a molecular programwith a unique phenotypic outcome. How its ex-tracellular molecular signature is activated andmaintained and the extent to which it influencesthe tissue milieu in healthy tissues, aged tissues,and diseased tissues are some of the many ques-tions that remain unanswered. However, evenwith our currently limited knowledge of theSASP and its potential effects on carcinogen-esis, promising new strategies for cancer ther-apies are possible. For example, restoring theactivity of tumor-suppressor proteins is anattractive, potentially powerful therapeuticapproach. Taking into account our present un-derstanding of the cell-nonautonomous effectsof tumor-suppressor genes such as p53, smallchemicals that can pharmacologically restoretheir normal function would help reestablishthe proper tissue and cell signals, therebystimulating cancer regression (147–150). Suchapproaches could stimulate cancer eliminationfor two reasons: First, they would limit inflam-mation and thus possibly allow proper tissuerepair; second, they would directly promotethe immune-mediated clearance of cells thatdrive cancer progression.
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DISCLOSURE STATEMENT
A.K. is CEO and CSO of StemLifeLine, Inc., a biotech company that uses stem cells for drugscreening and therapy. The other authors are not aware of any affiliations, memberships, funding,or financial holdings that might be perceived as affecting the objectivity of this review.
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The Pathogenesis of Acute Pulmonary Viral and Bacterial Infections:Investigations in Animal ModelsMary F. Lipscomb, Julie Hutt, Julie Lovchik, Terry Wu, and C. Rick Lyons � � � � � � � � � � � 223
